ADVANCES IN CANCER RESEARCH Volume I
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ADVANCES IN CANCER RESEARCH EDITED BY JESSE P. GREENSTEIN National Cancer Institute, US.Public HealthService, Bethesdu, Maryland ALEXANDER HADDOW Chester Beatty Research Institute, Royal Cancer Hospital, London, England
Volume I
ACADEMIC PRESS INC., PUBLISHERS NEW YORK, N.Y. 1953
COPYRIGHT
1963, BY
ACADEMIC PRESS INC. 126 East 23rd Street, New York 10, N.Y.
All Rights Reaerved No part of this book may be reproduced in any form, by photostat, microfilm, or any other means, without written permission from the publishers. Library of Congress Catalog Card Number: 52-13360
PRINTED IN THE UNITED STATES OF AMERICA
CONTRIBUTORS TO VOLUME I C. A. COULSON, Wheatstone Physics Department, King’s College, London, England E. V. COWDRY, Wernse Cancer Research Laboratory and Department of Anatomy, Washington University, St. Louis, Missouri L. DMOCHOWSKI, Department of Experimental Pathology and Cancer Research, School of Medicine, University of Leeds, Leeds, England
W. U. GARDNER,Yale University School of Medicine, New Haven, Connecticut R. J. C. HARRIS,Chester Beatty Research Institute, Royal Cancer Hospital, London, England CHARLES HEIDELBERQER, The M cArdle Memorial Laboratory, for Cancer Research, The Medical School, University of Wisconsin, Madison, Wisconsin
ELIZABETH C. MILLER,The McArdEe Memorial Laboratory for Cancer Research, The Medical School, University of Wisconsin, Madison, Wisconsin
JAMESA. MILLER,The McArdle Memorial Laboratory for Cancer Research, The Medical School, University of Wisconsin, Madison, Wisconsin
W. C. J. Ross, Chester Beatty Research Institute, Royal Cancer Hospital, London, England HERBERT SILVERSTONE, Department of Cancer Research, Medical Research Institute, Michael Reese Hospital, Chicago, Illinois ALBERTTANNENBAUM, Department of Cancer Research, Medical Research Institute, Michael Reese Hospital, Chicago, Illinois RICHARD J. WINZLER,Department of Biological Chemistry, University of Illinois College of Medicine, Chicago, Illinois
V
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PREFACE Cancer is a disease which has been recognized since ancient times, and which in every generation has claimed many victims of all ages and of all stations in life. Each generation through its medical practitioners has fought it with whatever ideas and tools were available at the time. It is a paradox that, as these ideas have become ever more clear and these tools ever more powerful, the proportion of individuals dying of cancer appears to have risen from year to year. Whatever the reason for these melancholy statistics may be, no matter whether they may be more apparent than real, it is not in the tradition of science to stand idly by in the face of this seeming failure. A society living in the midst of scientific miracles may rightly expect that this disease, like many others, should be comprehended and mastered. Although the comprehension and the successful therapy of a disease are not invariably related, it is the hope of rational men, confident in the scientific method, that if only a phenomenon were understood it could be controlled. Few more notable expressions of this faith in our time are evident than in the public and private support granted in many lands to the subject and field of cancer research. A host of scientific specialties has been marshaled to meet this challenge. As the search for the understanding of cancer proceeds, new scientific approaches develop and are emphasized, and older ones, for the time being perhaps, subside and diminish. The ebb and flow of ideas and experimental approaches in the field of cancer research, as in any of the creative areas of the arts and sciences, is a mysterious and inexplicable process. It is the purpose of the Editors that this and succeeding volumes of this series shall reflect this steady and inevitable march of the tides of our knowledge and increasing understanding. For this task, we must rely upon the generous cooperation of our colleagues in many lands, distinguished authorities in various branches of cancer research, t o review, synthesize, and interpret the advances made in their individual areas of investigation. It is our hope that these pages will reveal from year t o year the gallant and dedicated quest for comprehension and mastery of an ancient and elusive disease.
THE EDITORS
January, 1963 vii
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CONTENTS CONTRIBUTORS TO VOLUME 1. . . . . . . . . . . . . . . . . . . . . . .
V
EDITORS' PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . .
vii
Electronic Configuration and Carcinogenesis
BY C . A. COULSON, Wheatstone Physics Department, King's College. London. England I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 I1. Historical Survey . . . . . . . . . . . . . . . . . . . . . . . . . 3 I11. Valence-Bond, or Resonance. Method . . . . . . . . . . . . . . . . 8 . . . . . . . . . . . . . . . . . . . . . 20 V . Electrical Index for the K-Region . . . . . . . . . . . . . . . . . . 30 VI . Possible Mechanisms . . . . . . . . . . . . . . . . . . . . . . . 46 V I I . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Epidermal Carcinogenesis
BY E. V. COWDRY, Wernse Cancer Research Laboratory and Department of Antomy, Washington University, St . Louis. Missouri I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 I1. Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 I11. Sequence in Experimental Epidermal Carcinogenesis . . . . . . . . . . 66 IV. Microscopic Properties . . . . . . . . . . . . . . . . . . . . . . . 69 V . Chemical Properties of Whole Epidermis . . . . . . . . . . . . . . . 74 VI Integration of Data . . . . . . . . . . . . . . . . . . . . . . . . 85 VII . Indications Concerning Human Epidermal Carcinogenesis . . . . . . . 93 VIII . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
.
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The Milk Agent in the Origin of Mammary Tumors in Mice
BY L DMOCHOWSKI. Department of Experimental Pathology and Cancer Research. School of Medicine. University of Leeds. Leeds. England I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 I1 The Milk Agent and Genetic Factors . . . . . . . . . . . . . . . . 109 I11. The Milk Agent and Hormonal Factors . . . . . . . . . . . . . . . 119 IV The Milk Agent and Mammary Gland Structure . . . . . . . . . . . 127 V Inherited Hormonal Influence . . . . . . . . . . . . . . . . . . . . 129 VI . Properties of the Milk Agent . . . . . . . . . . . . . . . . . . . . 132 VII . Mammary Tumors in Hybrid Mice and the Milk Agent . . . . . . . . 148 VIII . The Nature of the Milk Agent . . . . . . . . . . . . . . . . . . . 156 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
. . .
Hormonal Aspects of Experimental Tumorigenesis
BY W. U . GARDNER,Yale University School of Medicine, New Haven. Connecticut I Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 I1. General Statements on Tumorigenesis . . . . . . . . . . . . . . . . 174 ix
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X
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CONTENTS
I11 Types of Experimental Hormonal Imbalances . . . . . . . . . . . . . IV . Influences of Differences in “Substrate” on Differences in Response . . V. OvarianTumors . . . . . . . . . . . . . . . . . . . . . . . . . VI Testicular Tumors . . . . . . . . . . . . . . . . . . . . . . . . . VII . Adrenal Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Pituitary Tumors . . . . . . . . . . . . . . . . . . . . . . . . . I X . Lymphoid Tumors . . . . . . . . . . . . . . . . . . . . . . . . . X . Uterine Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . XI. Mammary Glands . . . . . . . . . . . . . . . . . . . . . . . . . XI1. Hormones in Relation to Tumors of the Secondary Sex Organs of Males . XI11 Other Tissues or Organs in Which Sex or Sex Hormones Modify the Appearance of Tumors . . . . . . . . . . . . . . . . . . . . . . XIV Urinary Tract . . . . . . . . . . . . . . . . . . . . . . . . . . XV . General Discussion . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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. .
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178 180 184 194 198 200 204 207 211 219 220 221 221 223
Properties of the Agent of Rous No 1 Sarcoma
BY R . J . C. HARRIS,Chester Beatty Research Institute, Royal Cancer Hospital, London, England I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 11 Agent and Host . . . . . . . . . . . . . . . . . . . . . . . . . . 235 I11 Agent and Malignant Cell . . . . . . . . . . . . . . . . . . . . . 243 I V . Isolation and Properties of Rous No . 1 Agent . . . . . . . . . . . . . 250 V. Relationship of Rous Agent to Fowl Tumors and Leucoses . . . . . . . 261 V I Origin of Rous Agent . . . . . . . . . . . . . . . . . . . . . . . 264 VII Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
. . . .
Applications of Radioisotopes to Studies of Carcinogenesis and Tumor Metabolism BY CHARLES HEIDELBERGER, The McArdle Memorial Laboratory for Cancer Research, The Medical School, University of Wisconsin, Madison, Wisconsin I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 I1. Metabolism of Carcinogenic Hydrocarbons. . . . . . . . . . . . . . 279 I11. Other Carcinogenic Compounds . . . . . . . . . . . . . . . . . . . 290 IV Oxidative Metabolism of Tumors . . . . . . . . . . . . . . . . . . 293 V. Incorporation of Amino Acids into Tumor Proteins . . . . . . . . . . 301 V I Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 VII . Miscellaneous Compounds . . . . . . . . . . . . . . . . . . . . . 326 VIII Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
. . .
The Carcinogenic Aminoazo Dyes
BY JAMES A . MILLERand ELIZABETH C . MILLER,The McArdle Memorial Laboratory for Cancer Research, The Medical School, University of Wisconsin, Madison, Wisconsin I . General Introduction . . . . . . . . . . . . . . . . . . . . . . . 340 I1. Early Observations . . . . . . . . . . . . . . . . . . . . . . . . 341 I11 4-Dimethylaminoaeohensene and Its Derivatives . . . . . . . . . . . 342
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CONTENTS
xi
IV . Studies on the Hepato-Carcinogenicity of Other Azo Dyes . . . . . . . 379 V . On the Mechanism of Azo Dye Carcinogenesis . . . . . . . . . . . . 383 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
.
The Chemistry of Cytotoxic Alkylating Agents
BY W C. J . Ross, Chester Beatty Research Institute, Royal Cancer Hospital. London. England I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 397 I1. 2-Chloroethyl Sulfides (Sulfur Mustards) . . . . . . . . . . . . . . 399 I11 ZChloroethylamines . . . . . . . . . . . . . . . . . . . . . . . . 411 IV . 1.2-Epoxides . . . . . . . . . . . . . . . . . . . . . . . . . . . 429 V . Miscellaneous Agents . . . . . . . . . . . . . . . . . . . . . . . 436 V I . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446
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Nutrition in Relation to Cancer BY ALBERT TANNENBAUM and HERBERT SILVERSTONE. Department of Cancer Research. Medical Research Institute. Michael Reese Hospital. Chicago. Illinois Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 452 I . Some General Considerations . . . . . . . . . . . . . . . . . . . . 453 455 I1. Genesis of Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . I11 Growth of Tumors . . . . . . . . . . . . . . . . . . . . . . . . 481 IV . Nutritional State and Cancer in Man . . . . . . . . . . . . . . . . 487 491 V . Conclusions and Commentary . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497
.
Plasma Proteins in Cancer
BY RICHARD J . WINZLER.Department of Biological Chemistry. University of Illinois College of Medicine. Chicago. Illinois I . Some Methods of Study of Plasma Proteins . . . . . . . . . . . . . 506 I1. Alterations of Plasma Proteins in Neoplastic Disease . . . . . . . . . . 513 I11. Plasma Enzymes and Inhibitors . . . . . . . . . . . . . . . . . . . 529 IV . Protein-Bound Carbohydrate . . . . . . . . . . . . . . . . . . . . 535 V. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 538 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 539 AUTHOR INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
549
SUBJECT INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
573
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Electronic Configuration and Carcinogenesis C. A. COULSON Whealstone Physics Department, King’s College, London*
CONTENTS
Page I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 11. Historical Survey. . . . . . ............. 3 1. The K-Region.. . . . . ............. 3 2. Schmidt’s Box Mode ............................. 4 3. Svartholm’s Introdu ............. 6 7 4. The Work of Pullman, Daudel, and Others.. . . . . . . . . . . . . . . . . . . . . . . . . 111. Valence-Bond, or Resonance, Method.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1. u and IT Electrons.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2. Valence-Bond Structures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3. Some Complicating Features in the Valence-Bond Method.. . . . . . . . . . . 13 4. Derived Quantities.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 A. Bond Order.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 B. Charge Density and Distribution.. . . . . . . . . . . . C. Freevalence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 5. Methyl Substitutions, Hyperconjugation, . . . . . . . . . 6. Aza Replacement. .. ...................... 18 7. Penney Bond Orders.. . .......... . . . . . . . . 19 IV. Molecular-Orbital Method. . . . . . . . . . . . . . . . . . . . . . . . . 1. Molecular Orbitals.. ... .... . . . . . . . . . . . . . . 20 2. The LCAO Representation.. . . . . . . . . . . . . . . . . . . . . 3. Fundamental Magni ...................... 24 4. Some Particular Results.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 5. Polarizabilities. . . . . . . . . .................... 26 6. Hyperconjugation. .... ..................................... 27 7. Direct Tests of Theory ..................................... 28 V. Electrical Index for the .................. 1. Electrical Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Bond Orders in the 3. Free Valence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 4. Charge on the Atoms.. . . . . . . . . . . . . . . . . . 33 5. Pullman’s Work on 6. Molecular-Orbital Indices. . . . . . ........................ 40 7. Resonance Energy, 8. Electronic Excitation. . . . . . . .................................. 43 VI. Possible Mechanisms. . 1. Interpretation of Pr
* Now at the Mathematical Institute, Oxford. 1
2
C. A. COULSON
2. Advantages of the K-Region.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Significance of the Total Charge on the K-Region.. . . . . . . . . . . . . . . . . . . 4. Some Speculations... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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46 47 50 62 54
I. INTRODUCTION This account of the possible relationship between electronic configuration and carcinogenesis falls naturally into six sections. These are : (1) historical survey, (2) the quantum-mechanical resonance method of describing large unsaturated molecules, (3) the alternative molecularorbital method, (4) numerical applications of these two methods, ( 5 ) possible mechanisms of carcinogenic activity, and (6) conclusions. Of these (2) and (3) are not solely concerned with carcinogenic properties, but no reasonably simple and straightforward account of the two methods discussed seems to be available. This is particularly important if we bear in mind that both methods are approximations, whose reliability is not fully established. We shall see that although there is a considerable measure of mutual agreement, there are several places in which they disagree. As a result of this, there is still a certain amount of personal liberty possible in the interpretation of the calculations. Indeed, it may be admitted a t once that no final or complete account of the relation between electronic configuration and carcinogenesis has, or can, yet be given. The present account will therefore attempt to stress both the undoubted successes of the theory and at the same time some of its equally patent inadequacies. The whole field is a singularly interesting one, because it represents one of the very first serious attempts to relate what are obviously complex biological phenomena to quantum-mechanical principles. Its significance lies not only in the fact that carcinogenic potency (or otherwise) has been correctly predicted on purely theoretical grounds for quite a large number of molecules which had not, at that time, been investigated experimentally; but also in the fact that it opens up new fields of enquiry and discovery, and itself suggests new interpretations: this must inevitably lead to a better understanding of such phenomena as cocarcinogenesis, anticarcinogenesis, drug action, chemical mutations, and the mechanism of estrogenic and other hormone activity. There is little doubt but that in the next two or three decades we may expect enormous and far-reaching developments in these fields; and the developments are likely to be considerably facilitated if there is a fairly large body of experimenters who are familiar with the quantummechanical basis on which, quite evidently, the action of these chemicals must ultimately depend. It is for this reason that (2) and (3) have been
ELECTRONIC CONFIGURATION AND CARCINOOENESIS
3
written in their present form. Those who are already familiar with wave-mechanical methods can pass straight to the remaining sections of this review. 11. HISTORICAL SURVEY 1 . The K-Region
It was established about twenty years ago that certain polycyclic hydrocarbons have the property of inducing cancerous growths, either when painted on the skin or injected into the animal concerned. Nearly
Phenanthrene (1)
all these molecules could be regarded as derivatives of phenanthrene (I), though phenanthrene itself is not active. The staggered ring system of phenanthrene seems to be much more effective in promoting carcinogenic activity than does the straight type of annelation shown in anthracene (11),derivatives of which are hardly ever active. (An exception is 9,lOdimethylanthracene, which is the simplest known carcinogen among the hydrocarbon family. No satisfactory explanation of this has yet been suggested.) It is customary now t o distinguish between the anthracene -and phenanthrene-type skeletons by saying that in the latter the 9,lO-region has a character quite distinct from anything in the former. Following Mme. Pullman ( 1 9 4 6 ~1947c) ~ we shall call this the K-region. The K-region is easily recognized. Thus in the extremely important parent hydrocarbon 1,2-benzanthracene (111) there is one K-region, as shown by a thick line: in 3,4-benzphenanthrene (IV) and 1,2,5,6-dibenzanthracene (V) there are two K-regions. Our study of these molecules will largely consist of an enquiry concerning the electronic distribution in these regions and of the ways in which this distribution is affected by
1 ,l-Benxanthracene (111)
3,4-Benzphenanthrene (IV)
1,2,5,6-Dibenzanthracene (V)
4
C. A. COULSON
substitution (particularly methyl substitution), or by an aza replacement such as occurs when benzanthracene (111) is compared with benzacridine
(VI)-
8’
3,4-Benzacridine* (VI)
A t this stage reference must be made to the exceedingly valuable compilation of Hartwell (1941), who has listed all the available published (and unpublished) experimental results with molecules of the kind we are interested in. Practically all the experimental conclusions mentioned in this review are quoted from Hartwell’s quite invaluable tables. To this, and to the review article by Badger (1948) the writer is greatly indebted. 2. Schmidt’s Box Mode2 The first attempt to explain the significance of the K-region was due to Schmidt (1938, 1939a,b,c, 1941). In essence this amounted chiefly to an explanation, largely in classical terms (Schmidt’s wave-mechanical considerations were somewhat speculative, and have never gained general acceptance), to show why this region might be expected to behave differently from any other region in an aromatic system. Schmidt started with the hypothesis that there were certain regions, or groupings, of peculiar stability, which would try to preserve their character in any chemical reaction, so far as that was possible. Such groupings include benzene and naphthalene, but they do not include open-chain structures such as butadiene. The complete molecule can then be cut up into separate units as in Fig. 1, which suggests that phenanthrene and benzanthracene (a) should exhibit peculiarly strong reactivity precisely in their K-regions, but anthracene (a) should not. Rather is it that the reactivity of anthracene should reside in the meso, or 9,lO-positions. Now in the first and third of these molecules there is no doubt about the way in which the “boxes” should be drawn. Any attempts, such as those shown for anthracene (b), (c) to cut up the molecule in any other way, involves using an open-chain unit such as butadiene or ethylene. On the other hand, the rules do not prohibit the division shown in benz-
* Several distinct numbering systems are in use for this and other related molecules. For that reason we shall usually place our numbering scheme on the diagrams of the molecules in the text where they h t occur.
ELECTRONIC CONFIGURATION AND CARCINOGENESIS
5
anthracene (b), and we are left wondering whether there is, or is not, a special K-region in this molecule. It is certainly true that the units (or boxes) chosen by Schmidt are very stable ones, when they occur alone. Thus benzene has a resonance energy of 38 kcal./mole, and naphthalene nearly twice as much, compared with about 6 to 8 kcal./mole for butadiene. But there is no direct correlation between the behavior of the units when they occur as distinct molecules and when they occur as parts of a larger system. The evidence from ultraviolet absorption spectra suggests that molecules of this kind behave as single systems, whereas on Schmidt’s view we might have expected each component unit t o absorb separately from the rest. The
Phenanthrene
Benzanthracene (a)
Anthracene (a)
Anthraccne (b)
Benranthracene (b)
Anthracene (c)
Fro. 1. The “Box Model” of Schmidt.
theoretical basis for this theory is therefore destroyed, and, in view of the uncertainty about drawing the boxes and the almost complete lack of quantitative deduction that can be drawn from it, we can regard the theory merely as suggestive. It suggests (a) that the possession of a phenanthrene-type region is likely to make the behavior of a molecule different from that of molecules without this region, (b) that the K-region bond would be expected to be the seat of the enhanced reactivity, and (c) that in cases where no unique division into boxes is possible, there may be a double type of reactivity. This would mean that in benzanthracene, for example, both the K-region 3,4-and also the meso-positions 9,10 would have characteristic and important properties. We shall see later that all these suggestions correspond to the truth. In particular phenanthrene does differ from most aromatics in adding hydrogen at the 9,lO-positions (we shall discuss the so-called osmium tetroxide reaction later), and this addition is much easier than in benzene. Also the meso-positions in anthracene, and in benzanthracene too, are outstandingly important, both chemically (anthracene forms a photo-
6
C. A. COULSON
oxide in which -0-Olinks these two atoms) and carcinogenically (substituents at these positions tend to be more effective than at other places in the skeleton). 3. Svartholm’s Introduction of a Electrons
Schmidt’s work was unsatisfactory because it did not do justice to our knowledge of the electronic structure of molecules. The next stepand in some ways the most significant-was due to Svartholm (1941), though he does not appear to have fully appreciated the significance of his work. We shall not now describe in detail the analysis that he gave, because it has subsequently been developed much more fully by Mme. Pullman and R. Daudel, and will form the substance of Sec. 111. But, in essence, Svartholm accepted the description of an aromatic molecule in the form in which it had been given by Pauling and his co-workers. This treated the molecule as if it could be described as a simultaneous superposition of several so-called structures. These structures were nothing other than ways of drawing the necessary number of single and double bonds, as in the Kekul6 and Dewar structures of benzene. Wave mechanics showed that in the superposition some of these structures were more important than others, and thus that certain bonds were more nearly double bonds than were the others. Pauling, Brockway, and Beach (1935) had already used the concept of fractional bond order, which was implicit in Svartholm’s work. Svartholm showed that in phenanthrene and similar molecules the K-region did resemble a double bond much more closely than did any other bond. From this he concluded that this region could more easily add other groups or attach itself to the cell than could other regions. It was not unreasonable, therefore, to regard it as the seat of carcinogenic activity. A second conclusion followed, though it was less clearly stated. I n anthracene and other similar molecules there appeared to be a considerable unused bonding power a t the mesopositions 9,lO. These atoms would be expected also to be reactive for addition, so that, in cases such as benzanthracene where there was both a K-region and a pair of meso-atoms, we might expect one to be able to influence the other. The significant advance made by this work can be summarized: (a) it made use of a quantum-mechanical description of molecular structure ; (b) it showed that there really were certain special electrical properties associated with the K-region, and that these were capable of being calculated theoretically; finally (c) it threw emphasis on to the behavior of the electrons responsible for conferring double-bond character on the bonds of an aromatic molecule. These are the u electrons, which we shall describe more fully in Sec. 111. All this carried the problem into
ELECTRONIC CONFIGURATION A N D CARCINOGENESIS
7
the region of molecular structure, where, at this time, considerable advances both of a fundamental and a technical kind, were being made.
4. The Work of Pullman, Daudel and Others The next stage, which includes much the greater part of the work which we are reviewing, is due to the French school of theoretical chemists, Dr. and Mrs. Daudel, Dr. and Mrs. Pullman, and their colleagues. There has been a good deal of double publication, so that it is not easy always to be sure of the priorities, But the first full-scale report of the application to carcinogenic problems is given by Mme. Pullman (1945a,b, 1946a,c, 1947~). This includes the effects of aza substitution in the carbon skeleton and of methyl substitution around the periphery of the molecule. In order to develop this theory, however, it was necessary first to investigate the numbers of structures (in the sense used by Slater and Pauling-see Sec. 111)of given type; and in this work R. Daudel and Mme. Pullman largely published together (A. Pullman, 194613; B. Pullman, 1946; Daudel, 1946a; Daudel and Pullman, 1945b,c,d,e, 1946). More recently a review of some aspects of this problem has been given by Daudel and Daudel (1950), and a semihistorical account of much of the earlier work has been provided by R. Daudel (1946b). Very recently also a list of molecules for which carcinogenic potency has been predicted on theoretical grounds has been given by Daudel and Daudel and BuuHoi (1950). These workers effectively took Svartholm’s model, and made it quantitative. This involved, on the one hand, a very laborious solution of a large number of sets of simultaneous equations, and on the other hand, an estimate of the way in which the greater electronegativity of a nitrogen atom as compared with a carbon attracted electrons away from the K-region on to the nitrogen : and in which the electron-donating character of a methyl group replenished the supply of electrons in this region. Running through all this work was the conviction that some threshold existed for this region, and if what we may describe as the “electrical index” of this region exceeded the threshold value, the molecule would be carcinogenic: otherwise it would not be. The precise nature of what constitutes this electrical index is one of the major problems to be solved. Even now our judgment about it is continually changing as further experimental evidence accumulates. In Svartholm’s early work it was supposed that the bond order provided the effective index; in Pullman’s work on unsubstituted hydrocarbons it was the sum of bond order in the K-region and the two free valences at the carbons of this region; in substituted hydrocarbons and aza molecules it was the so-called total charge of this region (this is essentially the sum of bond order, free valences, and
8
C. A. COULSON
mesomeric charge migrations). More recently free valence alone has been suggested. But in no case is an unequivocal argument forthcoming that will deal with all possibilities. Some of the difficulties will be dealt with in Sec. V. They are of two kinds. I n the first place there is the definition of the electrical index in terms of the electronic distribution in and near the K-region. This is a matter of our own choice, and our object is clearly t o find that particular definition that seems t o fit the observed facts best. The second type of difficulty arises because, on account of the enormous complexity of a molecule with some twenty carbon atoms, the calculation of the quantities out of which the electrical index is built up (charges, bond orders, free valency, etc.) can itself be achieved only by making approximations. It is important, therefore, to distinguish between contributions to the first of these problems, which might be called the discovery of correlations, and contributions to the second, which may be called the improvement of technique. We shall see later that failure to recognize the inadequacies of some of the earlier techniques has not infrequently vitiated detailed numerical conclusions regarding the index. And the constant need to keep in mind the desirability of improving technic is shown by an entirely new discussion of charge migrations due to Nebbia (1950; Prato and Nebbia, 1950), and some much more detailed calculations of charge migration and bond order changes on substitution, recently completed by H. H. Greenwood (1951). We cannot, however, appreciate these arguments till we have explained the basis on which the bond orders, etc., are calculated. This we must now do. 111. VALENCE-BOND, OR RESONANCE, METHOD 1 . u and ?r Electrons*
The usual formulation of a chemical bond is to.describe it as the result of the pairing together of two electrons. In the isolated atoms from which the bond is formed, these electrons will occupy certain patterns, which may be calculated with reasonable accuracy. An approximate, though not quite rigorous, description of these atomic patterns (or atomic orbitals, as they are usually called) is obtained by imagining the electron spread out in the form of a cloud. The density of this charge cloud may be found by calculation. It is merely the square of the wave function, which is itself nothing more than the appropriate solution of the Schrodinger wave equation for the electron. The details of the whole pro-
* For further information Bee L. Pauling, Nature of the Chemical Bond, Cornell University Press, 1937, or C. A. Coulson, Valence, Oxford University Press, 1952.
ELECTRONIC CONFIGURATION AND CARCINOQENESIS
9
cedure do not matter; what does matter is that when the two atomic orbitals are “paired together,” there results a new charge cloud. This is the charge density for the bond, and with chemical single bonds it possesses two important properties: (a) It is almost completely localized in the region between the nuclei. (b) It is axially symmetrical around the line of the bond. These electrons are called u electrons. Normal single bonds (Hz,HC1, CHI) are of this type. They confer characteristic bond properties, such as length, dipole moment, ultraviolet absorption frequency, upon the bond in question, and the almost complete localization of the electrons makes each bond largely independent of the rest of the molecule.
(4
(b)
FIG.2. (a) An isolated atomic T orbital. (b) The formation of a R bond by pairing of two such orbitals.
But there is another type of electron cloud of much more interest to us. In the individual atom the density pattern consists (Fig. 2a) of two regions, or lobes, rather like the two halves of a complete dumbbell. In a normal double bond, such as occurs in ethylene, there is, first, a u bond of axially symmetric type, and then, superposed upon it, a ?r bond is formed (Fig. 2b) by the pairing together of two of these atomic patterns, in which the axes of the “dumbbells” are parallel and perpendicular to the plane of the CZH4 nuclei. In Fig. 2 the shaded regions denote those regions where the electronic charge cloud is chiefly concentrated. It can be seen from Fig. 2b that the *-bond pattern has zero density along the nuclear axis, which is just where the u-bond pattern is most concentrated. It is for this reason that we can attempt to deal with the two types of electron quite independently. The whole of our subsequent work will be concerned with the A electrons, because they confer aromatic character on a molecule, and, as Svartholm originally suggested, they give rise to the characteristic properties of the K-region. 2. Valence-Bond Structures
In ethylene (Fig. 2b) there was no difficulty in pairing the electrons in the formation of bonds. There were two u electrons to form a u bond,
10
C. A. COULSON
and two x electrons to form a ?r bond. But in an aromatic molecule like benzene this is no longer so straightforward. We can approach the matter most easily if we consider first the situation depicted in Fig. 3 where, on the left, we have three atomic dumbbell orbitals ABC on adjacent atoms. The three patterns are similar and parallel, pointing a t right angles to the line of centers. We are to make molecular patterns from these three atomic ones. Clearly one way is to link A and B, and so make a molecular pattern such as (3b), in which C is left as in an
(4
(4
FIQ. 3. (a) Three isolated unpaired atomic pairings to form molecular patterns.
(4 T
patterns. (b) (c) Alternative
@ ...
i
(4
(b)
...
..:
i
.
:
!
..
,
(4
FIG.4. Benzene pairings. (a) Separate unpaired r electron patterns. (b) (c) istinct alternative pairings to form molecular patterns.
isolated atom. But equally clearly there is another alternative (3c) in which we link B and C, and leave A isolated. Nobody can say that one of these pairing schemes is more favored than the other. In a word, the two possibilities (b) and (c), which Pauling calls valence-bond structures, or simply structures, are both equally possible. There is no unique pairing of the atomic electrons. And in particular, atom B must be supposed to pair with both of its neighbors A and C. This situation is enhanced in benzene and the aromatic systems generally. For in benzene, which is a planar molecule, we first allot electrons to the localized C-C and C-H bonds; after this we have six electrons left over, one on each carbon atom. In the isolated atoms they would occupy orbital patterns represented by the dumbbells of Fig. 4a. These are all parallel to each other and perpendicular to the molecular plane. Now there are
ELECTRONIC CONFIGURATION AND CARCINOGENESIS
11
obviously two distinct ways of joining these in pairs: just as we drew the patterns 3b and 3c, we can draw 4b and 4c. Neither of these is adequate by itself, and the mathematical reasoning of quantum theory tells us that the complete Schrodinger wave function for the molecule must be represented, at very least, by a sum of the functions representing the separate structures. These two structures are obviously the wavemechanical versions of KekulB’s bond patterns of 1865, and we should represent them by
WII)
respectively. The dynamic oscillation that he postulated is now replaced by the assertion that the complete wave function can be thought of as a simultaneous superposition of the two components. The result of this superposition (which we call resonance among the structures) is t o provide us with something quite different from either structure alone, though possessing certain characteristics of both of them, more or less in the way that a superposition of red and green gives a new color brown, distinct from either of its components. There are other ways of pairing these ?r electrons. For example we have three Dewar structures which we could represent as and
(VIII)
a
and
In these there is one long, or ineffective, bond. In such cases the pairing of these two orbits is purely formal since the internuclear distance is so large that no effective binding energy results. The significance to be attached to these structures is that, in them, the electrons a t the ends of a long bond may be treated as if they were almost free from constraint and could be used immediately for some other purpose such as starting a reaction. There are other structures that we could imagine, such as but it turns out (see later) that the 2 KekulB 3 Dewar structures
0,
+
provide a complete and sufficient collection and that any other structure could be expressed as some combination of these five. For that reason there is no point in introducing them, and we do best to adhere t o the chemically significant and relatively simple structures already described. It is convenient to have some terminology with which we may refer to these structures. We do this by the number of long bonds that they possess. Thus the Kekul6 structures of benzene are unexcited structures,
12
C. A. COULSON
the Dewar ones are singly excited, and so on. Figure 5 shows an assortment of structures for naphthalene, in which the total of forty-two independent structures divides into unexcited, singly, doubly, and triply excited members. The chief business at this stage is to determine the weights with which each of these structures enters the complete wave function. Each
Unexcited, or Kekule type
Fret excited
Total Number of This Type
Total Weight
3
0.55
a
@ J
0.39
Doubly excited
0.05
Triply excited
0.002
Total
PIO. 5.
1.oo
42
Structures in naphthalene.
structure is represented by a wave function. If these are the complete wave function is written
*
= a&+
+
*
'
$z
.- .,
*
(1) give the weights. They are usually normalized u2$2
The ratios uI2, u2), * - to unit sum. Thus benzene is written (in its ground state) 9 = 0.62 {$I
+ + 0.27 + +
(2) where $1 and +a are the Kekul6 structures, $8, $4, and $6 the Dewar structures. This means that the weights of a single Kekul6 and Dewar structure are (0.62)2 and (0.27)2, i.e., 0.39 and 0.07 respectively. The two Kekul6 structures amount in all t o 0.78, i.e., nearly 80% of the total, with the three Dewar structures contributing merely 21% in all. The corresponding values for naphthalene are shown in Fig. 5. In the process of determining the coefficients al, u2 - * in (l), the energy of the T electrons in the molecule is obtained. This is always less $2}
{$a
$4
$61,
-
ELECTRONIC CONFIGURATION AND CARCINOQENESIS
13
than that associated with the most stable structure (in benzene a Kekul6 structure). The amount by which the energy falls below that of the lowest component structure is the resonance energy of the molecule. With condensed aromatic hydrocarbons this amounts to about 36 kcal./mole per ring, being a little larger for the staggered rings like phenanthrene and chrysene than for straight ones like anthracene and napthacene. 3. Some Complicating Features in the Valence-Bond Method Several complicating features make this method much less easy in practice than might have been supposed. In the first place the choice of structures is not unique. Any set of indepenaent ones will do, though, if we are not careful later, they will give us quite different values for the desired quantities such as bond order and free valence. When there are odd-numbered rings, a8 in fulvene or azulene, no entirely satisfactory choice is possible. But when there are only even-numbered rings, as usually occurs, the difficulty is less severe. A systematic procedure has been developed by Moffitt (1949). The second difficulty lies in the enormous number of structures. For a molecule with 2n carbon atoms there are (2n)!/(n)!(n l ) !of these. Thus for benzene where 2n = 6, the number is 6!/3!4! = 5. For naphthalene we have 10!/5!6! = 42, and for anthracene 14!/7!8! = 429. This very soon introduces terrific complexity. Thus, to deal with the anthracene problem we require to solve a set of 429 simultaneous equations. This is a hopeless proposition. Some simplification must be introduced. Following a suggestion due to Pauling and Wheland (1933) all the Kekul6 structures are given the same weight. So are all the first excited, second excited, and so on. Without such simplification, Mme. Pullman (1947~)could never have dealt with molecules as large as beneanthracene. But it is important to realize that it is an approximation, which has been shown by Sherman (1934) for naphthalene to be correct only in its main conclusions; many details are incorrect. Certain other approximations turn out to be necessary even in setting up the simplified set of equations. Among these other approximations lies the question of which types of excited structures may be neglected. Figure 5 shows that in naphthalene the total weight of the triply excited structures is so small that these may evidently be neglected. But in naphthacene there are structures
+
Naphthacene
(1x1
C. A. COULSON
14
as much as sevenfold excited. It seems highly probable that in larger molecules the more highly excited structures become collectively more and more important. This makes the selection of structures a bit hazardous. Figure 6 (adapted from Pullman, 1947c) suggests that for molecules of carcinogenic interest, i.e., those containing four or five fused rings, the most important are the doubly and triply excited structures. 80r '
1
2
3
4
no. of condensed rings in molecule FIQ.6. Curve showing the total weight of all structures of a given class (degree of excitation) in terms of the number of rings in condensed polynuclear hydrocarbons. Curve 1 is for unexcited (KekulB) structures; curve 2 is for singly excited structures; curve 3 is for second-excited structures.
The number of these is prohibitively large (649 doubly excited structures in naphthacene), an unpleasant situation which adds greatly to the labor of computation. The third difficulty arises from the neglect of ionic structures. All the previous structures have been covalent, with exactly one n electron on each nucleus. But it was shown, a long time ago, by Sklar (1937),
0
that structures such as \ / play a part. These structures will occur in pairs, with reversed charges, and in the case of benzene will, by symmetry, not introduce any unevenness in the final charge distribution. But they are much more important in substituted molecules, or heteronuclear molecules like pyridine, where structures such as
ELECTRONIC CONFIGURATION AND CARCINOGENESIS
15
must play quite a large part. No satisfactory method yet exists for incorporating these ionic structures in systems bigger than benzene (Craig, 1950). The large but unavoidable amount of empirical character in this discussion of large molecules should now be apparent.
4. Derived Quantities Our chief interest in the calculations just described arises from the fact that they allow us t o derive certain magnitudes. It is with these magnitudes that any carcinogenic properties are to be correlated. The important magnitudes are bond order, charge and free valence. Let us describe them in turn. A. Bond Order. We have seen that our C-C bonds acquire a partial double-bond character as a result of resonance among the various allowed structures. Pauling, Brockway, and Beach (1935) suggested that a measure of this double-bond character could be obtained by adding together the weights of all the component structures in which the bond in question appeared as a double bond. An example will make this clear. A particular bond in benzene appears double in just one of the Kekul6 structures VII and just one of the Dewar structures VIII. The r-bond order is therefore 0.39 0.07 = 0.46. The total fractional bond order is 1.46; in other words, the percentage double-bond character is 46. Clearly all six C-C bonds in benzene are equivalent. But in naphthalene they are not. Using the weights referred to in Fig. 5 the total bond orders are calculated to be as in XI.
+
Bond orders in naphthalene (XI)
Here, and elsewhere, the bond order value is written along the relevant bond, and bonds not marked may be found by symmetry. In larger molecules where some of the approximations referred to on p. 13 have to be made, a good deal of analysis is needed t o determine in how many structures of each type a given bond appears double. But the requisite analysis has been provided by Wheland (1935) Pullman (1946c, 1947), and Daudel and Pullman (1945a,c,d,e, 1946). It should be mentioned here that in some of the early work of the French school, the term bond order was not used; instead values were given for the “charge de liaison.” This was simply twice the r-bond order. The justification for this term was that a complete pair of r electrons, as in ethylene, would give rise to a .rr bond of order unity.
10
C. A. COULBON
So, on a proportional basis, a fractional bond order p might be thought of as if it involved 2 X p electrons. The term bond order is now universally used. B. Charge Density and Distribution. In the case of hydrocarbons such as benzene, the ti charge must be evenly distributed by symmetry. This will always appear to be the case so long as we confine ourselves to structures such as VII, VIII, and those in Fig. 5. But as soon as we introduce structures such as XI1 or XI11 this is no longer true, and we
Naphthalene (ionic structures) (=I)
Azulene (ionic structures) (XIII)
do find certain charge migrations. Once again the total weight of structures of a particular kind will tell us how large these migrations are (Craig, 1950). Unless there are odd-numbered rings, however (Brown, 1948; B. Pullman et a2.,1950), these migrations are not very large, of the order of 1 or 2% of an electronic charge (f0.013e in butadiene). Except for some work by Nebbia (1950), which we shall discuss in Sec. IV, these shifts have generally been neglected. But with heteronuclear molecules and when methyl substitutents are present (see Sec. 11, 5, 6), this is obviously no longer the case, and structures analogous to X will appear, sometimes with a relatively large weight. C. Free Valence. Finally there is the free valence. On the old basis of Thiele and Werner, this could be regarded as the unused bonding of an atom. Thus it could be associated with the total weight of all the structures in which an ineffective, OF long, bond terminated on the atom. In benzene the only long bonds are in the Dewar structures, and each atom only has one such structure giving it a long bond. Thus the free valence at each carbon atom is 0.07. This was essentially the idea of Svartholm (1941), though it was considerably refined and systematized by Daudel and Pullman, (1945a) by Moffitt (1949), by R. Daudel and colleagues (1949), and by A. Pullman and B. Pullman (1949). In earlier work, on account of the fact that each long bond could be interpreted to mean an almost free electron a t each end, the term “charge du sommet” was used instead of free valence. But this latter term is now used exclusively. (An alternative definition of free valence will be mentioned in Secs. 111, 7 and IV, 3.) The combination of bond orders, charges, and free valences is often referred to as the “molecular diagram” of the molecule. The molecular diagrams of benzene and naphthalene* are shown in XIV and XV. On
* Calculated from the weights in Sherman (1934).
ELECTRONIC CONFIQURATION AND CARCINOQENESIS
17
account of the neglect of all ionic structures in the latter, all the 7r charges in both molecules are exactly equal to 1. When comparing molecular
(yJl 0.16
0.06
0"
1.46 0.07
Benzene (XW
?
1.59 0.10
Naphthalene
Molecular diagrams
(XV)
diagrams given by different authors it is most important to be sure of the particular approximations used in that diagram. Quite different values may be obtained by different approximations at various stages of the calculation. In general, however, the main chemical inferences (see Sec. VI) are unaffected by this unfortunate plurality of numerical values. 6. Methyl Substitutions, Hyperconjugation
It is known that methyl substitution of a hydrocarbon may profoundly alter its carcinogenic property. There are two mechanisms whereby a methyl group affects the electronic distribution in the rest of the molecule. Together they are described as hyperconjugation (Baker, 1951; Crawford, 1949; Mulliken et al., 1941). In the fist of those mechanisms, allowance is made for the well-known fact that a methyl group is an electron donor and is able to provide additional charge for the aromatic framework. Structures such as XVI(b) would be needed, in addition to normal structures XVI(a), to allow for this charge migration. In the second mechanism, no charge migration is allowed, but there is an increased conjugation (hence the name hyperconjugation). By analogy with NrC-, Mulliken argued that we might write H3=C-, in which the methyl group behaved as if it possessed a pseudo-triple bond. Thus there is a parallel between methyl cyanide Ha=C-C==N and cyanogen This parallel (which must not be pressed too closely) NrC-C=N. suggests that we could get conjugation of the pseudo-triple bond with adjacent aromatic groups. In the case of toluene we expect structures such as XVI(c) to express this additional conjugation. Structures (a)-(c) are, of course, only typical of many others thaf can be drawn. The only
(a) Normal
(b) Ionic (XVI) Structures in toluene
(c) Conjugation
difficulty now is to estimate the weights of these structures. Unfortunately this cannot be done by direct calculation, but must be inferred
18
C. A. COULSON
from evidence such as the C-CHs bond length and the dipole moment of the molecule. It will be noticed that all the new structures (b) and (c) show this bond as double. We should therefore expect that its length, which determines its double-bond character, should indicate the total weight of these additional structures. The results however can only be approximate since this length is not known sufficiently precisely and appears to vary somewhat erratically from molecule to molecule. Even this only tells us the sum of the weights of (b) and (c). By making certain assumptions about the distribution of net charge, A. Pullman (1947~)was able to estimate the separate weights of each type of structure. The resultant charge distribution is shown in XVII, where the figures shown at the various nuclei represent the net charge in electrons. Thus the methyl group in toluene would be supposed to carry a net positive charge O.lle. -0.037
6 -0.01
0
CHa + O N 2
-0.030
(XVII) (After Pullman)
Similar analysis may be used for methyl substitution in larger molecules: and the effects of two or more such substitutions may be treated as additive. There is no doubt but that this technique properly describes the phenomenon in a qualitative sense. But it can hardly be said to be satisfactory quantitatively. Thus as stated earlier, (1) the C-Me bond length is not known sufficiently accurately, (2) the argument which leads to the ortho positions carrying less charge than the para position is not completely convincing (see Sec IV), (3) the dipole moment calculated from XVII is about four times as large as the observed moment for toluene (0.4 D experimental, 1.4 D calculated). This suggests that the technique grossly exaggerates the charge migrations, which require to be scaled down by a factor of 3 or 4. But, on the other hand, the procedure is quite systematic, so that we may expect regular variations from one molecule to another to be exhibited by these calculations. Further applications of this analysis will be given in Sec. V. And in Sec. IV we shall show how the alternative molecular-orbital approximation attempts to deal with the difficulties which we have listed above. 6. Aza Replacement
A further effect of great importance is the replacement of a CH group in the aromatic framework by a N atom. In pyridine, for example, in addition to normal structures XVIII (a), (b) we have ionic structures
ELECTRONIC CONFIGURATION AND CARCINOGENESIS '
19
such as (c). Each structure (c) can be thought of as derived from an excited homopolar structure (b) by moving all the charge onto one end (i.e., the N atom) of a long bond.
(4
(b)
(4
(XVIII) Structures in pyridine
(4
(After Pullman)
Mme. Pullman made the hypothesis that the ratio of the weights of structures (c) and (b) was a constant, for all such pairs of corresponding structures, and for all molecules. If we accept this postulate, then it is not hard to estimate the charge distribution, since we need only work with homopolar, or covalent, structures, and these are evidently just the same as in the parent hydrocarbon (benzene). It is, unfortunately, impossible to test the validity of the postulate, because no one has yet succeeded in making any direct calculations of the weights in the different structures. Quite clearly this is a matter of considerable complexity. But it seems probable that here, as in Sec. 111, 5, systematic variations from one molecule, or series of molecules, to another, would be shown up by this analysis. It would, however, be foolish to claim anything like absolute accuracy for the final numerical values of the charges or bond orders. B. Pullman (1948) has endeavored to systematize the method of weighting a little more carefully. But several difficulties still remain. Thus his final charge distribution for the ?r electrons in pyridine is shown in XVIII (d). But the dipole moment associated with these charges is about 2.7 D, although the total molecular moment is known to be only 2.1 D, and the contribution from the u bonds is generally supposed to be in the region of 0.8 D. It looks as if Pullman's charge migrations are calculated t o be about twice as large as they should be (see also Daudel and Martin, 1948). 7. Penney Bond Orders One of the objections to the fractional bond orders described in Sec. 111, 4 arises from the fact that their precise values depend on which choice of structure we have made. We say that they are not true invariants. As a result they do not possess any very really fundamental theoretical significance, though in most cases, and particularly when no five-membered rings are present, the matter is not serious. It was shown by Penney (1937) some years ago that an alternative definition of bond
20
C. A. COULSON
order could be given, which did not suffer from this deficiency. Penney argued that in a molecule like benzene, if the spins of the two electrons associated with a bond were antiparallel (mutual angle of 180°), the bond would be pure double: and if they were quite randomly oriented relative t o each other (mutual angle SO”), it would be a pure single bond. Neither of these situations does in fact occur in large cyclic molecules. A situation arises in which we could think of a superposition of both possibilities. This could be described by saying that the mean value of angle between the spins had a value different from 90” or 180”. The magnitude of this mean value could be used as a measure of bond order. It turns out that this mean value can be calculated and hence the fractional bond order inferred. Such bond orders, of course, differ from those previously described. It is necessary, therefore, when comparing results by different authors, or by the same author at different times, to be sure which definition of bond order he has used. It may be said that the so-called Penney bond orders agree remarkably closely (Coulson et al., 1947) with those calculated according to the methods to be described in Sec. IV. The only unfortunate aspect of the matter is that a full Penney calculation is extremely lengthy. Attempts have been made with some success (Jean, 1948; R. Daudel, 1948c; Vroelant and Daudel, 1949a,c) under the title “method of spin states,’’ to simplify this procedure. And more recently, a much cruder method, taking account merely of the geometrical arrangement of the bonds in the vicinity of any selected bond, has been suggested by Vroelant and Daudel (1949b). All these distinct definitions give distinct bond orders, so that once again caution is necessary when quoting published values for comparison with other values. Further application of these ideas and techniques must be left till Sec. V. IV. MOLECULAR-ORBITAL METHOD 1. Molecdar Orbitals*
We must now describe the alternative method of representing the electrons in a conjugated compound. It is necessary to do so because this method has been used much more widely than the resonance method in recent years. Just as in Sec. 111, we consider merely the distribution of the ?r electrons. Since it is of the essence of these electrons that they are mobile, and resemble the conduction electrons of a metal, there is a certain advantage in recognizing this mobility right from the start. Now in a metal we do not attempt to localize individual electrons either in a bond or on an atom, but we think of each conduction electron as
* For further information, see C. A. Coulson, Valence, Oxford University Press. 1952.
ELECTRONIC CONFIGURATION AND CARCINOGENESIS
21
occupying an orbital which extends over the complete metal. I n the corresponding molecular problem we do precisely the same; that is to say, we suppose that each of the a electrons is assigned to a definite orbital, but these orbitals are polycentric and extend over all the resonating, or conjugated, part of the molecule. In this picture, there is no trace of the structures, or the resonance between them, which were the basis of the valence-bond method. In fact, the new method is fundamentally similar to the description of an isolated atom or of a block of metal; that is, we first calculate the possible orbitals (which we now call molecular orbitals, m.0.) and determine their energies. Next we “feed” the a electrons into these orbitals, two a t a time to allow for the Pauli exclusion principle which says that not more than two electrons may ever occupy the same space orbit, and then they must have opposed spins. We begin with the orbitals of lowest energy and continue until all the available a electrons have been disposed of in this way. The total energy is taken, rather approximately, to be the sum of the energies of the occupied orbitals. Excited levels occur by the promotion of one (or perhaps occasionally two) electrons from one of the occupied m.o.’s to one of the higher energy unoccupied m.o.’s. We can immediately see one important difference between the m.0. method and the resonance method. For now, each a electron may be found anywhere in the molecule. This means that each ?r electron contributes to the total a charge on each of the atoms and also to the bond order in each bond. In general any particular orbital will contribute different amounts to the charges on the various atoms and to the various bond orders. The greater part of the analysis referred to in this section is concerned with describing these orbitals and showing how a knowledge of the orbital enables us to compute the order of any bond and the total charge on any atom. The advantage of the m.0. method is that we can treat the electrons one a t a time, separately. Its chief disadvantage is that by not taking into account the simultaneous positions of any pair of electrons, we allow too many electrons to be in the same place simultaneously. The first statement of the m.0. method was due to Lennard-Jones; the greater part of the earlier theory was due to Bloch (for metals) and to Hund, Hilckel, and Mulliken (for molecules).
2. The LCAO Representation When one of the a electrons is in the close vicinity of a particular atom r, the forces acting on it are largely the same as the forces it would experience if it were attached to that atom. This means that the wave equation for this electron locally resembles the wave equation for this
22
C. A. COULSON
electron in a single atom-so also must the solution of this equation, i.e., the molecular orbital. Thus near atom T , $ resembles &, a known function; near atom 8, $ resembles 48, etc. Now +r and 4, are only of significant size in the neighborhood of their respective nuclei. A function, therefore, which satisfies the above conditions is $=
+
+
~141 ~ 2 4 2
*
. + Cn4n
(3)
where we suppose that there are n atoms in the resonating framework, and the cr are a set of constants yet to be determined. The m.0. method in its usual form asserts that by a proper choice of the c’s we can make (3) a good approximation to any of the real molecular orbitals. A definite technique (secular equations and secular determinant) exists for finding the appropriate c’s. It turns out that there are actually n distinct sets of c’s, and each set is to be associated with one m.0. Thus we have, in principle, a way of approximating to the required m.o.’s. In the process of determining the c’s we also determine the energy of this orbital, and we can therefore arrange the n orbitals in ascending sequence of energy. If there are 2m r electrons, then the lowest m of these m.o.’s willLbefully occupied in the ground state, and the total ?r energy can be found by a simple addition. The approximation used in (3) is the only one that has proved fruitful. It is called the LCAO (linear combination of atomic orbitals) approximation (Mulliken, 1932), since the molecular orbital $ is expressed as a linear combination of atomic orbitals 4,. There is a very simple interpretation of the coefficients Cr in (3). Since the square of $, rather than fi itself, has physical significance,and since $2
=
c12412
+ - + *
*
2ClC24142
+- *
we say that an electron in this m.0. is distributed among the atoms : cn2e If we normalize the orbital so that in the ratio c12:ca2 * cla cZ2 * * Cn2 = 1, we can say that an electron in $ contributes an amount c12 to the total r charge on atom 1, etc. Thus, by determining the sets of c’s, we can soon calculate the charge on each atom. There is a further point to be made here. In the density function 2. we have product terms such as ~ 1 ~ ~ 4 1 4These terms are quite insignificant unless atoms 1and 2 are neighbors, so let us restrict ourselves to such. We may say that the quantity clc2 ia a measure of the probability that the electron in question is associated with both atoms 1 and 2. But in such a case it is reasonable to suppose that it contributes to the bond between these atoms. We therefore define the contribution of an electron in this m.0. to the bond between atoms 1 and 2 as the product
+ +
+
--
ELECTRONIC CONFIGURATION A N D CARCINOGENESIB
23
clcz (Coulson, 1939). The total ?r-bond order is then the sum of products clcz for each of the a electrons. This definition can be shown to fit with the conventional single, double, and triple bonds; it is therefore a reason-
able definition to accept in other cases. It is obviously a matter of great importance to know what factors determine the coefficientscr and hence the other derived quantities (bond order, charge, energy, etc.) There are two sets of parameters on which everything else depends. As these will keep on coming up in later work, we must describe them carefully. They are the Coulomb terms a, of the atoms and the resonance integrals 0,. of the bonds. The Coulomb term arof atom T is a measure of the electronegativity (i.e., electron-attracting power) of this atom, or, more precisely, the electronegativity of this atom toward a electrons when in its proper position in the given molecule. It is defined as the energy of the a electron in the atomic orbital &, associated with nucleus T . It is nearly, but not quite, the same as the energy of the electron in the isolated atom. Although there are serious difficulties in determining exactly what a to use for any selected atom (e.g., nitrogen in pyridine) it is clear that a, which is negative, satisfies the inequality
I%(
< I a N I
<\a01
where the subscripts relate to carbon, nitrogen, and oxygen respectively. The usual convention is to take a to be the same for all carbon atoms in a pure hydrocarbon molecule and to take the value of a for any other atom to differ from a, by an amount proportional to the difference in electronegativity of the two atoms on Pauling’s scale (1937). No agreement has yet been reached on the constant of proportionality in this relationship; the constant first used by Wheland and Pauling (1935) is now believed to be too big, since it predicts dipole moments due to the a electrons which are two or three times as large as they should be. However, in earlier work, (see, for example, Coulson, 1946a; Coulson and Jacobs, 1949; Longuet-Higgins and Coulson, 1947) this constant was used, and it follows that charge migrations calculated in this way are always exaggerated. When comparing charge distributions in various molecules, it is essential to know what values have been taken for the different Coulomb terms. In this connection there are two particular points to look out for (Orgel et al., 1951) :the first is that a nitrogen atom in a 5-ring such as pyrrole XIX should have a different a from the nitrogen atom in pyridine. In fact la1 is greater for pyrrole than pyridine. Also 1.11 is greater for the pyridinium ion XXI than for pyridine (LonguetHiggins, 1950). Differences such as these have not always been sufficiently allowed for. They are important when discussing basic prop-
24
C. A. COULSON
erties of nitrogen heterocyclics. The second point to be noticed is that some writers (Dewar, 1949; Longuet-Higgins and Coulson, 1947; Wheland, 1941) introduce an inductive effect in which the enhanced
Q
GI
H
H
Pyridinium
(XXI)
electronegativity of, for example, a nitrogen atom, is shared in a decreasing degree with the atoms in its neighborhood. It is a great pity that despite thorough analysis of this problem (Mulliken, 1949) no satisfactory method yet exists for determining the appropriate a’s to be used in hetero molecules. We have still to describe the resonance integrals firs, which, with the ar, determine the details of the allowed molecular orbitals. It may be shown that Br8 (which is negative) is related to the difference in energy between a pure single and a pure double bond between the atoms T and s. If these two energies are E.inpls and &&Is, then, to fairly high accuracy Edoubls
- Ednpla
+
= [48ra2
(a,-
a8)2]1
(4)
In particular, for two similar atoms, where a, = a,, Edwble
- Eningle
=
28
(5)
Since Edouble and E.inple vary with the length of the bond, it follows that Bra will also vary. For most purposes, however, it is possible to neglect this variation; for large molecules it is essential to do so, in order to avoid quite unmanageable complexity in the calculations. Further details concerning the significance of the 8’s may be found in a paper by Mullilten (1949). More refined versions of the m.0. theory have been proposed (e.g., Coulson et al., 1951; Jacobs, 1949; Lennard-Jones, 1937; Mulliken, 1949) but we do not need to deal with them here, because they are quite inapplicable yet to large molecules. 3. Fundamental Magnitudes Once the appropriate values for the parameters a, and PI, have been chosen, it is fairly straightforward t o write down and solve the secular equations that determine the energy E and the coefficients cr in each molecular orbital. By summation of these and their squares and prod-
ELECTRONIC CONFIGURATION AND CARCINOGENESIS
25
ucts, as shown in Sec. IV, 2, we can derive the fundamental molecular magnitudes. These are: (i) total a energy &, which is simply Ze; (ii) total a charge qr at each nucleus, which is Z C r 2 ; (iii) total a bond order p,, in each bond, which is Z C , ~ , ; (iv) free valence F, a t atom r . This latter quantity is determined as follows (Coulson, 1946b). First the total bond orders (u bonds a bonds) of all the bonds that terminate on atom r is calculated. This, which we may call N,, measures the extent to which atom r is engaged in bonding. Now it may be shown (Moffitt, 1949) that for carbon atoms, which is the only case properly studied so far, there is a certain maximum possible value of N . If this is N,, then
+
N,,
=
3 f <3
=
4.732
(6)
The difference Nmax - N , represents the amount by which the actual bonding falls below the maximum possible. It is reasonable to call it the free valence (P,). Quite clearly this is a different definition from that used in the resonance scheme of Sec. I11 (though, in the Penney scheme of Sec. 111, 7 an analogous definition can be given, with almost identical numerical values). But fortunately, it turns out (Coulson, Daudel and Daudel, 1947, 1948) that despite the differences in definition, there is usually a close correlation between the numerical magnitudes obtained by the two methods of approximation. The values of qr, p,,, and Fr together comprise the molecular diagram of the molecule. Simplified ways of determining these quantities without recourse to calculating the sets of coefficients C , have been devised by Coulson (1948) and Longuet-Higgins (Coulson and Longuet-Higgins, 1947a,b, 1948a,b).
4. Some Particular Results Certain general results of later application can be proved. Thus (Coulson and Longuet-Higgins, 1947a) in all pure hydrocarbons with no odd numbered rings and no methyl groups present round the periphery, all the charges q, = 1. Also the presence of a single substituent in a long chain is to induce alternating increases and decreases in the q, as we move away from the place of substitution. These charge migrations rapidly diminish and are ineffective (Coulson and Jacobs, 1949) more than five or six atoms away from the substituent. The molecular diagrams for benzene, butadiene, and naphthalene are given below. They should be compared with XIV and XV obtained earlier by the resonance method. No charges are shown in XXII-XXIV, since these are all unity.
26
C. A. COULSON
Benzene (XXII)
Butadiene (XXIII)
Naphthalene (XXIV)
The enhanced free valence a t the end atoms in butadiene and at the a-position in naphthalene will be noticed; and also the relatively high bond order in the +bond of naphthalene. The result of aza replacement, where a CH group is replaced by a N atom, is shown by comparison of XXII and XXIV with XXV and XXVI (Longuet-Higgins and Coulson, 1947), though these latter diagrams were obtained with too large a range of Coulomb terms, as indicated in Sec. IX, 2. The charges shown in these diagrams are the net resultant charges. Thus in pyridine the nitrogen atom carries a net $“3
0.46
t
4-0.16
0.62
+;i6M
+0.06
0.U -0.59
+0.21
+ O M-0.04 )
0.63
o.ia
Pyridine (XXV)
Quinoline (XXVI)
charge of -0.59e and is therefore negatively charged. The other atoms are correspondingly all positive. 6. Polarizabilities
The difference between benzene and pyridine, in this view, resides chiefly in the different Coulomb term a for the nitrogen atom as compared with the carbon of CH. If the difference is not large it seems reasonable to suppose that an estimate of its effect could be obtained by a perturbation method. Here, starting with benzene, we can derive approximate charges, bond orders, etc., for pyridine quite simply. For if a change 6a, is made in the Coulomb term for atom r, there will be a corresponding change 6q, in the charge on atom s, where
re,+is called the mutual polarizability of atoms s and r. The word “mutual” is introduced because T8.r = rr,,. Coulson and Longuet-
ELECTRONIC CONFIQURATION AND CARCINOQENESIS
27
Higgins (1947a,b, 1948a,b) have shown how these coefficients can be calculated for the polycyclic hydrocarbons without too great difficulty. Once they have been calculated any number of changes 6a, may be allowed (if only they are not too large) and the resulting charge migrations determined by simple superposition of the separate contributions. This method has been used by Longuet-Higgins and Coulson (1949) to discuss aaa derivatives of naphthalene, anthracene, and phenanthrene, by Nebbia (1950) to introduce a difference in a! between the two internal carbon atoms 9 and 10 and the other carbon atoms in naphthalene and similar molecules; by Greenwood (1951) for converting known values for benzanthracene into previously unknown values for benzacridine ; and by Longuet-Higgins (1950) for discussing basicity in heteroaromatic amines and the effect of substitution on the transition state of a chemical reaction. It seems likely that these polariaabilities-and others of a similar kind which consider the effect of 6ar on bond orders and the effect of a change 6& on charges and bond orders-will prove increasingly useful when dealing with large molecules, which are rather intractable by direct means. 6. Hyperconjugation
A reference to Sec. 111, 5 shows that hyperconjugation between a methyl group and a cyclic hydrocarbon involves two main factors: the one is an enhanced conjugation path because the -C=H, group can be written so as to resemble a pseudo-triple bond: the second is an ionic effect due to the difference in electronegativity of a --CHI group and a H atom attached to a carbon of the cyclic framework. In the m.0. theory: these two effects are included in the analysis extremely easily, as was first pointed out by Mulliken, Rieke, and Brown (1941). Let us consider toluene as an example. In the first place it may be shown (see, for example, Crawford, 1949, and Coulson, 1947) that the -CHs group may be represented simply by the form -C-X. In this formulation X is a pseudo-atom, correspondis supposed to coning to the group HI, and the combination -C-X tribute two r electrons. Thus toluene would be written
c)-c-x and there would be a total of eight r electrons. These would fully occupy the four molecular orbitals of lowest energy. Then, in order to determine these molecular orbitals we have to decide on appropriate values for the Coulomb terms of the -Catom and the pseudo-atom X, and also on the appropriate resonance integrals 8.
28
C. A. COULSON
This is the weakest part of the theory at present, and plausible, though not strictly proved, estimates have to be made. When these are made, however, the calculation of the energy and the charge shifts can be carried through in an entirely straightforward manner. The most satisfying description of this kind has been given by Crawford (unpublished), whose final charge distribution is shown in XXVII. This set of charges
Charge distribution in toluene (V.A. Crawford) (XXVII)
leads to a dipole moment 0.37 D, which is very close to the observed value 0.4 D. It will be recognized that there is a net flow of charge from the --CHI into the ring, as required by chemical evidence of reactivity; but this flow is very much less than that shown in XVII by the early valencebond calculations of Mme. Pullman. The fact that the charge migrations into the ring are so small suggests that it should be possible to represent them as a suitable perturbation of unsubstituted benzene. Both Crawford and H. H. Greenwood have shown that this may be done, to considerable accuracy, by imagining that methyl substitution merely alters the Coulomb term of the carbon to which it is attached. Then the whole of the polarizability technique of Sec. IV, 5 may be used in just the same way as for aza replacement. This is what has actually been done in almost all the calculations reported in Sec. V. 7. Direct Tests of Theory This completes our description of the way in which bond order, charge and energy are calculated in the m.0. description, but before we discuss particular applications of these calculations, it is desirable that we should briefly refer to some of the evidence which supports the validity of the principles themselves. The most straightforward and, incidentally, the first test of this work was by comparison of the calculated energy of delocalization (or resonance) with the experimental value. We use the theoretical value for one molecule, e.g., benzene, as a means of estimating the parameter Pec for carbon-carbon bonds. When this is done it turns out (Wheland, 1934; Huckel, 1937) that the resonance energies for molecules such as naphthalene and anthracene are given correctly to within about 5 %. With heteromolecules the experimental evidence is still too meager, but the agreement for hydrocarbons, including hyperconjugation, is good enough to inspire confidence.
ELECTRONIC CONFIGURATION AND CARCINOGENESIS
29
The second test is concerned with bond lengths. Following an idea first suggested by Pauling we should expect that the order of a bond should determine its length by means of an order-length curve. Recent highly accurate x-ray measurement,s of lengths have allowed a full investigation of this relationship to be made for C-C bonds. As Fig. 7 shows [taken from a review prepared by the present writer (Coulson, 1951a)l there is a strong correlation between theoretical order and experimental length. This demonstrates beyond any doubt that for C-C
calciilatad bond order (molecular orbitals)
FIG.7. Curve showing the relation between calculated bond order (by use of molecular-orbital approximation) and observed bond length in conjugated hydrocarbons. A = anthracene, Be = benzene, Bu = butadiene, C = coronene, E = ethylene, G = graphite, N = naphthalene, 0 = ovalene, P = pyrene.
bonds the bond order has a real significance. For other types of bond (Cox and Jeffrey, 1951) the results are less conclusive, largely due to a scarcity of really accurate experimental analyses. Finally there is the charge distribution. This has already been referred to. With earlier values for the various fundamental parameters, the charge migrations were usually too large. More recent values give excellent agreement for such effects as methyl substitution or aza replacement. Certain difficulties still persist, but there is no denying that the theories correctly describe the character of the charge flow. This applies to its direction and the relative values at different parts of the molecule.
30
C. A. COULSON
Absolute values, however, are still somewhat in doubt. The molecularorbital method, because of its completely systematic and natural way of treating these effects, is almost certainly more reliable in this respect than is the valence-bond method. This concludes our description of the basic techniques that are used in studying large molecules. We are now ready to see how they apply to carcinogenic molecules, and with what success.
V. ELECTRICAL INDEX FOR
THE
K-REGION
1 . Electrical Index
We stated the main problem of this section in Sec. 11, 4. It is this: is there any electrical index, or combination of bond order, free valence, and charge distribution associated with the K-region, which provides a measure of carcinogenic potency? Historically the work of Mme. Pullman (1947~)was the first systematic attempt to establish a suitable index and discover the threshold value for carcinogenicity. It will, however, be most natural to discuss this work a little later (Sec. V, 5 ) rather than at once. We must begin with a recognition that the K-region is indeed significant in this way. Some of the arguments for this have already been discussed in Sec. 11, 1; to these we can add the facts: (1) The K-region is the only region in most of the molecules with which we are concerned, whose molecular properties are in any sense unusual; the only isolated atomic regions other than the K-region are the meso positions in anthracene and related molecules. All this can be seen very clearly from the molecular diagram for benzanthracene (Berthier et al., 1948) which we reproduce in XXVIII.
& & 0.40
#' .JM 1.608
1.732
1.584 1.495
1,447
1.!%lo
1.494
1.628
1.681 1.403
0.139
O.108
0.408
0.461
0.112
0.407
1.789
0.458
0.186
0.110 0.614
0.466
0.466
Bond orders Free valences (XXVIII) Benzanthracene (m.0. values)
These figures are calculated by the m.0. method. No charges are shown because, in this approximation, there is exactly one ?r electron on each atom. These diagrams reveal that the K-region bond is the only one with an anomalously high bond order and that the 9,lO-meso positions
ELECTRONIC CONFIGURATION AND CARCINOGENESIS
31
are the only ones with anomalously high free valences. A similar situation occurs in the valence-bond diagram, which will be given later. (2) Blocking the K-region by two methyl substitutions almost always destroys the carcinogenic activity. On the other hand, blocking by one methyl group often succeeds in activating the molecule, as in 3,4-benzphenanthrene, or as in l,a-bensanthracene, where the weak carcinogenic
1,2,3,4-Dibenzphenanthrene (XXIX)
activity (Steiner and Falk, 1951) is enhanced in this way. This argument, however, is not conclusive, for, as Badger (1948) and others have pointed out, although 1,2,3,4dbenzphenanthreneXXIX is carcinogenic, the 9- and 10-methyl derivatives are not. Despite all this confusion it does seem pretty clear that we ought to look for characteristic and significant features in the K-region rather than elsewhere. 8. Bond Orders i n the K-Region
The first, and simplest, view is that the bond order of the K-region bond must be high and exceed a certain minimum value. This does not appear to be the case. The third and fourth columns of Table I give the bond orders of the K-region bond (in naphthacene and triphenylene, where there is no K-region the highest bond order is given) for all the polycyclic hydrocarbons with four fused rings (A. Pullman, 1947c; Berthier et al., 1948). The only one of this series which is at all strongly carcinogenic is 3,4-benzphenanthrene, and this is not exceptional, whether viewed from m.0. or v.b. points of view (Steiner and Falk, 1951). The molecule with highest m.0. bond order, 1,2-benxanthracene, is only very slightly carcinogenic. We may therefore dismiss bond order alone as a satisfactory index of carcinogenicity. Apart from any other considerations the m.0. and v.b. columns tend, on the whole, to move in different directions. The only way in which we could suppose that bond order was significant would be to conclude that the necessary conditions for carcinogenicity were (a) that the bond order in the K-region was high and (b) that certain other conditions were also satisfied. These other conditions
32
C. A. COULBON
TABLE I Bond Orders and Other Properties of K-Region Molecule
Bond Order m.0. v.b.
Resonaece Energy (B)
Energy of Bond Addition (P)
7.10
1.05
6.51
1.06
1,2-Benranthracene
1.783
Pyrene
1.777
3,4Ben zphenanthrene
1.762
1.442
7.19
1.12
Chrysene
1.754
1.434
7.19
1.14
Naphthacene
1.741
1.452
6.93
1.21
1.690
1.446
7.27
1.4
rPriphenylene
1.440
would include a planar carbon skeleton, asymmetry around the K-region (though not necessarily in the molecule as a whole; see 3,4-benzphenanthrene) and unsubstituted carbons at the 1’, 2’, 3’ positions relative to the K-region. A further difficulty arises when we compare the five-ringed molecules with the four-ringed ones. No v.b. values are available, but Baldock, Berthier, and Pullman (1949) have made m.0. calculations for several of these pentacyclic systems. The K-region bond orders of 1,2,5,6-dibenaanthracene (XXX) and 1,2,7,8-dibenzanthracene(XXXI) are 1.778 and 1.780 respectively. The first of these is inactive and the second slightly active. Yet both bond orders are less than that (1.790) in pentaphene (XXXII) which is inactive. Quite clearly a high bond order alone is inadequate.
ELECTRONIC CONFIGURATION A N D CARCINOGENESIS
1,2,5,6-Dibenzanthracene
(XXX)
1,2,7,&Dibenzanthracene
(XXXI)
33
Pentaphene
(XXXII)
3. Free Valence
A second possibility (R. Daudel, 1948b; A. Pullman et al., 1950) is that the free valence, or sum of the free valences, in the K-region, is the determining factor. However, a glance at the benzanthracene diagram XXVIII shows that there is nothing outstanding about the free valence in this region. A further objection arises from the fact that aza replacement, such as converting benaanthracene to benzacridine, will increase the sum of the free valences, whereas such replacement is known to reduce carcinogenic power.
4. Charge on the Atoms A third possible criterion for carcinogenicity is the net charge on the K-region. Care is needed here because the term “total charge” has been widely used by the French workers in a rather different sense (see Sec. V, 5 ) . But if we confine it to its strict sense of meaning the actual T charge on the various atoms, we can see at once that in the conventional scheme (v.b. or m.0.) it can hardly be significant. For the ?r electrons distribute themselves equally over the aromatic framework in both theories. It is true (Coulson and Jacobs, 1951) that when more refined forms of the theory are used certain small charges do appear on some of the atoms, and it is remotely possible that these may be significant. But, neither in v.b. nor m.0. theories can the magnitudes of these charges be estimated for large molecules on account of the complexities of the mathematical calculations involved. This matter must therefore be left unsettled. Since, in any case, the charge is not likely to exceed that found a t other positions in the molecule, we are inclined to think that this is not a significant factor, at any rate whenconsidered by itself. To some extent these conclusions are reinforced by some recent calculations of Nebbia (1950) and Prato and Nebbia (1950), who followed an original suggestion of Mulliken (1948, 1949) to the effect that the “internal” atoms in a polycyclic molecule (i.e., the atoms which are common to
34
C. A. COULSON
two or three rings) should have a numerically smaller Coulomb term than the other carbon atoms. By using the polarisability coefficients described in Sec. IV, 5 , and treating these changes in Coulomb terms as a small perturbation, charge migrations could be calculated. Since the inner atoms are behaving as if they were less electronegative (i.e., less electron attracting) than the others, the electron migration is to the peripheral atoms. We reproduce in XXXIII the net charges calculated by these authors for phenanthrene. Additional charge is certainly brought to the K-region, but the amount is no greater than to certain other parts of the molecule. Further work will need to be done, particularly as regards the correct changes in the Coulomb terms to account for aza replacement and for methyl substitution before any convincing demonstration that the net charge is the dominating influence can be given. At present, judging by the diagram XXXIV for butadiene, these
&! -0.013
-0.013
-0.020
-o.om
+OW
-0.010
-0.0~1
Phenanthrene (AfterNebbia and Prato) (XXXIII)
charge shifts appear to be about five times as large as those calculated (Coulson and Jacobs, 1951) by other more refined methods. -0.071 -~
+ 0.071 -+ 0.071 ~
- 0.071
Butadiene (After Nebbia and F’rato)
(XXXIV)
One interesting suggestion has been made in this work. It arises from the fact that methyl substitution converts one of the peripheral atoms from being an external carbon to being an internal carbon. As a result it changes its Coulomb term in much the same way as was suggested in Sec. IV, 6, and it therefore permits alternative estimates t o be made of the resulting charge migrations on to the K-region. 6. Pullman’s Work on the
((
Total Charge”
This brings us to the original work of Mme. Pullman (1947~)on the “total charge” of the K-region. This work was done entirely within the valence-bond framework of Sec. 111, though in the last year or so
35
ELECTRONIC CONFIGURATION AND CARCINOGENEBIB
parallel calculations (Greenwood) have been made using the m.0. technique of Sec. IV. We shall discuss Mme. Pullman’s work first. Let us begin with l,2-benzanthracene. Using the various approximations described in Secs. 111, 3 and 111, 4 the molecular diagram was calculated to be as in XXXV (c.f., XXVIII for the m.0. values). Now a
&
1.383 1.460
1.449
1.366
1.371
1.440
0.137 0.182
o.,6*&1*
0.168 0.198
0.132
0.266
0.14t
0.204 0.2M)
Bond orders Free valences (XXXV) Benzanthracene (v.b. values, fter Pullman)
bond of mobile order p can be thought of rather loosely as involving 2 X p ?r electrons, though in view of the comment in Sec. I I I , 7 this is not an entirely correct interpretation. And a free valence F involves F electrons. Thus the total ?r charge on the K-region is 2p
+ Pi +
F 2
where FI and F 2 are the free valences at the two atoms of the K-region. This is called the total charge of the region or the “charge density,” though this latter is surely a rather unhappy choice of title. In the case of benzanthracene the numerical value is 2 X 0.440
+ 0.204 + 0.200
=
1.284
Similar values can be obtained for other unsubstituted hydrocarbons. When one or more methyl groups are substituted, then, as shown in Sec. 111,5, there will be additional ionic structures, which will convey net charges qr to the atoms. The same is true when aza replacement occurs, though here the charges are of opposite sign. We now define the total charge of the K-region as
+ Fi + Fz + +
2 ~ 1 2
~i
q2
(8)
Provided that suitable ratios are taken for the relative weights of ionic and long-bonded structures, these total charges can be obtained. Table 11, quoted from Badger’s review (1948) and corrected for certain recent alterations, shows the collected values. I n this table the molecules are arranged in order of increasing “charge density.” It is a t once obvious
36
C. A. COULSON
that a considerable degree of correlation exists between the calculated charge and the experimental activity. In particular, we can assume some sort of threshold value a t 1.291, above which carcinogenicity is almost certain to occur. It is rather gratifying t o find that the sequence of Table I for the four-ringed systems now gives way to a much more satisfactory sequence, in which 3,4-benzphenanthrene does come first, above the threshold value, and the others are all below it. It is also pleasant t o see how methyl groups in the right places can restore a potency which aza replacement had destroyed. The table is extremely suggestive, despite the fact, referred to in Sec. 111, that the calculations and definitions of the quantities used are far from being satisfactory. There are certain exceptions. Thus 9,10-dimethyl-1,2,5,6-dibenzanthracene XXXVI is either inactive or very slightly active, but the 9-methyl compound is strongly active. A possible way out of the difficulty is to assume that there is an upper limit as well as a lower limit to
Y,lO-Dimethyl-1,2,5,6-dibenzanthracene (XXXVI)
the charge density; presumably the compound XXXVI would have a considerably larger value than any of those in Table 11. But such an axiom is not very satisfactory and requires more examples t o justify it than are a t present available. Alternatively steric hindrance from the CH3 group may be invoked, even though it is not easy to see how this would operate in this case. A more serious difficulty arises with methyl substitution in the benz ring of 1,2-benaanthracene. Experimentally all these compounds are inactive, though some of them would have been expected to be active according to Pullman’s criterion. To obviate this difficulty it is assumed that two or more reactions have t o occur in order to produce a carcinogenic cell and that a methyl group in the side ring interferes with the second reaction, although it may possibly encourage the first. This is not very satisfying. A further difficulty has been discussed by Badger (1948). It appears that 10-substituted l12-benaanthracenesare carcinogenic, despite the low activity of the parent hydrocarbon, both when the substituent is electron attracting (e.g., -CN) and when it is electron repelling (e.g., -CHs). This can hardly be fitted into the original theory. In view of some of these difficulties, attempts have been made to alter
ELECTRONIC CONFIGURATION A N D CARCINOGENESIS
37
TABLE I1 Total Charge (or T Density) at K-Region in Pullman’s Theory Carcinogenic Activity
Total Compound Naphthacene Anthracene Triphenylene 3,PBenzacridine 1,2-Benzacridine Chrysene 5-Methyl-3,4-benzacridine Naphthalene 1,2-Benzanthracene 5,8-Dimethyl-3,4-benzacridine
5,7-Dimethyl-3,4benzacridine 5,9-Dimethyl-3,4-benzacridine Phenanthrene 8-Methyl-1,a-benzanthracene 5-Methylacridine 3,4Benzphenanthrene 7-Methyl-1,Zbenzanthracene 6-Methyl-1,a-benzanthracene 9-Methyl-1,Zbenzanthracene 5-Methyl-1,Zbenzanthracene 3-Methy1-lJ2-benzanthracene 4-Methyl-1 ,a-benzanthracene 5,7,9-Trimethyl-3,4-bensacridine 5,9-Dimethyl-lJ2-benzacridine 5,8-Dimethyl-1,a-benzacridine 5,7-Dimethyl-1,Pbenzacridine lO-Methyl-l,2-benzanthracene 5,6-Dimethyl-1,2-benzanthracene 5,9-Dimethyl-l,2-benzanthracene &Methyl-3,4benzphenanthrene 6-Methyl-3,4-benzphenanthrene 4,9-Dimethyl-lJ2-bensanthracene l-Methyl-3,4-benzphenanthrene ZMethy1-3,4-benzphenanthrene 5,7,9-Trimethyl-1,Pbenzacridine 7-Methyl-3,4benzphenanthrene 5,10-Dimethyl-1,2-benzanthracene 9,lO-Dimethyl-1,a-benzanthracene 4,lO-Dimethyl-1,a-benzanthracene 6,9,10-Trimethyl-1,2-benzanthracene
5,9,lO-Trimethyl-l,2-benzanthracene 5,6,9,1~Tetramethy1-1,2-benzanthracene
Charge 1.258 1.259 1.260 1.260 1.270 1.272 1.273 1.274 1.283 1.284 1.285 1.286 1.291 1.292 1.293 1.293 1.294 1.294 1.296 1.296 1.298 1.298 1.298 1.302 1.304 1.304 1.306 1.307 1.309 1.309 1.310 1.311 1.312 1.312 1.312 1.313 1.317 1.319 1.321 1.330 1.332 1.343
Skin
0 0 0 0 0
Subcutaneous Tissue -
0
-
+
+
+ +
+
0 0
0 0 0
+ +++ + ++ 4-4++ + + +++ +++
++++ ++++
+++ +++ + + ++ +++ +++ + -
++++ ++++ ++++ +++
-
0
-
+ -
0
-
++++ ++ ++ ++ 0 +++ +++ +++ ++++ ++++ 0 ++++ 0
+ ++ 0 ++++ +++ ++ ++ +++ + 0
38
C. A. COULBON
the definition of the electrical index which defines the carcinogenic power. Thus Buu-Hoi and colleagues (1947) have suggested replacing (8) by
+ + F2 + + 92 + p i + p2
2~12 Fi
Qi
(9)
where p12is the mobile bond order of the K-region bond, as before, and pl and p2 are the mobile orders of the two bonds which connect this bond with the rest of the molecule, as in XXXVII. The total charge of the K-region is now increased to a little over 2
(XXXVII)
electrons, but the general conclusions are unaltered. Figure 8, obtained by these workers and here quoted from Daudel and Daudel (1950) shows a clear correlation between the theoretical charge and the experimental index of carcinogenicity. It is notoriously difficult to establish a definite experimental measure, partly because the method of application and the
2.00
2.02 2.04 2.06 Total charge on K.region (in electrons)
FIG.8. Curve showing the relation between experimental index of carcinogenicity and the total charge on the K-region, when this latter is defmed according to equation (9). Each point represents a different molecule.
type of animal used affect the result, and it is seldom possible to apply the compounds to a sufficiently large number of animals to provide an adequate statistical conclusion. Furthermore the time interval before the appearance of tumors varies widely. But there is no doubt of the general
39
ELECTRONIC CONFIGURATION AND CARCINOGENESIS
significance of this curve on which the 22 points refer t o various methyl benzacridines and benzanthracenes. R. Daudel (1948b) has pointed out that a more careful scrutiny of these points suggests that, if an electrical index of this kind exists, it has a different threshold value for different series of related molecules and apparently decreases as the number of fused benzene rings in the main molecular skeleton increases. In particular, Fig. 9, taken from another paper by R. Daudel (1948a) dis150
-
100
-
Substituted benzanthracenes M -methyl
A
-
1 '3 5
* Substituted benzacridines M -methyl, E -ethyl - 5 6 benzacridine
3:lOdiM X X 2lOdiM
0
X 28 benzacridine
25
-
1.97
3M 10E 1:lOdiM
1.98
1
2.00 Total charge on K.region (in electrons)
13:lGtriM
t
2.02
I
)
FIG.9. Curves showing the relation between the experimental index of carcinogenicity and the total charge on the K-region, when this is defined according to equation (9). A different curve is required for the benzanthracene series (on top) and the benzacridine series (below).
tinguishes between the methyl benzanthracenes and the methyl benzacridines. In the first series carcinogenic activity appears to set in when the total charge exceeds about 2.015 electrons. However, in view of the approximations involved in these calculations and the relatively small changes in the total charge which appear to be necessary, it is doubtful whether very much can yet be inferred from a comparison of curves of this kind other than further confirmation of the general picture. Before concluding this paragraph reference must be made to a suggestion (A. Pullman, 1947a) that one of the exceptions t o the rules just
40
C. A. COULSON
described could be accounted for by a stabilization of one particular bond structure as a result of the Mills-Nixon effect. If the cyclopenteno rings in XXXVIII and XXXIX were as drawn, we might explain why the K-region bond order in the second molecule was lowered sufficiently to destroy most of the activity shown by the former. Unfortunately, as Longuet-Higgins and Coulson have shown (1946), bond fixation in the Mills-Nixon effect is very much smaller than was a t one time supposed and would, indeed, be likely to act in the opposite direction to that required for this argument. The explanation, therefore, must lie elsewhere.
I
(XXXVIII)
(XXXIX)
6 . Molecular-Orbital Indices
The work described in Sec. V, 5 was entirely valence-bond work.
It is clearly very desirable that equivalent calculations should be made using the alternative molecular-orbital technique. Such calculations have just been completed, largely by Greenwood. For reasons of convenience Greenwood made a very careful study of the various mono- and polymethylated benzanthracenes and benzacridines. The method of introducing methyl groups was substantially that discussed in Sec. IV, 6, and the calculations of the acridine compounds were based on the corresponding anthracene compounds and an extended use of the polarizability coefficients described in Sec. IV, 5. Great care was taken to insure that the validity of using perturbation methods in this way was established, and in many cases second order perturbation, as well as the usual first order, was applied. The quantities which were calculated were the charges flowing to or from the K-region (atoms 3 and 4), the bond orders of the K-region, and the free valences FS and F4. The conclusions to which Greenwood has come may be summarized briefly as follows: (1) Methyl substitution a t any point of the periphery of 1,2-benzanthracene leads to an increase in the sum q~ q4 (i.e., the net charge on the two atoms of the K-region) except when the substitution is a t position 8, where there is an extremely tiny decrease, or a t either of the atoms 3 and 4 of the K-region. This is in agreement with the v.b. calculations described earlier in this section. (2) An aza replacement which converts from a benaanthracene to a
+
ELECTRONIC CONFIGURATION AND CARCINOGENESIS
41
benzacridine removes charge from the K-region; though it may actually increase the charge on one of the atoms (atom 3 when the N is a t position 9, and atom 4 when it is at position lo), the decrease in charge on the other atom more than cancels this increase. So far as the total charge is concerned, this agrees with the v.b. calculations. It differs from it in predicting opposite behavior for the two ends of the K-region bond. (3) Methyl substitution at all positions except 3’ leads to a decrease in bond order in the K-region, so that there is a complete absence of correlation between high bond order and carcinogenicity. This conclusion had previously only been obtained for the unsubstituted hydrocarbons (see Sec. V, 2). (4) The change from benzanthracene to benzacridine lowers the bond order p34 of the K-region. ( 5 ) There is no correlation either between the change in free valence at atoms 3 or 4 of the K-region or between the sum of these free valences and the carcinogenic activity of the various derivatives. (6) Of the simple derived magnitudes, the only apparent correlation seems to be with the total charge 43 q4. But here, just as in Pullman’s v.b. work, it is necessary to exclude methyl substitution in the benz ring somewhat arbitrarily. It is necessary to select q8 44 and not either 43 or 44 separately, for which no correlation at all exists. To this extent Pullman’s original conception of the charge associated with the whole of the K-region seems t o find further support. The nature of the agreement with experiment may be illustrated by the six benzacridines shown below, for which Greenwood’s calculated charge migrations are shown as well as the experimental carcinogenic activity. The agreement here is
+
+
TABLE 111 Molecular Orbital Charges Molecule 3,4Bensacridine* 5-Methyl-3,4 benzacridine 5,7-Dimethyl-3,4-benzacridine l,%Benaacridine 5-Methyl-l,2-benzacridine 5,&Dimethyl-l,2-beneacridine
* The numbering Byatem ia aa in (VI).
Net Charge on K-Region -0.077 -0.075 -0.072 -0.005 +O ,033 +O. 036
Carcinogenic Potency
0 0 0 0
+++ ++++
evidently very comparable with that previously obtained by the v.b. technique. Before leaving the figures in Table 111,it should be said that they give the charge migration to the K-region for one particular value
42
C. A. COULSON
of the constant of proportionality described in Sec. IV, 2. This constant is the ratio of the electronegativity difference and the change in Coulomb term associated with the substituent. A change in this constant would merely multiply all the numbers in Table I11 by the same amount. Greenwood has verified that if the ratio of the assumed effects for a methyl group and a nitrogen atom is different from that assumed here (1:2) , the general conclusions are unaffected, though the actual charge shifts would be modified. It must not be presumed, from the above, that the criterion which we have described is universally applicable. We have already mentioned its failure to explain the deactivating effect of methyl substitution in the bena ring of bensanthracene. Another and equally serious exception arises when a -CN group is substituted a t the meso position 10. Without any doubt at all, this will have the effect of withdrawing charge from the K-region. According to Greenwood’s calculations it will also diminish the bond order. Yet the resulting molecule is strongly carcinogenic. One would have expected that, since -CN attracts electrons out of the aromatic framework, and -CHs pushes them into it, carcinogenic properties would vary smoothly in the sequence cyano-, unsubstituted, and methylbensanthracene. In fact the two extremes are active; the middle molecule is almost inactive. This example, which is not unique, warns us against any too simple explanation by means of an electrical index of this type. 7. Resonance Energy, etc.
It has occasionally been suggested that some correlation exists between the stability of the molecule (i.e., effectively its resonance energy) and its carcinogenic power. It is easy to see that no such correlation exists by a comparison of the six tetracyclic hydrocarbons of Table I. The fifth column of this table shows the resonance energy (Baldock et al., 1949; A. Pullman et al., 1950) in units of 0,which is the resonance integral for a C-C bond. It will be remembered that the only fairly strong carcinogenic compound in this table is the third one, but its resonance energy is in no way unusual. A further possibility is suggested by Pullman, Berthier, and Pullman (1950). It is often supposed (Boyland, 1948a) that one stage in the mechanism of these molecules is the addition of the molecule to some part of the cell and that this addition takes place at the K-region. The case of l,2-benzanthracene is illustrated in XL. It is reasonable to suppose that this attachment requires two bonds, such as those shown dotted. The conjugated framework that still survives is, in this case, a-phenyl-
ELECTRONIC CONFIGURATION AND CARCINOGENESIS
43
naphthalene. Its resonance energy can be calculated; and so, by subtraction from the original resonance energy, we calculate the loss of resonance energy in the process of attachment. Let us call this the energy of bond addition. The values of this energy for d l six tetracyclic hydrocarbons are shown in the last column of Table I. No correlation exists between these values and the carcinogenic activity of the six
molecules concerned. This means that no straightforward explanation is possible which is based on the idea either of total energy or of energy released on forming a complex with the cell. A slight correlation appears to exist between the resonance energy of the residue (a-phenylnaphthalene in XL) and carcinogenic potency. This resonance energy is simply the difference between the last two columns of Table I. The largest value is for the carcinogenic molecule 3,4-benaphenanthrene, and the next largest is for 1,2-benzanthracene9 which is slightly carcinogenic. Pullman, Berthier, and Pullman (1950) say that there are difficulties in extending this to five-ringed systems. But in any case it is extremely hard to see how the resonance energy, after formatiorl of the complex with the cell, could be physically significant. 8. Electronic Excitation
The last possibility which we shill envisage is the energy of electronic excitation. It appears, at first sight, quite plausible that the amount of energy which the molecule can absorb, when in its ground state, or emit, when in an excited state, should be significant. There is, unfortunately, considerable technical difficulty here. For no adequate calculations of excited states have been made by the v.b. method, so that all comparisons must be made with the m.0. method. But what the m.0. calculations predict is not strictly the energy of the electronic jump, but the average between an allowed jump t o a singlet level and a disallowed jump to a triplet level. The difference between these two jumps cannot readily be calculated. For that reason all calculations of molecular spectra are much more liable to error than calculations of the energy in the ground state. Fortunately, the rather limited theoretical evidence available
44
C. A. COULSON
agrees tolerably well with the experimental evidence. For that reason we shall concentrate on the former, though reference should be made to work by Jones (1940,1941,1943) for the experimental verifications. The theoretical calculations, due to Pullman, Berthier, and Pullman (1950) merely neglect the singlet-triplet difference just referred to and are therefore rather uncertain measures of the actual transition energy. First, as regards the set of six tetracyclic hydrocarbons in Table I, there appears to be absolutely no correlation whatever between carcinogenicity and energy of transition, though the range covered by the latter is from about 1.5 to 4.5 ev. (A. Pullman, 1949) (i.e. 30 to 100 kcal./mole). But secondly, there is a restricted type of correlation possible within one family. Pullman, Berthier, and Pullman (1950) have discussed the monomethylbenzanthracenes. Table IV is taken from this paper and shows that as the transition energy decreases the carcinogenicity increases. A notable point about this particular tsble is that substitution in the benz ring does not appear to alter the transition energy anything like so much as substitution in the anthracene part of the skeleton. This situation fits better with the experimental carcinogenicities than when, as in Secs. V, 5 and V, 6 we have arbitrarily to exclude the benz region. TABLE IV Excitation Energy and Carcinogenicity (See A. Pullman et al., 1950)
Compound 1,2-Benzanthracene 1'-Methyl 3'-Methyl 4'-Methyl 6-Methyl 2'-Methyl 7-Methyl 3-Methyl CMethyl 8-Methyl 5-Methyl 9-Methyl 10-Methyl
Excitation Energy (units of approx. 3 ev.) 0.9164 0.9164 0.9154 0.9134 0.9120 0.9116 0.9099 0.9067 0.9061 0.9059 0.9042 0.8984 0.8934
Carcinogenic Activity
+
+ ++ ++ + +++ ++++ +++++
It is obviously desirable that other calculations of this kind should be made, to establish whether a similar correlation holds in other series; and also whether it holds for multiple substitutions as well as for single
ELECTRONIC CONFIGURATION A N D CARCINOGENESIS
45
substitutions. No evidence is yet available to discuss the first of these problems, and with the second the evidence is not very satisfying. For, to the extent that the values in Table I V are valid, the effects of two or more methyl substitutions should be additive (steric hindrance not being assumed). This would imply that further substitution at one of the benz carbons should increase the carcinogenicity of any previous substituted molecule. In fact we know that it often destroys a carcinogenicity that already exists! (E.g., l’,10-dimethyl is inactive; 10-methyl is highly active.) Further objections can be raised against all this work. Recent more accurate work on naphthalene (Jacobs, 1949) has shown that the m.0. method in its simple form cannot give anything like an adequate account of the electronic spectrum, and a recent review (Coulson et al., 1951) of this field casts serious doubt on any calculation of molecular spectra using the simplified model which has so far been the basis of all the m.0. calculations for large molecules. This means that the quantity which is called the excitation energy is an almost entirely formal quantity, whose physical significance as a transition energy is by no means clear. Mme. Pullman (1947b) has shown that there is a close correlation between the differences in excitation energy as a result of methyl substitution (shown in Table IV) and the free valence at the position in the parent hydrocarbon at which the substitution takes place. Since, however, we have shown in Sec. V, 3 that free valence alone is unable to define carcinogenicity, it seems to follow from this that both the spectral effects in Table IV and the free valence effect of Mme. Pullman are secondary, and not primary, in the complete phenomenon. In concluding this section, it should perhaps be added that the correlations in Table IV do not persist if other alkyl groups than methyls are substituted. Also there is no apparent correlation between carcinogenicity and either the wavelength or the intensity of fluorescence (Bruce, 1941). We might also ask whether there is any notable difference in chemical reactivity of carcinogenic and related noncarcinogenic hydrocarbons. This is particularly important since it has been suggested by Haddow (1947) that the activity of the carcinogenic compounds might be due to an inhibition of normal oxidation processes in the cell. Quite recently, however, Magat and Bonkme (1951) have shown that although small traces of a carcinogenic are able t o inhibit the thermal polymerization of styrene, showing that they act here as antioxidants, these molecules behave in no sense differently from related noncarcinogens. This shows that in vitro the original suggestion is not valid; it may of course be different in vivo (but see the latter part of Sec. VI, 3).
46
C. A. COULSON
VI. POSSIBLE MECHANISMS 1. Interpretation of Previous Conclusions
We have now reached the stage where it should be possible to look for some interpretation of the conclusions obtained in Sec. V. Our present knowledge of the theory of chemical reactions is much greater than it was even a few years ago [for recent reviews of this field, see Coulson (1951b) and Coulson and Longuet-Higgins (1947c)], so that we might reasonably hope to be able to make inferences regarding the mechanism of chemical carcinogenesis from the numerical data described earlier in this account. If we could be sure of what constitutes the requisite electrical index, we should be able to say whether the mechanism was largely heterolytic, involving electrical charges, or whether it was largely homolytic, involving free radicals. There are other possibilities also, which we shall come to later. Our program therefore will be first to discuss the type of reaction and then to speculate, briefly, about what actually happens. In making our deductions about the type of reaction, we aha11 find the molecular-orbital data more useful than the valence-bond data. The explanation seems to be that the m.0. technique is more systematic and consistent from molecule to molecule than is the v.b. technique. %.Advantages of the K-Region
It is easy to see one great advantage possessed by the K-region. Its very shape makes it exceedingly accessible t o any approaching radical; it is less easily screened than most of the rest of the molecule. It might be called an “exposed bond.” In addition to this, we have seen that it does possess unusual electrical properties. The high bond order marks it out a t once as likely to be significant. And the relative ease with which charge can be moved to this K-region gives an explanation of the power of methyl substitution to enhance a carcinogenicity which is sometimes latent. There is a close parallel here between the chromophore which is necessary for color to be exhibited by a particular molecule and the auxochrome which enhances, or brings out, the latent color of the original chromophore. Thus in the dimethylbenzanthracene XLI we can distinguish what Boyland (1950) has called the carcinogenophore (the K-region) and the auxocarcinogens (the CHs groups). The parallel with color is well illustrated by comparing XLI with XLII, in the manner suggested by Boyland.
ELECTRONIC CONFIGURATION AND CARCINOGENESIS
chromophore
47
auxochrome
auxocarcinogens
(XLI)
(XLII)
3. SigniJicance of the Total Charge on the K-Region
We showed in Sec V, 3 that the free valence at the ends of the K-region did not appear t o exercise a dominant role in carcinogenicity. This conclusion enables us t o make one important inference. For it has been shown by R. Daudel and colleagues (1950) and by Burkitt, Coulson, and Longuet-Higgins (1951) that homolytic (i.e., free radical) reactivity is largely governed by free valence. It seems therefore as if we may provisionally dismiss this type of mechanism. In view of the very central part which such mechanisms are now believed to play in many biological (particularly radiological) phenomena, this is a valuable conclusion to have been reached. This brings us to the interpretation that should be placed on the total charge of the K-region. In the form (8) of Sec. V, 5 , Mme. Pullman’s valence-bond definition of charge, it is extremely hard to see what physical significance we can attach to the conclusion that a high charge implies carcinogenicity. This is because both the definition (8) and also the extended form (9) of Buu-Hoi and colleagues introduce all three quantities: bond order, free valence, and net charge. No simple interpretation of this mixture is possible, except the weak conclusion that a high value for the index will generally imply a high bond order, and this leads to a shorter bond and consequently an enhanced electronic density (electrons per unit volume). It looks as if any sound conclusions regarding the mechanism will have to follow more from the molecular-orbital calculations than from the valence-bond ones. In this connection we have already stressed that Pullman’s term “total charge” is not a completely happy one since it does not actually measure any observable net number of electrons. There was only one fairly reliable correlation which came from the m.0. work reported in Sec. V, 6. This was the conclusion that carcinogenicity appeared to be facilitated by a high K-region bond order and a certain small flow of charge to the K-region. Now the size of this flow of charge (see Table 111)is much less than that which we normally associate
48
C. A. COULSON
with heterolytic, or ionic, reactivity. Indeed, a very elementary calculation of the magnitude of the forces to be expected with charges as small as those suggested for a carcinogenic molecule such as 5-methyl1,a-benzacridine suggests that they are small compared with other forces, such as polarization forces, or the forces between two unit electronic charges. The unavoidable conclusion seems to be that the mechanism is not a normal heterolytic one. This argues very strongly in favor of the view that we are dealing with a bond reaction, in which the K-region bond reacts, as a largely neutral bond, with some enzyme associated with the living cell. Just what happens after the complex has been formed is a matter for speculation, to which we shall return in a later paragraph. For the moment it is important to recognize that our theoretical analysis has led us to conclusions already widely accepted by the experimental chemist. For it is well known that the K-region bond in phenanthrene (the 9,lO bond) behaves very much like an isolated double bond, and adds hydrogen far more easily than aromatic molecules usually do. It is very natural that it should add to the enzyme in the manner depicted pictorially in (XL). This suggestion fits in well with some experimental work by Badger (1949, 1950) and some theoretical studies by R. D. Brown (1948). Badger studied the rate of addition of OSOCto various carcinogenic molecules, and found that when he compared various unsubstituted hydrocarbons, there was a fairly close parallel between this rate and the bond order of the 3,4-bond (K-region) to which it seemed that the osmium tetroxide added, more or less as in (XLIII). This would agree well with
(XLIII)
the view that i t was preeminently a bond reaction, and the parallel with (XL) would argue that this too might be governed by the same factors. Methyl substitution however was found to increase the rate of reaction, although, as Greenwood showed (Sec. V, 6) it reduces the bond order. Thus the correlation, while suggestive, breaks down in detail. It is possible, of course, that the rate of reaction should not correlate in any but general terms with bond order, In fact as is well known, the rate of reaction will be governed by the energy required to activate the reacting
ELECTRONIC CONFIQURATION AND CARCINOGENESIS
49
molecules to the transition state. Brown has attempted to calculate this energy for unsubstituted hydrocarbons. He finds a fairly general parallel between activation energy and bond order. But the correlation is not perfect, so that “bond reactions” cannot be discussed in terms solely of bond order, even with unsubstituted molecules. With substituted molecules, it is almost certain that some combination of bond order and charge will be required. So far as the evidence goes, it appears t o support this view. Thus a reasonable correlation does exist, as Greenwood’s figures clearly show, between the charges which methyl substitution. transfers to the K-region and the rate of the OsOd reaction. No correlation, however, exists between this rate and the changes in bond order, or free valence, at the K-region. It is significant, here, that Badger notes a reduction in the rate on going from benzanthracenes t o benzacridines; here the charge migrations are proportionally much larger than the bond order changes. The conclusion to be drawn from this osmium tetroxide work is that bond reactions are largely determined by bond order, but that in any one series of closely related molecules the charge flow is also significant. There is thus no kind of conflict with the tentative view that the carcinogenic action begins by a bond addition to some enzyme. However there is other confirmation of this view from the fact that almost all strongly polar substituents deactivate carcinogenic molecules, either partially or, sometimes, completely. This suggests that we do not want too “violent” an initial attack, such as would be more likely from a heterolytic reaction. We can argue as follows, following Schmidt (1938, 1939a,b,c, 1941), Boyland, and Daudel (1948a). If the liaison with the enzyme is too weak, it will be broken up before the carcinogenic action has an appreciable probability of occurring; but if the liaison is too strong the hydrocarbon has too great a chance of being severely altered by the enzyme before it can react, and then because it was modified, it would cease t o be carcinogenic. A most significant observation was made by Boyland and by Berenblum and Schoental, and others. (A review is given in Boyland and Weigert, 1947.) This was that in the normal metabolism of polycyclic hydrocarbons an oxidation took place, converting first to a diol and then to a phenol, and that the point a t which the final remaining OH group was to be found was different from the point at which the oxidation would take place in a test tube. This suggests that in the living cell the usual point of attack is blocked and provides strong evidence that the initial reaction is a bond attack to the enzyme. This would naturally take place at the reactive K-region and thereby compel the subsequent oxidation to take place at a different region. The two phenols obtained
50
C. A. COULSON
metabolically with benzanthracene and with chrysene are shown in (XLIV). A discussion of this mechanism has been given by Daudel (19484.
(XLIV)
4. Some Speculations It is very tempting to indulge in guesses about the way in which the carcinogenic influence is exerted. But it is highly important to recognize that any such account is extremely speculative. To begin with, it seems fairly clear that the initial action is an attachment to some part of the cell. Presumably this must be merely the precursor t o more specific action. We shall mention three such possible actions, though there is no serious evidence to support any of them. The first is due to Anderson (1947), who put forward the hypothesis that chemiluminescence of the hydrocarbon was involved. Starting from the experimental facts that chemiluminescence was a phenomenon associated with the oxidation of all the major groups of chemical carcinogens and that oxidation did take place (Sec. VI, 3) in the course of their being metabolized by the living cell, he suggested that the role of the carcinogens was first t o be absorbed all over the framework of the cell; then, when they came in contact with a hydroxylating system, they would liberate the energy normally observed as chemiluminescence. This energy, just because it was liberated in the interior of the cell, would soon be absorbed and might be sufficient to cause some sort of change in the protein near to the point of absorption. This change is taken to be conversion t o a malignant form, and the parallel is stressed between tumors induced chemically and those induced by physical radiation. The idea that an energy barrier of some kind separated the normal and malignant cell, has been discussed by Haddow (1947). All that one can say is that the proposed mechanism is quite conceivable. If it should turn out to be correct, we require a chemical molecule which can diffuse fairly well through the cell and be absorbed a t the K-region, and then be oxidized and emit the right amount of energy. We have already said that there is no direct correlation between carcinogenicity and fluoremence, but this was fluorescence of the isolated molecule, and it would probably differ considerably from fluorescence of the adsorbed molecule. This is as far as the idea can be carried a t present.
ELECTRONIC CONFIGURATION AND CARCINOCENESIS
51
The second hypothesis is due to Schmidt (1938, 1939a,b,c, 1941), and it is based essentially on the experimental fact that radiation of characteristic energy hv greater than 3.4 ev. (i-e., 80 kcal./mole) is needed to induce cancer. This is regarded as some internal rearrangement of the atoms in one of the genes, or other significant region in the cell; Schmidt suggests as a conceivable example a keto-enol tautomerism in one of the protein chains, and he suggests that instead of requiring incident radiation, the same chemical change could be stimulated by a suitable catalyst. This is the role of the hydrocarbon molecule. The only light which is thrown on this suggestion by the electronic studies of this review arises from the fact that a keto-enol rearrangement is essentially a rearrangement of T electrons (XLV). It is quite probable that such a chemical
I
N-H
b=o Rb
RbH
I
I
N-H
N-H
(=V) change might be rendered more easily possible by a “catalyst” with characteristic properties arising from the T electrons in its K-region. The third suggestion is an expanded version of the second, and is due to Daudel (194613). He suggests that several such isomerizations are necessary before the cell becomes malignant. Further, the more of these changes that there are already, the less easy is it for the cell to revert to a normal form, and the more easy for further rearrangements to take place (as, for example, by conjugation of two fairly close enol groupings). The process continues until the ‘(mutation”reaches an irreversible condition. This theory certainly has some attractive points about it, but the stabilization referred t o will be very small, so that the reaching of a rearranged form from which the protein chain cannot revert to its original pattern is not very easily understood. One point about this suggestion is that the total energy barrier for mutation to a malignant form may be split up into a series of small energy jumps. Finally, the action of inhibitors may be thought of as a competition between the carcinogenic and noncarcinogenic compounds for available sites on the enzyme. If sufficient noncarcinogenic molecules are able to occupy suitable sites, then the irreversible mutation cannot occur. We can see that inhibitors, in order to compete with the carcinogenic compounds, should themselves possess a K-region. Such is frequently found to be the case.
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C. A. COULBON
VII. CONCLUSIONS The claim which has been made and which we have been discussing in this review is that there is a certain region, the K-region, in a condensed polycyclic molecule which, by virtue of a high concentration of r electrons, is able to possess carcinogenic power. The various arguments have all accepted the origin of this power as lying in the a-electron distribution, but they have differed in the particular combination of derived quantities (bond order, net charge, free valence, energy of excitation, resonance energy) with which a correlation is sought. It is extremely important, before coming to any final decision, to bear in mind four significant factors : (1) Opinion is steadily growing now that the T electrons may not be treated so entirely separately from the ordinary localized u-bond electrons, in the way that all the theories which we have described, have done. This opinion is supported by several distinct types of consideration : first, that when accurate electron density patterns are drawn (March, 1952), the *-electron cloud merges quite indistinguishably into the u-electron cloud: second, that as Lennard-Jones and colleagues have recently shown, (Lennard-Jones, 1949; Lennard-Jones and Hall, 1950; Lennard-Jones and Pople, 1950; Pople, 1950) alternative descriptions of conjugated molecules are often possible, in which no specific division into u and ?r electrons takes place; in fact this separation is not a rigorous quantum-mechanical one; finally, as Dr. Altmann (1952) has recently demonstrated, even when using the u and ?r classification, one must allow electron exchange to take place between members in the different classes. This is another way of saying that a electrons cannot be treated entirely on their own. (2) It must be recognized that the existence of a correlation as, for example, between carcinogenicity and charge, does not necessarily imply that the one determines the other, or vice versa. For both properties may be the result of some more fundamental characteristic, from which they are both derived. This is particularly evident from much of the work reported in Sec. V. (3) Another aspect of this correlation difficulty is shown by the restricted correlations which have so often been found, such as with the various methylbenzanthracenes, but not with different unsubstituted hydrocarbons. In such cases it seems almost certain that we have not * yet really found the fundamental factor. What we have actually found is a series of secondary factors derived from the unknown primary one whose relationship with each other will never be able, by itself, to tell us what the primary factor is. (4) The fourth consideration t o have in mind arises from the nature
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of the calculations which are being made. The discussions of method and basis in Secs. I11 and IV should serve t o show us how uncertain are the foundations on which the calculation of the various numerical magnitudes depends. This means that we shall probably never be able to get bond orders, charges, etc., with as great precision as we should like. For example, we shall never be able t o incorporate all the possible structures in a valence-bond calculation, or even determine the individual weights of those which are already incorporated in the wave function. Similarly, in the molecular-orbital calculations, we shall never know the Coulomb terms and resonance integrals with complete reliability. This means that, even supposing that one single electrical index did exist, it is most unlikely that we should ever find out just what it was or just how it was composed of its constituent elements, p , q, F , etc. But it seems exceedingly improbable that, in fact, one single electrical index does exist, since, as we have seen in Sec. VI, a satisfactory correlation with experiment, even in the simplest types of chemical reaction, involves more than one of the fundamental elements p , q, F. Such correlations with experimental measures of carcinogenicity are made vastly more difficult by the extreme effort necessary to make sufficient experiments to provide a statistically significant numerical value. It looks therefore as if we are obliged t o abandon any hope of a clear-cut description of the relation between electronic configuration and carcinogenesis. But having said this much by way of a warning, we must not allow the somewhat arbitrary nature of the electrical index to hide the fact that some definite progress has been made. There seems t o be hardly any reasonable doubt but that the total charge on the K-region, coupled with a relatively high bond order, plays a significant part in the activity of the carcinogen. The fact that there are some serious failures in this correlation suggests that there may be two or more ways in which the carcinogenicity is shown, or that there are two or more stages in the complete phenomenon, and our K-region analysis deals with only one of these stages-the remaining stages may be governed by an entirely different index, corresponding to an entirely different mechanism. It would be true to say that the old confident optimism of the years 1940-47-that the K-region was alone significant-has been abandoned and that we have given up the hope of finally establishing any one unique mechanism of this kind. But what has been achieved is exceedingly valuable. It is still a clue to be followed further, so that we have been able, with some confidence, to reject several plausible but invalid theories. Bearing in mind how stubborn the problem of cancer has turned out, from practically every angle, there is reason to be grateful for such progress as has been made.
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ACKNOWLEDGMENT I should like to acknowledge permission to quote unpublished work done in this department by Dr. H. H. Greenwood, Dr. S. Altmann, and Dr. V. A. Crawford. To them, and to Dr. and Mrs. Pullman and Dr. and Mrs. Daudel, I should like to offer thanks for the benefit of discussion and correspondence, and permission to reproduce published curves. I am similarly indebted to the editors of Endeavour and t o the Council of the Royal Society. REFERENCES Altmann, S. 1962. Proc. Roy. SOC.(London)A210,327. Anderson, W. 1947. Nature 160, 892. Badger, G. M. 1948. Brit. J . Cancer 2, 309. Badger, G. M. 1949. J . Chem. SOC.456. Badger, G. M. 1950. J. Chem. SOC.1809. Baker, J . W. 1951. Hyperconjugation. Oxford University Press, England. Baldock, G. R., Berthier, G., and Pullman, A. 1949. Compt. rend. 228, 931. Berthier, G., Coulson, C. A., Greenwood, H. H., and Pullman, A. 1948. Compl. rend, 226, 1906. Boyland, E. 1948a. Yate J . Biol. and Med. 20, 321. Boyland, E. 1948b. Biochem. J . 42, 27. Boyland, E. 1948c. Biochem. Sac. Symposia 2, 61. Boyland, E. 1950. J. chim. phys. 47, 942. Boyland, E., and Weigert, F. 1947. Brit. Med. BUZZ. 4, 968. Brown, R. D. 1948. Trans. Faraday SOC.44, 984. Brown, R. D. 1950. J. Chem. Sac. 3249. Bruce, W. F. 1941. J. Am. Chem. Sac. 63, 304. Burkitt, F. C., Coulson, C. A,, and Longuet-Higgins, H. C. 1951. Trans. Faraday SOC. 47, 553. Buu-Hoi, N., Daudel, P., Daudel, R., and Lacassagne, A,, Lecocq, J., Martin, M., and Rudali, G. 1947. Compt. rend. 226,238. Coulson, C. A. 1939. Proc. Roy. Soc. (London)A169, 413. Coulson, C. A. 1946a. Trans. Faraday SOC.42, 106. Coulson, C. A. 1946b. Trans. Faraday SOC.42, 265. Coulson, C. A. 1947. Quart. Revs. 1, 144. Coulson, C. A. 1948. J. chim. phys. 46, 243. Coulson, C. A. 1951a. Proc. Roy. Sac. (London)A207, 91. Coulson, C. A. 1951b. Research 4, 307. Coulson, C. A., Craig, D. P., and Jacobs, J. 1951. Proc. Roy. SOC.(London)A206, 297. Coulson, C. A,, Daudel, P., Daudel, R. 1947. Rev. sci. 86, 29. Coulson, C. A., Daudel, P., and Daudel, R. 1948. Bull. soc. chim. France 16, 1181. Coulson, C. A,, and Jacobs, J. 1949. J . Chem. SOC.1983. Coulson, C. A., and Jacobs, J. 1951. Proc. Roy. SOC.(London)A206, 287. Coulsonj C. A.,. and Longuet-Higgins, H. C. 1947a. Proc. Roy. Sac. (London)A 191, 39. Coulson, C. A,, and Lohguet-Higgins, H. C. 1947b. Proc. Roy. Sac. (London) Al92, 16. Coulson, C. A., and Longuet-Higgins, € C.I.1947c. Rev. Sci. 86, 929. Coulson, C. A., and Longuet-Higgins, H. C. 1948e. Proc. Roy. SOC.(London) AlB3, 447,456.
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Coulson, C. A., and Longuet-Higgins, H. C. 194813. Proc. Roy. SOC.(London) A196, 188. Craig, D. P. 1950. Proc. Roy. SOC.(London)A 200, 272, 390, 401. Crawford, V. A. 1949. Quart. Revs. 8, 226. Cox, E. C.,and Jeffrey, G. A. 1951. Proc. Roy. SOC.(London)A207, 110. Daudel, P., and Daudel, R. 1950. Biol. Med. 39, No. 4, 201. Daudel, P., Daudel, R., and Buu-Hoi, N. P. 1950. Acta union intern. contre cancer 7,Ql. Daudel, R., Jacques, R., Jean, M., Sandorfy, C., and Vroelant, C. 1949. J. chim. phys. 46, 249. Daudel, R. 1946a. Compt. rend. 222, 798. Daudel, R. 1946b. Rev. sn'. 84,37. Daudel, R. 1948a. BuZZ. Cancer 86, 319. Daudel, R. 194813. Compt. rend. SOC. biol. 142, 6. Daudel, R. 1948. Compt. rend. 226, 175. Daudel, R., and Martin, M. 1948. Bull. SOC. chim. France 16, 559. Daudel, R., and Pullman, A. 1945a. Compt. rend. 220,247. Daudel, R., and Pullman, A. 1945b. Compt. rend. 220,699. Daudel, R., and Pullman, A. 1945~. Compt. rend. 220,888. Daudel, R., and Pullman, A. 1945d. Compt. rend. 221, 201. Daudel, R., and Pullman, A. 1945e. Compt. rend. 221, 247. Daudel, R., and Pullman, A. 1946. Compt. rend. 222, 663. Daudel, R., Sandorfy, C., Vroelant, C., Yvan, P., and Chalvet, 0. 1960. BuZZ. soc. chim France 17, 66. Dewar, M. J. S. 1949. J. Chem. Soe. 463. Greenwood, H . H. 1951. Brit. J . of Cancer 6,441. Haddow, A. 1947. Brit. Med. Bull. 4,331. Hartwell, J. L. 1941. Survey of Compounds Which Have Been Tested for Carcinogenic Activity, Federal Security Agency, U.S. Pub. Health Service, Washington, D.C. Hilckel, E. 1937. 2.Elektrochem. 48, 752, 827. Jacobs, J. 1949. Proc. Phys. SOC.(London)A62, 710. Jean, M. 1948. Compt. rend. 227, 1239. Jones, R. N. 1940. J. Am. Chem. SOC.62, 148. Jones, R. N. 1941. J. Am. Chem. SOC.68, 151. Jones, R. N. 1943. Chem. Revs. 32, 1. Lennerd-Jones, J. E. 1937. Proc. Roy. SOC.(London)A188, 280. Lennard-Jones, J. E. 1949. Proc. Roy. SOC.(London)A198, 1, 14. Lennard-Jones, J. E., and Hall, G. G. 1950. Proc. Roy. SOC.(London)A202, 155. Lennard-Jones, J. E., and Pople, J. A. 1950. Proc. Roy. SOC.(London)A202, 166. Longuet-Higgins, H. C. 1950. J. Chem. Phys. 18, 275. Longuet-Higgins, H. C., and Coulson, C. A. 1946. Trans. Faraday SOC.42, 756. Longuet-Higgins, H. C., and Coulson, C. A. 1947. Trans. Faraday SOC.48,87. Longuet-Higgins, H. C., and Coulson, C. A. 1949. J. Chem. SOC.,971. Magat, M., and Boneme, R. 1951. Compt. rend. 232, 1657. March, N. H. 1952. Acta Crystallographica. 6, 187. Moffitt, W. E. 1949. Trans. Faraday SOC.46,373. Mulliken, R. S. 1932. Phys. Rev. 41, 49. Mulliken, R. S. 1948. Phys. Rev. 74, 736. Mulliken, R. S. 1949. J. chim. phys. 46, 497. Mulliken, R. S., Rieke, C. A., and Brown, W. G. 1941. J. Am. Chem. SOC.63,41.
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Nebbia, G. 1950. Ann. chim. applicala 40, 627. Orgel, L. E., Cottrell, T. L., Dick, W., and Sutton, L. E. 1951. Trans. Faraday SOC. 47, 113. Pauling, L. 1937. Nature of the Chemical Bond. Cornell University Press, Ithaca, N.Y. Pauling, L., Brockway, L. O., and Beach, J. Y. 1935. J. Am. Chem. SOC.67, 2705. Pauling, L., and Wheland, G . W. 1933. J . Chem. Phys. 1, 362. Penney, W. G. 1937. Proc. Roy. SOC.(London)A168, 306. Prato, M., and Nebbia, G. 1950. Le Sostanze Cancerigene, Bari. Pople, J. A. 1950. Proc. Roy. SOC.(London)A202, 323. Pullman, A. 1945a. Compt. rend. 221, 140. Pullman, A. 194513. Compt. rend. soc. biol. 130, 1056. Pullman, A. 1946a. Compt. rend. 222, 392. Pullman, A. 1946b. Compt. rend. 222, 736. Pullman, A. 1946c. Bull. Cancer 83, 120. Pullman, A. 1947a. Compt. rend. 224, 120. Pullman, A. 1947b. Compt. rend. 224, 1354. Pullman, A. 19470. Ann. chim. 2, 5. Pullman, A. 1949. Compt. rend. 220, 887. Pullman, A,, Berthier, G., and Pullman, B. 1950. Acta union intern. contre cancer 7, 140. Pullman, A., and Pullman, B. 1949. J. chim. phys. 46, 212. Pullman, B. 1946. Compt. rend. 222, 1396. Pullman, B. 1948. Bull. soc. chim. France 16, 533. Pullman, B., Mayot, M., and Berthier, G. 1950. J . Chem. Phys. 18, 257. Schmidt, 0. 1938. Z . physik. Chem. 39, 59. Schmidt, 0. 1939a. Z . physik. Chem. 42, 83. Schmidt, 0. 1939b. Z. physik. Chem. 43, 1&5. Schmidt, 0. 1939c. Z. physik. Chem. 44, 193. Schmidt, 0. 1941. Naturwissenschaften 29, 146. Sherman, J. 1934. J. Chem. Phys. 2, 488. Sklar, A. L. 1937. J. Chem. Phya. 6, 699. Steiner, P. E., and Falk, H. L. 1951. Cancer Research 11, 56. Svartholm, N. V. 1941. Arkiu Kemi Minerol. Geol. A M , No. 13. Vroelant, C., and Daudel, R. 1949a. Bull soc. chim France 16, 36. Vroelant, C., and Daudel, R. 194913. Bull soc. chim France 16, 217. Vroelant, C., and Daudel, R. 1949c. Compt. rend. 228, 399. Wheland, G.W. 1934. J. Chem. Phys. 2, 474. Wheland, G. W. 1935. J. Chem. Phys. 3, 356. Wheland, G. W. 1941. J. Am. Chem. Soc. 63, 2025. Wheland, G. W., and Pauling, L. 1935. J . Am. Chem. SOC.67, 2086.
Epidermal Carcinogenesis* E. V. COWDRY Wernse Cancer Research Laboratory and Department of Anatomy, Washington University, St. Louis, Missouri
CONTENTS
I. Introduction
1. Epidermal and Gastric Cancer Compared 2. Objectives 3. Cooperation 11. Experiments 1. Standard Series of Mice A. Strain of Mice B. Age C. Weight D. Sex E. Application of Carcinogen F. Time of Sampling G. Living Conditions H. Hair Follicle Cycle I. Controls 2. Special Series of Mice 3. Human Series 111. Sequence in Experimental Epidermal Carcinogenesis 1. Normal Epidermis 2. Precancerous Hyperplastic Epidermis 3. Cancers IV. Microscopic Properties 1. Number of Epidermal Cells 2. Volume of Epidermal Cells 3. Nuclei of Epidermal Cells 4. Histochemistry of Epidermis 5. Hair Follicles 6. Sebaceous Glands 7. Dermis V. Chemical Properties of Whole Epidermis 1. Minerals 2. Lipids 3. Vitamins 4. Enzymes
Page 58 58 61 61 62 62 62 62 63 63 63 64 64 64 65 65 66 66 69 69 69 69 70 71 71 72 73 73 74 74 74 75 76 76
* Aided by grants from many sources, especially the Charles F. Kettering Foundation, the National Cancer Institute, and the American Cancer Society. 67
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Page 6. Nitrogen Metabolism.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 6. PolarographicallyReducible Substance. .. . . . . . . . . . . . . . . . . . . 82 85 VI. Integration of Data., .............................................. 1. Chemical Composition of Epidermis Compared with That of Liver and 85 Muscle.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Alterations in the Relative Proportions of Epidermal Components
5. Nature of Malignant Transformation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Stages in Epidermal Carcinogenesis . . . . .
91
. . . . . . . . . . . . . . 93
VIII. Summary .......... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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I. INTRODUCTION 1 . Epidermal and Gastric Cancer Compared
There are several obvious reasons why so many workers have attempted to discover how cancer develops in epidermis. Some may have been influenced by the fact that epidermis probably leads all other tissues of the body in its tendency to undergo malignant changes. This leadership is not revealed by the number of deaths from skin cancer which are very few compared with those from cancer of the stomach, 3,568, as compared with 26,215 recorded in the United States during the year 1948. Data for 1949 have not yet been published. The difference is that most cutaneous lesions, of the kinds likely to become cancerous, are well known, easily recognized and treated before the cancers actually develop in them; whereas, in the stomach, precancerous lesions are little known, hidden and usually remain for a long time untreated, in consequence of which more of them become malignant. It is not possible accurately to estimate what the frequency of deaths from skin cancer would be if Nature took her own course. Large numbers of potentially dangerous epidermal lesions are removed every year of which no records are kept. Neither are records kept of actual skin cancers of which thousands are cured; because, before they spread, they are easier to cope with effectively than gastric cancers. Gastric cancers are the greatest killers and call for research from this standpoint, but it is easy to understand the lure held for some investigators by epidermis, the most prone of all tissues to undergo malignant transformations. All admit that cancer is a problem of cellular changes. The conditions of cell life in the epidermis are fascinating. Their investigation is tempting for cytologists interested in carcinogenesis. Briefly stated the cells making up the outer half of the thickness of the epidermal sheet are
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dead or dying while those making up the inner half are young and vigorous. Over most of the human body both together make up a sheet somewhat thinner than an ordinary sheet of carbon paper of the “onion skin” variety (40 p ) . The inner living cells are protected by the dead bodies of their predecessors firmly cemented together, chemically changed as they have aged, and kept pliable and impenetrable for most aqueous solutions by the secretions of sebaceous glands. They live a kind of frontier existence subjected to a wider range of vital hazards than any other cells of the body. Let us compare these hazards in the lives of such epidermal cells with those of gastric epithelial cells. We are concerned primarily not with the chances that they will suffer injuries from within the body, which chances are about equal for both, but with the possibilities of injuries from without, which are far from equal. Epidermal cells are obviously more directly exposed to radiations of all kinds than are deeply situated gastric epithelial cells. We think first of solar radiations and of the remarkably high incidence of skin cancer in Texas, but the direct effect of these rays on the stomach is nil since they do not penetrate more than a few microns. The more penetrating x-rays can produce epidermal cancer and seriously injure many deep-lying tissues, but apparently not the gastric mucosa. Epidermal cells are exposed to heat in hot baths and we drink hot fluids, but in passing toward the stomach through the mouth, pharynx, and esophagus the temperature is somewhat reduced. At any rate we do not drink fluids that burn the mouth. The stomach is not subjected, as the skin is, to severe and extensive accidental burns. Neither are the gastric epithelial cells subjected to the very low temperatures of the external environment. Epidermis and dermis exposed to air many degrees below zero can become stiff with cold. Some of the epidermal cells may freeze, an experience never suffered by the gastric ones. To mechanical injuries likewise, such as cuts and bruises, epidermal cells are subjected more. The protection afforded them by the thin but tough and oily sheet of dead cells may be more effective than that given t o the living surface epithelial cells of the stomach by mucus; but hard materials, capable of inflicting physical injuries, are for the most part denied entrance to the stomach whereas they affect the skin directly. Evidence that uncomplicated mechanical injuries are carcinogenic is lacking in these two situations. They are, however, t o be reckoned with as dangerous events in the lives of the cellular inhabitants in both locations. The exposure of living epidermal cells and of gastric epithelial cells to chemical injuries is different in kind as well as in degree. By the
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nonliving external epidermal investment the living epidermal cells are more protected against water-soluble ones than are the gastric epithelial cells, despite the fact that access to the latter is to some extent guarded against by the sensations of taste and smell and ability to regurgitate from the stomach. But the epidermal investment is not uniform. The principal weak points are the pits from which hairs project and the sebaceous glands discharging into them. In the pits substances are likely to collect instead of being washed or brushed away from the free epidermal surface. Remaining in these locations they can enter the sebaceous glands, pass on into the tissue fluid, and injure the living epithelial cells from within. Since epidermis is more liable to mechanical injury than the gastric mucosa, accidental avenues affording entry to harmful chemical substances are much more likely to be opened up. By the same token exposure of living epidermal cells to viruses and pathogenic microorganisms is greater than it is for the secluded gastric epithelial cells. Epidermis is in fact more plagued by viruses than any other tissue of the body, and the cells must combat more local infections. On the whole the lives of epidermal cells are far from being placid and uneventful. They must survive radiations of several kinds, extremes of heat and cold, and mechanical injuries galore affording entry to invading microorganisms and viruses. Injuries that may not themselves be carcinogenic may nevertheless promote the action of others which are carcinogenic. It could be argued that because epidermal cells are exposed to such a wide variety of noxious agents the influences of which must be considered, they should be dismissed as unprofitable subjects of investigation, despite their leading tendency to become cancerous. But to cytologists they are all the more interesting. Added t o this is the fact that they are all of ectodermal origin and are conveniently arranged in layers of increasing differentiation in one direction from perpetual youth in the basal layer (as long as the body lives), through maturity and senility to death, and that they serve an essential role in protection of the body after their death. In the gastric mucosa, where cancer is so much more deathdealing, cells of endodermal origin are specialized in four or more directions, which introduces complications. They are not conveniently arranged. Unlike epidermal cells they are mixed up with mesodermal components so that to unravel the role of each in carcinogenesis is a perplexing problem. Setting all this aside, the selection of epidermis for systematic investigation in carcinogenesis is justified by the fact that it is avascular and alymphatic. This cannot be said for gastric mucosa. It is therefore not necessary in making chemical analyses of epidermis to correct for the variable inclusion of blood vessels, blood, lymphatic vessels, and lymph.
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These attributes of epidermis are of compelling importance in the choice of material for study. There are also the facts that chemical and physical carcinogens can be directly applied to epidermis, that something can be seen of the modifications induced therein by naked eye inspection, and that material for analysis can be excised from animals employed in experiments and from patients. It is for these reasons that we, first in the Barnard Free Skin and Cancer Hospital (1938) and later in Washington University (1948), have concentrated our attention on epidermal carcinogenesis. 2. Objectives The objectives are: (1) to ascertain the properties of epidermis which in the course of carcinogenesis change and to measure the extent of the changes; (2) to identify the properties that remain constant; (3) to integrate these observations and thus to discover the sequences in events. 3. Cooperation
The following investigators, all of whom have published their results, have been or still are members of this team: V. M. Albers W. Andrew M. H. Au J. P. Baumberger J. T. Biesele M. M. Biesele A. L. Caldwell Perihan Cambel C. Carruthers C. H. U. Chu G. H. A. Clowes Z. K. Cooper C. J. Costello E. V. Cowdry W. Cramer H. Evans H. L. Firminger
S. Frankel H. C. Franklin B. B. Geren A. R. Gopal Ayengar M. D. Kamen S. K. Kung A. I. Lansing C. T. Li H. M. Liang C. E. Lischer C. K. Ma H. Miller P. E. Nielson F. X. Paletta G. B. Ramasarma H. C. Reller
M. G. Ritchie E. Roberts T. B. Rosenthal A. Schiff R. L. Simoes W. L. Simpson W. Smith R. E. Stowell V. Suntzeff E. L. Tatum H. C. Thompson, Jr. G. H. Tishkoff M. H. Toosy L. F. Wicks J. H. Van Dyke D. K. Ziegler
Credit is given by them where it is due. A spirit of generosity and of mutual helpfulness without recognition reduces the number of authors on scientific contributions to reasonable limits. In this report I have the privilege of describing for them the results to date of our study of experimental epidermal carcinogenesis in mice, and of our less systematic investigation of naturally occurring epidermal carcinogenesis in man, which will be presented and discussed in relation to researches along these lines by many other workers. The responsi-
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bility for the description is none the less mine because it has not been feasible for them to correct the manuscript. 11. EXPERIMENTS 1 . Standard Series of Mice
All properties of epidermis during carcinogenesis cannot be determined in single specimens no matter how closely they are taken in sequence. The amounts of epidermis are too small and the techniques are too complicated. It has been necessary therefore to reduce variables to a minimum by standardizing the process of epidermal carcinogenesis in mice. The idea is to be able to produce a t will when required particular stages in carcinogenesis so that data concerning a given stage, obtained by different workers at different times by different techniques, can be superposed, so t o speak, and will supply comprehensive information concerning the properties of epidermis in the stage of investigation. In other words, it has been our ambition that all stages from beginning to end of our standardized series should be superposable, permitting the observations to be added up all along the line. Consequently in the standard series attempts are made to achieve as much uniformity as possible in the following experimental conditions, but in the course of thirteen years some variation has unavoidably crept in as one observes from noting the specifications in each published paper. A. Strain of Mice. The various inbred strains of Albino mice do not respond uniformly by epidermal carcinogenesis to methylcholanthrene similarly applied to the skin. For instance, Cowdry and Suntzeff (1944) found that the epidermis of CBA mice is more resistant than that of New Buffalo mice to the production of cancer by this carcinogen. In our early experiments we employed Strain A mice. We then shifted to New Buffalo mice but were forced by inadequate supply of them to concentrate in all subsequent experiments on Swiss mice. B. Age. Influence of the age factor has been examined. Cowdry and Suntzeff (1944)found that young New Buffalo mice (3-4 months) responded by cancer formation earlier and in a higher percentage of cases than did older mice (12-13 months) to the same methylcholanthrene treatment, a phenomenon not observed in CBA mice in which the inherited resistance wiped out expression of the differential age factor. No correlation was noted between the decrease in total epidermal lipid and this difference in response between the two strains (Suntzeff et al., 1948). Newborn Swiss mice (2-10 hours) were found to be refractory to cancer production by methylcholanthrene, a fact correlated with thick epidermis and the total absence, or the rudimentary condition, of hair
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follicles and sebaceous glands (Suntzeff et al., 1947). Later Cowdry et al. (1948) returned to the difference in susceptibility of New Buffalo and CBA mice and discovered that it is not paralleled by a difference in the calcium, copper, and zinc contents of epidermis. Therefore, though age is a factor to be considered, if it is kept reasonably uniform for mice of a given strain, results of different experiments can be integrated. C. Weight. The average weight of mice in a11 series is specified as evidence of condition of nutrition, strain and age remaining uniform. Occasional runts are of course not used. D. Sex. Each series must be of mice of the same sex. It is known that sex is a modifying factor in the production of liver tumors by carcinogens in rats (Rumsfeld et al., 1951). Changes in the properties of epidermis in a series made up of individuals of one sex are not expected to be duplicated exactly in a series consisting of the other sex even though all other controllable factors may be the same. Perhaps sex difference is accompanied by a difference in the disposal within the body of methylcholanthrene and/or its products. Wicks (1941) has made the interesting observation that male mice of several ages and strains normally exhibit a considerable degree of proteinuria, which is present t o a much smaller degree or absent in female mice. Bullough (1943, 1946, 1948, 1950) has described epidermal sex differences in mice. In females the thickness of epidermis and the frequency of mitosis therein are both rather more than doubled in the proestrus period and decrease quite suddenly after ovulation-phenomena absent in male mice. There is the further complication that these changes are more marked in the anterior dorsal epidermis than in posterior dorsal epidermis. Production of androgenic hormone in males is more continuous and less subject to cyclic variations than that of estrogenic hormone in females. Male mice are therefore theoretically preferable to female mice in these studies on epidermal carcinogenesis. But they fight more and frequently suffer epidermal injuries before the experiments are commenced. I n our early work we employed mice of both sexes indiscriminately; then we used males; now we recognize that females are the best. E. Application of Carcinogen. A 0.6% solution of methylcholanthrene in benzene is uniformly employed (other solvents having been studied by Stowell and Cramer, 1942). This solution is referred to simply as “the carcinogen.” It is obviously unsafe to attempt closely to integrate our results with those of others using different carcinogens. The carcinogen is applied uniformly by one stroke of a No. 4 camel’shair brush. The site of application has unfortunately not been the same in all
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E. V. COWDRY
our experiments. The mitotic counts have been made of ear epidermis, whereas all other properties have been measured in back epidermis. Differences in the extent of the area of application introduce a confusing factor preventing logical superposition of the properties estimated in carcinogenic series because the amount of carcinogen delivered differs. The ear probably received less than the back. However, the large extent of the area of the back treated with carcinogen was uniform in all the series yielding material for chemical analysis. Another factor productive of diversity in the response is that in some experiments, preliminary to the application, the skins were shaved, while in others they were not shaved. The ear epidermis was not first shaved. In some cases the back epidermis was also unshaved. But preliminary shaving was essential for all the chemical studies because, in the early stages before epilation due t o the carcinogen, the hairs would otherwise have confused the issue. It is recognized that shaving creates many small injuries so that its effect has been studied by Banyen (unpublished). To allow the epidermis of many mice to recover about equally, the treatment with carcinogen is postponed for five days after shaving. F. Time of Sampling. Difference in the time of day, when epidermal samples are taken for study, could be a confusing factor in the estimation of some properties in epidermal carcinogenesis. It has been observed in mice that the maximum frequency of mitosis is by day (Cooper and Franklin, 1940). The difference between minimum and maximum is significant, especially in relation to cellular replacement. G . Living Conditions. All mice are kept on the same diet in an airconditioned room, first at Barnard Hospital and later a t Washington University. Special efforts have not and are not being made to equalize their exposure to visible light. This is mentioned because Doniach and Mottram (1940) have reported that absence of light enhances epidermal carcinogenesis in Swiss mice. More recently Morton et al. (1951) observed the same phenomenon in DBA mice. But in both experiments the difference in illumination was far greater than that which could occur by chance in our animal room. H. Hair Follicle Cycle. A greater source of variability in our standard series, which we did not at first appreciate, is possible differences in the stage of the hair cycle of the mice a t the time the applications of carcinogen are started. Attention was directed to alterations in the hair follicles by Liang (1948) who employed for the first time, in the study of hair follicles, the technique of making whole mounts of the epidermal sheet with the attached hair follicles separated from the underlying dermis by maceration in dilute acetic acid. By this method both the distribution of the follicles and some of their individual characteristics
EPIDERMAL CARCINOQENESIS
65
can be ascertained much more accurately and easily than by the examination of many stained sections. More recently Wolbach (1951), on the basis of the literature and his own observations, has emphasized the importance of the hair follicle cycle in the study of sequences in experimental epidermal carcinogenesis. According to Dry (1925-26) the first generation of hair growth in mice is completed after about one month, the second after two months, and the third after approximately three months. In Wolbach’s experiments the second generation is well advanced in mice 28 days old, which would indicate a shorter period of growth of the first two generations than that given by Dry. Wolbach points out that in the stage of activity hair follicles with open hair canals are more vulnerable to the carcinogen than are the quiescent ones with plugged canals. To achieve as much uniformity as possible in the phase of the hair cycle by appraisal of the length, quality, and number of hairs in the area studied is as important as it is to select mice of the same sex, age, and weight in the hope that they will respond more or less uniformly to the carcinogen. In practice we find that young mice of the same litter are generally fairly uniform in respect to stage in hair follicle cycle. There is a pattern in the cycle of hair follicular activity, some regions being in advance of others; but this is not a confusing factor when observations are restricted to a single area viewed in the same direction. The patterns in the rat are best described by Haddow and Rudall (1945) and by Haddow et al. (1945). Wolbach thinks that they are similar in mice. In our group they have been studied by Perihan Cambel in mice (unpublished). I. Controls. Epidermises of the standard series have been most used in providing material for chemical analysis. The same properties of epidermises in the standard series are also compared with those in control standard series subjected only to treatment with benzene without any carcinogen. In addition a few observations were made of epidermal hyperplasia in pathological controls obtained from healing wounds (Paletta et al., 1941) and from responses to scarlet red (Carruthers, 1950). In these cases, as after benzene treatment, the alterations though they may be present in epidermal carcinogenesis, are not essential steps in cancer formation. 2. Special Series of Mice
I n addition to these basic observations on standard series resort has been made to supplementary investigations on special series for particular purposes. These include special series designed to reveal the influence of age, of increased environmental temperatures (Kung, unpublished), of treatment with estrogen (Paletta and Max, 1942), of x-ray exposure
66
E. V. COWDRY
(Toosy, 1951) of a single dose of carcinogen (Cramer and Stowell, 1943)) and of different hereditary endowments (Cowdry and Suntzeff , 1944). The purpose of such special carcinogenic series is to gain perspective on the events in the standard series by alterations in selected factors. 3. Human Series To establish epidermal carcinogenic series a t the human level is naturally still more difficult. The first task was to study normal epidermis, which if nature had not been interfered with, might conceivably have undergone malignant transformations. In one study antecubital skin was excised from many volunteers and examined (Evans et al., 1943). In another, samples of skin removed after death from many regions of the body were investigated (Cowdry et al., 1947). Numerous specimens of approximately normal skin were obtained from surgical operations. Senile keratotic lesions, representing “precancerous conditions,” were studied (Cowdry and Andrew, 1950), likewise specimens from many squamous cell carcinomas of epidermal origin. It is a case of hunting for opportunities to secure material and of gradually accumulating sufficient material to cover the main stages in human epidermal carcinogenesis of the so-called spontaneous variety, because the etiological factors remain obscure. Much remains for us to do in this direction before data on our standard and special carcinogenic series in mice can be integrated with those on human epidermal carcinogenesis.
111. SEQUENCE IN EXPERIMENTAL EPIDERMAL CARCINOGENESIS It was soon discovered that the “biological equation” epidermis chemically pure carcinogen +. epidermal cancer in a high percentage of cases is a highly artificial simplication. We have considered the epidermal cells of the Albino mice employed to be essentially of a single type though of increasing stages of differentiation in a proximo-distal direction from vegetative intermitosis t o fixed postmitotics as described by Cowdry (1950). Billingham (1948) has concluded that “the dendritic cell must be regarded as a cellular element constantly present within epidermis, which is therefore a compound tissue composed of cells of at least two distinct races.” He has reported the occurrence of “white dendritic cells” in the nonpigmented skins of guinea pigs. These are said to resemble the pigmented dendritic cells in all respects save melanogenesis. They are regarded by him as truebreeding variants of the same race of cells. According to Rothman et al. (1940) the white, or nonpigmented, dendritic cells of white human skin contain the enzyme syatem required for pigment formation, but this is inactivated by an inhibitor (see also Billingham, 1949). The possibility
+
EPIDERMAL CARCINOQENESIS
67
is not denied that “white dendritic cells” may be present in the epidermis of mice in our standard series; but the tumors developed in response t o methylcholanthrene are neither typical melanomas nor amelanotic melanomas. Moreover, it is unlikely that such cells occur in sufficient number when included with the chemical examination of the remainder to modify the results. The carcinogen is added to the surface of the epidermis; but it is questionable whether it reaches the surface of living epidermal cells which are themselves capable of undergoing the malignant transformation. Tracing the methylcholanthrene in by its characteristic fluorescence in ultraviolet light Simpson and Cramer (1943) found that in two minutes, and possibly earlier, methylcholanthrene can be detected by its fluorescence in the keratin layer of epidermis and in the sebaceous glands. From these glands some carcinogen enters the dermis and may influence the epidermis from within. Other carcinogen is expelled onto the epidermal surface in the sebum. The glands are the portals of entry. Simpson and Cramer observed t h a t six to ten days after a single application the fluorescence due to this carcinogen completely disappears from all parts of the skin. I n their opinion “there is no evidence that the unchanged carcinogen is taken up directly by the epithelial cells of normal epidermis.” They attribute the presence in later stages in some specimens of fine fluorescent globules in circumscribed areas of epidermis to be the expression of a degenerative process. The assumption is that the carcinogen (or its products) is taken up by the fat. Graffi (1942) described fluorescent material as present within the basal cells and Mottram and Weigert (1942) as present in the Malpighian layer of epidermis. Ahlstrom and Berg (1948) agree with Graffi. A physiological barrier against entry of carcinogens in relatively insusceptible species has been demonstrated by Cambel (1951). Entry of 3,4-benzpyrene has been investigated by Miller (1951). She found that fluorescent substances apparently derived from it and combined through chemical bonds with protein appear within three hours after a single application and disappear after two weeks. It could be that the unchanged carcinogen influences the epithelial cells, through same kind of surface action, without being taken up directly by them. Another possibility is that some carcinogenic product (or products) of methylcholanthrene, passing unnoticed in the experiments of Simpson and Cramer, does reach the epithelial cells and acts either a t their surface or within them. However this may be, the assumption that epidermal cells become malignant simply because methylcholanthrene, or some product (or products) of methylcholanthrene, is added to them is unwarranted. The problem is more com-
68
E. V. COWDRY
plicated. There is no reason to think that all epidermal cells, having malignant potentialities, are equally exposed. In fact, of thousands of epidermal cells presumably exposed in the area treated with the carcinogen, only a very small minority in a tiny focus (or foci) eventually become cancerous. This statement, like almost all others usually made in such an attempted analysis, requires qualification. Perhaps both the degrees of exposure and of the susceptibility of the exposed cells are unequal. Moreover, it does not necessarily follow, as implied by the equation, that some of the cells constituting the epidermis, to which the chemically pure carcinogen is added, themselves give rise to epidermal cancers in a high percentage of cases. Most of them are replaced by others before recognizable cancers appear. The normal rate of replacement of epidermis in the area treated is not known. In the plantar epidermis of adult Albino male rats weighing about 250 g., Storey and Leblond (1951), employing the colchicine method of arresting mitoses in the metaphase and ennumeration in stained sections, calculated the replacement time for the Malpighian layer of epidermis to be 19.1 days, “of this time 16.9 days was taken for renewal of the stratum germinativum and 2.2 days for that of the stratum granulosum.” In our terminology the stratum germinativum includes the stratum basale and spinosum. Since, in our whole mounts of epidermis, the number of mitoses per 15,000 nuclei increases from 17 (Cooper and Reller, 1942), to 149.7 in the first twentythree days of carcinogenesis, while the number of layers of cells in the epidermis increases only six times, it would appear that replacement is somewhat accelerated. In the special series, in which the carcinogen is applied only once, most of the cells affected by it are probably replaced at least once before cancers are observed to develop. In the standard series, with multiple applications, it is possible, however, that some of the contacted cells become malignant. The biological equation is also inadequate for it does not take into consideration the epidermal appendages, hair follicles, and sebaceous glands, as well as the underlying dermis. Entry is by the sebaceous glands, the living epithelial cells of which appear to bear a kind of charmed life for they seldom become cancerous. The carcinogen sinks into and is concentrated in the pits about the hairs. The hair follicular epithelial cells play a role in replacement of the epidermis as they shift outward and spread laterally. These should be considered as a possible source of the cancers in addition to epidermal cells in the limited sense included in the equation. To evaluate the modifications that take place in epidermal carcinogenesis, whether microscopic or chemical, data are required at three principal levels in the sequence in mice.
EPIDERMAL CARCINOQENESIS
69
1. Normal Epidermis The properties of normal epidermis measured are basic in so far as the same properties of epidermis reacting to the carcinogen are unchanged, increased, or decreased in relation to those of normal epidermis. 2. Precancerous Hyperplastic Epidermis
The properties are here measured in epidermises on the road to cancer development. All the epidermis exposed t o the carcinogen is not precancerous in the sense that i t is in a stage preliminary to cancer formation because cancer develops only in small and limited parts of the treated area. Consequently all the properties measured may not be actual stages in the carcinogenic sequence. Somewhere in each such specimen it is likely that the stage is being set for the malignant transformation. The designation “precancerous hyperplastic epidermis ” is convenient and not objectionable in view of this explanation. “Epidermis in the latent period” would be a more accurate phrase except for the fact that in the first day after the first application it is doubtful whether the epidermises of all treated mice really are latent in respect to ultimate cancer formation, because, if there are no more applications, the percentage of these epidermises that do yield cancers is small. 3. Cancers Under this heading we include the investigation of the properties of squamous cell cancers which constitute the vast majority. Basal cell carcinomas and sarcomas of the skin are of rare occurrence and are disregarded. In our early studies we examined and included in the standard series primary squamous cell cancers. But these exhibit considerable individuality, each tending to be a little different from the rest. After the establishment of strains of transplantable methylcholanthrene squamous cell carcinomas in mice by Cooper et al. (1944),all our attention has been focused on them. Use of transplants has the great advantage of providing a su5cient amount of material made up of tiny tumors of uniform size complicated by minimum necrosis for chemical analysis. The properties of these cancers, both microscopic and chemical, are compared with those of precancerous hyperplastic epidermis in attempts to discover the sequences in epidermal carcinogenesis. All chemical analyses before use of the polarograph were concentrated on transplantable Tumor No. 1. Thereafter many other strains were examined.
IV. MICROSCOPIC PROPERTIES It is within the changing structure of the epidermis that alterations in chemical properties take place.
70
1. V. COWDRY
1. Number of Epidermal Cells
Hyperplasia sets in early. It depends not only upon the mitotic rate but also upon the death rate and the speed of removal of the dead cells. Only the mitotic rate has been measured with a fair degree of accuracy. In our experience the best method is to separate the epidermis as a complete sheet from the underlying dermis by macerating in 0.5% acetic acid, to stain lightly with hematoxylin, and to count the number of mitoses per 15,000 non-dividing nuclei, thus avoiding the study of sections. Each stage recorded supplied an average of three such counts. Six to 12 hours after an application of carcinogen the mitotic count was slightly lower than that of normal epidermis (17/15000); a t eighteen hours it was slightly above normal, four reached a peak of 149.7/15000 at twenty-three days, then subsided to 72.7/15000 at sixty-five days, when cancers began to appear (Cooper and Reller, 1942; Reller and Cooper, 1944). It may be significant that between the ninth to the fifty-first day the mitotic frequency waa maintained a t a fairly even high level. Orr (1938)observed that the epidermal hyperplasia after a single application of methylcholanthrene reaches a maximum a t the end of the first week and thereafter shows little change until immediately before appearance of tumors. No determinations have been made of mitotic and intermitotic time in epidermal carcinogenesis. Perhaps the x-ray technique advocated by Knowlton and Winder (1950) could be utilized. They report that in normal epidermis of CF female mice 8 to 10 weeks old mitotic time is 30.2 f 12.0 minutes, and intermitotic time is 670 f 300 hours. Of the seven tissues examined, only one, the adrenal, is described as having longer intermitotic time. The long duration of intermitotic time described by Knowlton and Winder in normal epidermis may explain the infrequency of mitosis (17per 15,000nuclei). It is not feasible to determine the normal mitotic frequency in different layers of ear epidermis of mice as a starting point because the epidermis, having no organized spinous layer, is thin. But in the many-layered epidermis of the foot pads of mice Cowdry and Thompson (1944)observed that the maximum mitotic frequency is centered in the spinous later. This is in sharp contrast to its localization in the basal layer in hyperplastic pre-cancerous epidermis (Cowdry et al. , 1946)and is considered to be significant (see discussion of “Localization of Carcinogenic Action ’,). Localization of hyperplasia in relation to hair follicles will be mentioned later. Pullinger (1940) has directed attention to cells with paired nuclei (binucleated cells) which she found to be more numerous one day after an
EPIDERMAL CARCINOGENESIS
71
application of 1 % methylcholanthrene in acetone than mitoses. She interpreted their presence as indicative of a transitory dominance of direct division over mitosis a t this time. In the following nine days the ratio of binucleated cells to mitoses decreased. It is possible that in these cells the nuclei divided either by a mitosis or amitosis while the cytoplasm failed t o divide so that the result is not an increase in number of cells. If this is what happens, there is an element of arrest in normal cell function. Hemprova Ghosh is therefore making a special study of binucleated epidermal cells in our series.
3. Volume of Epidermal Cells Pullinger (1940) examined the early responses of the clipped skin of the nape of the neck of mice t o 1% methylcholanthrene in acetone. An increase in size of epidermal cells and nuclei was observed at one day. This reached a maximum at three days. From four to ten days, when observations were discontinued, decreases were noted in size of cells and nuclei. Alterations in the volumes of basal and spinous cells have been computed by Cowdry and Paletta (1941a). The measurements made a t 13, 19, 31, 39, 46, and 62 days suggest that a progressive increase takes place in the volume of both. The size of spinous cells was, however, relatively constant at 31, 39, and 46 days, whereas a noticeable increase took place in.the basal cells. Only the basal cell volumes in carcinogenesis could be compared with normal ones because spinous cells are absent or difficult to recognize in normal mouse epidermis. 3. Nuclei of Epidermal Cells According to Cowdry and Paletta (1941a) there is a progressive increase in the volumes of spinous and basal nuclei; but the stages observed were quite far apart. They found that the nucleo-cytoplasmic ratios were decreased to approximately the same degrees in both kinds of cells and that a t 13, 19, 31, 39, 46, and 62 days these ratios held to about the same level as are the mitotic frequency and the cell volume. It is feasible t o compute nuclear sizes quite accurately from whole nuclei separated from cytoplasm and collected for measurement by Ziegler’s method (1945). She observed (Dorothy Ziegler Kraemer, 1946) that “A 50% increase is mean nuclear volume, over that of normal untreated tissue, occurs 10 days after the first painting, and nuclear size is maintained at this level despite repeated paintings with the carcinogen.” A remarkable change in epidermal carcinogenesis is the increasing ease whereby nucleoli and nuclear chromatin can be displaced by ultra-
72
E. V. COWDRY
centrifugal force (Cowdry and Paletta, 1941b). This was first noted in epidermal cells eleven days after the first treatment, but may have occurred earlier. It is evident in benign papillomas as well as in squamoue cell carcinomas. The ease of this displacement resembles that observed in embryonic epidermal cells. Two days after the first application of carcinogen the first instance of enlarged and possibly doubled chromosomes was observed in fifty metaphases examined. At three days the frequency increased to 13%. It was somewhat reduced in the lo-, 20-, 29-, and 57-day specimens examined and increased to about 50 % in the cancers. Not only diploidchromosomes were seen but also some tetraploidchromosomes. No such modifications could be detected in the benzene controls (Biesele and Cowdry, 1944).
4. Histochemistry of Epiderma's By the elaboration of an ingenious photometric method for the measurement of thymonucleic acid (desoxyribosenucleic acid) revealed in sections by the Feulgen technique and by its careful use, Stowell (1942) has made interesting observations. The thymonucleic acid content is reduced in precancerous hyperplastic epidermises and in control specimens treated only with benzene. It is significantly increased in some carcinomas. By differential centrifugation Ayengar and Cowdry (1947) collected chromosomes for chemical analysis and found an average of 0.129 mg. of desoxyribosenucleic acid per milligram of dry weight in normal epidermis; 0.091 mg. ten and eleven days after three applications of carcinogen; 0.106 mg. sixteen days after six and seven paintings; and 0.208 mg. in the carcinomas. It is not known whether the decrease took place before ten days. Attempts by C. Ruangsiri (unpublished) to demonstrate alterations in mitochondria1 content revealed no significant modifications but were not carried throughout the carcinogenic series. By differential centrifugation quite pure samples of epidermal mitochondria were obtained but the yields have been insufficient to permit chemical analysis. Biesele (1944) has reported that the cytoplasmic ribonucleic acid concentration was notably increased twelve hours after the first treatment with carcinogen, attained a maximum from the third to the tenth day and dropped to an intermediate value by the fifty-seventh day only to rise again in the one cancer examined. No modifications were detected in benzene controls. Biesele and Biesele (1944) discovered considerable alkaline phosphatase activity the second and third days after the first treatment in
EPIDERMAL CARCINOGENESIS
73
the basal epidermal cells. Thereafter it was reduced in the epidermis but became very intense in the carcinomas. Microincineration preparations show that in the precancerous hyperplasia, as well as in benign hyperplasia of a healing wound, there is considerable demineralization especially in the distal part of the spinous layer. In the former, however, there are local variations in mineral content not found in the latter (Paletta et al., 1941). Greater use of histochemical techniques is clearly indicated because it is highly desirable t o localize microscopically within the epidermis the alterations antecedent to the malignant transformation. Many techniques of proved value await use, especially those of Giroud and Bulliard (1935) and Giroud and Leblond (1951) for SH groups and the qualitative analysis of keratin. 5. Hair Follicles
Liang (1948) has attempted to localize the sequence of microscopic changes in carcinogenesis by the detailed study of whole mounts of epidermis, in many respects a more advantageous technique than the laborious examination of serial sections. His studies were limited to the thirty days after the first application period. It was found by him that the first indication of local epidermal change was the appearance of cell clusters in a radiating pattern usually a t the junction of the hair follicles and the basal epidermal layer. Extensive multiplication of these cells caused disruption of the follicular pattern and eventual ulceration in the center of the proliferative area. Degenerating, enlarged, and fused follicles were observed but their role, if any, in epidermal carcinogenesis remains to be determined. However, the technique of whole mounts is being utilized in further unpublished observations by this member of the team. We do not know to what extent the epithelium of hair follicles participates in the replacement of epidermis. Their role in epidermal carcinogenesis is a fruitful field for further research. 6. Sebaceous Glands Reference has already been made to the entry of the carcinogen into the sebaceous glands, which, having later discharged their sebum, collapse and disappear four to six days after a single application of carcinogen (Simpson and Cramer, 1943). Kung (1949) found a noticeable decrease in the lipase activity of sebaceous glands one day after the penetration into them of methylcholanthrene. After three days lipase activity is no longer demonstrable in the few remaining sebaceous glands. According to Kung, vestiges of a few of these glands persist somewhat longer than other workers have reported. It is remarkable that the
74
IJ. V. COWDRY
living cells constituting their walls, though more directly exposed than any others to the carcinogen, very rarely indeed give rise to cancers. 7. Dermis The action of the carcinogen in dermis is shown by a temporary decrease in lipase activity of the fat cells (Kung, 1949), and by local modifications in alkaline phosphatase activity (Biesele and Biesele, 1944). Cramer and Simpson (1944) were impressed by alterations in the number of mast cells in the dermis. In their view the increase in number of mast cells before the development of a carcinoma is a reaction related to the development of the epidermal hyperplaaia and apparently conditioned by it. They think that it is a defense process against the development of skin cancer. Progressive modifications in elastic and collagenic fibers (determined microscopically and chemically) have been reported by Ma (1949).
V. CHEMICAL PROPERTIES OF WHOLEEPIDERMIS Epidermises were separated from dermis by the heat method of Baumberger et al. (1942). A sufficient number of each stage were combined together in lots each containing an adequate amount of tissue for chemical analysis. 1. Minerals The alterations in these, determined by Carruthers and Suntzeff
(1943, 1944, 1945, 1946) and Suntzeff and Carruthers (1943, 1944) in a series of investigations, are represented in Fig, 1. It will be noted that
the calcium and iron-nucleoprotein phosphorus ratios decreased to about 50% of normal and that these modifications were observed ten days after a single application of carcinogen. After ten days, during which there were three applications, the decreases in both were of about the same magnitude. After twenty-four applications within sixty days the same depression in calcium was observed while that in iron was further increased by approximately 10%. In the cancers (CA in the figure) all minerals except iron are decreased further. Modifications during carcinogenesis in citric acid metabolism have been described and related to the calcium changes (Miller and Carruthers, 1950). Similar reductions in minerals were not observed in control mice treated only with benzene; but Carruthers (1950) has reported a drop in calcium content of nearly 40 % in benign epidermal hyperplasias caused in mice by scarlet Ted. The relative amounts of ultrafilterable (free) and nonultrafilterable (bound) calcium have been determined by Lansing, Rosenthal, and Au
75
EPIDERNAL CARCINOGENESIS
(1948). In normal and precancerous hyperplastic epidermis the ultrafilterable calcium was 38% of the total calcium while in the transplantable carcinomas it was reduced to 29%. Lansing, Rosenthal, and Kamen (1948) have shown that in normal epidermis calcium45 is rapidly +50
1
+40
No. of applications of methylcholanthrene 3 6 12 24
2
+30
+20 10
+
0
- 10 -20
.-E .c
K
!!
-30 40 50
-60 -70
-80 -90
1
0
Zn
Fe
1 10
10
10 20
30 40 50 Time, days
1
cu
Ca
60
70
FIG.1. Indicates the percentage of changes whether increase (+)or decrease (- ) in minerals. The general shift is toward decrertse. Note prompt decrease in calcium and iron-nucleoprotein phosphorus ratios (from C. Carruthers and V. Suntaeff, 1946, J . Biol. Chem. 159-651).
replaced. In early stages of the precancerous epidermal hyperplasia there is a decrease in the capacity for calcium storage. Epidermis in later stages and in the transplantable carcinomas has apparently completely lost its ability to replace or exchange calcium. The turnover of calcium becomes practically nil. It is suggested that this may result from an alteration in the calcium-binding mechanism operating a t the cell surface. 2. Lipids Interesting alterations have been detected in the total lipid-protein nitrogen and total cholesterol-protein nitrogen ratios by Wicks and Suntseff (1942, 1945). Examination of Fig. 2 will show that reductions in these ratios reached a maximum a t about five days after a single application of the carcinogen. Moreover as soon as these reduced ratios were established, they were maintained for a considerable period of time during which there were many applications. Weitkamp (1945) and
76
E. V. COWDRY
Weitkamp et al. (1947) have found that the fatty acid components of lanolin are remarkably complex. Epidermal lipids therefore must contain a mixture of many components any one of which might be of Days
Number of applications
FIG.2. Indicates prompt decrease in total lipid and total cholesterol-protein
nitrogen ratios and maintenance without much further change (from L. F. Wicks and V. Suntzeff, 1946, Cancer Research 6, 464-468).
importance in this connection. Much more refined analyses of the individual constituents of the lipids of epidermis are needed.
5. Vitamins Tatum et a2. (1946) and Ritchey et a2. (1947) have determined the quantities of pyridoxine, choline, inositol, p-aminobenzoic acid, vitamin Ba complex, and- biotin in the epidermis of mice undergoing carcinogenesis by assay with mutant strains of neurospora. The outstanding modifications are : (1) Decrease in biotin in the precancerous hyperplastic epidermis to approximately 64 % of that of normal epidermis. (2) Little or no change in choline in the precancerous hyperplastic epidermis, but increase to 2.5 times normal in the cancers. (3) Little change in inositol in the precancerous hyperplastic epidermis but increase to 2.19 times normal in the cancers.
4. Enzymes Succinic dehydrogenese and cytochrome oxidase (Carruthers and Suntzeff , 1947), adenylpyrophosphatase (Roberts and Carruthers, 1948),
77
EPIDERMAL CARCINOQENESIS
arginase (Roberts, 1948; Roberts and Frankel, 1949b), and cytochrome c (Carruthers, 1947; Carruthers and Suntzeff, 194th) have been studied in the carcinogenic series. The activities of the five enzymes listed in Fig. 3 are indicated by the heights of the columns representative of each. 180
I
160
140 -
rn
1Apyrase
1
a Succinic dehydrogenase 120 Cytochrome C a Cytochrome oxidase 100 - 0 Arginase
80
60
Appl. .MC.
Fro. 3. Levels of enzyme activity. There is marked increase in the activities of succinic dehydrogenese, apyrase and arginase in the carcinomas, and of cytochrome oxidase in late hyperplastic epidermis (from C. Carruthers, 1950, Cancer Research 10, 255-265).
The control columns on the left indicate the levels of activity of these five enzymes in normal epidermis. The estimations after three, six, and twelve applications of the carcinogen are grouped together asare those after eighteen and twenty-four applications. The activities of all these enzymes, save cytochrome oxidase, the only component which rises significantly prior to cancer formation, remained practically unchanged
78
hl. V. COWDRY
in hyperplastic epidermis. No great significance can be attached to the absolute values of the individual enzyme activities except the cytochrome c studied because of uncertainty in correctly expressing the results. The relative changes of the enzyme activities are of greater interest since the ratios of the &-values (&-value; quantity of substrate used or decomposed per hour expressed in microliters of gas per milligram of wet weight of tissue) of the enzyme activities are independent of the basis of reference. The ratios of the &-values for arginase, cytochrome oxidase, adenylpyrophosphatase, and succinic dehydrogenase, respectively, are 6.2/1/0.85/0.15 for normal epidermis and 97.6/1/3.3/0.39 for the tumors. It is thus readily apparent that the enzymatic balance in the tumors must be entirely different from that in the normal epidermis. Although the activities of the enzymes measured in epidermis were lower, with the exception of arginase, than those generally found in other tissues, the values for the carcinomata were similar to those found for tumors derived from other tissues (Schneider and Potter, 1943; Greenstein, 1947). 6. Nitrogen Metabolism
Urea and ammonia were the first nitrogenous constituents to be studied (Roberts and Frankel, 1949a). Epidermis of the normal adult mouse was found to contain extremely high concentrations of urea and preformed ammonia. Even higher levels of these substances were observed in the epidermis of mice during the first nine days of postnatal development. Maximum levels for urea and ammonia were attained a t nine days and were two and eight times higher, respectively, than those found in the adult tissue. These high levels may be associated with the metabolic activities taking place during the formation of the first hair coat. It is not yet known whether the urea and ammonia are formed in the epidermis itself or are concentrated from the blood. The only thoroughly established mode for the formation of urea in the mammalian organism is by the action of arginase on arginine. A suitable method was developed for the study of arginase activity in homogenates of epidermis and tumors (Roberts, 1948). It was found that epidermis possessed appreciable arginase activity. A comparison of the dermis with the epidermis revealed that the high levels of urea were characteristic only of the epidermis. An investigation was then made of the content of total nitrogen, trichloroacetic acid soluble-nitrogen, urea, preformed ammonia, and arginase activity in the tissues of the standard carcinogenic series and of suitably selected control tissues (Roberts and Frankel, 194913). (See Table I.) The painting of the epidermis with methylcholanthrene in benzene produced changes in the various quantities studied. However,
TABLE I Nitrogen Content and Arginase Activity
Property Total nitrogen % fresh weight TCA*-soluble nitrogen % fresh weight % total N Urea nitrogen % fresh weight % fresh N Ammonia nitrogen % fresh weight % total N Arginase activity (7 urea liberated per milligram total N in standard assay)
Benzene Paintings
Methylcholanthrene Painting
Norma1
3
6
4.56
5.39
0.79 17.4
0.74 13.7
24
Tumor
4.76
4.66
2.28
0.82 17.2
0.95 20.3
11
0.23
10.1
3
1.06 24.8
6
0.92 24.3
11
-
Croton Oil 6
1.01 18.2
0.077 1.65
0.044 0.82
0.042 0.88
0.040 0.86
0.022 0.97
0.116 2.77
0.054 1.29
-
0.042 0.76
0.024 0.53
0.020 0.37
0.028 0.59
0.025 0.54
0.008 0.35 6,6W
0.025 0.60
0.035 0.84
-
0.036 0.65
586
1,540
421
19,4001
-
1,244
1,167
598
* Tricbloroacetic acid.
t Transplantable squamous cell Carcinoma I.
X Transplantable squamous cell Carcinoma 11.
675
-
$ 2
3 0
M
2
80
E. V. COWDRP
(B) FIG.4. Paper chromatograms of alcoholic extracts of 75 mg. of normal mouse (A), and human epidermis (B), and of mouse squamous cell carcinoma (CD). Key to numbers on chromatograms: phenylalanine, 1; leucine and isoleucine, 2; tyrosine, 3; valine, 4; methionine (as sulfone), 5 ; proline, 6 ; histidine, 7; hydroxyproline, 8 ;
E P I D E R M A L CARCINOGENESIS
81
the alterations so produced could also be brought about by other means. For example, although the application of the carcinogen produced a decrease of approximately 50% in the content of urea nitrogen, six paintings with benzene containing 0.1% croton oil produced a decrease of the same magnitude. The croton oil has been shown conclusively not to be carcinogenic. Although three paintings with methylcholanthrene in benzene caused a threefold increase in arginase activity, similar increases were found when benzene alone was applied. The distribution of the nitrogenous substances in the squamous cell carcinoma, however, was completely different from that found in any of the epidermal samples. The arginase activity was also much greater in the tumor than in any of the samples of normal or precancerous epidermis examined. It would, therefore, appear that the changes from normal observed in the precancerous st,ages were not associated with carcinogenesis per se and that the metabolic equilibrium in the tumor was entirely different. Determinations were made (Roberts et al., 1949) of the content of twelve amino acids in hydrolyzates of whole tissue samples of normal epidermis and of the epidermis of mice receiving varying numbers of standardized applications of methylcholanthrene in benzene and of
(C)
alanbe, 9; threonine, 10; taurine, 11; 0-alanine, 1 2 ; glutamine, 13; glycine, 14; serine, 15; arginine, 16; lysine, 17; glutainic acid, 18; aspartic acid, 19; cystine (cysteic acid?, 20; unknown ninhydrin reactive material which disappears on acid hydrolysis, 21; glutathione, 22.
82
E. V. COWDRY
benzene alone. The transplantable squamous cell carcinoma was also studied. Changes in amino acid distribution were produced by the application of benzene alone. However, the carcinogen in the benzene caused changes different from those found to result from this solvent alone. The tumor tissue as a whole did not show a distinctive pattern of amino acids which would set it completely apart from all the nonmalignant precancerous hyperplastic samples. I n particular, the amino acid distribution in the carcinomas was very similar to that found in the epidermis which had received three paintings of the carcinogen in the standard series. The changes which take place in total amino acid content of tissues may result from alterations in the ratios of cell types and quantitative and qualitative changes in intracellular constituents. In continuation i t is necessary to characterize the various cell fractions with respect to amino acid content and to study the carefully fractionated tissue proteins. Examination was then made by paper partition chromatography of free amino acids in mouse epidermis in various phases of growth (Roberts and Tishkoff, 1949). This showed that the carcinoma differed from all the samples of the epidermis studied by the distribution of the free amino acids in the alcoholic extracts (Fig. 4). Normal epidermis was found to have the highest overall concentration of free amino acids of all twenty normal tissues examined (Roberts and Frankel, 1 9 4 9 ~a) ~fact in keeping with the high concentration of urea, ammonia, and nonprotein nitrogen. Tissue made hyperplastic by the application of the carcinogen and the epidermis of newborn mice had greater concentrations of detectable constituents than normal epidermis, while the tumors showed a striking decrease. The distribution of the free amino acids in the resulting squamous cell carcinomas was very similar t o that found in a large variety of different transplantable mouse tumors carried in various strains of mice. Each normal tissue examined had a distribution of free amino acids typical for it while all the tumors, regardless of derivation, showed similar patterns. It is questionable whether the large concentrations of easily available nitrogenous const,ituents in epidermis are related to its remarkable regenerating capacity or whether they represent the end products of degradative mechanisms involved in the discarding of old epidermal cells and the formation of keratin. Studies by tracer techniques may be helpful. 6. Folarographically Reducible Substance The existence of tb qualitative difference in a polarographically reducible substance in epidermis as compared with that in induced and
83
EPIDERMAL CARCINOCIENESIS
transplantable squamous cell carcinomas was discovered by Carruthers and Suntzeff (1948b, 1949, 1950, 1951). This difference was found in the current-voltage curves obtained by the electrolysis of the reducible substance in a solution of 50% dioxane by volume, 50% buffers by volume to which was added sufficient tetrabutylammonium iodide to make the latter 0.1 molar. A polarogram of the reducible substance from epidermis at pH 6.5 is shown in Fig. 4A in which the current in microamperes is plotted against Ed.e.the potential of the dropping mercury electrode. The midpoint of the S-shaped curve is the half-wave potential, a characteristic constant for a compound or group of compounds, and for the first wave this potential is -1.62 volts vs. the saturated calomel electrode (S.C.E.). Figure 4B shows a polarogram of the substance from the carcinomas a t pH 4.0, and the half-wave potential is - 1.28 volts.
TABLE I1 Polarographic and Ultraviolet Absorption Characteristics of the Reducible Substances in Epidermis and Squamous Cell Carcinomas
Tissue Normal epidermis
PH 4.16
5.00
6.50 6.85 9.2
Hyperplastic epidermis
Squamous cell carcinoma
E 3 Volt vs . S.C.E.
Absorption Maxima mr
-1.39 -1.44 -1.62; -1.68 -1.71
268 -
No wave
4.21 5.00 6.50 6.85 9.2
-1.38 -1.43 -1.60; -1.66 -1.71
4.15 5.03 6.55 6.96 9.2
-1.28 -1.33 -1.43 -1.49
No wave
No wave
-
272 278 270
-
273 276 260 -
-
2 60 260
The pronounced difference in the half-wave potentials of the substances from epidermis and carcinoma are shown in Table 11. At pH 4.16, the half-wave potential of the reducible substance of the epidermis is -1.39 volts, whereas that from the carcinoma is -1.28 volts. At pH 6.5 the half-wave potential of the substance from the carcinoma is - 1.43 volts whereas that from the normal epidermis is - 1.62 and - 1.68 volts; that is, in the substance from the latter tissue two waves are
a4
E. V. COWDRY
present at pH 6.5, whereas only one is given by the substance from the carcinoma. Finally a t about pH 6.9 the half-wave potential of the substance from the epidermis is over 200 millevolts more negative than that from the carcinoma. Since the half-wave potentials are significantly different for the reducible substances in the carcinoma and in the epidermis, a structural difference is indicated in the reducible compounds. Later experiments revealed that the reducible compounds absorbing specifically in the ultraviolet are different. The substance characteristic of the epidermis, both normal and hyperplastic, absorbs maximally a t 280 mp in alcohol, and the absorption maximum is pH dependent (Carruthers and Suntzeff, 1950 and Table 11). On the other hand the substance from the carcinomas absorbs maximally a t 260 mp, and the absorption maximum is pH independent (Table 11). Therefore differences in polarographic behavior are correlated with those in the ultraviolet, and both properties of the reducible compounds change abruptly as sQonas the carcinomas arise following the application of methylcholanthrene. Furthermore the reducible compounds present in the epidermis of the mouse, rat, and man are indistinguishable polarographically and by their absorption characteristics in the ultraviolet, and yet in carcinomas of the epidermis of these species the altered compound appears to be the same. It is important to know whether the altered compound in the carcinoma and the compound characteristic of the epidermis are related. The following data prove that these compounds belong to the same class of substances. In the first place the polarographic waves (Fig. 5A and B) disappear if the electrolysis is carried out above pH 8.0. Secondly these compounds need iodide ion, or some other property of tetrabutylammonium iodide, the supporting electrolyte, for their reduction polarographically. Finally there is a one electron transfer in their reduction. Data have also been presented which prove that the properties of absorption in the ultraviolet and polarographic reducibility are common to the substance characteristic of epidermis (Carruthers and Suntzeff, 1951). This was done with the use of countercurrent distribution technique of Craig and with paper partition chromatography. On the other hand, the substance present in the carcinomas appeared to be cleaved into one component which was reducible and another which absorbed in the ultraviolet. The manner by which the carcinomas are able to alter structurally the reducible compounds is probably enzymatic. If this is true, the enzyme is absent or inhibited in the epidermis since the reducible substance in this tissue does not contain properties of that found in the carcinoma or vice versa. Recent studies have demonstrated that there are also polarographi-
EPIDERMAL CARCINOGENESIS
85
cally reducible substances characteristic for muscle and liver, and in malignant growths of these tissues, the altered compound is identical by our measurements with that substance found in squamous cell carcinomas.
FIG.5. A. Polarogram of the reducible substance characteristic of epidermis in a solution of 50% dioxane, 50% citrate buffer of pH 6.4and containing sufficient tetrabutylammonium iodide for a 0.1 molar solution. B. Polarogram of the reducible substance from squamous cell carcinomas in a solution of 50 % dioxane, 50 % citrate buffer of pH 4.0 and containing sufficient tetrabutylammonium iodide for a 0.1 molar solution. (Courtesy of Dr. C. Carruthers.)
VI. INTEGRATION OF DATA 1. Chemical Composition of Epidermis Compared with That of Liver and
Muscle
Epidermis is a unique tissue, and, for this reason, the alterations reported will not necessarily have equal significance in the appraisal of carcinogenesis in other tissues. In Table I11 some properties of epidermis side by side with those of liver and muscle. This comparison is qualified by the admission that the figures given for these three tissues do not relate equally to the chemical composition of the cells in each capable of undergoing a malig-
86
E. V. COWDRY
TABLE I11 Chemical Compoeition of Epidermia Compared with Liver and Muscle Epidermis Cytochrome oxidase* Qo, Succinic dehydrogenase* Qo, Apyraset Arginaae$ Cytochrome c, pg./gw. w.0 Urea, pg./gw. w. Ca, pg./gw. w. Mg, pg./gw. w. Na, p@;./gw.w. K, pg./gw. w. Ascorbic acid, pg./gw. w. Biotin, pg./g. dry weight Choline, pg./g. dry weight Inositol, pg./g. dry weight Pyridoxine, pg./g. dry weight
24.9 3.7 3.0 32.0 62.0
770.0 440.0
190.0 168.0 847.0 240.0 0.196 2471 .O 626.0 2.46
,
Liver
392.0 r 87.7r 12.9 r 2463.0m 90.0 r 300.0 r 90.0 r 190.0h 306.0 h 72.0 h 320.0 m 1.87 m 6280.0 r 933.0 m 4.66 m
Muscle
180.0r 36.0r 23.3 r
-
97.0r 100.0h 270.0h 72.0 h 365 .O h 43.0m 0.086r
-
466.0r 3.03r
* Qo, oxygen uptake per milligram dry weight per hour.
t Miorograms P liberated under standard wsay conditions.
t Miarogram urea liberated under atandard aaaay conditions.
-
-
1 Wet weight of tissue. All epidermis analyaed is from mice. The iourcea of the liver and musale are indicated by the lettern h human, m moue, r rat. Table prepared by Dr. C. Carruthers.
-
nant transformation. Epidermis of mice is made up of cells of a single epithelial type but a considerable proportion of them are senile or dead and incapable of becoming cancerous. Liver and muscle contain cells of several sorts though hepatic epithelial cells and muscle cells in which we are interested greatly predominate. Moreover, unlike epidermis] they include blood and larger volumes of tissue fluid. Nevertheless some substantial differences exist and are worthy of consideration. Since the malignant change is much more frequent in liver than in muscle, the chemical composition of liver is more directly involved in our orientation than is that in muscle. With these contrasting chemical properties in mind an examination of the properties of squamous cell carcinomas compared with those of normal epidermis given in Table IV is helpful. Choline, for instance, increases 50% or more in epidermal carcinogenesis. Its amount, however, to start with in normal epidermis was less than half that in normal livers. One would not expect a similar increase in the already high choline content of liver to be found in hepatic cancer. Calcium, on the contrary, is present in normal epidermis in a t least four times the concentration present in normal liver. This epidermal
EPIDERMAL CARCINOQENESIS
87
calcium is decreased 50% or more in epidermal cancer. One would not expect, and one does not find, a similar decrease in the already low calcium content of liver in the production of hepatic cancer. TABLE IV Properties of Transplantable Squamous Cell Carcinomas of Mice Compared with Those of Normal Epidermis (Modified from Cowdry, 1949) Increase 50% or more
Less than 50%
No change Decrease Less than 50%
50% or more
Lipid phosphorus: dry wt. ratio, succinic dehydrogenase activity, adenylpyrophosphatase activity, arginase activity, lysine, tryptophane isoleucine, choline, inositol and Bs complex (dry wt. bases). Water content, choline, leucine, methionine, valine, phenylalanine, threonine. Ascorbic acid: NP ratio, specific activity of Pal in phospholipid fraction, histidine, glutamic acid, cystine, arginine. Potassium/NP ratio, sodium/NP ratio, magnesium/NP ratio, cytochrome oxidase, desoxyribonucleic acid (wet wt. basis), cytochrome c, p-aminobenzoic acid, biotin (wet wt. basis). Calcium/NP ratio, iron/NP ratio, copper/NP ratio, zinc/NP ratio, nonprotein nitrogen (wet wt. basis), urea (wet wt. basis), ammonia (wet wt. basis), total free amino acids (wet wt. basis; chromatography), Ca45uptake and retention, free calcium.
It is well known that the chemical composition of malignant tissues is more uniform than that of their normal tissues of origin (Greenstein, 1947). When the tissue of origin is unique in having very high or low concentrations of any particular substanc.es, marked alterations are to be expected in carcinogenesis. Concentrations which are not so conspicuous in either way are likely t o be less modified in carcinogenesis. Knowledge of the individual properties of many normal tissues of origin gives perspective in evaluating alterations in carcinogenesis. 2. Alterations in the Relaiive Proportions of Epidermal Components
As the epidermis responds to the carcinogen in the standard series subjected to chemical analysis changes in the relative numbers of different classes in the cellular population may introduce factors that should not be ignored in the interpretation of the results of chemical analysis of pooled epidermises. If, for example, cells of the spinous layer are by nature low in calcium it could be that the remarkable decrease in total calcium is occasioned only by increase in the number of the spinous cells compared with the others.
88
E. V. COWDRY
It is therefore essential t o discover the changes ih relative numbers of different cells within the cellular epidermal population during epidermal carcinogenesis. Basal cells are defined as those in actual contact with the basement membrane, spinous cells as those immediately distal to them having spines more or less developed, and granular cells as those still more distal in position possessed of granular cytoplasm and constituting the layer of living fixed post mitotic epidermal cells nearest to the surface. Because the basal cells are thus artifically limited to what constitutes in effect a single layer in contact with the basement membrane throughout epidermal carcinogenesis, it is to be anticipated that their number relative to that of the total epidermal population will decrease as the epidermis becomes hyperplastic with increase in the number of layers of cells. The spinous cells are barely redognizable in normal untreated epidermis made up only of two or perhaps three layers of cells. A t this time the granular cells are inconspicuous since the surface cells pass through the granular phase abruptly and become keratinized. Glucksmann (1945) attacked this important problem of changes in relative numbers of cell 'types in epidermal carcinogenesis and made important observations; but he employed benzpyrene, while we have consistently used methylcholanthrene, and his classification of cell types is even less suitable than ours as a check on the chemical analysis. Consequently Banyen (unpublished) has examined the epidermal cellular population in our standard series. His results confirm the impression, given by a casual examination of sections, that there is a larger increase in spinous cells than in basal and granular cells. Keratinized epidermis, in which individual cells are not recognizable, is increased, but this is difficult to measure quantitatively and it is not certain what alteration, if any, takes place in the ratio of keratinized to nonkeratinized epidermis. Some keratinized epidermis is unavoidably lost in making histological preparations. Therefore part of the chemical changes reported can be attributed to the increase in the proportions of spinous cells, and perhaps in a minor degree t o increase in the keratinized epidermis, relative to other components included in the material analyzed. But the askewness of the chemical properties of the cancers in contrast to those of normal epidermis summarized in Table IV is difficult to explain merely on the basis of such differences in proportions of component materials analyzed. Some chemical substances are unchanged while others increase tremendously and others decrease to an equally remarkable degree. The same factor of possible alterations in the proportions of tissue components is to be considered not only in epidermal carcinogenesis but
EPIDERMAL CARCINOGENESIS
89
in the chemical study of carcinogenesis in all tissues. If the relative volumes of cells subject to the malignant transformation, of fibrous components, of tissue fluid, indeed of almost anything, are subject to significantly large modifications during cancer production, the assignment of chemical changes demonstrated in the whole tissue to the cells of primary interest becomes difficult. Some of these complicating considerations do not apply to epidermis because it is avascular, alymphatic, and devoid of all mesodermal components. 3. The Latent Period in Epidermal Carcinogenesis In our experiments the latent period, between the first application of carcinogen and the appearance of cancers, extends for several weeks. Such a latent period is present in carcinogenesis in all tissues. It has a definite relation to the life span of the species being shorter in mice than in man. During this period in our standard series there is promptly established and maintained a new kind of equilibrium covering the period described as “precancerous hyperplastic epidermis.” The properties that remain fairly constant are both structural and chemical. The following are typical. (1) The mitotic rate is held a t a fairly high level from 9 to 51 days. (2) A 50% increase in mean nuclear volume occurs a t 10 days and persists at this level despite repeated paintings with carcinogen. (3) The frequency of doubled chromosomesin the metaphase is about the same in lo-, 20-, 29-, and 57-day specimens. (4) Sebaceous glands are absent from about 6 days onward. (5) The calcium content of epidermis decreases at 10 days is held at the same level to 60 days (Fig. 1). (6) The iron content decreases at 10 days and shows slight further decrease to 60 days (Fig. 1). (7) Reductions in total lipid and total cholesterol protein nitrogen ratios noted at 5 days after a single application persist (Fig. 2). In this latent period repeated applications of the carcinogen may not be as effective as the first few applications, because entry is soon barred by early disappearance of the hair follicular pits and destruction of the sebaceous glands and increase in thickness of the stratum corneum of the epidermis. There may, however, be some scattered areas of thinness of hyperplastic epidermis after repeated application of carcinogen (Pullinger, 1943). The results of chemical analysis should not be interpreted t o mean that the new equilibrium is equally spread throughout the tissue. As previously stated, the chemical data have been obtained by analysis of pooled precancerous hyperplastic epidermises from several mice. Since
90
E. V. COWDRY
the data relate to the properties of many epidermises combined together the degree of uniformity existing in a given property among the epidermises thus lumped together is unknown. It is possible, for instance, that in the reacting epidermis of some individual mice the calcium content is reduced much more, or much less, than the value obtained for the pooled epidermises indicates. In other words, the procedures employed indicate statistical trends in groups of mice, not individual responses. In addition to this possibility of lack of uniformity between different mice, considered to be a t approximately the same stage in epidermal carcinogenesis, there is the fact that within the precancerous hyperplastic epidermis of a single mouse chemical differences may well occur in different areas which would not be revealed by our analyses. Thus, the decrease in biotin is not necessarily uniform throughout any of the epidermises combined together for purposes of analysis. It could be that one or more foci within the epidermis are far behind, or far ahead, of the rest in undergoing all the chemical modifications determined by examination of pooled epidermis (Table IV).
4. Localization of Carcinogenic Action The vast majority of cancers produced are squamous cell carcinomas, not basal cell ones, so one would be inclined to look first for their site of origin in the spinous layer. Microscopic evidence is accumulating that the effects of carcinogenic action are most evident in cells of the spinous layer. This layer does not exist as such in the extremely thin normal epidermis of mice. It makes its appearance when epidermis becomes hyperplastic. Since a single application of carcinogen applied to normal epidermis can produce cancer (Mider and Morton, 1939; Cramer and Stowell, 1943), it is doubtful whether the cells primarily affected are of the spinous variety, though their descendants may be spinous. In the standard series, with epidermis repeatedly subjected to applications of carcinogen, cells already characteristically spinous in nature may nevertheless be primarily directly or indirectly affected by it. Four observations seem significant: (1) Demineralisation, shown by microincineration, is greater in the spinous than it is in the basal or corneal layers. (2) Displacement of chromatin and nucleoli, under the influence of ultracentrifugal force, is also most marked in the spinous layer. (3) Chromosome abnormalities, observed in smears of the cells, appear also to be most evident in the spinous layer. (4) Maximum mitotic frequency is located in the basal layer of epidermis, not in the spinous layer where it is concentrated in the manylayered epidermis of the foot pads of normal mice.
EPIDERMAL CARCINOQENESIS
91
6 . Nature of Malignant Transformation
It is tempting to assume, purely as a working hypothesis, that the living conditions of the new chemical equilibrium have become more unfavorable for the spinous cells than they have for the basal ones, which, by contrast, are nearer t o the source of supplies in the blood stream of the dermis and from which waste can more readily be eliminated. Perhaps the shifting of the stratum of maximum frequency of mitoses from the spinous to the basal layer is not unrelated to the better conditions presumed to exist in the basal layer which is nearer to regulation exercised by the blood stream in the underlying dermis. Under the more rigorous living conditions of cells in the spinous layer than in the basal layer, there may be not only more chromosome abnormalities than elsewhere in the epidermis but also a greater tendency to mutations occasioned by alterations in nuclear, or in plasmagenes, or in both. Strong (1947) has discovered that methylcholanthrene can produce a t least seventy-nine different germinal mutations in the epidermis and epidermal appendages of mice. The same carcinogen may also bring about mutations that condition malignancy since it is so highly mutagenic. The occurrence of the malignant change in one, or in a very few, of many thousands of cells living generation after generation for over sixty days subjected to such hardships is what one would expect numerically if the change is actually a mutation. However, mutations are quick, sudden changes. If cancer cells arise through mutation why do they not manifest their presence immediately? Instead of doing so, if they actually arose under the influence of the carcinogen, they appear to remain quiescent throughout the latent period. Such hypothetical cancer cells, displaying no recognizable malignant properties, are commonly designated "latent cancer cells." These have been more discussed in studies on epidermal carcinogenesis than in carcinogenesis in other tissues because the epidermal cancers and papillomas are surface modifications that can be easily detected and the evolution of which can be followed even by gross inspection. Berenblum (1949) has presented much of the evidence concisely. 6 . Stages in Epidermal Carcinogenesis
The usual view is that carcinogenesis is a two-step phenomenon, and the steps have been given many names. Of these there is least objection to the initiating process and promoting process suggested by Rous and Kidd (1941). The initiating process could be a mutation produced, as it appears in our carcinogenic series, either by the hardships suffered by the spinous
92
E. V. COWDRY
cells, or, more directly, by the mutagenic action of methylcholanthrene. In other cases the initiating process could be a virus action in which case analysis of the situation is also difficult, for evidence cannot be ignored of the existence of active and latent viruses and of the change of a given virus from activity to latency and vice versa. The promoting process is equally difficult to analyze. There are many promoting factors. It was observed, in our special carcinogenic series, that cancer development is accelerated by injections of estradiol benzoate (Paletta and Max, 1942), by increasing environmental temperature (Kung, unpublished) and is modified by age, being accelerated in the young mice 3 4 months, as contrasted with 12-13 months (Cowdry and Suntzeff, 1944). Almost every new condition imposed on the carcinogenic response modifies to some extent in one way or another the production of tumors. Consequently whether a cancer develops or not, after operation of the initiating process, does not depend solely upon the action, or absence of action, of a promoting process. A third kind of process merits due consideration, namely an inhibiting process. Returning to what may happen during the period of latency in our standard series of epidermal carcinogenesis the “critical size ” concept of Fisher and Hollomon (1951) is attractive. According to this, the solitary cell or tiny group of cells, first to undergo the malignant transformation, is located in a tissue fluid environment dominated by a vast predominance of normal cells. In this environment the cell, or cell group, is unable to display malignant potentialities. Gradually the cells increase in number and attain a size, or volume, which is “critical” in the sense that they now manage to overcome the control of adjacent normal cells on their tissue fluid environment and establish one in which they can behave as cancer cells. Implicit in this concept is the idea that we are dealing in the latent period with true cancer cells restrained during the period of latency by unfavorable fluid surroundings. When single cancer cells, or small clumps of them, spread by metastasis to other normal environments are they therein subjected to similar restraint? We do not know. The period of latency, if it exists in these other environments, is almost impossible to measure. The tissue fluid environment of epidermal cells has special attributes. The amount of tissue fluid in contact with epidermal cells, subject to the malignant transformation, is minimal, and the cells themselves are more closely bound together than in any other tissue. James M. Weaver, in a personal communication, has called attention to the fact that in epidermis rendered hyperplastic by the methylcholanthrene there is a slight but noticeable separation of cells in the spinous layer which lends prominence
EPIDERMAL CARCINOGENESIS
93
to the spines and involves a local increase, and possibly alteration, in the tissue fluid about them, a process superficially not unlike edema. This may be in part correlated with the increase in water content of whole hyperplastic precancerous epidermis compared with that of whole normal epidermis (Suntzeff and Carruthers, 1946). The chances are, however, that most of this slight increase in water is intracellular. How widespread this sheet of increased fluid may be remains to be discovered. Apparently it is of wider extent than the small focus, or foci, in which cancers make their appearance. As intimated, the alteration in fluid about the cells, hypothecated by Fisher and Holloman, may be more in quality than in amount and quite undetectable by our crude techniques. Consequently, the evidence that we can bring forward is not particularly helpful. Another line of inquiry, bearing on a possible loosening of the binding together of latent cancer cells and normal epidermal cells, should be explored. Chambers and Zweifach (1947) have referred to the work of several early investigators who stressed the presence of a reversible calcium salt which serves as a cohesive substance for binding cells together. In their own experiments they found that calcium-free perfusates act by softening and dissolving the interendothelial cell cement. The finding by Carruthers and Suntzeff (1946) that in our standard series the total calcium content of precancerous hyperplastic epidermis is reduced to about 50% of normal epidermis is of interest in this connection. Coman (1947) has discovered that the application of methylcholanthrene to epidermal cells reduces their adhesiveness, and, further, that the actual measured adhesiveness of cancer cells is definitely less than that of normal cells. All this adds up to the thought that the decrease in calcium unlocks the bindings of latent cancer cells, if they really exist, for they have not been seen, permits them to move, to invade the dermis, and to exhibit malignant behavior. As has been pointed out, we think the principal decrease in calcium is in the spinous layer; but it has not been possible sharply to localize it in one or two foci in epidermis responding to the carcinogen. I am not in agreement with Shubik (1950) that “undoubtedly the use of repeated applications of carcinogen would result in a conversion of most of, if not all, the cells in the area to latent tumor cells.” If this were the case, the mechanism suggested should unlock them in more than a few isolated foci.
VII. INDICATIONS CONCERNING HUMAN EPIDERMAL CARCINOGENESIS Our observations on mice have been supplemented to a small extent by studies on human epidermis. The obvious difference is that we cannot apply methylcholanthrene to human skin and note the resulting
94
1. V. COWDRY
alterations in the precancerous hyperplastic epidermis. It is possible to examine normal human epidermis, hyperplastic epidermis, and senile keratotic lesions which are considered to be precancerous; but with even more qualification is required than was stressed in the definition of “precancerous hyperplastic epidermis ” in mice, for the probability of eventual development of “spontaneous ” cancers in human epidermis is definitely less. We can frequently examine human primary squamous cell carcinomas of epidermal origin, but these are not attributable to a particular carcinogen. Moreover chemical analysis cannot be made of many uniform transplants, having a minimum of complicating necrosis, as can so profitably be done with mice. At the human level one must be opportunistic, strive to cultivate the cooperation of clinical friends, make the best use of specimens collected and be on the alert for the radical differences between mice and man in making interpretations. In general the same factors, as in mice, are likely to produce lack of uniformity, but their relative values are somewhat different. The epidermises of individuals of different races, ages, and sexes under different conditions of nutrition and environment may not respond uniformly to the same carcinogen. Sunlight is probably the most frequent carcinogen for human beings and the least frequent for mice. Human epidermis is probably exposed to a wider range of carcinogens, and of promoting and inhibiting factors, than is that of mice. Regional differences, magnified by inequality in exposure to the environment, and in protection by hair, or the lack of it, are much greater in human beings. Human epidermis is thicker and in all races contains two types of epidermal cells, ordinary ones and dendritic cells (melanoblasts). It is unnecessary here to express an opinion as to the relation of “clear cells” to melanoblasts. It is sufficient to recognize the fact that the epidermal cellular population is less uniform in human beings than in mice, Where hairs are scarce, as over large stretches of the human body, the contribution of cells of hair follicles to the epidermis is less than in mice. The presence of many sweat glands is probably not of great significance as contributing cells prone to become malignant in human skin. Knowledge of the chemical composition of human epidermis lags far behind that of mouse epidermis. In both accurate investigation commenced with the utilization of a technique for the separation of epidermis from dermis in a condition suitable for chemical analysis (Baumberger et al., 1942). Previously mixture of dermis in variable amounts with the epidermis analyzed constituted a factor difficult to apprise. Some data from our series in mice are contrasted with data for man in Table V and Fig. 4. In most cases the same investigators applied the same techniques to the tissues of mice and man, while in the remainder
95
EPIDERMAL CARCINOQENESIS
TABLE V Comparison of Epidermal Carcinogenesis in Mice and Man Mice
Man
Whole mounts in ear and Examined many locations. back. Minor differences Greater differences (Cow(Cooper & Reller, 1942; dry et al., 1947) Liang, 1948) Thickness in section. Age changes in structure Not determined Great individual differof epidermis ences (Evans et al., 1943). Structure in whole mounts (Cowdry et al., 1947) Age changes in minerals Increase in calcium Alterations not noticed in a small series of specimens in old epidermis (Suntzeff et al., 1945) (Suntzeff & Carruthers) Mitotic rhythm in nor- Maximum by day (Cooper Maximum by night (Cooper & Schiff, 1938; & Franklin, 1940) mal epidermis Cooper, 1939) Displaceability of nucleoli Not noticeable in normal Not noticeable in normal; and chromatin by ultra- epidermis; marked in pre- marked in hyperplastic centrifugal force cancerous hyperplastic epidermis of healing epidermis and greatest in wound and in papillomata (warts), and greatest in carcinomas (Cowdry & Paletta, 1941b) squamous cell carcinomas (Cowdry & Paletta, 1941b) Hyperplastic epidermis less Thymonucleic acid con- Reduced in precancerous tent determined photo- hyperplastic epidermis and than normal epidermis, carcinomas more than metrically per unit area in control specimens treated only with benzene. normal epidermis (Stowell Significantly increased in & Cooper, 1945; Stowell, some carcinomas (Stowell, 1946) 1942) Ribosenucleic by pyronin- Cytoplasmic concentration Increased per unit area in ribonuclease method increased to maximum some carcinomas (Stowell, 10th day, then dropped. 1946) Increased in one carcinoma (Biesele, 1944) Nucleo-cytoplasmic ratio Larger in carcinomas than Larger in primary carciin spinous and about same nomas than in spinous and as in basal cells of precanabout the same as in basal cerous epidermis (Cowdry cells of hyperplastic epi& Paletta, 1941a) dermis (Cowdry & Paletta, 1941a)
Regional differences in structure of epidermis
96
E. V. COWDRY
TABLE V (Continued) Mice Minerals, pg. per 100 mg.
K Na Ca Mg Zn cu
Mineral residue after microincineration
Total lipid Protein nitrogen ration Total lipid Protein nitrogen ration Lipid phosphorus Protein nitrogen
Man
Normal Normal Epidermis Carcinoma Epidermis Carcinoma 347 326 322 123 168 141 44.0 9.0 16.0 8.5 18.0 19.0 18.0 5.2 1.7 2.4 1.7 0.58 0.10 0.54 0.16 (Carruthers & Suntzeff, (Carruthers & Suntreff, 1945) 1946) Patchy reduction in spinous Considerably less in senile layer of precancerous hy- keratotic lesions than in perplastic epidermis normal epidermis (Cowdry (Paletta et al., 1941) & Andrew, 1950) Normal Normal Epidermis Carcinoma Epidermis Carcinoma 21.3 28.6 19.1 1.66
0.66
0.95
-
0.16 0.31 0.12 (Wicks & Suntzeff, (Carruthers & Suntzeff, 1942-45) unpublished) Urea and ammonia mg. of Normal Normal N per 100 g. fresh tissue Epidermis Carcinoma Epidermis Urea 77 22 33 Ammonia 24 24 8 (Roberts & Frankel, 1949) (Roberts & Frankel, 1949) Polarographically reduci- Characteristic for normal Same, the mouse subble substances epidermis and different yet stances being indistincharacteristic for carci- guishable from the human ones (Carruthers & nomas (Carruthers & Suntzeff, 1949) Suntzeff, 1949) Increase of mast cells in In precancerous hyperplas- In precancerous hyperkeradermis tic epidermis (Simpson & tosis and Bowen’s disease Cramer, 1944) (Simpson & Cramer, 1944) Concentrations of free Chromatograms Concentrations of free amino acids increase in amino acids are high in hyperplasia and decrease normal human epidermis in squamous cell carci- (Roberts, unpublished; see noma when same fresh Fig. 4) and in benign papillomas (Awapara, weights are compared personal communication), (Roberts & Tishkoff, and decrease in squamous 1949) cell carcinomas in a manner similar to mouse (Awapara)
EPIDERMAL CARCINOdENESI6
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different members of the same research team applied the same techniques to the tissues of mice and man. For this reason the comparison is fairly direct. The incompleteness of the data is obvious. The comparison is merely a beginning in the extension of observations by methods proved useful in our experiments on mice to human epidermal carcinogenesis. It supplies only a few features possessed in common at the mouse and human levols relative to the tissue environment in which the malignant change occurs and the properties of the cancers. To establish the direct sequence of events in epidermal carcinogenesis in mice is extraordinarily difficult. It is even more difficult in man because the latent period is much longer giving greater opportunity for the operation of promoting and inhibiting factors. VIII. SUMMARY
As was expected, epidermis proves to be on the whole a very suitable tissue in which t o investigate carcinogenesis for the reasons stated in the introduction. However, many unexpected difficulties have been encountered in producing uniformities in standard methylcholanthrene carcinogenic series in mice. The measurements of properties of epidermis that remain constant, or that increase, or decrease quantitatively, in one such series can only be stacked up with those measured in another series approximately because the series are far from identical. Nevertheless it is clear that profound modifications take place. The chemical properties of the carcinomas are askew compared with those of normal epidermis (Table IV). This cannot be altogether attributed to alterations in the relative proportions of basal, spinous and supraspinous cells or to destruction of the sebaceous glands and consequent lack of sebum. Evidence is presented that conditions of cell life in the spinous cell layer are more profoundly modified that in the basal and granular layers by the carcinogen or by its products. The data are consistent with the hypothesis that the malignant transformation is one of probably several mutations. X-rays are, like methylcholanthrene, highly mutagenic. The changes as far as they have been followed in epidermis produced by x-rays very closely resemble those caused by methylcholanthrene (Toosy, 1951). It is probably that the mutation takes place early in the period of latency and that decrease in calcium content is one factor in loosening the binding of epidermal cells together so that the cancer cells can display their properties. Isolated observations on human epidermal carcinogenesis indicate many similarities in the conditions of cell life with those in mouse epidermis (Table V). It is possible that human squamous cell carcinomas
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originate in the same spinous layer of epidermis in consequence of mutations resulting from unfavorable conditions of many sorts. Further progress requires more accurate microscopic localization and seriation of the structural changes leading to the malignant transformation in the epidermis of mice and human beings and of the accompanying chemical modifications. Improvements in technique are essential. The difficulties met with in the interpretation of results of chemical analyses of whole area8 of the epidermises of several mice responding to the carcinogen pooled together to supply sufficient amounts of material are recognized. It is necessary to devise methods whereby the hyperplastic precancerous epidermis can be split into layers which will permit the collection for chemical analysis separately of the basal cell and spinous cell fractions each uncomplicated by variable amounts of other material. Because the amounts of material thereby made available will be even smaller than those with which we have been dealing, techniques will have to be made still more ultramicro. This is being done in the laboratories of Linderstrgm-Lang, D. Glick, 0. H. Lowry, and others. Concentration of attention on types of cells capable of the malignant transformation, if it can be achieved, will increase the value of chemical analyses of isolated parts of cells, such as chromosome threads and mitochondria, since these will be collected from particular cells types rather than from the whole epidermis. Much is to be expected from the careful use of rapidly improving microchemical reactions and physical techniques for the microscopic localization and measurement of chemical changes in epidermal carcinogenesis. The techniques already proved helpful in this investigation of epidermal carcinogenesis, can be of assistance in the still more difficult problem of carcinogenesis in deep-lying epithelium especially that of the stomach, first in animal experiments and later in man. The conditions of cell life, the degree of exposure to carcinogens, and the chances of operation of promoting and inhibiting factors require study in all tissues of the body. Advances in our understanding of any one of these tissues may light the way to better understanding of others. Francis Bacon has truly said “knowledge is power.” Our goal is knowledge of cancer cells sufficient to enable us to overcome their malignant attributes or to kill them without killing the entire cellular community in which they live. We cannot expect ever to prevent all cancers, but some reduction in the frequency of cancer is within our reach. REFERENCES Ahlstrom, C. G., and Berg, N. 0. 1948. Acta Path. Microbiol. Scad. 26, 1-21. Ayengar, A. R. Gopal, and Cowdry, E. V. 1947. Cancer Research 7 , 1-8.
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Fisher, J. C., and Hollomon, J. H. 1951. Cancer 4, 916-918. Giroud, A,, and Bulliard, H. 1935. Arch. d'Anat. Micr. 81, 271-290. Giroud, A., andLeblond, C. P. 1951. Ann. N . Y . Acad. Med. 68, 613-626. Glucksmann, A. 1945. Cancer Research 6, 385-400. Graffi, A. 1942. Z. Krebsforsch. 62, 165-184. Greenstein, J. P. 1947. Biochemistry of Cancer. Academic Press, New York. Haddow, A., Elson, L. A., Roe, E. M. F., Rudall, K. M., and Timmis, G. M. 1945. Nature 166, 379-381. Haddow, A., and Rudall, L. M. 1945. Endeauoui 4 (16) October. Knowlton, N. P. Jr., and Winder, W. R. 1950. Cancer Research 10, 63. Kraemer, Dorothy Ziegler. 1946. Anat. Record 94, 289-311. Kung, 9. K. 1949. J. Natl. Cancer Znst. Q, 435-438. Lansing, A. I., Rosenthal, T. B., and Au, M. H. 1948. Arch. Biochem. 16,361-365. Lansing, A. I., Rosenthal, T. B., and Kamen, M. D. 1948. Arch. Biochem. 19, 177-183.
Liang, Hsu-mu. 1948. Cancer Research 8, 211-219. Ma, Chung K. 1949. Cancer Research 9, 481-487. Mider, G. B., and Morton, J. J. 1939. Am. J . Path. 16, 299-302. Miller, E. C. 1951. Cancer Research 11, 100-108. Miller, H., and Carruthers, C. 1950. Cancer Research 10, 636-641. Morton, J. J., Mider, G. B., Luce-Clausen, E. M., rind Mahoney, E. B. 1951. Cancer Research 11, 559-561. Mottram, J. C. 1944. J. Path. Bad. 66, 181-187. Mottram, J. C., and Weigert, F. 1942. Nature 160, 635. Orr, J. W. 1938. J . Path. Bact. 46, 495-515. Paletta, F. X., Cowdry, E. V., and Lischer, C. E. 1941. Cancer Research 1,942-952. Paletta, F. X., and Max, P. F. 1942. J . Natl. Cancer Znst. 2, 577-581. Pullinger, B. D. 1940. J. Path. Bact. 60, 463-471. Pullinger, B. D. 1943. J. Path. Bact. 68, 287-288. Reller, H. C., and Cooper, Z. K. 1944. Cancer Research 4, 236-239. Ritchey, M. G.,Wicks, L. F., and Tatum, E. L. 1947. J. Biol. Chem. 171, 51-59. Roberts, E. 1948. J . Biol. Chem. 176, 213-222. Roberts, E., Caldwell, A. L., Clowes, G. H. A., Suntzeff, V., Carruthers, C., and Cowdry, E. V. 1949. Cancer Research 9, 350-353. Roberts, E., and Carruthers, C. 1948. Arch. Biochem. 16, 239-255. Roberts, E., and Frankel, S. 1949% Arch. Biochem. 20, 386-393. Roberts, E., and Frankel, S. 194913. Cancer Research 9,231-237. Roberts, E., and Frankel, S. 1949c. Cancer Research Q, 645-648. Roberts, E., and Tishkoff, G. H. 1949. Science 109, 14-16. Rothman, S., Krysa, H. F., and Smiljanic, A. N. 1946. Proc. SOC.Exptl. Biol. Med. 62, 208-209.
Rous, P., and Kidd, J. G. 1941. J . Exptl. Med. 78, 365-389. Rumsfeld, H. W. Jr., Miller, W. L. Jr., and Baumann, C. A. 1951. Cancer Research 11, 814419. Schneider, W. C., and Potter, V. R. 1943. Cancer Research 8, 353-357. Shubik, P. 1950. Cancer Research 10, 713-717. Simpson, W. L., and Cramer, W. 1943. Cancer Research 8, 362-369. Simpson, W. L., and Cramer, W. 1944. Cancer Research 4, 601-616. Storey, W. F., and Leblond, C. P. 1951. Ann. N . Y . Acad. Sci. 68, 537-545. Stowell, R. E. 1942. J . Natl. Cancer Inst. 8, 111-121. Stowell, R. E. 1945. J . Investigative Dermatol. 6, 183-189.
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Stowell, R. E. 1946. Cancer Research 6, 426-435. StoweI1, R. E., and Cooper, 2. K. 1945. Cancer Research 6, 295-301. Stowell, R. E., and Cramer, W. 1942. Cancer Research 2, 193-197. Strong, L. C. 1947. Fourth International Cancer Congress, St. Louis, p. 42. Suntzeff, V., and Carruthers, C. 1943. Cancer Research 9, 431-433. Suntzeff, V., and Carruthers, C. 1944. J . Biol. Chem. 169,521-527. Suntzeff, .V., and Carruthers, C. 1945. J . Biol. Chem. 160, 567-569. Suntreff, V., and Carruthers, C. 1946. Cancer Research 6, 574-577. Suntzeff, V., Carruthers, C., and Cowdry, E. V. 1947. Cancer Research 7,439-443. Suntaeff, V., Cowdry, E. V., and Carruthers, C. 1946. Cancer Research 6, 179-182. Tatum, E. L., Ritchey, M. G., Cowdry, E. V., and Wicks, L. F. 1946. J . Biol. Chem. 169, 675-682. Toosy, M. H. 1951. Cancer Research 11, 361-365. Weitkamp, A. W. 1945. J . Am. Chem. SOC.87,447-454. Weitkamp, A. W., Smiljanic, A. M., and Rothman, S. 1947. J . A m . Chem. SOC. 69, 1936-1939.
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The Milk Agent in the Origin of Mammary Tumors in Mice L. DMOCHOWSKI Department of Experimental Pathology and Cancer Research, School of Medicine, University of Leeds, Leeds, England
CONTENTS
Page I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. The Milk Agent and Genetic Factors.. . ........................ 109 1. Introduction.. ................................................. 109 2. The Influence of Genetic Factors on Susceptibility t and Its Production.. .......................... A. Mice of High-Breast-Cancer Strains. . . . . . . . . . . . B. Mice of Low-Breast-Cancer Strains. ............................ 11 1 3. The Influence of Genetic Factors on Transmission of the Milk Agent.. 113 A. Mice of High-Breast-Cancer Strains, . . . . . . . . . . . . . . . . . . . . . . . 113 B. Mice of Low-Breast-Cancer Strains. ..... . . . . . . . . . . . . . . . . . . . . . . . 113 4. The Milk Agent and Transplanted Breast Tumors.. . . . . . . . . . . . . . . . . 115 A. Sarcomatous Transformation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 5. Spontaneous Breast Tumors in Low-Cancer-Strain Mice. . . . . 6. Conclusions.. ................................. 111. The Milk Agent and Hormonal Factors.. . . . . . . . . . . . 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The Milk Agent and Estrogen-Induced Breast Tumors in Male Mice.. 121 Methylcholanthrene-Induced Breast Breast Tumors Tumors in in 3. The Milk Agent and Methylcholanthrene-Induced Mice.. . . . . . . . . . . . ...... .. . .. . ., .. ............ . . . .. . . ...,........ .. . .. . . ............. .. .. . . . 123 123 rance of of Carcinogen-Induced Carcinogen-Induced Breast Breast Tumors. Tumors..... 125 A. Histological Appearance . . 125 4. Conclusions.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 IV. The Milk Agent and Mammary Gland Structure.. . . . . . . . . . . . . . . . . . . . . 127 V. Inherited Hormonal Influence. . . . . . . . . . . . . . . . . VI. Properties of the Milk Agent.. . . . . . . . . . . . . . . . . 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Distribution of the Agent.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 3. Physical and Chemical Properties of the Agent. . . . . . . . . . . . . . 135 4. Attempts a t Isolation of the Agent.. . . . . . . . . 5. Behavior of the Agent in vivo.. . . ............................ 140 6. Immunological Studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 VII. Mammary Tumors in Hybrid Mice and the Milk Agent. 1. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 2. Recent Investigations. .................... 3. Histology of Mammary Tumors in Hybrid M 4. Conclusions.. ................................. ......... VIII. T h e Nature of the Milk Agent.. . . . . . . . . . . . . . . References. .................... .............................. 159 103
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I. INTRODUCTION A survey of the progress of our understanding of the development of mammary cancer in mice presents a gratifying example of collaboration of specialists of many branches of science such as genetics, pathology, biology, virology, and biophysics, and of achievements through the application of their methods to experimental cancer research. The study of structure of mammary tumors in mice; the development of genetically homozygous stiains of mice combined with the study of the influence of genetic factors on the origin of these tumors; the investigations of the part played by hormonal factors in the development of breast cancer in mice; the discovery of an extrachromosomal factor and the study of its properties, have all combined t o produce a better understanding of the origin of mammary tumors in mice. The first morphological descriptions and classifications of breast cancer in mice were carried out largely by English (Bashford et a/., 1905; Bashford, 1906, 1911; Bashford and Murray, 1907; Murray, 1908a,b) and German workers (Apolant, 1906; Ehrlich, 1906, 1907; Ehrlich and Apolant, 1905), whose work led to the recognition of these tumors as true malignant growths. An excellent review of these studies has been presented by Dunn (1945). Further attempts a t classification of mammary cancer arising in various inbred strains and their hybrid progeny and at correlation of their appearance with the presence or absence of the milk agent will be presented in this review. The development of inbred or homozygous strains of mice, initiated by Little, has been one of the greatest contributions to cancer research. Comprehensive accounts of the many inbred strains of mice are now available (Snell, 1941; Strong, 1942; Law, 1948). These strains, in which the breast tumor incidence may vary from 1% to practically loo%, are described either as low-breast-cancer or high-breast-cancer strains. However, the name low-breast-cancer strain does not imply complete resistance to the development of mammary tumors, and it does not indicate the incidence of other types of tumors. These strains are subject to changes which occur, although infrequently, and can be ascertained by transplantation (Little, 1941a). Variations in the tumor incidence of a strain were first shown by Bittner (1941c,d) and Burrows (1941), and reviewed by Bittner (1945). The data on the tumor incidence and age at which tumors develop in various inbred strains provided a proof that hereditary factors play an important part in the oiigin of breast cancer in mice. Since the original observation of Murray (1911a,b) of the importance of heredity in the development of these tumors, the early work on genetic factors stressed a recessive manner (Slye, 1927, 1937, 1941) and
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later studies (Little, 1931; Bittner, 1940b,c, 1942c, 194413) suggested a dominant way in which these factors are inherited in mice. This was made possible because of the use of homozygous strains of mice. The study of inbred strains of mice has shown that different tumors are inherited in different and separate ways. This later work finally led to our understanding that genes and not characters are inherited and that breast cancer as a physiological character is not inherited but is the result of interaction of genic and enGironmenta1factors (Heston, 1944, 1945). The studies on the influence of hormonal factors in the origin of mammary tumors in homozygous strains of mice confirmed and extended the original observations on these factors (Lathrop and Loeb, 1911, 1913, 1916; Loeb, 1919, 1924). The influence of breeding on the incidence of breast cancer in mice of inbred strains was confirmed (Andervont, 1941a; Bittner, 1939g; Murray, 1928;Little et al., 1939) and the influence of rapid breeding with prevention of nursing on the origin of breast cancer in mice of these strains disputed. The original observation of Lathrop and Loeb (1916) on the effect of the removal of ovaries on the development of breast cancer in mice was confirmed and extended to various inbred strains. The induction of breast cancer in male mice (Lacassagne, 1932) by estrogenic hormones was followed up by studies which established the importance of estrogens in the induction of breast cancer in mice. These studies, reviewed by Shimkin (1945b), demonstrated that estrogenic hormones induce breast cancer in male mice of a strain in approximately the same incidence as that of breeding females of the same strain. Further the appearance of breast tumors following treatment with estrogens was shown to depend on the presence of the so-called milk influence (Lacassagne, 1939, and others). Lathrop and Loeb (1918) first recorded the part played by the maternal parent in the development of breast tumors in hybrid mice. The correct interpretation of these results was made possible only after the establishment of inbred strains of mice by cross-breeding experiments with highand low-cancer-strain mice and the observation on the tumor incidences in the resulting hybrid mice (Staff of the Roscoe B. Jackson Memorial Laboratory, 1933; Korteweg, 1934, 1936a,b; Murray and Little, 1935a,b, 1936). The predominance of the maternal influence in genetically identical hybrids was ascertained in these experiments and the transmission of this influence was shown not to take place through the chromosomes. Of three possible ways in which this maternal or extrachromosomal influence could be transmitted (Korteweg, 1936b), the cytoplasm of the ovum (Korteweg, 1936b) has not been proved to be one of them, the intra-uterine transmission, put forward (Fekete and Little, 1942) in spite of previous evidence to the contrary, has not received confirmation
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(Dmochowski, 1949d), and the third, the mother’s milk was found to be the indisputable route by which the influence passes from mother t o the progeny. Bittner (1936a) was first to point out the presence of this influence in the milk of high-breast-cancer-strain female mice by showing that mice of high-cancer strains suckled by foster mothers of low-breastcancer strains develop only a few tumors, while low-cancer-strain mice nursed by high-breast-cancer strain mothers show a higher incidence of mammary cancer than that normally observed in these mice. Bittner named this extrachromosomal factor, the milk influence or milk factor. Many investigators established the general validity of the part played by the milk factor or agent in the origin of spontaneous breast cancer in inbred strains of mice (Andervont and McEleney, 1938, 1939, 1941b; Andervont, 1940b, 1943; Andervont et al., 1942; Barnes and Cole, 1941; Bittner, 1937a,b,c, 1939a,b,d,f, 1940b, 1941b; Bittner and Little, 1937; Fekete and Little, 1942; Murray, 1941b; De Ome, 1940; van Gulik and Korteweg, 1940b; Furth et al.,1942; Dmochowski and Gye, 1943; Bonser, 1944b; Miller and Pybus, 194513; Haagensen and Randall, 1945). Contrary t o some overemphasis on the influence of the milk agent (Murray and Little, 1939), Bittner (1939c,f, 1940a,c, 1941d,f, 1942a,b,c,d, 1943, 1945b) has repeatedly stressed that at least three factors are involved in the origin of breast cancer in mice, namely the genetic factor or inherited susceptibility, the hormonal factor or hormonal stimulation, and the milk agent, and that a low incidence of mammary cancer is the result of any one of these factors missing. The milk factor has also been described as the mammary tumor agent or inciter (Woolley, Law, and Little, 1941; Bittner, 1942c,d) or as a virus (Visscher, Green, et al.,1942). It has recently been pointed out (Heston, 1945; Heston et al., 1949, 1950; Andervont, 1950b) that there may be a quantitative balance between these three factors, as some mammary tumors may develop either in the absence or subthreshold amounts of the milk agent, the deficiency of the agent being overcome by increased hormonal stimulation and genetic susceptibility (Shimkin and Andervont, 1945; Bittner, 1946-47; Andervont, 1949~). The milk agent is present during the entire period of lactation (Andervont and McEleney, 1939; Bittner, 1939a), but its concentration may vary during the life span of a female mouse as revealed in the higher incidence of tumors in mice of the fourth to seventh litters than in the preceding two or the following litters (Bittner, 1941b, 1942c,d, 1943, 1944a, 1945a,b). In view of this, the removal of young mice from uteri of high-cancer-strain female mice has been introduced in an attempt t o obtain mice free of the agent (Andervont and McEleney, 1941b). There exists a quantitative correlation between the amount of milk ingested and
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the time of appearance and incidence of breast cancer in susceptible mice (Andervont and McEleney, 1939; Bittner, 1940a; Andervont and McEleney, 1941b; Andervont et al., 1942). The susceptibility to the agent is influenced by the age of mice. Adult female mice develop few or no tumors after administration of the agent in quantities which induce a high incidence of breast cancer in young female mice. Administration of sufficiently large amounts of the agent overcomes the decreased susceptibility of adult mice (Dmochowski, 1945a). The susceptibility to the agent is a strain characteristic and may vary from strain to strain and even in different sublines of the same strain maintained in various laboratories (De Ome, 1940; Andervont, 1945b). The milk agent is widely distributed throughout the body of highbreast-cancer-strain mice and can be transmitted to susceptible mice by extracts of various tissues (Bittner, 1939e). In spite of the wide distribution of the milk agent in the body of high-cancer-strain mice, it is not transmitted through body contact (Andervont, 194513). The agent does not seem to change the genetic constitution of mice carrying it (Bittner, 1941f). The successful transmission of the milk agent is conditioned by genetic factors and variations within these factors in different high- and low-breast-cancer strains (Heston et al., 1945), by the hormonal factors which in turn are influenced by the genetic factors, and by the amount of the agent supplied (Bittner, 1939f, 1940b). Transmission of the milk agent is not necessarily connected with tumor development (Bittner, 1942~). The fate of the agent introduced into bothhigh-and low-cancer-strain mice is unknown. A characteristic point is the long latent period, from six to twenty months, before the development of breast cancer takes place following the introduction of the agent into mice. It has been suggested that during this period the agent may influence the hormonal metabolism producing :an exces8 of carcinogenic hormones compared with the amount produced by low-cancer-strain mice (Bittner, 1950b). Attempts to shorten the latent period have so far proved unsuccessful (Borgess, 1949). The introduction of the agent into the body of mice is not followed by any characteristic changes in the various physiological manifestations of hormones (Shimkin, 1945b). Although the amount of alveolar budding of the ducts of mammary glands was attributed to the action of the milk agent (van Gulik and Korteweg, 1940b), this was disputed by other investigators (Huseby and Bittner, 1946) who found hormonal factors to be responsible for these changes. There exists however an agreement about the presence of nodules of alveolar hyperplasia in high-breastcancer-strain mice which differ from inflammatory nodules present in both high- and low-breast-cancer strains. All three factors, known to play a part in the origin of breast cancer, are required for the development
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of these nodules (Huseby and Bittner, 1946). Further work along these lines may perhaps lead toward the development of a more rapid test for the presence of the milk agent. Mammary tumors of high-cancer-strain mice seem to differ from those occasionally arising in low-cancer-strain mice by the apparent absence of the milk agent in tumors of low-cancer-strain mice (Dmochowski, 1951b). Mammary cancer of mice resembles enzymatically other tumors more than normal mammary glands (Greenstein, 1945, 1947). The study of biochemical changes following foster nursing and introduction of the agent into the body of mice has, so far, not yielded any agreement (Khanolkar and Chitre, 1944; Shimkin et al., 1944), although a great deal more of research should be carried out along these lines before reaching any final conclusions. It must be emphasized after Heston (l946,1948a,b) that breast cancer as a character is the result of a chain of events directed by genetic, hormonal, and environmental factors, none of which can be the only cause of cancer, and this makes it impossible to state which of the factors is more important (Bittner, 1945b; Heston, 1945). In addition, the influence of other factors, such as abnormal environmental temperature (Fuller et al., 1941; Wallace et al., 1944, 1945; Mills, 1945; Tannenbaum and Silverstone, 1949b); different diets, caloric restriction, decrease or increase of various essential nutritional constituents, and other environmental factors (Bittner, 1935c; Tannenbsum, 1945a,b, 1947; Tannenbaum and Silverstone, 1946, 1947a,b, 1949e; Silverstone and Tannenbaum, 1950; White, 1944; White and White, 1944a,b; White et al., 1944, 1947; Dubnik et al., 1950; Morris, 1945a,b, 1947; Huseby and Ball, 1945); even overcrowding (Andervont, 1944; Muhlbock, 1950b,c)have been extensively studied and shown t o influence the development of mammary cancer in mice. The action of these factors may be through the endocrine system (Huseby et al., 1945; Morris, 1945a; King el al., 1949) by alteration of hormonal secretions which influence the growth of mammary glands (Ball et al., 1946; Casas et al., 1947; Dalton el al., 1948; Morris et al., 1946a,b; Trentin and Turner, 1941;Visscher, Ball, et al., 1942) and thus have an inhibitory effect on the development of mammary cancer in mice. The effect depends on the degree and type of dietary restriction (Boutwell et al., 1949;Tannenbaum, 1945a,b, 1947; Tannenbaum and Silverstone, 1949a,b,c,d). Any discussion on the part played by the milk agent in mammary tumor development in mice involves therefore of necessity a consideration of other factors which are known to influence the origin of breast cancer. The final result (appearance of a tumor) depends on the interplay of all the factors, although the quantitative relationship between these factors may
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vary, and increased amounts of one or more of them may successfully replace decreased quantity of other factors. As an example of the interplay of some of the factors in the origin of breast cancer in mice, theinfluence of genetic factors will first be presented.
11. THE MILK AGENTAND GENETICFACTORS I. Introduclion After the initial tendencies to assign either a minor part t o the genetic factors (Little, 1941b) or a major part to the milk agent (Murray and Little, 1935a,b, 1939), it is now known that the genetic factors influence the development of breast cancer in mice by controlling the response of mammary tissue to hormones and the agent. In spite of suitable hormonal stimulation and the presence of the milk agent, breast cancer will not develop in mice whose mammary tissue is not suseeptible to the agent (Bittner, 1940~). Variations in the response of mammary tissue t o the action of hormones and of the agent, seen in various strains of mice, are the result of differences in the genetic susceptibility of breast tissue of these mice. The nature of inherited susceptibility to breast cancer was investigated by Andervont (1940a), Andervont and McEleney (1941a), Bittner (1939b,c, 194213, 1944a,b), Murray (1941a,b), and it probably depends on multiple genetic factors (Bittner, 194413). Studies on inbreeding, hybridization, even studies on the relationship between genetic factors and enzymes (Beadle, 1945) demonatrated that genes play an intimate part in the physiological processes leading to tumor formation (Heston, 1946). I n any precise description of genetic factors one should refer to genes which alone are inherited and are subject to mutations. The genetic factors can be divided into primary genes and background, and the action of genes may be only studied by taking the cytoplasm as a medium through which they act (Heston, 1944). Studies on inbred strains of mice, which showed genic differences or differences in the genetic factors of various inbred strains, were followed by attempts to locate them and to discover how they are manifested (Heston, 194813). It is now known that specific genes are involved in specific types of tumors and that the gene for brown color (Bittner, 194410, 1950b), the lethal yellow gene (Little, 1934), and the agouti gene (Heston and Deringer, 1948) are linked with the development of breast cancer in mice. There may of course be many more of them. After the introduction of the milk agent into mice, the development of breast cancer depends on the type of genes with which the agent will be associated (Heston, 1946). It is also conditioned by hormonal factors which in turn are influenced by the genetic factors (genes). The agent gives strikingly different results according to the genetic
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constitution of mice on which it acts (Bittner, 1940b,c; De Ome, 1940; Shimkin and Andervont, 1942). The greater the susceptibility of a strain of mice, the higher is the resulting tumor incidence and the earlier is the appearance of breast tumors after the action of the milk agent on such a strain (Heston, 1945, 1946). The age of mice influences the susceptibility to the agent and susceptible mature mice are quite resistant t o the milk agent (Andervont and McEleney, 1941b; Bittner, 1942c,d, 194513; Andervont et al., 1942). Nevertheless a high incidence of breast cancer is obtained after administration of large amounts of material containing the agent t o adult susceptible mice of the strains tested (Dmochowski, 1945a). 2. The Injtuence of Genetic Factors on Susceptibility to the Milk Agent and Its Production A. Mice of High-Breast-Cancer Strains. The resistance of susceptible mice t o breast tumor development after they reach maturity can be overcome by the agent if present in large quantities (Dmochowski, 1945a, 1948a). It is not necessary therefore for the milk agent t o be present from the time the mammary glands of susceptible mice start t o develop in order to prepare them for the cancerous change and induce a high incidence of breast cancer as reported previously (Bittner, 1942d). It seems that while estrogens induce breast cancer in susceptible mice if their mammary glands have developed in the presence of the agent, the latter may act even ifithe glands have developed in its absence. Mammary tumors can be induced in susceptible mice which have an entirely different genetic constitution from that of the strain which supplied the agent (Dmochowski, 1944b, 1948a). Further, equal amounts of tumor material from two different high-cancer strains containing the agent give a different incidence of breast tumors in the same susceptible mice. Thus there exist differences in either quantity and/or quality of the agent present in two different high-cancer strains (Dmochowski, 1945b, 1948a). Reciprocal crossings of two high-breast-cancer strains and observation of the tumor incidence and tumor age in the resulting virgin hybrid mice have similarly shown variability of the agent present in these high-cancer strains (Murray, 1941a; Bittner et al., 1944; Bittner and Huseby, 1946). Similar experiments on different sublines of the same strains, however, have failed to show a difference in the milk agent of these strains (Heston and Andervont, 1944). Observations on virgin hybrid mice of two other high-breast-cancer strains led to the conclusion that the agent was more concentrated in one of the strains (Warner et al., 1945). In breeding hybrids, owing to increased hormonal stimulation which may increase the sensitivity of breast tissue or produce more agent, the difference between the agent from different strains may not be seen (Bittner and
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Huseby, 1946),although thisis not always the experience (Bittner, 1936b). Cross-breeding experiments theref ore may not always give conclusive evidence of existing differences between the agent present in various high-cancer strains. They do, however, show clearly that genetic factors influence breast tumor development through hormonal mechanism (Heston, 1945). The different tumor ages and tumor incidences in virgin mice foster nursed by female mice of various high-cancer strains also point to differences in the milk agent, although the difficulty in assessment of the activity of the agent may be great in view of the milk agent being active in high dilutions which may give a higher tumor incidence than lower dilutions (Bittner, 194813). It has been pointed out some time ago that the different tumor incidences of various inbred strains of mice may be the result of variability or difference in concentration of the agent which in turn is produced by the genetic factors in these strains (Murray, 1941a,b; Murray and Little, 1939). Microscopical studies of breast tissues of three inbred strains have also led to the conclusion that the agent in these strains appears to vary in quantity and/or quality (Khanolkar and Ranadive, 1947). However, it has been rightly pointed out that the ultimate histologic appearance of breast tissue is determined also by genetic factors which influence the response of the breast tissue to the agent and by hormonal factors (Khanolkar andRanadive, 1947). Heston eta,?.(1945) by employing susceptible hybrid mice derived from high-cancer-strain mothers and low-cancer-strain fathers and by backcrossing these hybrid mice t o high- and low-cancer-strain males showed that differences in the agent were produced by differences in the genetic constitution of the mice from which the agent originated. They also advanced the view that the action of genes in controlling the susceptibility of mice t o breast cancer is located in breast tissue cells. Murray and Warner (1947) attributed the changes in the tumor incidence of a strain to variations in the potency of the agent transmitted by the females of the strain. They were inclined to accept changes in the agent itself rather than any changes in the genetic factors. The variations in the agent of one strain may, however, be attributed to changes in the genetic factors which led to a constitution less susceptible to the agent or less prone to propagate the agent to the point above the threshold of tumor development. This again may be due to hormonal changes or t o a milk agent inhibitor produced by genetic changes, although the latter has not yet been shown. Thus the genetic factors influence the susceptibility or resistance of mice t o the milk agent and the production of the agent. B. Mice of Low-Breast-Cancer Strains. The influence of genetic factors on the degree of susceptibility or resistance of mice to the milk agent
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may also .be seen in the behavior of low-breast-cancer-strain mice. While large doses of the agent or, rather strictly put, material containing the agent can break down the resistance or overcome the low susceptibility of breast tissue of adult susceptible mice, the agent, in amounts far exceeding those required to induce breast cancer in young or even adult susceptible mice, has no influence on 4 to 16 weeks old low-breast-cancerstrain mice (Dmochowski, 1948a). Although there seems to be little doubt therefore that the genetic constitution of these mice influences the reaction of breast tissue to the agent, the possibility of a milk agent inhibitor present in low-breast-cancer-strain mice must be borne in mind. Differences in the susceptibility of various sublines of the same low-cancer strain also have to be taken into consideration. The tumor incidences in the foster-nursed breeding females of various sublines of the CWBlack strain may vary from 10% (Andervont, 1940b; Dmochowski, 1946b; Bittner, 1940b; Murray, 1941b; van Gulik and Korteweg, 1940b), 20% (De Ome, 1940), 50% (Bagg, 1939; Fekete and Little, 1942), to 63% (Andervont, 1943) or 76% (Haagensen and Randall, 1945). However the possibility of the influence of the different sublines of high-cancer strains used as foster mothers must not be overlooked because of the probable strain differences in the agent. The low susceptibility of a Ca7Black subline (Dmochowski, 194th) may, at least partially, be responsible for the failure of large doses of the milk agent to induce breast cancer in mature mice of this low-cancer strain, even if combined with forced breeding, that is, bearing of a number of litters in quick succession. It would be of interest to examine the behavior of other sublines which proved more susceptible to the agent following nursing by high-cancer-strain female mice. There is no reason to assume that different results with varying incidences of tumors would not be found following the administration of large doses of material containing the agent. Mating of high-cancer-strain females to low-cancer-strain males and backcrossing the progeny to low-cancer-strain male parent brought elimination of the agent from such mice after eight generations of backcrossings (Murray and Little, 1939). This may serve as another example of the influence of genetic factors on the milk agent. De Ome (1940) suggested that the different tumor incidences in lo w-cancer-strain females following introduction of the agent during their early life may in part be the result of their low susceptibility and in part the outcome of their constitutional inability to produce the agent. Although one subline of CE,,Black strain has been reported tumor free following foster nursing (Andervont, 1945c), no strain should be called nonsusceptible until breeding mice which obtained the agent have been observed (Bittner, 1950b).
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3. The Influence of Genetic Factors on Transmission of the Milk Agent A. Mice of High-Breast-Cancer Strains. Heston et al. (1945) demonstrated that backcross-hybrid female mice, derived from mating highcancer-strain females (C3H)'to low-cancer-strain males ((26,) and backcrossing the hybrid females either to high- or low-cancer-strain males, which have the same milk agent but different genetic constitution according to the type of male parent, develop significantly different tumor incidences. These two groups of backcross-hybrid females, when used as foster mothers, induced a different tumor incidence in the same susceptible mice, according to the origin of the male parent to which their mothers were backcrossed. A higher tumor incidence developed in the test mice nursed by backcross-hybrid females with high-cancer-strain fathers than in those nursed by backcross-hybrid females with low-cancer-strain fathers. Further, in both types of backcross-hybrid females the tumorous mice transmitted the agent effectively, while nontumorous females showed considerable variation in the transmission of the agent. Heston et al. (1945) concluded that there may be two sets of genetic factors, one controlling the susceptibility to the agent and the other its propagation and ability t o transmit the agent. Thus female mice with both sets of genetic factors would transmit the agent and develop breast tumors; females without genes for susceptibility of tissue to the agent would not show tumors but transmit the agent; and females without genes for propagation of the agent would not transmit it and would not develop cancer. According to Heston (1946) this genetic influence on the transmission of the agent cannot be localized in any tissue, in view of the presence of the agent throughout the body. The existence of two sets of genetic factors was disputed by Bittner (1946-47) in view of the lack of significant difference in the tumor incidence in susceptible mice nursed by the tumorous females of both types of backcross-hybrid mice, and also because the nontumorous backcrosshybrid mice, regardless of their genetic constitution, failed to propagate the agent as well as the tumorous backcross-hybrid females. It seems that more experiments along these lines are required to find out whether the genetic factors do segregate into two separate sets, but there is no doubt that they influence both the susceptibility to the agent and its transmission. B. Mice of Low-Breast-Cancer Strains. The influence of the genetic factors in low-cancer-strain mice on the propagation and transmission of the milk agent may also have to be considered. While adult mice of one low-cancer strain (S) will transmit the agent to their susceptible hybrid progeny, although themselves remaining free of cancer (Dmochowski,
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1948a), adult mice of another low-cancer strain (I),given the agent, fail to develop breast tumors and do not transmit the agent to their hybrid progeny, obtained by mating these females to high-cancer-strain males (Andervont, 1945~). Still other low-cancer-strain females (C), if given the agent, will develop a high incidence of mammary tumors (Andervont, 1940b, 1946) and will transmit the agent to their progeny and remain a high-cancer strain (Andervont, 19494. Some low-cancer strains transmit the agent through one generation only (Andervont, 1945~). Bittner (19424 has also demonstrated that low-cancer-strain females, foster nursed by high-cancer-strain mothers, can transmit the agent to their progeny, although they themselves remain tumor-free. It seems that mice, at least of some low-cancer strains, may differ not only in their susceptibility to the agent but also in their ability to transmit the agent. The ability to transmit the agent may vary from 100% as in strain C (Andervont, 1940b, 1941b, 1945c), through Cay Black strain (Fekete and Little, 1942), strain CBA (Miller and Pybus, 1945b), down to I strain (Andervont, 1945e). The differences in the ability of these strains to transmit the agent correspond approximately with their susceptibility. In the CBA low-breast-cancer strain which has been shown to have a high susceptibility combined with the ability to transmit the agent (Miller and Pybus, 1945b), treatment with estrone (Miller and Pybus, 1942) or breeding as such (Miller and Pybus, 1945a) did not increase the incidence of tumors. In the C67 Black strain whose sublines show considerable variation in the susceptibility to the agent, with tumor incidences varying from 10% to 76%, the progeny of some foster-nursed sublines developed a similar or even higher incidence of tumors (Haagensen and Randall, 1945; Fekete and little, 1942), the sublines thus showing the ability to tranl4mit the agent. Other c 6 7 Black sublines with low tumor incidence following foster nursing either did not transmit the agent to their progeny (Andervont, 1943) or only the fostered mice would transmit the agent but not their progeny (Andervont, 1945c), thus showing a poor ability t o transmit the agent; still other sublines propagated the agent (Bittner, 1948c), although on foster nursing by high-cancerstrain females they showed a low incidence of tumors. There is no doubt that the genetic factors in low-cancer-strain mice influence not only their susceptibility to the agent but also their ability t o transmit it to their progeny or to susceptible test mice. The possibility of two sets of genetic factors, one controlling the susceptibility of breast tissue cells to the agent either directly or through hormonal factors, the other controlling the propagation of the agent, must also be considered in low-cancer-strain mice, but up till now there is not sufficient evidence
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against the probability of these two influences being the expression of the same set of genes.
4. The Milk Agent and Transplanted Breast Tumors Experiments on transplantation of tumors were the basis of the genetic theory of transplantation (Little and Tyzzer, 1916; Strong, 1922; Strong and Little, 1920; Little and Strong, 1924). According to this theory the genetic relationship between the host and the inoculated tumor cell is responsible for the reaction of the host to the transplanted tumor cell. After the establishment of homozygous strains of mice, experiments on transplantation of tumors made it possible to detect genetic changes in the transplanted tumor cells (Bittner, 1931; Cloudman, 1932a,b; Strong, 1926,1929), to determine the homozygoaity of different strains or different sublines of the same strain (Little, 1931;Strong and Little, 1920;Little and Strong, 1924), and to discover sex linkage and linkage with color or other genes (Strong, 1929; Little and Strong, 1924; Bittner, 1934; Gorer, 1947) as well as a relationship between some of the genes required for the growth of transplanted tumors and those determining the presence of an antigen (Gorer, 1937, 1938,1942). Tumors which developed in the same animal were found not to require identical genes for progressive growth (Bittner, 1931; Cloudman, 1932a, 1946; Strong, 1929). Transplantation studies of mammary tumors arising in hybrid mice obtained from matings of high-cancer strains revealed their successful growth in any of the hybrids and only occasional growth in either of their parent strains (Little, 1931; Bittner, 1933a,b,c,d). The progress of transplantation studies and implications arising from these studies have been reviewed by Little and Gorer (1943) and Little (1941a, 1947). Transplantation of spontaneous breast tumors arising in various high-cancer strains has shown the existence of sublines within these homozygous strains, indicated also by the different tumor incidences (Andervont, 1940b; Bittner, 1943, 194813; Woolley et al., 1939). The use of homoeygous strains made the transplantation of spontaneous and induced breast tumors a simple procedure dependent entirely on a simple technique; it also made possible the use of only a few mice for each transplant, thus providing an opportunity for the simultaneous propagation of many tumors. In the early days of the classical experiments carried out by Bashford, Haaland, Murray, it was an arduous procedure which required many scores of mice for each tumor to be transplanted. The availability of transplanted breast tumors provided an opportunity for testing for the presence of the milk agent in these tumors. The agent was demonstrated in cells of a transplanted spontaneous breast
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tumor of a high-breast-cancer strain following ten serial passages in mice free of the agent but susceptible to it (Bittner et al., 1945), although in another case it was not found after three serial transplants (Andervont, 1945a). The presence of the agent could not be ascertained in a so-called 63Ca transplantable breast carcinoma which had been transplanted for over 420 generations (Dmochowski, 1948a), but it is not certain whether the agent was lost during the course of serial transplants or whether it was absent in the primary tumor. The agent was found present in tumor cells following ten passages of a mammary tumor which developed in a lowcancer strain (CW) after foster nursing by a high-cancer-strain female (Bittner, 1948c) and after 42 passages of a similarly induced tumor (Dmochowski, 1949a), and also in high-cancer-strain tumor cells after thirty-one passages in fertile eggs (Armstrong and Ham, 1950). It is not possible to say what part, if any, is played by the milk agent in the propagation of the transplanted breast tumor cells, whether the presence of the agent is a permanent feature of the transplanted cells and whether the tumor cells will continue to multiply in the same way should the agent disappear from the cells. However, it is known that growth on transplantation is not connected with the presence of the agent in the host mice of either sex and the tumors grow equally well in mice whether they lack the agent or possess the same or different agent from that present in the transplanted tumor (Bittner, 194713; Dmochowski, 1949a). The reaction of the host to the transplanted tumor cells depends on the genetic relationship between the host and the transplanted cells and not on the presence of the milk agent in the host. Although it is possible to induce breast cancer by supplying the agent to mice of an entirely different genetic constitution from that of the mice which served as a source of the agent, the induced tumors will grow on transplantation only in mice, in which the tumor originated in spite of the absence of the agent in the host mice, but not in the mice which supplied the agent, because of their different genetic constitution. While the development or disappearance of spontaneous mammary tumors in mice may be the result of the presence or absence of the agent, the genetic constitution of the host mice which remains unaltered (Bittner, 1941f, 1943) and not the milk agent is responsible for the successful transplantation of breast tumors induced by the milk agent. The susceptibility for breast cancer differs from the susceptibility for the growth of transplanted breast cancer, and different sublines of the same strain (Cw) which show different susceptibility to the development of mammary tumors may have the same susceptibility for the growth of transplanted breast tumors, or as in another strain(dba) the sublines may show different susceptibility to transplanted breast tumors (Bittner, 1947b).
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Grafting of mammary tumor tissue with the agent into susceptible mice has either failed to transfer the agent (Andervont, 1949b) or has induced only a low incidence of tumors (Hummel and Little, 1949b). This may have been due to the comparatively short time during which the tumor cells were grown in the hosts and also to transplanted tumor tissue being a comparatively poor source of the agent (Barnum et al., 1947). It may not be therefore a satisfactory method for testing a tumor for the presence of the agent. A. Sarcomalous Transformation. It was first observed in transplanted mammary tumors many years ago (Apolant, 1906; Bashford and Murray, 1907; Ehrlich and Apolant, 1905; Haaland, 1908; Loeb, 1906; Russel, 1909) and interpreted as a change of normal connective tissue into sarcoma (Haaland, 1908) or attributed to a change of glandular cells into spindle type cells (Ewing, 1940). At first it was found t o take place only occasionally (Bashford, 1911) and later in studies on transplanted mammary tumors of high-cancer-strain mice to be of common occurrence (Ludford and Barlow, 1945). In view of the ability of mammary tumor cells t o stimulate the growth of fibroblasts in vitro (Ludford and Barlow, 1944) and of the greater ability of stromal cells to survive when transplanted into homozygous strains of mice, Ludford and Barlow (1945) interpreted sarcomatous transformation as a change of the stroma of transplanted mammary cancer. This transformation was also observed in a high-cancer-strain tumor followingcultivation on eggs and attributed to the action of the milk agent (Taylor, 1948; Taylor and Carmichael, 1947). In an attempt to find an association between the agent and sarcomatous transformation, Dmochowski (1950b) examined a series of spontaneous and induced tumors of high- and low-cancer-strain mice. No sarcomatous change was observed in six spontaneous breast tumors in Cs7Black strain, two spontaneous tumors in Y and P strains, and in three methylcholanthrene-induced tumors in CW and IF low-cancer strains, during transplantation for up t o forty passages. Sarcomatous transformation was however observed in one tumor in each of the different high-cancer-strain (RIII, A, CsH) breast tumors examined and comprising together a total of nine high-cancer-strain tumors, while the others remained unchanged in spite of over thirty serial passages. Sarcomatous transformation was not demonstrated in an agent-induced C67 Black strain mammary tumor transplanted for over 100 serial transfers. Any final conclusions as to the part played by the milk agent in sarcomatous transformation must therefore be based on the results of an examination of a larger number of agent-induced tumors in low-cancer-strain mice and also of agent-free tumors such as have been observed in some agent-free high-cancer-strain mice (Heston et al., 1950).
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6. Spontaneous Breaat Tumors in Low-Cancer-Strain Mice
It is now known that low-cancer-strain mice are not entirely resistant to breast tumor development and that they as well as their sublines develop varying, although low, incidences of breast cancer. For example in strain CBA, the incidence of breast tumors may vary from 2.8% (Strong, 1936), 13.5% (Bittner and Little, 1937) to 22% (Strong, 1938); in strain CS, Black it may be less than 0.5% (Little et al., 1939) or 1.7% (Korteweg, 1936b). To explain the occasional appearance of breast cancer in mice of lowbreast-cancer strains, Bittner (1942c,d) postulated the existence of two types of breast cancer in mice, the so-called inherited and noninherited type of breast tumor. The first develops in mice which have the agent, the susceptibility, and the hormonal factor; the second type arises either in mice which have the agent and are not susceptible or in mice which do not possess the agent and are either susceptible or nonsusceptible. Hormonal factors according to Bittner are present in the noninherited type of breast cancer. The progeny of mice with the inherited type of mammary tumor develops a high incidence of cancer, while the progeny of mice with the noninherited type show only a low incidence of tumors. This classification has been disputed by Heston (1945), who pointed out that a highcancer-strain female mouse, deprived of the agent, but with a tumor, would therefore carry the noninherited type of cancer, while her littermate with an identical genetic constitution but with the milk agent would develop the inherited type of tumor, and suggested therefore that the difference between these two mice may be only a quantitative difference in the stimulation of the milk agent. Biological tests of low-breast-cancer-strain tumors (Car, P, Y, strains) failed to reveal the presence of the milk agent in these tumors, in spite of repeated inoculations into susceptible mice (Dmochowski, 1950c, 1951b). It may still be argued that the agent is present in a latent form in these tumors, but it is also possible that under certain, as yet unknown, conditions, genetic factors in some mice may stimulate reactions which lead to breast tumor development. Should these reactions involve the milk agent in amounts sufficient t o induce breast cancer in these mice but not large enough to pass to the progeny and induce tumors, biological tests of these low-cancer-strain tumors, as might be expected, would a t least reveal the agent in these tumors. In view of the failure to detect the agent, it is possible that genetic and hormonal factors may be responsible for the origin of these tumors. Experimental evidence obtained from the study of carcinogen-induced breast tumors in low-cancer-strain mice supports the conclusion that the milk agent need not be involved in the origin of all mammary tumors in mice.
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6. Conclusions Genetic factors influence the susceptibility of breast tissue cells, both in high- and low-cancer-strain mice, to the action of the milk agent. The decreased susceptibility of adult mice of susceptible strains t o the breast tumor development following introduction of the agent may be overcome by large doses of the agent which fail to have a similar effect on adult low-cancer-strain mice, even if combined with increased hormonal stimulation. Variations in genetic factors may influence the agent present in different high-cancer strains, as revealed in the different tumor incidences induced in the same susceptible mice by the agent from various high-cancer strains, or by the tumor incidence in hybrids derived from crosses of different high-cancer strains. It is not known, at present, whether this is due t o differences in quality or quantity of the milk agent present in various strains. Thus both the production of the agent and the susceptibility of tissue to the agent are controlled by the genetic factors, either directly or indirectly through hormonal and other influences. Transmission of the agent by both high- and low-cancer-strain mice is also influenced by the genetic factors as revealed by hybridization experiments, and the observation of breast cancer incidence in low-cancer-strain mice and their progeny, following administration of the agent to these mice. It still remains t o be decided whether the genetic factors are segregated into separate sets of factors responsible for the various manifestations of their influence on the milk agent. The agent is not responsible for the successful transplantation of mammary tumors which depends entirely on the genetic relationship between the host and transplanted tumor cells. It has still t o be ascertained whether the presence of the agent is required for the continuous growth of transplanted breast tumor cells. At least some breast tumors which develop spontaneously in lowbreast-cancer-strain mice do not possess the milk agent, as shown in biological tests, and therefore, other factors such as the genetic and hormonal may probably be responsible for the origin of these tumors.
111. THE MILK AGENTAND HORMONAL FACTORS 1. Introduction After the original observations on the influence of hormones on the origin of mammary tumors in mice (Lathrop and Loeb, 1913, 1916, 1918; Loeb, 1919, 1921)) a great deal of work along these lines was carried out and presented in a number of reviews (Gardner, 1939; Loeb, 1940; Allen, 1942; Shimkin, 194513; Burrows, 1949). The difference in breast cancer incidence in virgin and breeding mice of some strains (Bittner, 1939b) 194413) was suggested to be the result of
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the influence of genetic factors on the rate of production and elimination of hormones. No difference in the breeding behavior between various highand low-cancer-strain mice could be found which would explain the different tumor incidences (Bittner, 1935a,b; Bittner and Murray, 1936; Murray, 1934), and the number of litters born to females of one strain was found to influence the tumor incidence (Jones, 1940), but this was not confirmed in other strains (Bischoff, 1945; Miller and Pybus, 194513; Bittner, 194647). Forced breeding, that is, bearing of litters in quick succession without nursing them, was reported to increase the tumor incidence in some high-cancer strains (Bagg, 1936a,b; Muhlbock, 1950d) as well as in some low-cancer strains (Bagg and Hagopian, 1939; Bagg and Jackson, 1937). This was found not to apply t o other high-cancer strains (Bischoff, 1945; Bittner, 1946-47) or low-cancer strains (Bagg, 1939; Little and Pearsons, 1940; Fekete, 1940). Similarly no increase in breast tumor incidence was found in other low-cancer strains (JK, P, Y, Cm Black) in spite of an intensive course of forced breeding (Dmochowski, unpublished). In connection with these experiments the influence of prolonged lactation or stagnation of milk contrary to some reports (Bagg, 1925, 1927; Bagg and Hagopian, 1939), was shown not t o play any part in breast tumor development (Fekete and Green, 1936; Murray, 1930; Fekete, 1940; Bittner, 1946-47) in either high- or low-cancer strains, and pregnancy and lactation as such not to influence the origin of breast cancer (Loeb, 1919; Law, 1941b). In connection with the observations of the decrease in breast tumor incidence following ovariectomy (Lathrop and Loeb, 1916; Loeb, 1919; Murray, 1927, 1928, 1932; Cori, 1926, 1927) it was demonstrated that in some high-cancer strains similar procedure only lowered the tumor incidence to that of intact virgin females because of estrogenic stimulation (Fekete el al., 1941; Woolley el al., 1939, 1940, 1941, 1943), while in other strains (Ce) it led to adrenal cortical carcinoma (Dorfman and Gardner, 1944; Woolley and Little, 1945a,b,c,d,e, 1946) which was also observed in castrated hybrids of these and other strains (Bittner, 1948b). Ovariectomy with the resulting hyperplasia of the adrenals led to breast tumor development in mice with the agent while in mice without the agent only adrenal hyperplasia was observed (Smith, 1945, 1946, 1948; Smith and Bittner, 1945; Bittner, 1948a). In this way as well as by adrenalectomy (Cramer and Horning, 1939; Shimkin and Wyman, 1945) or by the administration of adrenal cortical extracts (Gardner, 1940; Shimkin and Grady, 1941) the part played by adrenal cortex in the origin of breast oancer in mice was shown. The important part which the pituitary gland plays in breast tumor development was demonstrated by a number of workers on hypophysectomized mice bearing pituitary and ovarian
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grafts or on castrated mice treated with pituitary and other hormones (Loeb et al., 1944; Korteweg and Thomas, 1946; Frantz et al., 1947; King et al., 1948). Thus it was shown that the same hormonal factors which take part in the normal development of mammary glands exert also an influence on the development of malignant changes in these glands. In spite of many attempts, no correlation was found between the degree of susceptibility of different strains of mice to the development of breast cancer and the various physiological manifestations of the hormonal factors such as estrus cycle, excretion of estrogens, changes in various glands of the endocrine system (for literature see Gardner, 1939; Loeb, 1940; Shimkin, 1945a,b,c). Any differences, which had been observed, were found to be only strain characteristics not connected with the development of bread cancer. The presence or absence of the milk agent was found not to influence the various physiological manifestations of hormones as demonstrated by reciprocal crossings of high-cancer strains or by foster nursing experiments (Deringer et al., 1945; Armstrong, 1948). Similarly the degree of sensitivity of the genital tract to estrogens bears no relation to the milk agent, being genetically determined (van Gulik and Korteweg, 1940a; Shimkin and Andervont, 1941; Trentin, 1950). The greater resistance of the high-cancer strains examined t o estrogen stimulation, as demonstrated in the vaginal and uterine lesions, shown by van Gulik and Korteweg (1940a); Gardner and Allen (1939); Allen and Gardner (1941); Muhlbock (1947, 1948b), and the comparatively higher sensitivity of low-cancer-strain mice in this respect as well as of their mammary glands (Korteweg, 1935; Korteweg et al., 1948; Muhlbock, 1948a) may be the result of differences in estrone production (van Gulik and Korteweg, 1940a), mice of low-cancer strains producing smaller amounts of estrone compared with high-cancer-strain mice (Korteweg, 1948a; Fekete, 1946). According to Korteweg (1948a) a t least part of the genetically determined susceptibility to breast cancer may therefore be connected with the production of estrone. Strain differences in the response of mammary glands to estrogens were also found in agent-free ovariectomized mice of other strains (Pullinger, 1947). However, the results of foster nursing experiments (Shimkin and Andervont, 1941) and of the study of a large number of high- and low-cancer strains and their reciprocal hybrids (Trentin, 1950) indicate that the differences in tumor incidences of various strains are not influenced by the different amounts of estrogen produced by these mice. 2. The
Milk Agent and Estrogen-Induced Breast Tumors i n Male Mice
Experiments on castrated high-cancer-strain males bearing ovarian grafts led to the successful induction of breast tumors in male mice (de
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Jongh and Korteweg, 1935; Murray, 1928). A similar or even a higher incidence of tumors in castrated susceptible males, with the milk agent and bearing ovarian transplants, than in virgin females of these strains was reported by Huseby et al. (1946b) and Huseby and Bittner (1948c), while noncastrated susceptible males with ovarian grafts showed a lower incidence of tumors at a later age than virgin mice of the same strains. Thus the influence of ovarian estrogens on the origin of breast cancer was shown in males, susceptible and harboring the milk agent. The age factor in the development of breast tumors in males was demonstrated by the higher tumor incidence in males which at an early age had been castrated, grafted with ovaries, and given the agent than in males, castrated and grafted with ovaries a t a similar early age, but given the agent several months later (Huseby and Bittner, 1948b). This relative lack of susceptibility was attributed to a general increased resistance to the milk agent shown also by susceptible adult female mice. The successful induction of breast tumors in males and the part played by the milk agent in the development of these tumors with a similar influence of age on the tumor incidence was also demonstrated on agent-free but susceptible males treated with estrogens and given the agent a t various ages (Silberberg and Silberberg, 1948), on agent-carrying susceptible males treated with estrogens and castrated a t various ages (Silberberg and Silberberg, 1951), and also on susceptible males castrated at different ages and bearing ovarian and hypophyseal grafts (Silberberg and Silberberg, 1949). Breast tumors were also induced in estrogen-treated susceptible but deprived of the agent males following transplantation of breast tissue from agent-carrying females of the same strain (Shimkin et al., 1946). There is therefore no doubt that in males as in females the development of breast tumors takes place, provided they possess suitable genetic susceptibility, hormonal stimulation, and the milk agent. Hormonal stimulation was considered as necessary for creation of a suitable substrate for the agent (Shimkin and Andervont, 1941) or for all types of stimuli (Loeb, Suntzeff, et al., 1944) or as a developing factor on breast tissue already sensitized by the agent (Bonser, 1945). While doubts may be expressed as t o sequence of action of various factors on the breast tissue, there is no doubt of their equal importance. Lacassagne (1932) first induced breast cancer in male mice by estrogenic hormones. A whole series of studies which followed this important observation established the importance of estrogens in breast cancer of mice. Natural and synthetic estrogens were found to induce breast tumors in males of strains in which female mice showed a high incidence of these tumors. The importance of the milk agent in the development of estrogen-induced breast tumors was established by the observation of
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tumor incidences in high- and low-cancer-strain males following estrone treatment and even more conclusively by the increase in the incidence of estrone-induced tumors in low-cancer-strain males foster nursed by high cancer-strain females (Lacassagne, 1939; Lacassagne and Danysz, 1939; Twombly, 1939, 1940; Bittner, 1940b, 1941e; Gardner, 1941a; Shimkin and Andervont, 1941,1942; Dmochowslu and Gye, 1944; Bonser, 1944a,b; Dmochowski and Orr, 19494. Thus agent-carrying susceptible males developed breast cancer only if treated with estrogens, and male mice of low-cancer strains developed none or only a few tumors following treatment with estrogens, unless given the milk agent. The presence of the milk agent in estrogen-induced tumors in high-cancer-strain males, although presumed, was first demonstrated by Dmochowski and Orr (194913). 5. The Milk Agent and Methylcholanthrene-Induced Breast Tumors in Mice
The accelerating influence of methylcholanthrene and other carcinogenic hydrocarbons on the appearance of breast cancer in mice was first observed by Maisin and Coolen (1935) and Perry and Ginzton (1937). The induction of breast tumors or acceleration of their appearance was conclusively demonstrated in a series of studies which followed these first observations (Mider and Morton, 1939a,b; Orr, 1939, 1943, 1946; Bonser and Orr, 1939; Strong and Smith, 1939; Bonser, 1940; Strong and Williams, 1941; Engelbreth-Holm, 1941; Engelbreth-Holm and LefBvre, 1941; Law, 1941a; Kirschbaum and Strong, 1942; Shimkin and Bryan, 1943; Kirschbaum et al., 1944; Kirschbaum, Williams, and Bittner, 1946; LefBvre, 1945; Kirschbaum and Bittner, 1942, 1945; Strong, 1945; Engelbreth-Holm and Rask-Nielsen, 1947; Kirschbaum, 1949). The induction of breast cancer in low-cancer-strain female mice and acceleration of its appearance in high-cancer-strain females was demonstrated and it was shown that estrogenic hormones are required for the induction of breast cancer with carcinogenic hydrocarbons since only females developed these tumors with breeding females showing a higher incidence than virgin mice, and males only if treated with estrogens at the same time. The development of mammary tumors in mice of high- and low-breastcancer strains following treatment with carcinogenic hydrocarbons made the explanation of the origin of such tumors as a result of the estrogen-like action of methylcholanthrene inadequate, and necessitated a study of the part played by the milk agent in the origin of methylcholanthreneinduced breast tumors in both high- and low-cancer-strain mice. Dmochowski and Orr (1949a) induced mammary tumors in high(C3H)and low-breast-cancer-strain ((36, Black) males following combined treatment with estrogens and methylcholanthrene. Methylcholanthrene
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alone failed to induce tumors both in high- and low-cancer-strain males, whereas estrone treatment gave rise to breast tumors in high-cancerstrain males but not in low-cancer-strain males. Treatment with methylcholanthrene alone induced breast tumors in low-cancer-strain females, both breeding and virgins. Thus again hormonal factors were shown to play a part in the development of tumors induced by methylcholanthrene. However, a case of induction of breast cancer in a lowcancer-strain male (CBA) in the absence of estrone treatment was reported by Orr (1943). According to Kirschbaum and Bittner (1945) methylcholanthrene-induced breast tumors may appear earlier and more frequently in mice of the same genetic constitution if the milk agent is present, although they pointed out that its presence is not essential in the development of these tumors. The induction of breast tumors in low-cancer-strain males also indicated that the agent need not take part in the origin of these tumors, but it was not known whether treatment with methylcholanthrene may not lead to the production of the agent or activation of a latent agent. Andervont and Dunn (1950a) reported that combined treatment with the agent and the carcinogen failed t o accelerate the development of breast tumors in a susceptible but agent-free (dba) strain. However, carcinogen alone induced fewer tumors than the milk agent alone or methylcholanthrene combined with the agent; estrogenic stimulation did not increase the effect of methylcholanthrene. In an assessment of these and the previous results, the technique of application of the carcinogen, the dosage, the solvent, and furthermore the age of mice and possible variations in susceptibility to carcinogen between various sublines of the same strain must be considered. This would explain the induction of tumors in one subline of a low-cancer strain (CS,Black) by Dmochowski and Orr (1949a) and the failure in another subline of the same strain (Kirschbaum, Williams, and Bittner, 1946), or the difference between the appearance of breast tumors in agent-free mice of a high-cancer strain after treatment with methylcholanthrene (Kirschbaum, Williams, and Bittner, 1946; Andervont and Dunn, 1950a) and the absence of tumors in other sublines of the same strain even in the presence of the milk agent (White and Mider, 1941; White et al., 1943). Treatment with varying amounts of the carcinogen may lead to the appearance of different types of tumors (Andervont and Dunn, 1950a), or only delay the appearance without altering the incidence of carcinogen-induced breast cancer (Orr, 1950). The incidence of breast tumors in agent-free mice may also be increased by gamma rays (Lorenz et al., 1949) and possibly by x-radiation (Andervont and Dunn, 1950b) and by application of 2-acetylaminofluorene (Armstrong and Bonser, 1947). Biological tests for the presence of the milk agent in breast tumors
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induced in high- and low-cancer-strain mice by methylcholanthrene alone or by estrone combined with methylcholanthrene have shown a difference in these tumors according to the strain in which they originated. The milk agent was found in breast tumors induced in high-cancer-strain male mice with estrone and methylcholanthrene combined but not in breast tumors induced in low-cancer-strain male and female mice following similar treatment (Dmochowski and Orr, 1949b), Similarly, methylcholanthrene-induced tumors in agent-free susceptible mice were found not to contain the agent (Bittner and Kirschbaum, 1950; Andervont and Dunn, 1950a). The development of breast tumors in high- and low-cancer-strain mice following treatment with methylcholanthrene, the differences in susceptibility to the development of these tumors in various high-cancer strains (Kirschbaum and Bittner, 1945) as well as between low-cancer strains (Kirschbaum, Williams, and Bittner, 1946; Strong, 1945; Orr, 1946; Dmochowski and Orr, 1949a), and the absence of the agent in induced breast tumors of low-breast-cancer-strain mice support the conclusion that the action of methylcholanthrene is independent of the milk agent. This conclusion strengthens the opinion (Strong, 1945) that the type of susceptibility involved in the origin of carcinogen-induced tumors differs from that involved in the origin of spontaneous breast cancer, all the more as mice with low susceptibility t o the milk agent may show high susceptibility to methylcholanthrene and vice versa. Methylcholanthrene does not induce the appearance of the agent in mice which do not possess it, and while few or no breast tumors develop in agentfree, even susceptible, mice following treatment with estrogens, methylcholanthrene induces breast cancer in agent-free mice, both susceptible and resistant to the milk agent. A. Histological Appearance of Carcinogen-Induced Breast Tumors. The greater variation in structure of carcinogen-induced breast tumors in agent-free mice compared with that of agent-induced tumors was first pointed out by Strong and Williams (1941). The presence of squamous metaplasia in these tumors was emphasized by Strong (1945) and by Kirschbaum and Bittner (1945). The presence of squamous metaplasia in the precancerous hyperplastic nodules induced in mammary tissue by the carcinogen both in the presence and absence of the milk agent led Kirschbaum, Williams, and Bittner (1946) to conclude that they differ from normal hyperplastic nodules and that the origin of methylcholanthrene-induced tumors is different from that of spontaneous breast tumors. The nature of these nodules, however, may be disputed as Huseby and Bittner (1946) observed nodules consisting of squamous metaplasia of glandular epithelium in mice of both high- and low-cancer strains and described them as inflammatory rather than precancerous.
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Similar nodules in a high-cancer strain have been reported to regress and disappear during the period of observation (Browning, 1948). Squamous metaplasia was also described by Orr (1946) and Dmochowski and Orr (19494 as a characteristic feature of carcinogen-induced breast tumors in low-cancer-strain mice, appearing less frequently in similarly induced tumors in agent-carrying high-cancer-strain mice. Although a variety of histological structures in methylcholanthrene-induced tumors and a more uniform type of structure in agent-induced tumors were described by Andervont and Dunn (19504, some carcinogen-induced breast tumors whether in susceptible mice with or without the agent or in low-cancerstrain mice may not be distinguished from agent-induced breast tumors (Dmochowski and Orr, 1949a; Andervont and Dunn, 1950a). Although a rigid classification of breast tumors in mice and in man must not be adopted and has only a descriptive value (Dunn, 1945; Willis, 1948), the more frequent appearance of squamous metaplasia in carcinogen-induced tumors and the more often observed structures in breast tumors of agentfree mice only seldom encountered in agent-carrying mice (Andervont and Dunn, 1950a) deserve attention. Further, strain or subline differences must be remembered if one considers the number of tumors with squamous metaplasia following carcinogen treatment, reported by various workers (Orr, 1946; Dmochowski and Orr, 1949a; Andervont and Dunn, 1950a). Carcinogen-induced tumors show greater invasiveness of neighboring tissues and metastasize more frequently than agent-induced tumors (Strong, 1945). Squamous metaplasia of ductal epithelium in the carcinogen-induced and acinar proliferation in the agent-induced breast cancer (Kirschbaum, Williams, and Bittner, 1946) may be the result of the carcinogen acting on different elements of the mammary gland or on several elements of the gland (Andervont and Dunn, 1950a) thus producing greater variety of tumors. Kirschbaum (1949) has pointed out that the histological structure of spontaneous breast tumors in agent-free mice is similar to that of carcinogen-induced, although this is by no means a rule (Dmochowski, 1950c; Heston e2 al., 1950). According to Bonser (1949) the differences in structure between spontaneous, hormone-induced, and carcinogen-induced tumors may be of degree rather than of kind, the intraduct type of tumor occurring more frequently in carcinogen-induced than spontaneous breast cancer, characterized by intra-acinous changes and acinar proliferation, because of the affinity of the agent for acini.
4. Conclusions The induction of breast tumors in both male and female mice by natural and synthetic estrogens depends on the susceptibility of a strain
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to mammary tumor development and on the presence of the milk agent. The induction of breast cancer in both high- and low-breast-cancer-strain mice by carcinogenic hydrocarbons is governed by susceptibility to carcinogen-induced tumors, which appears to be different from that to the milk agent, and is also influenced by estrogenic hormones, which is particularly shown by the absence of breast tumors in high- and lowcancer-strain males treated with methylcholanthrene. The histology of agent-induced, estrogen-induced, and carcinogen induced breast tumors is not identical. It appears that different etiological factors may induce histologically different types of breast cancer in mice, although no sharp distinctions can be drawn, as some breast tumors may have the same appearance whether induced by the milk agent, estrogens, or carcinogenic hydrocarbons. It appears that methylcholanthrene induces the development of breast cancer as a separate agent. Studies on the induction of tumors in mice with carcinogenic hydrocarbons indicate that the milk agent is not essential in the development of all breast tumors in mice. IV. THEMILK AGENTAND MAMMARY GLANDSTRUCTURE The study of the correlation between mammary gland structure and breast tumor development was started by Apolant (1906) and Haaland (1911), who described the more frequent occurrence of small adenomatous areas in glands of mice with breast cancer, compared with that of similar changes in mice without breast cancer, and considered them to be inflammatory in origin. Following the development of inbred strains of mice, these studies were taken up again. At first no correlation between the structure of glands and mammary tumor development could be ascertained (Gardner and Strong, 1935). Soon, however, localized areas of acinar hyperplasia, similar to the previously described adenomas, were found in the glands of breeding females of a high-cancer strain (Fekete, 1938) as well as in breeding females of other high-cancer strains with only infrequent occurrence in the glands of low-cancer-strain mice (Gardner et al., 1939). Hormonal as well as hereditary factors were considered t o influence the development of these changes (Gardner et al., 1935, 1939) and their precancerous character stressed and confirmed by their progressive growth in hypophysectomieed mice (Gardner, 1942). Similar observations with confirmation of the precancerous nature of these changes were made by van Gulik and Korteweg (1940b) and Taylor and Waltman (1940). The first attempts to correlate changes in the mammary glands with the action of the milk agent were reported by van Gulik and Korteweg (1940b) who attributed the formation of lateral buds along the ducts
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of glands of some strains to the influence of the agent. These observations were confirmed by Shimkin et al. (1941), and treatment with estrogens was found to influence the increase of lateral budding in agentcarrying mice (Shimkin, 1945b). Further investigations, however, led to a conclusion that the difference in the amount of lateral budding in the glands of various strains should be attributed t o strain peculiarities (Gardner, 1945). Similarly, Huseby and Bittner (1946) could not correlate the increase of lateral budding with the action of the milk agent, but reported the correlation in virgin mice between the degree of lateral budding and the so-called inherited hormonal influence, because of its infrequent occurrence not only in low- but also in high-cancer strains with low incidence of cancer in virgin mice and its constant occurrence in high cancer strains whose virgins show a high incidence of mammary tumors. A thorough study of areas of acinar hyperplasia by Bittner et al. (1944) and by Huseby and Bittner (1946) showed all three factors influencing the origin of breast cancer to be of importance in the formation of these areas of acinar proliferation. They were not found either in high-cancer-strain mice deprived of the milk agent, or in virgin mice of high-cancer strains lacking adequate hormonal stimulation, or in lowcancer-strain mice, that is, mice with low susceptibility to breast cancer. They were further shown to be different from inflammatory nodules, which occur with equal frequency in high- and low-cancer-strain mice (Huseby and Bittner, 1946). The influence of hormones on acinar hyperplasia was also demonstrated by Gardner (1946). No nodules were observed in other agent-free high-cancer strains or in breeding low-cancer-strain mice (Kirschbaum, Williams, and Bittner, 1946). Nodules of acinar hyperplasia in the glands of high-cancer-strain females and estrogen-treated males and their absence in these mice deprived of the agent was also reported by Khanolkar and Ranadive (1947), and also by Smith (1948) in castrated agent-carrying mice of strains with the inherited hormonal influence because of the adrenal hyperplasia acting as a source of hormones. Pullinger (1947) succeeded in accelerating the appearance of these nodules by administration of measured amounts of estrogens to ovariectomized high-cancer-strain mice and demonstrated their persistence in the presence of the agent after the withdrawal of hormonal stimulation. These foci of nodular proliferation of acini differ from squamous foci which can be seen in old breeding agent-free female mice of high-cancer strains or are induced in the same strains with methylcholanthrene (Pullinger, 1949; Kirschbaum, 1949). Morphological evidence of the presence of the milk agent in mammary glands prior to the appearance of these precancerous lesions, that is,
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nodular proliferation of acini, has not yet been found. In view of the dependence of these lesions on the milk agent and the possibility of accelerating their appearance in the presence of the agent by a suitable application of hormones, an enquiry into devising a more rapid test for the milk agent seems worth while.
V. INHERITED HORMONAL INFLUENCE The observation of differences in the mammary tumor incidence between virgin females of various high-cancer strains (Bittner, 1939b; Andervont, 1941a; Heston and Andervont, 1944; Bittner et al., 1944) and the demonstration that they are caused by genetic factors (Heston and Andervont, 1944; Bittner, 1945b; Heston, 1945) has led to the introduction, in addition to the three factors known to influence the origin of breast cancer, of yet another factor, described as “inherited hormonal influence” (Bittner, 194513; Smith, 1945). The part played by this factor is seen in the development of breast cancer in virgin females, provided the hormonal stimulation is not increased. This factor was demonstrated by reciprocal crossings of two high-cancer strains, strain A with a low incidence of breast cancer in virgins and strain CaH with a high incidence of breast tumors in its virgin mice, which resulted in virgin hybrid females with a high incidence of tumors (Bittner et al., 1944; Heston and Andervont, 1944; Bittner and Huseby, 1946). This influence is based on control of hormonal stimuli by genetic factors (genes), which appear to be different from those determining the inherited susceptibility to breast cancer (Bittner and Huseby, 1946). Although this influence is transmitted as a dominant factor because of the similar tumor incidence in the progeny and the parent with the influence, it is not entirely dominant because of the difference in the tumor age between virgin hybrid mice and their male parent transmitting the influence (Huseby and Bittner, 1948a). Multiple genes are probably involved in the inherited hormonal influence (Bittner and Huseby, 1946) as they are in the genetic susceptibility for breast cancer (Bittner, 1944a,b), although the exact relationship between these two susceptibilities still remains to be determined, especially in view of the opinion held in some quarters that these two susceptibilities should not be treated separately (Korteweg, 1948b). There are now several inherited factors discernible which influence the origin of breast cancer, inherited susceptibility to breast tumors, inherited hormonal influence, inherited influence on the propagation and transmission of the agent. However, in spite of the presence of inherited susceptibility to breast cancer and of the inherited hormonal influence, and adequate hormonal stimulation, the presence or absence of
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the milk agent determines whether breast cancer will develop (Bittner and Huseby, 1946). A relationship exists in certain strains between the changes in the adrenal cortex following castration, originally reported by Woolley et al. (1939, 1940, 1941) and the inherited hormonal influence (Smith, 1945, 1946; Smith and Bittner, 1945). Castrated mice of strains (CaH) with the influence as well as their castrated hybrids obtained from matings with strains (A) without the influence were found to develop hyperplasia of the adrenal cortex and adrenal cortical tumors and, if the milk agent was present, breast tumors as well, while mice of strain A showed no adrenal changes after ovariectomy. Hyperplastic adrenal cortex was shown to be the source of hormones, similar t o those of the ovaries, which led to breast cancer in castrated mice or their castrated hybrids in the presence of the milk agent (Smith, 1948). Castrated mice without the hormonal influence, in spite of genetic susceptibility to breast cancer and the presence of the agent showed no significant changes in the adrenals and no signs of hormonal stimulation and no breast tumors (Smith, 1948). A further correlation between the inherited hormonal influence and the age of the opening of vagina, being earlier in mice with the influence, was reported by Deringer et al. (1945). This influence is acting, at least partially, through the ovaries, as demonstrated by the tumor incidence in spayed virgin hybrid females of two high-cancer strains bearing transplanted ovaries from parental strains with and without the influence (Huseby et al., 1946a,b), and also probably through the pituitary (Smith, 1948). It may also influence the response of mammary gland cells to hormones (Deringer et al., 1945). It seems now established that more than one endocrine organ are affected by this influence. Bittner (1948a) reported that other high-cancer strains (dba) which were found to possess the inherited hormonal influence also develop adrenal hyperplasia following castration. The presence of the inherited hormonal influence is not limited to high-cancer strains. Some lowcancer strains (strain I) which show a low incidence of breast tumors in breeding females even with the milk agent present (Huseby and Bittner, 1948a) and others, which have a high tumor incidence even in virgin females if the agent is present (strains DI, C, De, Bittner, 1951), have been found to transmit the influence and to develop hyperplasia of the adrenal cortex following castration. Still other low-cancer strains ( ( 3 6 1 Black) without the influence do not develop adrenal hyperplasia (Huseby and Bittner, 1948a). Virgin hybrid females, obtained by matings of strain A females to males of low-cancer strains with the hormonal influence, showed a high incidence of breast cancer, and when castrated developed adrenal hyperplasia (Huseby and Bittner, 1948a).
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Further investigations revealed that adrenal hyperplasia and the inherited hormonal influence do not always appear together and that mice of different strains may possess various hormonal mechanisms with different influence on the origin of breast cancer in virgin mice. Males of the Ce low-cancer strain, which develops adrenal hyperplasia and adrenal cortical carcinoma after castration (Woolley et al., 1941; Woolley and Little, 1945a,b,c,d, 1946), and which develops a high incidence of breast tumors in breeding and a low incidence in virgin mice if given the milk agent (Bittner, 1948b), when mated with strain A females gave virgin hybrids with adrenal cortical tumors following castration (Bittner, 1950b). These virgin hybrids developed an incidence of breast tumors higher than virgin strain A mice but lower than virgin hybrids from crosses of the same strain A mice with males of other strains showing adrenal hyperplasia. Even the breeding hybrid females had a lower incidence of breast tumors than breeding mice of either strains. Males of the JK low-cancer strain, which develops adrenal hyperplasia after castration and has a low incidence of breast tumors in breeding females with the agent (Bittner, 1950b), and males of the N H low-cancer strain, which develops spontaneous adrenal hyperplasia and adrenal cortical carcinoma (Gardner, 1941b; Kirschbaum, Franta, and Williams, 1946; Frantz et al., 1947; Frantz and Kirschbaum, 1949), when mated with A strain females, gave hybrid progeny whose virgins showed low incidence of breast cancer in spite of the presence of postcastrational adrenal hyperplasia (Bittner, 1951). Therefore, not all low-cancer strains which show adrenal changes following castration transmit the inherited hormonal influence. While some low-cancer strains develop adrenal tumors spontaneously, other low- as well as high-cancer strains (Bagg albino, CBA, C3H) show these tumors only after castration (Kirschbaum, 1948). It would be of interest to study the behavior of other high-cancer strains in respect to the parallelism between inherited hormonal influence and the development of adrenal hyperplasia. The presence or absence of postcastrational adrenal hyperplasia and also the histological appearance of these changes were found t o be a function of genetically controlled adrenal responsiveness (Huseby and Bittner, 1951). According to Huseby and Bittner (1947, 194th) there may still be another hormonal difference between the A and C3H strains, as the milk agent significantly alters vaginal smears of the strain A females and (AxCaH)Fl hybrids but not of C3H drain mice. Further, the excretion of 17-ketosteroids in feces of both strains and their hybrids is decreased by the action of the agent (Samuels and Bittner, 1947), although no correlation could be found between the excretion rates and the tumor incidences in virgin and virgin hybrid mice of these strains. There are indi-
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cations of an inverse relationship between the level of the hyaluronidase inhibitor in sera and the presence of the inherited hormonal influence (Glick et al., 1948) and of a direct relationship between the influence and the porphyrin level (Bittner and Watson, 1946). Thus information is gradually accumulating which may lead t o a better understanding of the mode of action of the milk agent and its relationship with other factors, such as hormonal and genetic, and the various ways in which they may influence the origin of breast cancer in mice. VI. PROPERTIES OF THE MILK AGENT 1 . Introduction
The discovery of the milk agent in the milk of high-cancer-strain females was soon followed by numerous attempts to ascertain its presence in various tissues and organs of normal mice of high-cancer strains and also in breast tumors of these strains. For this purpose either implantation of various organs into suitable susceptible mice was used or extracts of these organs and tissues were given either by feeding or injecting into the test mice. Susceptible high-breast-cancer-strain mice, deprived of the agent by foster nursing by low-cancer-strain mice or by removing them from the uterus of their own mothers and nursing by low-cancerstrain mothers, susceptible low-cancer-strain mice (Andervont and Dunn, 1948a,b), or susceptible hybrid mice obtained by mating low-cancerstrain females with high-cancer-strain males, may be used as test mice. In this way the agent was found in the spleen (Bittner, 1937a, 1939a,e; Andervont et al., 1942; Dmochowski, 1944a), thymus (Bittner, 1939e, 1940a), lactating mammary tissue (Bittner, 1939e, 1940a; Andervont el al., 1942; Andervont and Bryan, 1944; Barnum et al., 1946), spontaneous breast tumor tissue (Bittner, 1941a, 1942a; Bryan et aZ., 1942; Visscher, Green, et al., 1942; Barnum et al., 1944, 1947; Andervont and Bryan, 1944; Dmochowski, 1944b, 1945c), transplanted breast tumor tissue (Bittner, 1945b; Bittner et al., 1945; Barnum et al., 1947;Dmochowski, 1949a), and in Harderian gland (Bittner and Watson, 1946). As far as is known all tissues tested were those of high-cancer-strain females. The agent was also found in whole blood (Woolley, Law, and Little, 1941) where it appears to be more concentrated in cellular elements than in the serum (Bittner, 1945a). The presence of the agent in some organs such as liver was doubtful (Bittner, 1941b, 1947b) or was not ascertained as in the stomach milk, and there existed contradictory evidence of the transmission of the agent in the uteri of high-cancer-strain females (Fekete and Little, 1942), because it was demonstrated (Andervont,
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194513) that susceptible mice do not develop breast cancer if removed from the uterus of their high-cancer-strain mothers and nursed by low-cancerstrain females. 2. Distribution of the Agent
During recent years more data have been accumulated on the distribution of the milk agent in the body of mice. It was found to be widely distributed in the body of high-cancer-strain males because of its presence in the liver, lungs, kidneys, spleen, thymus, and heart of these mice (Dmochowski, 1948b,c, 1949c,d), in the seminal vesicles (Andervont and Dunn, 1948a), in the sperm from cauda epididymis (Muhlbock, 1950a), and also the whole blood of high-cancer-strain males (Hummel and Little, 1949a). A study of the presence of the agent in the whole blood of high-cancer-strain mice and its fractions, globulins, albumins, and washed red cells showed the agent to be either present there in small quantities or absent (Graff et al., 1946). There are indications that the agent can be absorbed by red blood cells of agent-free mice from the serum of high-cancer-strain mice (Bittner, 1947a) and that it is more concentrated in cellular elements (Bittner, 1945a) than in the plasma (Humme1 and Little, 1949a), but is not present in clotted blood (Hummel and Little, 1949a). In young mice the agent seems to be more concentrated in cellular elements of the blood than in plasma, and in older mice with tumors this is reversed (Bittner, 1945a). The agent has also been found in the liver of high-cancer-strain males (Dmochowski, 1948b, 1949c,d; Hummel and Little, 1949a). It may be there in small quantities either by virtue of blood which the liver contains or it may be there in an attenuated form. The part played by the liver in the removal and destruction of the agent requires further investigation. Organs of low-cancer-strain females do not harbor the agent (Dmochowski, 1948d, 1949d) as ascertained in biological tests based on repeated injections of tested material into susceptible mice (Dmochowski, 1945a). Similar results with the blood, liver, and spleen of other lowcancer-strain mice have been reported by Hummel and Little (1949a). Fekete and Little (1942) and Little (1944) reported intra-uterine transmission of the milk agent on the basis of the appearance of tumors in the progeny of high-cancer-strain females born from fertilized ova transferred t o low-cancer-strain mice. This observation contradicted the reports on the low incidence of breast tumors in up to twenty generations of high-cancer-strain mice foster nursed by low-cancer-strain females (Bittner, 1945b) and on similarly low tumor incidence in several generations of mice descended from high-cancer-strain mice removed from the uteri of their mothers and nursed by low-cancer-strain females (Ander-
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vont, 194513). Dmochowski (1948d, 1949d) found no evidence of the agent in high-cancer-strain embryos before their birth and only very small quantities of the agent in the placenta of high-cancer-strain females. It is not known, as yet, whether this is due t o some unknown factors which neutralize the agent in the placenta. Hummel and Little (1949a) reported the absence of the agent in embryo blood and in the placenta of high-canceljstrain females. The latter observation may be due to smaller quantities of material tested compared with those of Dmochowski (1949d). According to Hummel et al. (1949), the placenta may be the site of neutraliaation or inhibition of the agent. It seems therefore well established now that there is no intra-uterine transmission of the agent, a t least in its active form (Dmochowski, 1948c, 1949b,d). This was already indicated by the results of cross-suckling of high- with low-cancerstrain mice as well as by the results of cross-matings of high- and lowcancer strains (Bittner, 1944b) and also by the decrease in tumor incidence following the removal of young high-cancer-strain mice by cesarean section (Andervont and McEleney, 1941b; Shimkin et al., 1946) or after searing the nipples of high-cancer-strain mothers (Andervont et al., 1942). Stomach milk of young 5- to 11-day-old high-cancer-strain mice has been shown to be a good source of an active agent (Dmochowski, 1949d; Hunimel and Little, 1949a). Blood is a poor source of the agent in comparison with spontaneous tumor tissue or lactating mammary glands (Bittner, 1947a), and blood from mice with spontaneous breast cancer is a better source than blood from pregnant or even normal high-cancer-strain females (Hummel and Little, 1949a). The small yield of the agent from blood of pregnant mice may be due t o the inhibitory effect of the placenta (Hummel and Little, 1949a), which has been shown to neutralize the agent in uitro following incubation with extracts of breast tumor tissue (Hummel and Little, 1949b). Although it is now known how widely the agent is distributed throughout the body of mice, it is still not ascertained in which tissues it is specifically propagated. Comparison of the activity of the milk agent present in extracts of lactating mammary tissue and of tumor tissue indicated greater activity in the former than in the latter (Barnum et al., 1946). It must be remembered however that the concentration of the agent in the tumor tissue may depend on the age of,the mouse and the histological structure of the tumor. These differences between the extracts of these two types of tissue may not be observed in other test mice with a different genetic constitution (Bittner, 1947a). Great care is required in this type of experiment as it is conditioned by the age and genetic constitution of the test mice as well as by the concentration of the extracts tested. Extracts of breast tumor tissue may give a higher incidence of tumors
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when tested in higher dilutions than in more concentrated dilutions, although in younger mice this may not be so evident (Bittner, 194647). A thousand-fold dilution of material containing the agent has been reported to give a higher incidence of tumors than a ten-fold dilution, while a ten-thousand-fold dilution induces a lower incidence of tumors a t an earlier age (Bittner, 194813). Agent-harboring tissues even in a million-fold dilution have been shown still to induce a comparatively high incidence of cancer (Bittner, 1945a, 1946; Barnum et al., 1946). This may be the result of removal of autolyzing enzymes. All these observations must be borne in mind while testing any material for the presence of the agent. Excreta of mice, urine, and feces have been shown not to contain the agent (Dmochowski and Passey, 1951; Muhlbock, 1951), in spite of repeated injections of Berkefeld filtrates of urine of high-cancer-strain mice into susceptible mice (Dmochowski and Passey, 1951). The presence of the milk agent does not influence the amount of porphyrins present in the Harderian glands (Bittner and Watson, 1946), in spite of the reported parallelism between susceptibility and the porphyrins (Strong, 1944). Differences in the antiproteolytic activity of sera of various strains (Clifton et al., 1949) as well as in the titer of antibodies against sheep and human red blood cells (Davidson and Stern, 1949,1950; Davidson et al., 1950), and in the antibodies in mice against antigens of high-cancer-strain mice (Gorer, 1947) have been found to be strain peculiarities not connected with the milk agent. Attempts to introduce a test based on hemolytical properties of tumor extracts (Gross, 1947b, 1948) also have not been successful. Although the milk agent is not involved in the origin of other tumors (Allen and Gardner, 1941; Bittner, 1940d; Andervont, 1940b; Shimkin et al., 1941) some influence of cross-suckling on leukemia in mice (Barnes and Cole, 1941; Kirschbaum and Strong, 1942; Furth et al., 1942; Kirschbaum, 1944) was demonstrated. A maternal influence on the growth of transplantable tumors has been reported by Cloudman (1941), Law (1942), and Barret and Morgan (1949), but it seems to differ from the milk agent . 3. Physical and Chemical Properties of the Agent
Freezing and desiccation of breast tumor tissue preserves the activity of the milk agent for some time, varioudy reported to be six months (Bittner, 1941a), less than one year (Bittner, 1945b), or two years (Dmochowski, 1946a). Desiccation a t room temperature (Bittner, 1945b) or storage of tumor tissue in 50% glycerin (Bittner, 1942a; Andervont and Bryan, 1944) preserve the activity of the agent for one
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week only. The agent remains active following filtration of tissue extracts through Seitz filters (Bittner, 1942a) and Berkefeld candles (Bittner, 1942a,c; Andervont and Bryan, 1944). Filtrates of breast tumor tissue show stronger activity in higher dilutions than in lower (Bittner, 1945b). The agent was found stable within a wide range of hydrogen ion concentrations of tissue extracts from pH 5.0-10.2, and is not inactivated by petroleum ether or acetone and not appreciably soluble in them (Barnum et al., 1944). Heating of tissue extracts for one hour at 60°C. or of milk for thirty minutes at 66°C. destroys the activity of the agent (Andervont and Bryan, 1944; Barnum el al., 1944). The agent survives in mouse milk kept at 8°C. for fourteen days and in saline extracts of tumor tissue kept at 37°C. for one hour (Bittner, 194513). Treatment of tissue extracts with trypsin in small concentrations for thirty minutes a t 37°C. and at pH 6.0-8.0 does not destroy the activity of the agent, although it may decrease it (Passey et al., 1947, 1950). Extraction of dried tumor tissue with petroleum ether of low boiling point inSoxhlet apparatusfor one hour leaves the activity of the agent unaltered (Passey, Dmochowski, Astbury et al., 1950b). Dialysis by stirring for three hours of Berkefeld filtrates of tumor tissue against distilled water brings no diminution of activity of the agent (Dmochowski and Stickland, 1949). Treatment of mouse milk with chymotrypsin leaves the action of the agent unaltered (Graff et al., 1948, 1949). Claims of liberation of the agent from breast tumor tissue stored a t -79°C. (Gye et al., 1949; Mann, 1949) or from frozen a t -79°C. and then dried tumor tissue (Mann and Dunn, 1949) with the resulting increase of activity of the agent, have been demonstrated to be based on the survival of tumor cells themselves (Passey and Dmochowski, 1950; Passey, Dmochowski, Lasnitzki, and Millard 1950a,b; Dmochowski and Millard, 1950; Bittner and Imagawa, 1950), and the activity of the agent in cellfree filtrates of tumor tissue, whether frozen or fresh, has been shown to be similar (Bittner and Imagawa, 1950). It can be concluded from these experiments that the activity of the milk agent in tumor tissue, stored for any length of time a t -79°C. remains unaltered, but the behavior of the agent in cell-free extracts stored under similar conditions still remains to be determined.
4. Attempts at Isolation of the Agent Ultracentrifugation of extracts of lactating mammary tissue of highcancer-strain mice (Visscher, Green et al., 1942) and of breast tumor tissue and milk (Bryan et al., 1942; Kahler and Bryan, 1943; Kahler and Andervont, 1948) indicated that the milk agent may be a protein of high molecular weight. However, complete sedimentation of the active principle
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was not obtained even after centrifugation at 110,000 times gravity for one hour (Bittner, 194513). Ultraviolet absorption spectrograms showed the presence of ribose nucleic acid in the sedimentable and active fractions of tumors and milk of high-cancer-strain mice (Kahler and Bryan, 1943). Similar ribose nucleic complexes with a molecular weight of 3 to 5 million were also found in normal tissues and milk (De Ome et al., 1942; Kahler and Bryan, 1943,1945), and it was concluded that further purification of the virus-like nucleoproteins is required in order to remove normal constituents from the active fraction. An assessment of purity of a virus preparation is based on physical homogeneity as shown by ultracentrifugation, electrophoresis, and electron microscopy combined with biological activity of the preparation (Beard, 1945). In the case of the milk agent in spite of the long latent period, the biological test of activity is just as important, and preparations of the agent yielding material with high activity are more important for the final conclusions as to the purity of the material than any other preparations. In connection with the encountered difficulties in spinning down the active principle in extracts of various tissues, a series of titration studies of various fractions obtained by ultracentrifugation of agent-containing material have been reported (Barnum et al., 1947, 1948; Barnum and Huseby, 1950; Huseby et al., 1950). Studies of serial dilutions of a fraction, obtained by differential centrifugation (Claude, 1946) of extracts of tumor and mammary gland cells, led t o a conclusion that sediments after centrifugation at 23,000 times gravity for one hour contained most of the activity, while the supernatants were “essentially free” of it, because further centrifugation of the supernatants a t 70,000 times gravity for one hour resulted in sediments giving 14% of tumors and in supernatants inducing 5% of tumors in the test mice, compared with 65% incidence induced by sediments obtained after centrifugation a t 23,000 times gravity (Barnum et al., 1947). In similar titration studies of a microsome fraction of a mammary gland extract, obtained by centrifugation at 23,000 times gravity for ninety minutes it was shown that the end point of activity of that fraction was 5 X loF6g. equivalent of the original extract, while the supernatant was calculated to contain only 0.25 % of the activity of the microsomal fraction (Barnum and Huseby, 1950). On the basis of these titration experiments and also in view of the high activity of extracts of lactating breast tissue in dilutions of up to loe6 (Huseby et al., 1950) it was concluded that the milk agent is essentially sedimented at 23,000 times gravity during a period of ninety minutes. It was claimed therefore that the milk agent is associated with two cytoplasmic fractions of lactating mammary glands or breast tumor tissue, the large granules
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sedimented after five minutes a t 23,000 times gravity and microsomes sedimented after ninety minutes at 23,000 times gravity (Barnum et al., 1948; Huseby et al., 1950). The chemical composition of microsome fractions from both agentcarrying and agent-free mice of genetically identical strains was found to show no difference (Barnum el al., 1947, 1948). No impairment of activity of the agent followed the removal of a t least 95% of pentose nucleic acid from the microsome fraction which appeared to be essentially a lipoprotein complex (Barnum and Huseby, 1950). In spite of the high activity of this fraction, even in doses containing only 0.000223 pg. of nitrogen (Barnum and Huseby, 1950), no difference could be seen in the electron microscope' between the microsome fractions of agent-carrying and agent-free mice (Huseby et al., 1950). It is probable that the microsome fractions prepared by Barnum et al. (1947, 1948)) Barnum and Huseby (1950), and Huseby et al. (1950) were for the most part composed of . normal: tissue constituents, and any characteristic for the agent particles were obscured by normal tissue microsomes as it was pointed out by Huseby et al. (1950). Attempts at isolation of the milk agent from milk of high-cancer-strain (RIII) females treated with chymotrypsin and centrifuged for thirty minutes at 120,000 times gravity (Graff et al., 1948, 1949) showed that a considerable amount of activity still remained in the supernatant. The fraction sedimentable at this speed showed two components also seen electrophoretically, while in the electron microscope i t contained spherical particles of 500-1500 A in diameter and of an average size of 980 b. No such material could be seen in similarly prepared fractions from milk of low-cancer-strain ( C S ~mice, ) but could be observed (with a smaller 720 A diameter) in the milk of mice of the same strain after foster nursing by high-cancer-strain females. These fractions showed tumor activity in dilutions containing 0.008 pgN . It is not certain whether demonstration of activity in much higher dilutions of this material would help in assessment of purity of this material compared with material of Huseby et al. (1950), as suggested by Barnum and Huseby (1950), in view of the different method of preparation of the respective materials. Passey et al. (1947, 1949, 1950) and Passey, Dmochowski, Astbury, Reed, and Johnson (1948, 1950a,b,c, 1951) reported spherical particles about 300 8 average diameter, ranging from 200-1200 b, in extracts of either dried or fresh normal and malignant tissues of high-cancer-strain mice, which had been treated with petroleum ether, distilled water, trypsinized, and filtered through Berkefeld candles. These extracts were biologically active, while similarly prepared extracts of low-cancer-strain tissues contained comparatively few similar size particles and were not
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active biologically. Ultracentrifugation of both trypsinized and nontrypsinized extracts of spontaneous breast tumor tissue for two hours a t 60,000 times gravity or for one hour at 113,000 times gravity showed in the electron microscope the presence of these particles in the supernatants (Passey et al., 1948). These supernatants showed also considerable biological activity (Passey, Dmochowski, Astbury, Reed, and Johnson, 1950a,b, 1951). Following centrifugation a t 120,000 times gravity for two hours, no particles were seen in the supernatants which showed no biological activity (Passey, Dmochowski, Astbury, Reed, and Johnson, 1951). The difference in size between the particles of Graff et al., and those of Passey et al., as well as the difference in the resistance to the action of trypsin, may be the result of differences in preparation of tissue extracts, which may also have caused considerable change in the physical properties of the particles. In this connection, the studies of Hoster et al. (1950) may be of interest. They studied nuclear, mitochondrial, and microsomal fractions from heparinized extracts of normal and Hodgkin’a lymph glands in phosphate buffers and obtained particles ranging from 100-2800 A in fractions from normal and malignant glands, with the predominant size of 100-200 A only present in malignant lymph glands. Chemically, however, these fractions from both normal and malignant tissues showed no significant differences. These studies may give a valuable insight into the structural composition of tissues possessing the agent. It is possible that electron microscope observations of human tumors and milks (Gessler and Grey, 1947; Gessler el al., 1948; Gessler et al., 1948a,b; Gessler et al., 1949; Gessler, McCarty, Parkinson, and Bardet, 1949; Hellwig, 1949; Passey et al., 1950c, 1951; Passey, Dmochowski, Astbury, Reed, and Eaves, 1951) may have a similar bearing on the understanding of the composition of human tumors. Biological activity is an essential test of any preparation of the milk agent tested for purity. Serial dilutions of various preparations of material containing the agent or of fractions obtained by differential, centrifugation have been examined in suitable test mice (Barnum et al., 1947, 1948; Barnum and Huseby, 1950; Huseby et al., 1950). In this way differences in the quantities of the agent present in lactating breast glands and tumor tissue have been found. On the same basis the presence of the agent in mitochondrial and microsomal fractions sedimented at 23,000 times gravity for sixty or ninety minutes has been suggested, and its distribution throughout the slower sedimenting fractions of tissues disputed. Although there is no doubt about the necessity of such tests and about the statistical significance of the difference of activity between the microsomal fraction sedimented at 23,000 times gravity compared
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with that sedimented at 70,000 times gravity, the adequacy of the method of serial dilutions may be disputed for the following reasons. Some of the tested material has shown no activity a t lower dilution but has been active at higher dilution; some tested material has given an approximately equal distribution of tumors throughout the whole series of dilutions, as in the case of lactating breast tissue which was shown to be a very good source of the agent (three tumors in six mice a t dilution and three in seven mice a t lo-* dilution); lower incidence of tumors induced by higher dilutions of the material and higher incidence by the still higher dilutions of the tested material. Should there be an inhibitory factor, it would be expected to fall off in progressive dilutions. Attempts a t purification of the milk agent by chromatography have shown that while the starting material of high-cancer-strain tumor tissue, treated with petroleum ether, extracted with distilled water and filtered through Berkefeld N candle, was active at a dilution of 0.03 pg. of N per dose, one of the fractions was still active at a dilut,ion of 0.004 pg. N per dose (Dmochowski and Stickland, 1950), that is smaller than that reported by Graff et al. (1949). Attempts a t isolation of the milk agent have thus so far not yielded uniform results, and a great deal more study will be required before an agreement is reached about the size and other physical properties of the particulate components with tumor-inducing activity. This line of studies is even more interesting as i t may lead to the beginning of our understanding of the biogenesis of the milk agent and similar agents. The observation of spherical particles of double structure in cells of mammary carcinomas grown in vitro with 1,300 A external and 750 A internal diameter is of great interest in this respect (Porter and Thompson, 1948) as it may lead to our understanding of the appearance of the agent in tumor cells and of the host-agent relationship 6. Behavior of the Agent in vivo
Filtration through Seitz and Berkefeld filters and ultracentrifugation experiments have demonstrated that the agent can be transmitted by cellfree material. Experiments on the transmission of the agent implied propagation of the agent. When introduced into susceptible mice (Andervont, 1945b) the agent induced a high incidence of breast cancer which was maintained by successive generations of these mice. If the agent is not self-reproducing, there was at least no loss of the agent in spite of the very small initial dose given to susceptible mice. It is still a matter of conjecture at the present moment whether the mice produce more of the agent or the agent reproduces itself. Successfulgrowth of mammary tumor8 on the developing chick embryo
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(Taylor et al., 1942, 1943, 1948) led to claims of induction of tumors in mice within a few days after the injection of cell-free filtrates of tumorbearing yolks (Taylor, 1943; Taylor et al., 1945). These claims with filtrates of diluted yolk after the growth of breast tumors in the yolk sac were not substantiated (Heilman and Bittner, 1944; Bryan et al., 1945; Twombly and Meisel, 1946), and the tumor-producing property of yolk was found to be due t o the presence of viable tumor cells, especially as various procedures which decreased the number of cells in the yolk of tumor-bearing eggs led to a decrease in tumor-inducing activity, and tumors grown on the allantois gave yolk free of tumor-inducing activity (Hungate et al., 1945). The recovery of the agent from breast tumors grown in chick embryos after eleven passages (Bittner and Heilman, 1945) is not surprising, but the increasing mortality of the embryos in later passages (Armstrong and Ham, 1947) is of interest. Bittner e l al., (1945) reported the recovery of the agent from filtered and unfiltered yolk after twelve days, following the injection into the yolk sac of 5-dayold chick embryos either of suspensions of tumor cells or of filtrates of tumors, as well as from unfiltered yolk after nine passages. It has been reported that the milk agent, after inoculation of the yolk sac with filtrates of breast tumors, may become more concentrated in the yolk sac and induce an earlier appearance of tumors (Bittner, 1945b). It is difficult to form an opinion about the multiplication of the agent on the basis of these studies and more experiments along these lines are no doubt needed to settle this important question. The increased mortality of chick embryos and various abnormalities and lesions following repeated passages of breast tumors in the yolk of developing chicks, interpreted as the result of the agent’s activity (Taylor and Carmichael, 1949) could only be accepted as produced by the agent, after a failure to observe similar changes following the growth of agent-free breast tumors. It is possible that serial transmission of the amniotic fluid over a series of passages (ten or twelve) and comparison of the activity of the milk agent after the first and the last passage may go some way towards clarifying the question of multiplication of the agent. The milk agent has been reported to appear suddenly in mice of a susceptible strain which was deprived of the agent by foster nursing, and it may be transmitted to their progeny (Bittner, 1941~). By excluding contamination it may be reasoned that the agent has arisen de novo or what is more likely that a weak agent has become activated by as yet unknown factors. Serial transfers of the agent through highly susceptible mice by means of intraperitoneal injections of filtered extracts of lactating breast tissue of mice originally given material containing the agent has resulted in the disappearance of the agent after the third passage (Green
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et al., 194613). This may have been due to a transfer different from natural transmission or a premature transfer of the agent, as smallness of the dose cannot be considered in view of the reported activity in dilutions of one in a million. It is possible that the second alternative may have produced the disappearance of the agent and not the first, because of the reported high incidence of tumors after artificial feeding (Haagensen and Randall, 1945). Although even one nursing is sufficient to induce a certain number of tumors in susceptiblemice (Andervont and McEleney, 1941b; Shimkin and Andervont, 1942), i t may not be enough to maintain an appreciable tumor incidence in the progeny of these mice if after the first nursing by their own mothers they are foster nursed by low-cancer-strain females. Gradual diminution of the agent in these mice was reported by Bittner (1939d, 1940b). This suggests that the amount of the agent required to maintain a high incidence of breast cancer in successive generations of susceptible mice must be higher than that necessary to induce tumors in only one generation of these mice. How this observation affects the problem of self-propagation of the agent is not quite certain. A similar disappearance of the agent in the third generation of high-cancer-strain mice each of which had remained with their mothers for only forty hours was reported by Andervont (1948). Individual variation in susceptibility to the agent may have played a part, since some mice lost the agent in the third and some in the fourth generation. What part in the disappearance of the agent, if any, was played by changes in the agent itself or by its transmission in subinfective quantities, is not known. It is probable that individual variation, in what should be a uniformly susceptible strain, to small amounts of the agent does play a part, in view of the observation (Andervont, 1949b) that some of the progeny of the susceptible mice, which had remained tumor-free after only a short period of nursing by their mothers (three hours), developed tumors and some did not. Susceptible strains may vary in their response to the experimental procedure in which mice of one or two litters of each successive generation of females which had remained with their own mothers for a period of twenty-four to forty hours, were divided into two groups, one which remained with their own mothers for a similarly short period before foster nursing and the other, controls, which remained for the whole period of nursing with their own mothers (Andervont, 194913). Mice of a high-cancer strain ( G H ) showed a decrease of tumor incidence from 85 % (and 75 % controls) in the first generation, t o 16% (and 17% in the controls) in the fifth generation; mice of another highly susceptible strain (C), although showing a lower incidence of cancer in breeding females than the former strain, showed only a small decrease in the
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incidence of tumors in the fourth generation compared with the first. Transmission of the agent took place in some breeding females of this strain for four successive generations without tumor development (Andervont, 1949b). This strain may also be transferred into a high-cancer strain for many generations after one foster nursing by high-cancer-strain females, provided the mice remain with their high-cancer-strain foster mothers for the whole period of nursing (Andervont, 1941b, 1945~). It is therefore possible for a strain to transmit a weak agent without the appearance of tumors and for another to transmit a strong agent with similar outcome because of the genetic resistance or difference in susceptibility or lack of hormonal stimulation. Further, it seems, on the basis of these observations, that in some susceptible mice, although they obtain the agent early in life, the agent may not increase sufficiently during the two or three months before the birth of a litter or may not propagate a t all during this time, or the hormonal stimulation may not be sufficiently long to create a favorable medium for its propagation. In this respect the milk agent does not seem to resemble a virus which increases in concentration in each susceptible host. It would have been instructive to test the niice of strains used by Andervont (194913) in their later life for the presence of the agent, because Bittner (1942b) suggested that the agent may be more concentrated in older females. It is quite possible, although by no means certain, that these observations may put the agent into a separate category of disease-producing agents. It is now known that the amount of the agent is not constant in agent-carrying mice (Hummel and Little, 1949a; Hummel et al., 1949). The disappearance of the agent from a high-cancer-strain female and fifteen generations of her descendants was also observed by Murray and Warner (1947). The inhibitory effect of low-cancer-strain milk must also be borne in mind as a possible cause of the decrease in tumor incidence. In contrast with observations of the slow rate of multiplication of the agent (Andervont, 1949b), rapid multiplication of the agent during the growth of mammary cancer in successive transplants was reported by Bittner (1948~). Similar observations were reported by Huseby et al. (1950), who found similar concentrations of the agent in the milk and in the cells of the mammary glands, which, in view of the high daily rate of milk production amounting to three times the weight of the glands, indicated a rapid multiplication of the agent. It is difficult a t the moment to assess these seemingly contradictory findings. It is possible that strain variations and dosage of the agent as well as the age of the mice may a t least in part be responsible for this discrepancy between the two sets of Observations. A further point of interest is the long latent period of
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tumor development in spite of the reported rapid multiplication. It may well be that hormonal factors may have to act for a certain length of time before the action of the agent becomes evident in the rise of a tumor. Attempts to shorten the latent period have so far met with no success (Bennison, 1949; Borgess, 1949). Further experiments along these lines may well be helpful in revealing at least in part the mode of action of the milk agent. 6. Immunological Studies
Numerous attempts have been carried out to investigate the antigenic properties of the agent in order to develop a test which would serve as a rapid answer whether the agent is present in the material examined. The biological test, still the only practical, requires up to twenty-four months for completion and even longer (Andervont, 1950b) if designed t o ascertain small quantities of the agent, as then it requires the observation of a t least one generation of descendants of the test mice, which may become infected without showing tumors and transmit the agent to their descendants which develop tumors. It must be stressed that throughout the literature dealing with immunological studies of the agent, the term agent has been used too freely when describing the antigenic properties of the agent, because the term should have meant not the agent itself but material containing the agent. Not until a purification procedure is established and what constitutes a purified agent is agreed, it should be remembered that it is not and it has not been up till now the agent, but material containing the agent which had been investigated. Andervont and Bryan (1944) first described neutralization of the agent in breast tumor extracts by rabbit immune sera induced with crude or partially purified tumor extracts. These Bera also had an inhibitory effect on the development of breast tumors in susceptible mice given material containing the agent after they had been injected with the immune sera. Similar sera failed to prevent the development of breast tumors in mice which had obtained the agent during nursing (Bittner, 1948b). It is difficult to interpret the specificity of this neutraliaation effect as controls with rabbit immune sera against similarly prepared extracts of tumors and normal tissues without the agent were not used. Green et al. (1945, 1946a) and Green and Bittner (1946) also reported that rabbit and rat sera elicited with material prepared by centrifugation of tumor extracts neutralized the agent in material prepared in the same way, Antisera against material from normal tissues had no neutralizing effect, while normal rat and rabbit sera exerted a delaying and to a certain extent inhibiting effect on the development of tumors in suscepti-
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145
ble mice for, as yet, unexplained reasons. They concluded that the milk agent is antigenic and different from normal tissue constituents and also extrinsic in origin. An opinion whether these antibodies were specific to the agent itself or to the tumor tissue cannot be given, as it was not made clear by Green et al. (1946a) whether the normal mouse tissues were obtained from the same mice which had served as the source of breast tumor tissue but were deprived of the agent, or from mice of another genetically different agent-free strain. Material, obtained from breast tumor tissue without t,he agent which originated in the same strain but free of the agent, would have gone even farther toward assessment of the specificity of the neutralizing antibodies. Green (1946) reported the inhibition of the growth of tumor cells by rabbit immune sera against material prepared by ultracentrifugation of mammary tumor extracts, while similarly prepared antisera against normal lactating tissue without the agent had a growthdelaying effect, and even normal rabbit sera had a slight growth-inhibitory effect. Again it is not clear whether the observed neutralizing effect was produced by antibodies to malignant tissue constituents or to the milk agent since the normal tissue was obtained from mice genetically not identical with those from which the tumor material was derived. In contrast with these observations, Law (1949) reported growthinhibitory effect of rabbit immune sera against normal lactating tissue of genetically identical mice, both with and without the agent. Antisera against spontaneous breast tumor tissue had no effect. Gorer and Law (1949) failed to demonstrate neutralizing antibodies against the milk agent in normal sera of high- and low-cancer-strain mice or in sera of low-cancer-strain mice, hyperimmunized against material containing the agent. No complement fixing antibody to highly purified preparations containing the agent material, obtained by ultracentrifugation, could be detected in sera of high- and low-cancer-strain mice, whether normal, bearing tumors with the agent, or immunized with material containing the agent (Dmochowski, Hoyle, and Passey, 1952). It seems probable that mice do not react to the milk agent as a foreign agent, even if the mice are of strains which do not carry the agent; even immunization of mice does not elicit antibodies against the agent. Growth-inhibitory effect on tumor cells by guinea pig immune sera elicited with breast tissue containing the agent and only a slight similar effect by guinea pig sera induced by mammary tissue without the agent were reported by Imagawa et al. (1950). Again it is not clear whether the effect was due to the agent, as tissue from the same strain but agent-free mice was not used as control. They also reported that sera of agentcarrying mice or mice bearing transplanted tumors had no influence on
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growth of breast cancer cells. Guinea pig immune sera against tumor tissue of one high-cancer strain were found to inhibit the growth of tumors of another high-cancer strain (Imagawa et al., 1951). It was concluded that this effect is independent of the strain of mice and inseparably related, but not necessarily dependent on the presence of the agent. It seems that this cannot be finally accepted until control immune sera against tumors in the same strains but without the agent are tested for any similar effects. High titer precipitation tests between rabbit immune sera against ultracentrifugally prepared material from breast tumor and normal mammary tissues with the agent and normal and malignant tissues containing the agent and low titer precipitation tests with normal tissues free of the agent were reported by Imagawa et al. (1948). Antisera against normal tissue with the agent gave a high titer, and antisera against similar tissue without the agent gave a low titer precipitation test with mammary tissue containing the agent. Absorption of anti-breast cancer sera with normal breast tissue lacking the agent failed to remove the antibody which gave strong precipitation with the antigen in normal and malignant tissues of agent-carrying mice. The presence of a common antigen in normal and malignant tissues of mice carrying the agent and its absence from normal tissues of agent-free mice was put forward as explanation of the precipitation tests, but no controls of tumor tissue of agent-free mice were used. Quantitative differences in complement fixation tests with rabbit immune sera against ultracentrifugally prepared material from extracts of spontaneous breast tumor tissue and extracts of spleen from agent-carrying and agent-free mice, the latter giving a lower titer, were reported by Bennison (1947, 1948). Malmgren and Bennison (1950) found no differences in complement fixation tests between mitochondria from normal and malignant tissues and antisera against material from low- and high-cancer strains. Mitochondria from livers and spleens of mice with and without the agent showed no significant differences in complement fixation tests with rabbit antisera against mitochondria from mammary tumors. Similarly, mitochondria from spleens of normal and leukemic mice showed no qualitative differences (Dulaney et al., 1949). In all these experiments antibodies against material containing the agent were induced in animals of various species and studied by means of various tests. The specificity of these antibodies was not entirely analyzed. The results of some investigations showed lack of qualitative differences between antibodiesinduced by tissues of agent-carrying and those of agent-free mice. Dmochowski, Hoyle, and Passey (1952) observed no quantitative differences in complement fixation tests between rabbit
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147
immune sera induced by ultracentrifugal biologically active deposits of differentially spun breast tumor extracts and extracts of malignant tissues of genetically identical mice both with and without the agent, or between these sera and extracts of normal tissues of genetically identical agent-carrying and agent-free mice. It was concluded that the milk agent is serologically similar to material present in normal tissues of mice. It is not known whether more purified material would show any differences, but it is at least doubtful that it would. Similar results on Rous sarcoma were reported by means of complement fixation tests (Dmochowski, 1948e), precipitation tests (Kabat and Furth, 1940), and neutralization tests (Gye and Purdy, 1933). I n the case of fowl leukosis agent (Kabat and Furth, 1941), antisera to normal host proteins failed to neutralize the agent but gave strong precipitation tests with the agent. Again, in the case of influenza virus, antisera to normal host material failed to neutralize the virus, but gave strong complement fixation tests with the best purified influenza virus preparations (Knight, 1946). It is known that physical homogeneity is not an adequate basis for serological or chemical differences between the virus particles and normal host material. Characteristic particles with specific biological activity and specific physical properties may still contain proteins of the host. It is possible that similar reasoning may be applied to the various milk agent preparations. 7. Conclusions The milk agent is widely distributed in organs of high-cancer-strain males and is not present in the excreta of high-cancer-strain mice. Its absence in embryos of high-cancer strains has finally settled the problematical transmission of the agent through the uterus. The discovery of the agent in the stomach milk of young high-cancer-strain mice drew attention to its possible resistance to digestive enzymes which was confirmed by the results of treatment with trypsin and chymotrypsin of extracts of normal and malignant tissues and of milk of high-cancer-strain mice. These treatments were used in attempts a t isolating the agent from tumor tissue and milk. Ultracentrifugation procedures combined with electron microscopy have not given clear-cut data as to the physical properties of the agent, but have opened an interesting field which promises to be most fruitful in the investigations of the host-agent relationship and perhaps also in the biogenesis of the milk agent. The study of the multiplication of the agent has thrown an interesting light on the behavior of the agent in vivo which in some respects seems to differ from that of other known viruses. The similarity of the serological behavior of various preparations of the agent and those of other tumor-inducing viruses has been
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demonstrated by the observed lack of qualitative serological differences between normal and malignant tissues of mice without the agent and normal as well as malignant tissues of genetically identical but agentcarrying mice.
VII. MAMMARY TUMORS IN HYBRIDMICE AND
THE
MILK AGENT
1. Introduction
Hybrid mice derived from mating of low-breast-cancer-strain females to high-cancer-strain males have for many years been employed as test mice for the pressure of the agent (Bittner, 1939f, 1940a, 1941b; Murray, 1941b; Andervont et al., 1942). In these hybrids a low incidence of breast cancer was frequently reported (Bagg, 1936a,b; Bagg and Jackson, 1937; Murray and Little, 1935a, 1939; Bittner, 193913; Gardner, 1941a; Strong, 1943; Dmochowski, 1944a, 194th; Andervont, 1940a, 1945d; Foulds, 1947). Several interpretations of the appearance of breast tumors in these hybrids were put forward, such as the presence of the agent in the low-breast-cancer strain whose mammary glands are resistant to the agent or to hormonal stimulation, or the development of a different type of breast tumor in which the milk agent does not take part. Extensive experiments were therefore carried out in recent years in an attempt a t getting some insight into the origin of breast tumors in these mice. 8. Recent Investigations
The importance of the genetic constitution in the origin of breast tumors in hybrid mice was demonstrated by the different tumor incidences observed in hybrids of various derivations, obtained by crossings of various low-cancer strains differing in their susceptibility to breast tumor development in the presence of the agent and also by matings of these strains to high-cancer strains. Mating of low-cancer-strain susceptible females (strain C) to high-cancer-strain males resulted in hybrids with a tumor incidence of up to 60% at a late age of 20 or more months (Andervont, 1945d; Andervont and Dunn, 1948c, 1950b). Hybrids, obtained by crossing low-cancer-strain females with low susceptibility to tumors in the presence of the agent (c67 Black) with (RIII) high-cancer-strain males, gave hybrids with a much lower incidence of 13% to 15% at an average age of 10 to 14 months (Foulds, 1947, 1949; Dmochowski, 1950b, 1951a). A low incidence of tumors was also observed in hybrids from cross-matings of (C67, I) low-cancer strains (Andervont and Dunn, 1948a). The presence or absence of the milk agent in the male parent was also observed to influence the incidence of tumors in hybrid mice, as demonstrated by the consistently lower tumor incidence in hybrids born to
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males of the same but agent-free strain (Andervont and Dunn, 1948c; Andervont, 1950a; Foulds, 1947, 1949). It is not certain, however, whether the genetic constitution of agent-free males was not sufficiently different from that of agent-carrying males to influence the difference in the tumor incidence (Andervont and Dunn, 1948c; Foulds, 1949). Hybrids from the susceptible strain C females and agent-free high-cancerstrain males had a higher incidence of tumors than those from the same females and low-cancer-strain males, thus again revealing the influence of the genetic constitution (Andervont and Dunn, 1948a). The hormonal stimulation in all the various hybrids was comparable, and Andervont (1950a) stated that there exists a parallelism between a genetic tendency to tumor development and susceptibility to the milk agent, The influence of hormonal stimulation on the development of breast tumors in hybrid mice was demonstrated by the higher tumor incidence in hybrids from susceptible (C) low-cancer-strain females and agent-carrying or even agent-free (CIH) males, as hybrids bearing seven to eightlitters showed more tumors than those with three to five litters (Andervont and Dunn, 1948c; 1950b; Andervont, 1950a). The influence of hormonal stimulation was more pronounced in hybrids from agent-carrying than in those from agent-free males (Andervont and Dunn, 1948~). Similarly, forced breeding of (CS,X RIII)Fl hybrids induced an incidence of 15% of tumors in these mice, compared with only 3% in normally bred littermate controls (Dmochowski, 1950b, 1951a). A search for the presence of the milk agent in the hybrids of various derivations revealed the agent in tumors of hybrids from C strain females and agent-carrying C3H males, which developed a t an early age, while all tumors arising a t a late age failed to reveal the agent; hybrids from the same strain females and agent-free males developed tumors a t a late age and none was found to harbor the agent (Andervont and Dunn, 1948c, 1950b; Andervont, 1950a). The agent was also found in tumors of (CW X R1II)Fl hybrids (Foulds, 1947, 1949) and in tumors of similar derivation hybrids but at a later age (Dmochowski, 1951a) than that at which the agent was found in tumors of hybrids of other derivations (Andervont and Dunn, 1948~). A number of ways in which the milk agent has appeared in the hybrids have suggested themselves and have been subjected to a close scrutiny. In view of the possibility of the low-cancer-strain mothers carrying either small quantities of the agent or a weak agent (Andervont, 1945d), the C strain females were tested, but none of the employed tests revealed the presence of the agent in their milk or mammary glands (Andervont and Dunn, 1948~). Treatment with stilbestrol also failed to reveal the presence of the agent in these females (Andervont, 1950a). Any conta-
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gion of these as well as other females and their hybrids could be discounted in view of the previous observations (Andervont et al., 1942, 194513) and of the absence of the agent in the excreta of mice (Dmochowski and Passey, 1951; Muhlbock, 1951). Evidence against the possibility of an increase of the agent in older C strain females was obtained by the observation of similar tumor incidences in hybrid mice and their progeny from both older and younger C strain mice (Andervont and Dunn, 1948~). Attempts to enhance the activity of the agent in C strain females with x-radiation produced no conclusive results (Andervont and Dunn, 1950b), as tumors appeared in irradiated and nonirradiated C strain females, and the average tumor incidence and age were similar in the hybrid progeny of both types of females. The agent was found in the majority of females which had developed tumors after irradiation, although there was a discrepancy between the presence of the agent and the age of tumor appearance, as some females, in spite of the presence of the agent, developed tumors a t a late age. Further, there was no correlation between the presence of the agent in C strain females and its presence in their hybrid progeny with CaH high-cancer-strain agent-carrying males, females with the agent giving rise to hybrid progeny with only late tumors free of the agent or to progeny with both early tumors harboring the agent and late tumors without the agent. It would have been of interest to test the nonirradiated tumorous C strain females as well as to examine for the agent the irradiated females before mating t o high-cancer-strain males or after mating to similar strain agent-free males. It might have helped to separate the influence of radiation from that of the agent-carrying males. Previously Andervont and Dunn (1948a) reported that C strain females do not carry the agent and suggested the tumors in the hybrid progeny of these females may be the result of the transmission of the agent by high-cancer-strain males, although none of the original females, from which the hybrids had been derived, had developed breast cancer. Transmission of the agent by high-cancer-strain males may be a possible way of the access of the agent into the hybrid mice, because of the presence of the agent in various organs of these males (Dmochowski, 1949c,d; Andervont and Dunn, 1948a; Muhlbock, 1950a). It is known that “scrapie” virus is transmitted by the sperm of the ram t o the progeny, without the ewes being affected (Greig, 1940). Similarly cocks may pass the virus of fowl paralysis (Blakemore, 1934). The possibility of the embryos passing the agent to some of the mothers may be questioned because of the reported neutralization of the agent by the placenta (Hummel et al., 1949) although it is by no means excluded. The transmission of the agent by high-cancer-strain males to the embryos may also be questioned, in view of the failure to find the agent in high-cancer-
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strain embryos (Dmochowski, 1949a; Hummel and Little, 1949a). The low incidence of tumors in the progeny of C strain females which had previously been mated to C3H agent-carrying males and then mated to C strain males, even after treatment with stilbestrol, points against the possibility of the agent passing from C3H males to C strain females. However, the development of tumors in strain C females after mating with high-cancer-strain males, with the agent present both in these tumors as well as in the tumors of their progeny, was reported by Bittner (1950a). The tumor incidence in the hybrid progeny was low in first litters and in later litters about 90% (Bittner, 1950a). There is a t the moment contradictory evidence about the transmission of the agent by high-cancer-strain males to low-cancer-strain females after mating. However, in view of the high incidence of tumors in several generations of the progeny of one C strain female mated for the second time to a highcancer-strain male (Andervont and Dunn, 1948c), it would be of interest to compare the incidence of tumors in hybrids following repeated matings of these and other strain females to high-cancer-strain males. The increasing incidence of tumors in the hybrid progeny observed by Bittner (1950a) may indicate eventual passing of the agent to females repeatedly mated with agent-carrying males. Attempts to enhance the activity of the agent by backcrossing the hybrids from C strain females and C3H agent-carrying males with their fathers proved unsuccessful, the backcross-hybrid progeny showing a distinctly lower tumor incidence than their hybrid mothers, the tumors developing a t a similar late age and lacking the agent (Andervont and Dunn, 1948c), in spite of a favorable genetic constitution and adequate hormonal stimulation. Brother to sister matings of the progeny of (C X C3H)FI hybrid females, in spite of increased hormonal stimulation (forced breeding), also resulted in a low, gradually decreasing tumor incidence in successive generations of these hybrids (Andervont and Dunn, 1948~). In one case, however, it probably led to the' appearance of the agent as one litter of the hybrids showed early tumors and their progeny showed a high incidence of cancer at an early age in all successive generations. As a rule, the tumors in the (C X C3H)F1 hybrids were not limited to certain low-cancer-strain mothers, and the tumors in the backcross-progeny appeared independently of the presence or absence of tumors in their hybrid mothers (Andervont and Dunn, 1948~). Similarly, the presence of the agent in C strain females was not correlated with its presence in their hybrid progeny (Andervont and Dunn, 1950b). The tumor-bearing hybrid mice may however be concentrated in particular families (Foulds, 1949), and their progeny may show a high tumor incidence in successive generations.
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L. DMOCHOWSKI
The response to intensive hormonal stimulation may also vary in the progeny of hybrids of different derivations. Contrary to Andervont and Dunn’s experience ( 1 9 4 8 ~ forced )~ breeding of the progeny of tumorous as well as nontumorous original (C67 X RIII)F1 hybrids was shown by Dmochowshi (1950b, 1951a) to induce a high incidence of tumors in the progeny of both tumorousand nontumorous hybrid mothers, the only difference being in the lower tumor age in the progeny of the original tumorous hybrids. The difference in tumor age decreased gradually in each successive generation and became approximately equal in the sixth generation of the progeny. The incidence of tumors in litters of several hybrid females before they developed tumors was similar to that in the litters born after the hybrids showed tumors, which indicates that the hybrid mothers obtained the agent early in life or i t appeared early in their life. Tumors in the progeny of both tumorous and nontumorous hybrids were found to possess the agent, which was also presumed from the high incidence and the early age at which the tumors developed. The appearance of tumors in some of the original (c67 X RIII)F1 hybrid mice only after forced breeding could be explained by the transmission of a weak agent by Cb7Black strain mothers, as none of them developed cancer although they lived for over twenty months, and its activation in their hybrid progeny by increased hormonal stimulation of a susceptible genetic constitution or by the transmission of the milk agent by the highcancer-strain males to their hybrid progeny. Individual variation in the genetic constitution of the original hybrid females may have been responsible for some of them not showing tumors, although they transmitted it to their progeny, which developed tumors containing the agent. While the first generation hybrid progeny had to bear at least six litters before any female developed cascer, in the sixth generation more than half of the number of tumors appeared in females which had fewer than five litters. This may have been in part due t o the forced breeding and also in part to the influence of the genetic constitution following brother to sister matings. Sudden appearance of the agent de novo (Bittner, 1941c, 1943) was also considered as a possible cause of the appearance of the agent in some of the younger hybrids (Andervont and Dunn, 194%), although Burnet (1945) maintained that evidence of any virus appearing in this way is lacking. The more likely possibility of some of the embryos acquiring the agent from their agent-carrying fathers would serve to explain the random distribution of the agent in hybrids of some derivations. Infection with the virus of lymphocytic choriomeningitis does occur in utero and mice may carry it as a latent infection (Traub, 1938, 1939). According to Bittner (1950a) hybrid mice, born from two different strains, neither of
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which possessed the agent, may develop tumors which have the agent. The appearance of the agent in these tumors leads to an interesting speculation on the influence of the genetic constitution as an outcome of crossing of two different strains, possibly combined with hormonal factors. The decision whether tumors which appear in old hybrids as the result of the combined action of their genetic constitution and hormonal stimulation without participation of the agent (Andervont, 1950a) are in fact independent of the milk agent may well have to be suspended. Andervont and Dunn (1948~)pointed out possible adverse effect of extraction procedures on the agent or the influence of an inhibitor present in these tumors. Heston et al. (1950) observed an incidence of tumors of 38% at twenty months in their breeding agent-free line of C3H mice and only 5 % incidence in nonbreeding mice, and none of the tumors tested revealed the agent. They rightly stressed that absolute proof of the absence of the agent has not been provided, but should it be present, it would be of decreased potency, as agent-carrying C3H mice show 98% incidence of tumors. Thus, small quantities of the agent may have to be reckoned with, whether in these tumors or in those appearing in older hybrids. It is not known whether the technique of repeated injections of a tested tumor (Dmochowski, 1945a) would be helpful, but it seems worth trying, although it is possible that even this test may fail to reveal small quantities of the agent. In view of the discovery of the agent in some C strain females (Andervont and Dunn, 1950a; Bittner, 1950a) in experiments on irradiation and/or simple mating to agent-carrying males, it could still be argued that either of these procedures or their combined action have led to activation of a latent agent in these females or successful transmission of the agent from the male which has resulted in tumor development in some of the C strain females. The amount of the agent activated or transmitted may not have been sufficient to induce a high incidence of tumors in all their hybrid progeny, possibly also as a result of individual variation in susceptibility among the hybrid progeny. Similar variations in susceptibility among the C strain females may have resulted in some females showing the agent’s presence and transmitting it to their progeny, and others not showing tumors but still transmitting the agent to their hybrid progeny, of which again some developed tumors early and revealed the agent and others developed tumors late and showed no presence of the agent, because of individual variations in genetic susceptibility. Thus a more favorable genetic make-up may account for the increased amount of the agent and the ease of its detection. There is also the possibility of the agent being present in a constant amount and its detection and behavior being entirely dependent on the genetic background and hormonal stimulation encountered in various individuals.
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L. DMOCHOWSKI
I n conclusion it may be stated that the experimental evidence (Andervont and Dunn, 1950b; Bittner, 1950a; Foulds, 1949) indicates that hybrid mice may acquire the agent from high-cancer-strain male parents;possibly through the milk of their mothers infected by the highcancer-strain males. The failure to detect the agent in tumors of old hybrids may be interpreted by supporters of virus theory that the agent has changed into a masked form as the age of the mice progressed, in opposition to the possibility of these tumors arising entirely by hormonal stimulation of a suitable genetic background (Andervont, 1950a). There is no doubt that the problem of the origin of tumors in hybrid mice is very complicated, but it seems to unfold gradually the quantitative interaction of the three factors and the milk agent-host relationship, and lead to our better understanding of the nature of the agent. 3. Histology of Mammary Tumors in Hybrid Mice
The high degree of morphological variation encountered in mammary tumors of mice was interpreted as a series of phases of the same pathological process (Dunn, 1945). In connection with the studies of the origin of breast tumors in hybrid mice of various derivations, new attempts a t classification of these tumors and correlation of their appearance with the presence or absence of the milk agent and the age a t which they appear were reported (Andervont and Dunn, 1948a,c, 1950b). The tumors of hybrids were a t first classified in two so-called I and I1 types (Andervont and Dunn, 1948a) and later into four A, B, C, and D types (Andervont and Dunn, 1950b). According to this classification, type A was of predominant acinar structure; type B, with little or no acinar structure but solid groups of epithelial cells or cystic spaces lined with cuboidal cells or papillary projections into cysts, or cords of epithelial cells separated by an abundant stroma, types A and B corresponding with type I; type C (or type 11) with small cysts lined with cuboidal epithelium and spindle cells; type D with large areas of stratified squamous epithelium (adeno-acanthoma). No difference in the appearance of mammary tumors in hybrids and that of tumors in high-cancer-strain mice was reported by Foulds (1947), and the predominant structure of the majority of tumors in hybrid mice of similar derivation was also found to be similar to that of agent-carrying tumors by Dmochowski (1951a). This may have been the result of the presence of the agent, ascertained in tumors of both sets of hybrids. A correlation between the type of tumor and the age of hybrid mice was reported by Andervont and Dunn (1950b), in younger mice type A appearing more frequently (63%) than type B (33%), and in older mice type B being encountered more frequently (48%) than type A (33%)
MILK AGENT AND MAMMARY TUMORS
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The predominance of type A in younger mice with the agent agrees with the observation of the influence of the agent on the development of acinar hyperplasia (Bonser, 1945, 1949). There was no correlation however between the types of tumors in C strain females and those in their hybrid progeny, or between the presence of the agent and any particular type of tumor in the hybrid mice or in their agent-carrying C strain mothers, except for the squamous type D which was very rare in the younger agent-carrying hybrid mice and more frequently encountered in tumors of older hybrids, which like all the other types in older hybrids failed to reveal the agent (Andervont and Dunn, 1950b). The same type of tumor may arise, therefore, in mice with and without the agent, because of the similarity in structure of many tumors developing in breeding C strain mice (Andervont, 1945d), in hybrids from mice presumably free of the agent (Andervont and Dunn, 1948a), and in older hybrid mice from C strain females and agent-carrying males (Andervont and Dunn, 1948c), in spite of the difference in frequency of the particular types encountered in these mice (Andervont and Dunn, 1950b). The type of tumor may also depend on the genetic constitution of the mice and the type or amount of hormonal stimulation (Andervont and Dunn, 1948~). According to Kirschbaum (1949) the greater frequency of squamous metaplasia in tumors of old hybrids may indicate their development independent of the agent. The greater frequency of squamous type (D) was also reported in agent-free high-cancer-strain mice (Gardner, 1947; Heston, 1948a; Heston et al., 1950) than in similar mice with the agent (Heston et al., 1950), while in ten spontaneous tumors of a subline of Cs7Black presumably agent-free mice which appeared a t an average age of 480 days, the predominant structure was similar to type B of Andervont and Dunn, with one tumor showing squamous metaplasia (Dmochowski, 1950a,b). There appears to be a greater variety of structure in tumors of mice of some strains in which the agent was not revealed (Andervont and Dunn, 1950a,b; Dunn, 1950; Heston el al., 1950). It has been rightly stressed that a great deal more study of tumors in various strains is required before definite conclusions are reached as to possible strain differences in the structure of tumors (Andervont and Dunn, 1950b).
4. Conclusions The study of the development of mammary tumors in hybrid mice has shown that there is still a great deal to learn about the propagation and transmission of the milk agent. It may also help in promoting our understanding of the origin of human breast tumors, especially in view of the similarity of structure between many of them and the breast tumors in
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L. DMOCHOWSKI
man. Repeated attempts, by means of various tests, to discover the agent in tumors arising in hybrid mice have shown that those developing late in life do not seem t o contain it, while those appearing in younger hybrid mice invariably possess the agent. The possibility of mammary tumors of older hybrid mice harboring an attenuated agent or only small quantities of it should be borne in mind. The influence of increased hormonal stimulation combined with a suitable genetic background on the origin, acceleration, and increased appearance of tumors in hybrid mice has been demonstrated. The combined influence of these two factors, as well as other factors such as radiation, may activate a latent or a weak agent in low-cancer-strain mice, which still remains open to experimental evidence, or it may expose and facilitate its transmission by high-cancerstrain males, which has already been demonstrated. There is a wider morphological variation in mammary tumors in which the milk agent could not be detected, although strict correlation between the appearance of tumors and the presence or absence of the agent has not been established.
VIII. THENATUREOF THE MILE AQENT Numerous attempts, based either on recent advances in virology or on theoretical considerations from recent advances in genetics, biochemistry, and general biology, were made in recent years to explain the nature of the agent. The milk agent, because of its similarity in behavior and properties, has been considered a true virus (Rous, 1946, 1947; Green, 1947; Gross, 1947a, 1949;Andervont, 1949c; Duran-Reynals, 1950; Andrewes, 1950a,b; Smith, 1951). In its latent form it may become activated by various factors such as hormonal (Andrewes, 1950a) or by genetic selection toward the development of breast cancer in mice (Andervont, 1949~). According to Andrewes (1950b) it is an independent parasite like other viruses and not a self-duplicating cellular constituent which on becoming free would affect other cells. Recent discovery of the transmission of the virus of encephalomyelitis in the milk of mice (Magnus and Magnus, 1949), the long latent period in the infection with “scrapie” virus (Greig, 1940), and its similarity of transmission to that of the milk agent in tumors of hybrid mice have certainly strengthened the comparison of the agent with other viruses. The milk agent has also been described as a cytoplasmic constituent which may have arisen by a transformation of a normal cell constituent. This comparison has been based on attempts to identify viruses with particulate cytoplasmic components, related to genes and taking part in cellular differentiation, such as plastogenes in plants, plasmagenes in
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animals, or to enzymes which also have a particulate substrate (Haddow, 1944; Darlington, 1944, 1948; Dixon, 1945; Potter, 1943, 1945), that may appear suddenly through the action of the nucleus, various carcinogens, or the competition between authosynthetic proteins or enzymes. These attempts were based on the observations that certain particulate components of the cytoplasm are ribonucleoproteins and can also be identified with enzymes (Claude, 1940, 1944). They were further based on the studies of chemical reactions these components promote (Potter, 1941), on experiments with paramecia (Sonneborn, 1943a,b), and with plants (Du Buy and Woods, 1943; Woods and Du Buy, 1943, 1945). These experiments and also studies on yeasts (Spiegelman and Kamen, 1946; Spiegelman et al., 1945; Spiegelman, 1947) provided an explanation for the non-Mendelian type of inheritance of the cytoplasmic constituents. The milk agent has therefore been compared with viruses arising by mutation of cytoplasmic determinants, plasmagenes, and the cytoplasmic factor “kappa” of paramecia (Haddob, 1944; Darlington, 1948; Heston et al., 1945; Heston, 194813; Andervont, 1949~). According t o Heston (1946, 194813) studies on the gene-agent relationship point out the necessity of looking a t the milk agent not as an entity in itself, but as an entity among the physiologically coordinated nuclear and cytoplasmic units which comprise the cell. Hence the value of the comparison of the agent with cytoplasmic factors. The killer or nonkiller character of paramecium is determined by the presence of the “kappa” factor and a pair of allelic genes K and k, the first being dominant for the killer character. Recent work on this cytoplasmic factor (Sonneborn, 1945a,b; Sonneborn et al., 1948; Preer, 1948) has shown that suitable characters may be determined by the interaction of nuclear genes and cytoplasmic substances. It is now known that the nucleus plays a part in the propagations of the milk agent (Heston et al., 1945), and therefore the genetic control of the “kappa” factor has been compared with the genetic control of the milk agent, which would like the “kappa” factor be transmitted by cytoplasmic heredity and which would be responsible for differentiation from normal into malignant cell (Andervont, 1949~). There is a similarity between the dependence of the propagation and transmission of the “kappa” factor on the genotype of paramecium and the influence of genes on variations of the milk agent in different strains of mice, demonstrable by the different tumor incidences induced in susceptible test mice, and the influence of genes on the variations in the cytoplasm of the mammary cell, demonstrable by the effectiveness with which the milk agent is transmitted. High-cancer-strain mice, which have a suitable genotype show a high incidence of tumors and transmit the milk
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agent, but repeated backcrossing to low-cancer-strain mice, which removes the genotype, removes the agent and no tumors result. Low-cancerstrain mice lack a suitable genotype and therefore the agent, but repeated backcrossing to high-cancer-strain males in itself does not produce the agent. The change of a low-cancer but susceptible strain into a permanent high-cancer strain by the introduction of the milk agent may be compared with the change of a nonkiller strain of paramecium into a killer strain after the introduction of the “kappa” factor into a paramecium, with either two dominant or one dominant and one recessive genes, originally without the factor. Therefore, the K or dominant gene is essential for the synthesis of the “kappa” factor, but the gene without the factor is unable to synthesize more of the factor. Further, the introduction of the factor into paramecium with two recessive genes will change the strain into a killer strain for a few generations only. This is similar to the introduction of the milk agent into low-cancer-strain mice without a suitable genotype with the resulting tumor development in one or only few generations. Thus the milk agent with many properties common with viruses may also be considered a cytoplasmic agent dependent on the action of genes, the tumor development being controlled by the variations in the genotype of both mice, that is, of the mouse which receives and of the mouse which supplies the milk agent. It is obvious that a great deal more work will have to be done before all these considerations can be accepted. Nevertheless a start has been made which promises a t least a progress in our understanding of the nature of the milk agent in mice. Milk Agent in Relation to Breast Cancer in Man. Studies on the origin of breast cancer in mice have at least indicated how complicated must be the problem of human breast cancer. If in the chain of events leading t o the development of breast cancer in man, one or other of the links could be ascertained, it might then perhaps be possible to look for preventive measures. I n some quarters prevention of nursing has already been recommended (Gross, 1944, 1949; Hammett, 1946), in others because of lack of evidence it has not been accepted (Bittner, 1941b; Horne, 1950; Macklin, 1946). There is no doubt that a search for an agent similar t o the milk agent should be instituted, and it already has taken place (Gross et al., 1950; Passey et at., 1950c, 1951), but the results so far obtained are far from conclusive. The discovery of an agent, either a virus or cytoplasmic factor in character, may well take place as a result of the study of such factors as the genetic and hormonal influences. It is now known from the studies on mouse breast cancer (Heston, 1944, 1946, 1948a,b) that cancer a8 a multiple factor character
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may fluctuate about a physiological level; the nearer these factors are to the point above which cancer appears, the more likely may the individual collect nongenetic factors which will give rise to an early appearance of cancer. This alone, to quote Heston (1944), illustrates that it is impossible to predict cancer from ancestry data, whether cancer will appear in an individual which is part of a heterogenous population. Should there be an agent, it would probably be even more closely related to the genetic constitution (Heston et al., 1945), and i t would show extreme variability in transmission because of heterogeneity of the human population. From this alone can be seen the importance of studies on the milk agent and other factors operative in the origin of mammary cancer in mice, and it seems hardly required to stress the necessity of their continuation and extension. REFERENCES Allen, E. 1942. Endocrinology SO, 942-952. Allen, E., and Gardner, W. U. 1941. Cancer Research 1,359-366. Andervont, H. B. 1940a. J. Natl. Cancer Znst. 1, 135-145. Andervont, H. B. 1940b. J. Natl. Cancer Znst. 1, 147-153. Andervont, H. B. 1941a. J. Natl. Cancer Znst. 1, 737-744. Andervont, H. B. 1941b. J. Natl. Cancer Znst. 2, 307-308. Andervont, H. B. 1943. J. Natl. Cancer Znst. 3, 359-365. Andervont, H. B. 1944. J. Natl. Cancer Znst. 4, 579-581. Andervont, H. B. 1945a. A.A.A.S. Research Conference on Cancer. American Association for the Advancement of Science, Washington, D.C. p. 101. Andervont, H. B. 1945b. A Symposium on Mammary Tumors in Mice. American Association for the Advancement of Science, Washington, D.C. pp. 123-139. Andervont, H. B. 1945c. J. Natl. Cancer Inst. 6 , 383-390. Andervont, H. B. 1945d. J. Natl. Cancer Inst. 6, 391-395. Andervont, H. B. 1945e. J. Natl. Cancer Znst. 6, 397402. Andervont, H. B. 1946. Yale J. Biol. and Med. 18,333-344. Andervont, H. B. 1948. Acla union intern. contre cancer 8, 179-182. Andervont, H. B. 1949a. J. Natl. Cancer Znst. 10, 193-200. Andervont, H. B. 1949b. J. Natl. Cancer Znst. 10, 201-214. Andervont, H. B. 1 9 4 9 ~ . Natl. Cancer Znst., Natl. Znst. Health, Federal Security Agency, 1-16. Andervont, H. B. 1950a. J. Natl. Cancer Inst. 11, 73-80. Andervont, H. B. 1950b. J. Natl. Cancer Znst. 11, 545-553. Andervont, H. B., and Bryan, W. R. 1944. J. Natl. Cancer Znst. 6, 143-149. Andervont, H. B., and Dunn, Th. B. 1948a. J. Natl. Cancer Znst. 8, 227-234. Andervont, H. B., and Dunn, Th. B. 1948b. J. Natl. Cancer Znst. 8, 235-240. Andervont, H. B., and Dunn, Th. B. 194% J. Natl. Cancer Znst. 9, 89-104. Andervont, H. B., and Dunn, Th. B. 1950a. J. Natl. Cancer Znst. 10, 895-926. Andervont, H. B., and Dunn, Th. B. 1950b. J. Natl. Cancer Inst. 10, 1157-1190. Andervont, H. B., and McEleney, W. J. 1938. U.S. Pub. Health Repts. 63, 777-781. Andervont, H. B., and McEleney, W. J. 1939. U.S. Pub. Health Repts. 64, 15971603. Andervont, H. B., and McEleney, W. J. 1941a. J. Natl. Cancer Znst. 2, 7-11. Andervont, H. B., and McEleney, W. J. 1941b. J. Natl. Cancer Znst. 2, 13-16.
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Strong, L. C. 1944. Proc. SOC.Exptl. Biol. Med. 67, 78-79. Strong, L. C. 1945. Proc. SOC.Exptl. Biol. Med. 69, 217-220. Strong, L. C., and Little, C. C. 1920. Proc. SOC.Exptl. Biol. Med. 18, 45-48. Strong, L. C., and Smith, G. M. 1939. Yale J. B i d . and Med. 11, 589-592. Strong, L. C., and Williams, W. L. 1941. Cancer Research 1, 886-890. Tannenbaum, A. 1945a. Cancer Research 6, 609-615. Tannenbaum, A. 1945h. Cancer Research 6,616-625. Tannenbaum, A. 1947. Ann. N.Y. Acad. Sci. 49, 5-17. Tannenbaum, A., and Silverstone, H. 1946. 37th Ann. Meeting Am. Assoc. Cancer Research. Cancer Research 6, 499. Tannenbaum, A., and Silverstone, H. 1947a. Cancer Research 7, 567-574. Tannenbaum, A., and Silverstone, H. 1947b. 38th Ann. Meeting Am. Assoc. Cancer Research. Cancer Research 7, 711. Tannenbaum, A., and Silverstone, H. 1949a. Cancer Research 9, 162-173. Tannenbaum, A., and Silverstone, II. 1949b. Cancer Research 9, 403-410. Tannenbaum, A., and Silverstone, H. 1949c. 40th Ann. Meeting Am. Assoc. Cancer Research. Cancer Research 9, 607. Tannenbaum, A., and Silverstone, H. 1949d. Cancer Research 9, 724-727. Tannenbaum, A., and Silverstone, H. 1949c. Cancer Research 9, 577-579. Taylor, A. 1943. Science 97, 123. Taylor, A. 1948. Acta Union intern. contre Cancer 6, 670-673. Taylor, A., and Carmichael, N. 1947. Cancer Research 7, 78-87. Taylor, A., and Carmichael, N. 1949. Cancer Research 9, 498-503. Taylor, A., Carmichael, N., and Norris, T. A. 1948. Cancer Research 8, 264-269. Taylor, A., Hungate, R. E., and Taylor, D. R. 1943. Cancer Research 3, 537-541. Taylor, A., Kynette, A., and Hungate, R. E. 1945. Univ. Texas Pub. 4607. Cancer Studies, 13-22. Taylor, A., Thacker, J., and Pennington, D. 1942. Science 96, 342-343. Taylor, H. C. Jr., and Waltman, C. A. 1940. Arch. Surg. 40, 733-820. Traub, E. 1938. J . Exptl. Med. 68, 229-250. Traub, E. 1939. J . Exptl. Med. 69, 801-817. Trentin, J. J. 1950. Cancer Research 10, 580-583. Trentin, J. J., and Turner, C. W. 1941. Endocrinology 29, 984-989. Twombly, G. H. 1939. Proc. SOC.Exptl. Biol. Med. 40, 430-432. Exptl. Biol. Med. 44, 617-618. Twombly, G. H. 1940. Proc. SOC. Twombly, G. H., and Meisel, D. 1946. Cancer Research 6, 82-91. Visscher, M. B., Ball, Z. B., Barnes, R. H., and Silvertsen, I. 1942. Surgery 11, 48-55. Visscher, M. B., Green, R. G., Bittner, J. J., Ball, Z. B., and Siedentopf, H. A. 1942. Proc. SOC.Exptl. Biol. Med. 49, 94-96. Wallace, E. W., Wallace, H., and Mills, C. A. 1944. Cancer Research 4, 279-281. Wallace, E. W., Wallace, H., and Mills, C. A. 1945. Cancer Research 6,4 7 4 8 . Warner, S. G., Reinhard, M. C., and Goltz, H. L. 1945. Cancer Research 6,584-586. White, F. R. 1944. J . Natl. Cuncer Inst. 6, 119-158. White, F. R., and White, J. 1944a. J . Natl. Cancer Insl. 4, 413-415. White, F. R., and White, J. 1944b. J . Natl. Cancer Inst. 6, 41-43. White, F. R., White, J., Mider, G. B., Kelly, M. G., and Heston, W. E. 1944. J . Nall. Cancer Inst. 6, 43-48. White, J., and Mider, G. B. 1941. J . Natl. Cancer Inst. 2, 95-97. White, J., Mider, G. B., and Heston, W. E. 1943. J . Natl. Cancer Inst. 3, 453-454. White, J., White, F. R., and Mider, G. B. 1947. J . Natl. Cancer Inst. 7, 199-202.
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Willis, R. A. 1948. Pathology of Tumors. C. V. Mosby Co., St, Louis. Woods, M. W., and Du Buy, H. G. 1943. Phytopathology 88, 637-655. Woods, M. W., and Du Buy, H. G. 1945. Science 102, 591-593. Woolley, G. W., Fekete, E., and Little, C. C. 1939. Proc. N d l . Acad. Sci. 26, 277-279. Woolley, G. W., Fekete, E., andLittle, C. C. 1940. Proc. SOC.Exptl. BioE. Med. 46, 796-798. Woolley, G. W., Fekete, E., and Little, C. C. 1941. Endocrinology 28, 341-343. Woolley, G. W., Fekete, E., and Little, C. C. 1943. Science 97, 291. Woolley, G. W., Law, L. W., and Little, C. C. 1941. Cancer Research 1,955-956. Woolley, G. W., and Little, C. C. 1945a. Cancer Research 6, 193-202. Woolley, G. W., and Little, C. C. 1945b. Cancer Research 6, 203-210. Woolley, G. W., and Little, C. C. 19450. Cancer Research 6, 211-209. Woolley, G. W., and Little, C. C. 1945d. Cancer Research 6, 321-327. Woolley, G. W., and Little, C. C. 19450. Cancer Research 6, 506-509. Woolley, G. W., and Little, C. C. 1946. Cancer Research 6, 712-717.
Hormonal Aspects of Experimental Tumorigenesis* W . U. GARDNER Yale University School of Medicine, New Haven, Connecticut CONTENTS Page
I. Introduction
11. General Statements on Tumorigenesis 111. Types of Experimental Hormonal Imbalances IV. Influences of Differences in “Substrate” on Differences in Response V. Ovarian Tumors 1. Ovarian Tumorigenesis under Experimental Conditions 2. Transplantability and Hormonal Functions of Ovarian Tumors 3. Ovarian Tumorigenesis in Primates VI. Testicular Tumors VII. Adrenal Tumors VIII. Pituitary Tumors IX. Lymphoid Tumors X. Uterine Tumors XI. Mammary Glands XII. Hormones in Relation to Tumors of the Secondary Sex Organs of Males.. XIII. Other Tissues or Organs in Which Sex or Sex Hormones Modify the Appearance of Tumors 1. Liver 2. Bone XIV. Urinary Tract XV. General Discussion References
173 174 178 180 184 184 193 194 194 198 200 204 207 211 219 220 220 221 221 22 1 223
I. INTRODUCTION The following is in part a review and in part an interpretation based on the reported observations of others as well as those of the writer.
* The investigations of the writer which have permitted familiarity with some aspects of the problems discussed have been supported by the Anna Fuller Fund, the Jane Coffin Childs Memorial Fund, and the U.S.Public Health Service, as well as special research funds of the Yale University School of Medicine. The writer is furthermore grateful for the associations with the late Professors E. Allen and G. M. Smith and with present and former associates (C. A. Pfeiffer, L. C. Strong, C. W. Hooker, J. J. Trentin, A. Kirschbaum, A. Gorbman, M. H. Li, S. C. Pan,H. C. Chang, J. Boddaert, J. T. Wolstenholme, J. Rygaard) and students who independently or in collaboration have worked on certain aspects of the problem and have undoubtedly contributed directly or indirectly to the points of view expressed. 173
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Prejudices of the writer will undoubtedly be reflected in both the selection of the materials to be cited and in the interpretation made from the citations. But so many reviews (1,2,3,4,5,6,7,8,9,10,11) have been written on different aspects of this topic and the reported investigations are so numerous that it seems unfortunate to reiterate previously reviewed facts. InterpretaOions and hypotheses change somewhat with additional facts and hence have a temporal value-they are outmoded periodically. The exposition mill relate primarily to tumorigenesis or carcinogenesis and not to the effects of hormones upon tumors. The factors associated with the origin of tumors may not be the same as those associated with compatibility to the environment provided by their host for progressive growth. Although admittedly we may never determine how many cells of the body become “malignant” and yet fail to progress to a stage of recognition, it is not improbable that such frequently occurs. It’s those neoplastic foci that possess more or less autonomy that progress to produce recognizable symptoms. Therefore it is somewhat fallacious to attempt a study on tumorigenesis without consideration of tumor growth, because in a certain sense this cannot be done. But, in the sense that hormones may be chemotherapeutic agents, a division of the subject can be made. This aspect of the problem will not be considered here even though hormones (1) might be considered to contribute significantly to the environment in which a neoplasm is growing and hence adversely affect or stimulate its growth, (2) might act upon the hosts’ tissues to increase their resistance to the tumor, or (3) might modify the tumor cells so that they would “differentiate” and hence be more orderly or restricted in their activities.
11. GENERALSTATEMENTS ON TUMORWENEBIS If the quality designated “carcinogenicity” can be ascribed to any agent, chemical, biological, or physical, which, when appropriately applied to animals results in the appearance of cancers that would not otherwise have appeared, then some hormones must be assumed to be carcinogens. More simply this means that agent plus animal gives tumor. But the accuracy of a simple mathematical abstraction does not prevail. The animal is a composite of many different types of cells that are influenced by the multiple factors in their environment, of cells possessing unexplained specific intrinsic qualities that differentiate one type from another. Although rigidly controlled in a genetic sense a metazoan is still a population rather than an individual, and the intrinsic environmental conditions to which diff went cells are exposed may range widely. The quality “tumor” or “cancer” which represents the crude summation in the above formula can be described only in superficial
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terms as a proliferation of cells unrestricted by the usual regulating mechanisms and detrimental t o the life of the host. Thus only the agent is known specifically; only the agent may merit consideration as a reproducible and consistent quality. A consideration of a formula in the terms of three nouns, however, neglects the action processes. At the present time the mechanisms by which cancer is produced and designated by the “plus” are unknowns in the simple formula. Theories of genic and enzymatic mutation prevail. The “gives” also hides unknowns except superficially. The history and methodology of the studies on the relation of hormones to carcinogenesis do not differ greatly from those used in the study of other carcinogenic agents. The descriptive stage or “natural history” stages preceded the stages of experimental approach. The experimental approach involves the standardization of biological tests for the agent, range of its activity, purification, its mechanism of action, methods of neutralization, and finally demonstration of involvement or attempts to demonstrate involvement of the agent under the conditions in which “spontaneous ” tumors arise. Responses to carcinogens (cancers) may be local, that is, a t the site of application of the carcinogen; for example, skin tumors at the site of application of methylcholanthrene, skin tumors a t sites exposed to ultraviolet irradiation, or local tumors at the sites of injection of specific viruses. Responses to carcinogens may be remote from the site of application, for example, lung or lymphoid tumors, in mice of some strains, following the injection of carcinogenic hydrocarbons. Remoteness here, however, must certainly be questioned as the agent is undoubtedly widely distributed throughout the body. Hormones might be considered to act only remotely as carcinogenic agents, or they might rather be considered not to act as carcinogens to skin or subcutaneous tissues t o a significant extent because following subcutaneous or percutaneous injection epidermal or subcutaneous tumors rarely occur, whereas tumors of other organs do occur. Tissue or organ specificity to carcinogens frequently exists as indicated above. To cite a few: urethan shows a particular capacity to induce lung tumors; 6-napthalamine, bladder tumors; butter yellow, liver tumors; and Rous virus, connective tissue tumors. The predominance of tumors in specific tissues elicited by any one agent may be due to quantitative differences of contact of the tissues with the agent or its metabolic derivatives in some instances. The appearance of tumors in specific tissues or organs of mice subjected to estrogens is not necessarily unique; other carcinogenic substances may elicit similar specific responses. The prolonged interval between the original exposure to a carcinogenic
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stimulus and the manifestation of the response by the symptoms of cancer is characteristic of most of the responses to agents other than some of the viruses. The mammary tumor virus in mice, however, affords an example that is not unlike exposure to carcinogenic doses of x-rays, or small doses of carcinogenic hydrocarbons. In all instances, the carcinogenic actions of hormones are manifest only after prolonged exposure of the tissues to these substances. It is not particularly surprising, therefore, that most of the hormones that have been shown to be carcinogenic are those that are produced cyclically, those to which the body’s tissues are not normally exposed continuously for prolonged periods, or are not exposed for prolonged periods in the absence of some modifying hormone. The above general discussion is an attempt to emphasize some similarities of the actions of some hormones and other carcinogenic agents before hormones in carcinogenesis are considered specifically. Actually there is no proof that hormones are carcinogenic. There is only proof that tumors or cancer occur in appropriate animals when they are subjected to treatments with adequate amounts of certain hormones. Metabolic derivatives of the hormones may be carcinogenic rather than the hormones themselves. Hormones may be indirectly carcinogenic by inciting cells to produce substances that are carcinogenic. It is not surprising that our knowledge here is so inadequate. We do not know how the hormones to be considered produce within the responding tissues or “end organs’’ those responses that are considered t o be normal; the “plus” and the “gives” of the formula represent unknowns in relation to their physiological activities as well as the pathological responses. The major differences are (1) that the “sum” in the normal responses is not so frequently modified by genetic differences among animals of the same species, or by species differences, as it is in the cancerous responses, and (2) the “normal” responses are relatively immediate. In other words we know no more about how estrogens provoke proliferation and cornification of the vaginal epithelium than we do about how they provoke cervical cancer in mice. The former response occurs promptly, the latter becomes evident only after prolonged periods of exposure to the unusual environment. To the endocrinologist, the hormone (inciter) and the responding organ or tissue (responder or end organ) are not isolated systems within the body-they are modified by other factors, both sometimes by other hormones. At least four major variables must be considered in investigations on the actions of steroid hormones of intrinsic and also extrinsic origin in experimental carcinogenesis, and each of these may be modified by multiple variables or possible variables (Table I). Although all the variables listed may not coexist, instances can be cited in which they
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have been demonstrated or have been postulated as a result of interpretation of appropriate experiments. TABLE I Hormonal Imbalances-Factors That May Contribute to Them Changes or differences in rate of pro- Genetic influences duction of hormones Agmntogenic stage Nutritional status Reciprocal glandular interactions Disease Other factors? Modifications of or differences in rate Genetic influences of destruction, utilization or excretion Agmntogenic stage of hormones Nutritional status Specific inhibiting, augmenting or competing factors Disease Differences in the capacity of end or- Genetic influences gans to respond Agmntogenic stage Nutritional status Inhibiting and augmenting hormones Duration and continuity of stimulus Modification*inthe quality of hormone Genetic influences Age-ontogenic stage produced Nutritional status? Disease ?
The hormones that, at this time, can be considered to be carcinogenic (bearing in mind the qualifications mentioned above) are essentially growth stimulating hormones, namely the estrogens and gonadotropins; those growth hormones that normally act cyclically in the female mammal. To this might be added thyrotropic hormone. The classification of these hormones as essentially stimulators of growth is controversial in that they also produce differentiation of responsive tissues ;for example, cornification of the vaginal epithelium and secretion of uterine cervical and fundic glands represent functional differentiation of these tissues. Nevertheless the stimulation of growth of specific gonadal tissues and of the female accessory genital tissues or their male homologues is a striking manifestation of the gonadotropic and estrogenic hormones respectively. The ovaries and genital tissues of most mammals are not subjected to the unrestricted action of these growth-stimulating hormones for prolonged periods. The quality of intermittent production of specific hormones and intermittent response of certain tissues characterizes mammalian female reproductive phenomena. The internal environment to
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which specifically responding tissues are exposed is rhythmic, not constant. The imposition of a constant stimulation would impose an abnormal environment and would create an imbalance in the normal physiology of the animal. Most of the experiments in which either hormones have been administered or operative procedures conducted that have lead to cancer, have been in this sense experiments in which the normal balances of hormones have been altered, or at least maintained at a stimulating level for an abnormal period of time. The abnormality of balance may be the duration of stimulation rather than an abnormal level or quality of exposure to hormones. Hormonal imbalances are at present largely a matter of interpretation and are difficult to define because of difficulties attending definition of the normal hormonal interrelationships. 111. TYPESOF EXPERIMENTAL HORMONAL IMBALANCES For many yeara it has been known that ovaries when transplanted into castrated males of some species fail to function in a cyclic manner as they do when transplanted into ovariectomized females. Ovaries in castrated male rats, for example, do not form corpora lutea, presumably because of a sex difference in the pituitary gland. The sex difference in the function of the pituitary gland of the rat apparently is determined early on ontogency by the testis and, once established, is not reversible. When testes of newborn rats were transplanted into their littermate sisters, the hosts failed to show cyclic ovarian function when they became mature; for the greater part they showed continuous estrus as indicated by vaginal smears (12,13). Even when the grafts were removed a few weeks after transplantation, and hence in rats only a few weeks old, the functional changes of the pituitary persisted throughout the life span of the temporary host. In this instance a hormonal imbalance induced persistent and irreversible changes. Furthermore, the amounts of hormone produced by the early postnatal testes evoked this response. Newborn female rats given injections of testosterone during the first few weeks of life showed similar persistent physiological disturbances (14). Some other hormonal imbalances are reversible after the removal of imbalancing influences-completely so, apparently, if not maintained over too long a period, or if not imposed upon the body at critical periods in ontogeny. Hormonal imbalances of this type are most readily produced by the injection of hormone-containing extracts or specifically active chemicals. They may be imposed by conditions that modify the production and inactivation of intrinsic hormones. The [technic of parabiosis has been used often in experiments of this type. For example,
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when an intact female rat is placed in parabiosis with a gonadectomized rat, the intact parabiont shows prolonged periods of estrus, marked uterine and ovarian stimulation, and even hypophyseal hyperplasia (Fig. 2). Experiments of the latter type have the advantage of revealing that endocrine glands in the body can produce amounts of hormone that result in abnormal hyperplasias. They provide some evidence that “ physiological” levels of hormones can produce abnormal responses. NEWBORN Q
ADULT $
Continuous estrus
FIG. 1. Scheme of an irreversible type of endocrine imbalance in rats. The testis of a newborn male so modified the endocrine interrelationship of the newborn female host that upon maturity the host failed to show estrous cycles. Adapted from 11.
The argument has been raised that “pharmacological ” doses of estrogens, for example, are required before tumors appear. This assumption is doubtful; in most instances the abnormal aspect is the continuity of stimulation rather than abnormally high concentrations. Abnormal responses have been produced experimentally by two different methods of modification of the hormonal balances within the body, namely, (1) the addition of extrinsic hormones or biologically active substances, and (2) manipulations of the animals to secure altered production or destruction of hormones, two examples of which have been cited above.
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W. U. QARDNE1R INTACT PARABIONT
CASTRATE PARABIONT
FIG.2. Endocrine imbalances produced by parabiosis to indicate the abnormalities that can be produced by endocrine glands if mechanisms for the production of hormones are not controlled. Adapted from 11.
IV. INFLUENCES OF DIFFERENCES IN “SUBSTRATE ” ON DIFFERENCES IN RESPONSE The statement has been made above that neoplastic responses to hormones are frequently limited to animals of some species or even to animals of some strains within a species, and this is certainly true. Species differences in response are not restricted to neoplastic responses. “Normal” responses are also sometimes species limited; mammary growth does not occur in estrogen-treated dogs (15), pubic separation does not occur in rabbits or rats but does occur in guinea pigs and mice (16,17), and hypercalcemia is associated with ovulation in pigeons and probably ofther birds (18J9). The major differences are that the neoplastic responses are more commonly species limited or strain-limited. Investigators do not expect the pubic bones of estrogen-treated rats to be separated by an interpubic ligament and to be partially resorbed, but such ti response does occur in mice. We might assume that the “substrate,” the pubic symphysis and pubic bones, of rats is not capable of the osteolytic and fibroblastic response. Hormones in general initiate few or no new responses in the body-they regulate rates of responses for which cells possess intrinsic or potential capacities. The intrinsic
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capacities sometimes differ in animals of different species or strains. Until more is known we must assume that genetic or transmitted factors limit these capacities. The vaginal epithelia of mice of different inbred strains require different amounts of estrogens for vaginal cornification as shown in Table I1 (20). Six hundredths microgram of estradiol benzoate induced vaginal cornification in 50% of the mice of one strain and almost five times this amount was required to induce a similar response in mice of another strain. Such experiments are objective in themselves but must be interpreted to indicate strain differences (possibly genetic differences) TABLE I1 Minimum Amount of Estradiol Benzoate Giving Positive Vaginal Smears in Approximately 50 % of the Mice (20) Strain* or Hybrid Group C67
JH CIH BL ,’ ~
cc1 cc2
CBA ABz N A
AL
Micrograms of Estradiol Benzoate 0.06 0.08 0.1 0.08-0.1 0.08-0.1 0.1 0.15 0.2 0.25-0.27 0.25-0.27 0.27
*CCI, CsrO X CBAc?; CCS. CBAV X c67b; AB:, CBAV X A d ; BL, CaH without mammary tumor agent; AL,A without mammary tumor agent.
of either (1) threshold requirements of the end organ, (2) rates of destruction or excretion of the hormone administered, or (3) presence of different amounts of or absence of hormones that may oppose the action of the hormone being studied. To some extent the differences in threshold of the vaginal epithelium can be checked by the direct application of the hormone to the end organ, A fivefold difference in threshold was observed (Table 111) when estrone was applied directly to the vaginal mucosa (21). These observations indicate that the differences in threshold of the vaginal epithelium are not due to strain differences in destruction or excretion of estrogens and that they are attributable either to differences in the vaginal epithelia of mice of different strains or t o the third factor mentioned above, namely, differences in the amounts of or absence of antagonistic or modifying hormones in the body. Experiments to differentiate the above two influences have not been reported.
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TABLE I11 Per Cent of Ovariectomized Mice Showing Vaginal Cornification after Intravaginal Instillation of Estrone Dissolved in 50 Glycerol ~~
Doses in Micrograms 0.0001
Strain
-
Cs7Black
20
020 Leeuwenhoekhuis
P20 dba
-
0.0005
0.001
0.005
21 4 0
72 87 93
-
5
77
Certain qualities associated with cancer susceptibility have no influence on the vaginal response to estrogens. Reciprocal hybrid mice of parents “susceptible ” and “resistant I’ to mammary adenocarcinoma (CC, and CC1 in Table 11) have similar vaginal thresholds indicating that no maternally transmitted influence existed (20). Furthermore, foster nursing did not modify the vaginal sensitivity to estrogens, indicating that the mammary tumor agent transmitted through the milk was not a modifying influence (20,22). Furthermore, the above observations indicate that the hybrids have a vaginal threshold intermediate to that of the parental strains. The strain differences in the responses of the mammary glands of mice to estrogen treatment also have been studied and again marked differences have been noted. Castrate males of three different inbred strains required 1, 2, and 3 pg. of estrone for a minimal mammary response and intact males, 50, 10, and 10 pg., respectively (23). Ovariectomized female mice also showed strain differences in mammary thresholds (Table IV). The rate of response of the mammary glands of castrate TABLE IV Strain Differences in Response of Mammary Gland and Vaginal Epithelium of Ovariectomized Mice to Estrone Data from Miihlbock (24) Strain*
1
Csr Black
2
020
10 1
Riet dba
* Csr strain taken
Mammary Glands
&B
unity.
Vaginal Epithelium
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and intact male mice of several inbred strains and hybrid groups ranged widely (25). Although the range within each group was rather wide among mice of most strains, the differences between the means of the responses among the several groups were quite wide and significant. Furthermore, the number of rudimentary mammary glands either persisting or differentiating embryologically differed greatly among the male mice of the several strains. Male mice of one strain had very few mammary rudiments and few of those present responded when estrogens were administered. Other types of studies of a somewhat different and probably less objective nature have also indicated strain differences in vaginal sensitivity and mammary sensitivity of mice (26). Such experiments must be carefullly and critically designed in order to present valid conclusions, and the amount of material must be adequate to indicate the range of response in each group. Some experiments have indicated an increased sensitivity to estrogens of the mammary tissue of female mice with the mammary tumor agent and susceptibility to mammary cancer. These experiments were on a small amount of material and were not analyzed statistically (27). The presentation of some quantitative differences in ((normal” responses of some end organs and some of the qualitative differences in response among animals of different species constitutes an attempt to emphasize the need of caution in the extrapolation of observations derived from experimentation with any limited sample. Each experiment means only that a specific result was obtained with the use of specific materials and under specific circumstances. Tumors of a t least ten different tissues or organs have shown a tendency to appear more frequently in one sex or the other or with more or less controlled and reproducible hormone changes in one or more species of laboratory animals (Table V). All the conditions have not been studied to the same extent a t this time. In all instances the number of species in which the hormone-tumor relationship has been demonstrated is limited. Occasionally tumorous responses are limited to one or more strains or stocks within a species. A listing of the possible tumorhormone associations as in Table V unfortunately does not give adequate consideration to those studies in which negative results have been reported. An analysis of why tumors do not appear in some strains or species, while in others they do appear under somewhat homologous circumstances, is of much greater significance than omission from the table would imply. It is the writer’s opinion that adequate consideration has not always been given to (‘why tumors do not appear.”
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TABLE V Tumors That Have Been Associated with Sex or Hormonal Changes in Laboratory Animals of Some Species Tissue or Organ
Mammary gland Uterus or uterine cervix Testis Lymphoid tissues Liver Pituitary gland Bone Kidney Ovary Adrenal Thyroid
Hormones Involved*
Species
A. Related to gonadal hormones Estrogens “pituitary ” MOW, t rat, t rabbit t Estrogens “pituitary” Moue, rabbit, guinea pig Estrogens “pituitary” Mouse? Mouse t Estrogens “pituitary ” Androgens ” Mouse, rat Estrogens-‘Lthyroid deficiency” Mouse t Female Mouse t Estrogens Hamster B. Related to hypophyseal hormones Mouse, rat, rabbit Pituitary-gonadotropic ” Absence of gonads “pituitary ” Mouse ?-guinea pig Mouse, rat “Pituitary thyrotropic ”
*The quotation marka indicate the desirability of considerably more direct evidence of the assumption. t Probably limited to certain strains or stocks of animala within the species.
V. OVARIAN TUMORS 1 , Ovarian Tumorigenesis under Experimental Conditions
Ovarian tumors arise among rats, mice, and probably rabbits under experimental conditions that indicate a hormonal etiology. Mice exposed to x-rays and gonadectomized mice bearing ovarian grafts in sites drained through the hepatic portal system acquire ovarian tumors. The tumors appear in mice of all strains and in so far as is known, at a similar rate. Both in irradiated mice and in mice bearing grafts that have their venous drainage through the hepatic portal system it is assumed that either prolonged and probably elevated amounts of, or abnormal qualities of, gonadotropic hormones are responsible for the neoplastic response, These tumors do not appear in animals with intact gonads or that have received adequate doses of appropriate sex steroids. Up to this time, however, ovarian tumors have not been reported to occur in animals to which extrinsic gonadotropic hormones have been administered-the direct experiment has not been successful. Furthermore, ovarian tumors occur rarely in untreated mice. The hormonal imbalance assumed to be associated with ovarian tumorigenesis in rats or mice bearing ovarian grafts in sites draining their venous blood through the liver results from (1) the alteration of the inactivation of ovarian hormones and (2) resulting modification of
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gonadotropic secretion. Hepatic tissue of rats was first shown to inactivate estrogens in experiments undertaken by Zondek (28). Many investigators have confirmed these observations. Golden and Sevringhaus (29) noted that hepatic tissues also inactivated estrogens produced by the ovaries; ovaries transplanted into the mesenteries or spleen persisted and showed follicular growth without their hosts exhibiting the vaginal responses characteristic of estrus. Aschheim and Zondek reported that human urinary gonadotropins were elevated postmenopausally. Subsequently Evans and Simpson (30) and Engle (31) reported the elevation of hypophyseal gonadotropin subsequent to castration. The experiments in which parabiotic pairs of rats consisting of one castrate and one intact female indicate that the circulating gonadotropins are also elevated (32). Urinary gonadotropins in women, serum gonadotropins in rats or mice, and hypophyseal gonadotropins of rats (30), mice (33), and guinea pigs (34) all increase in the absence of ovaries or ovarian hormones. Because hepatic tissue destroys estrogens, especially the normal occurring estrogens, presumably produced by the ovaries of animals of most species, an ovary draining into the hepatic portal system can exist in an animal that is physiologically castrated. In such an environment ovarian tumors appear, These tumors are predominately granulosa cell tumors, although luteomas or luteinized granulosa cell tumors appear occasionally. The incidence of tumors in rats bearing such grafts for periods in excess of 157 days was approximately 86% (Table VI). Seven rats TABLE VI Ovarian Tumors in Gonadectomized Rats Bearing Intrasplenic Ovarian Grafts
No. of Animals Biskind and Biskind ( 3 6 ) Peckham et al. (37) Biskind and Biskind (35) Total
9
12 50 71
No. of Tumors 3 large, 2 small g.c.t.* 12 g.c.t. 46 61
Age of Grafts 265+ days 265f days 157+ days
* g.o.t., grsnuloss cell tumor. bearing intrasplenic ovaries that were adherent to the body wall and that were observed more than 155 days had no tumors, and nineteen rats which survived 158 days or more with ovarian grafts in their kidneys had no ovarian tumors. The grafts in these sites survived for almost two years. Rats of three different stocks were used in the above experiments, and no mention was m-de of differences in tumor incidence among ani-
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mals of the different stocks. In intrasplenic ovarian grafts in rats follicles were found in grafts up to 150 days old. Thereafter lutein tissue, designated luteoma because of its hyperplastic state, predominated, and granulosa cell tumors were frequent only in grafts 350 days old or older. Ovarian tumors, primarily of the granulosa cell type, but with occasional luteomas or mixed tumors, appeared among gonadectomized mice bearing intrasplenic or intrapancreatic ovarian grafts (Table VII). The TABLE VII Ovarian Tumors in Gonadectomized Mice Bearing Intrasplenic or Intrapancreatic Ovarian Grafts Investigator
Li and Gardner (38) Furth and Sobel (39) Li and Gardner (40)
No. of Animals
68*t 43*t 22*t
+ methylcholanthrene
No. of Tumors 17 29 21
* Ovariectomised.
t Orchieotomimd.
tumors first appeared in grafts about 200 days old, and the incidence and size of the tumors increased with advancing age to attain approximately 100%in old grafts. The tumors appeared in mice of all strains or hybrid groups that were studied. Intrasplenic ovarian grafts in ovariectomized guinea pigs were observed after periods as long as 654 days (34). Some of the grafts were large, exceeding 1 cm. in diameter. Some follicles persisted in the grafts, but the older grafts consisted predominately of irregularly shaped luteinieed cells “somewhat similar to the luteinized theca cell tumors of the ovary of the woman.” Two ovariectomized rabbits that survived 512 to 513 days with intrasplenic ovarian grafts had solid, yellowish tumor masses measuring 6 and 6.4 mm. in diameter. Histologically these tumors were of the granulosa cell type with some evidence of luteinization. Animals surviving for shorter periods did not show ovarian tumors (37). In a t least three species of rodents ovarian tumors arose in castrated animals bearing ovarian grafts in areas drained by the portal vein. The observations would seem to fit the hypothesis mentioned above. But many observations were required before the hypothesis could be established with reasonable definiteness. Intrasplenic grafts that became adherent to the body wall or the uterus did not become tumorous, and their hosts more frequently showed estrous cycles indicating a systemic effect of ovarian hormones (35,40). Ovaries transplanted intraspleni-
HORMONES AND EXPERIMENTAL TUMORIGENESIS
187
cally in unilaterally gonadectomized mice did not become tumorous (40). That the gonadal hormones produced by the intact go'nad or the adherent grafts inhibited ovarian tumorigenesis was indicated, so experiments were undertaken in which estradiol benzoate and testosterone propionate were injected subcutaneously in castrated mice bearing intrasplenic grafts (40). Twenty-seven mice with intrasplenic ovarian grafts that were given 16.6 pg. of estradiol benzoate or 1.25 mg. of testosterone propionate weekly did not have ovarian tumors. Weekly injections of 1 mg. of progesterone did not prevent ovarian tumorigenesis, but such an amount is less than is required daily to maintain pregnancy in the mouse. The daily injection of 25 i.u. of gonadotropin (from pregnant mare's serum) did not increase the incidence of tumors in the intrasplenic grafts but most of the tumors were luteomas. All the above data are compatible with the interpretation that the ovarian tumors are attributable to the increased intrinsic gonadotropic hormones associated with decreased gonadal function. If this interpretation is true, it might account for the rare spontaneous granulosa cell tumors. But why are such tumors so rare? Ovarian function decreases in animals of several species some time before death. I n such circumstances the urinary gonadotropins increase, in man a t least. Such an environment might predispose to ovarian tumorigenesis if the aged ovary were susceptible to tumorigenesis. Old ovaries are susceptible to tumorigenesis. When relatively old ovaries were transplanted intrasplenically into old mice few tumors developed, but when transplanted intrasplenically into gonadectomized young mice they did become tumorous (Table VIII). The old ovaries were not refractory t,otumorigenesis when in a young environment. The incidence may be slightly lower and the latent period slightly longer. If old and inactive ovaries could survive in their hosts for a prolonged TABLE VIII Incidence of Tumors in Intrasplenic or Intrapancreatic Ovarian Grafts in Mice of Different Ages Investigator Li and Gardner (41) Klein (42)
No. of Mice 8
12 9 23 40 24
Age of Donor
Age of Hosts
No. of Tumors
8-14 204-369 340-491 29 147 799
32-49 204-369 54-105 134 123 128
5 4 8
Age of Grafts
180-182 252-375 152-388 21 (91%) 205 av. 35 (87%) 204 16 (67%) 221
188
W. U. GARDNEE
period, and if the levels of gonadotropin persisted a t high levels, more ovarian tumors would be expected. The hormone production of aged ovaries is decreased as indicated by irregular estrous vaginal smears of old mice. Mice of some strains acquire adrenal cortical tumors as do castrated mice, indicating that failure of ovarian function in this instance is compensated by adrenal hormonal activity (43). The amount of gonadotropin in the pituitaries of aged intact and gonadectomiaed mice is lower than in younger adults (33). The possibility exists that the aged pituitary gland produces less gonadotropin than does that of the mature adult because, in general, levels of hormone within the glands tend to parallel those in the circulation. The low incidence of ovarian tumors in old mice is therefore probably due to the decrease in gonadotropic function of the pituitary coincident with prolonged failure of ovarian function. Hepatic tissue of mice destroys estrogens in vitro (44,45). In vivo, hepatic tissue of mice, as well as rats, destroys estrogens. Cornified vaginal smears rarely occur in ovariectomized mice bearing intrasplenic ovarian grafts. Intact female mice in parabiosis with ovariectomized mice bearing intrasplenic ovarian grafts show unusual stimulation of their uteri (46). Larger uteri occurred in mice in parabiosis with mice that had carried intrasplenic grafts for periods of thirty to fifty days than in parabionts in which all operations were performed simultaneously (Table IX).
TABLE I X Production of Gonadotropins in Castrated Mice Bearing Intrasplenic Grafts and in Roentgen-Irradiated Mice as Indicated by Parabiosis Weights of Uteri or Ovaries, mg. Intact Investigator
Treatment
Ovaries
Miller and Pfeiffer Intrasplenicgrafts. All 7 . 8 (46) operationson same day Miller and Pfeiffer Intrasplenic grafts. 10.7 (46) Castrated 30-SO days before parabiosis Boddaert (47) X-rayed. Parabiosia 16.8 2 months later
Castrated or x-rayed
Uteri
Ovaries
Uteri
41.9
-
33.9
73.3
-
30.4
238.8
2.6
58.7
Many investigators have used parabiotic unions of castrated and intact rats or mice. All have concurred in the transmission across the
HORMONES AND EXPERIMENTAL TUMORIGENESIS
189
parabiotic union of augmented amounts of gonadotropins from the castrated to the intact member as determined by the response of the ovaries and uteri of the nongonadectomized member. The Wisconsin group have determined how much gonadal hormones must be injected into the castrated member to prevent the stimulation of the genital tissue of the intact member (48,49,50). I n rats amounts of estrogen too small to stimulate the uterus of the gonadectomized parabiont prevented the increase of gonadotropin production as indicated by the failure of the ovarian and uterine stimulation in the intact parabiont. Approximately thirty times as much estrogen was required to pass the parabiotic union and thus stimulate the uterus of the intact parabiont as was required to prevent increase in the gonadotropins. The follicle-stimulating hormone (FSH) is the gonadotropin that increases in ovariectomized rats (51,52) in so far as can be determined and presumably is therefore the gonadotropin responsible for the ovarian tumors. The increase in FSH is not as great as in t&e castrated rats with intrasplenic grafts as without (Table X). Furthermore, gonadectomized TABLE X Weights of Ovaries and Uteri of Hypophysectomized Recipients of Pituitary Glands of Rats under Different Conditions Assays undertaken in the presence of constant and excessive amounts of LH (53) Average Weight Condition of donor
No. of Tests
Left ovary
Uterus
Ovariectomized; without grafts Ovariectomized;intrasplenic grafts with adhesions Ovariectomized;intrasplenic grafts, no adhesions 0
16
104
168
10
37
113
14 14
26
110 64
15
rats given estrogen injections did not have the FSH content of the pituitary glands reduced below normal levels, but the increase following castration was prevented by such treatment (54). The above observations could be interpreted t o indicate (1) that small amounts of ovarian hormone may escape hepatic destruction and keep the pituitary mechanisms under control, (2) that the grafts produce a nonestrogenic substance that acts on the pituitary gland (56), or (3) that the grafted ovaries utilized gonadotropin and prevented its accumulation (53). These observations are significant to the problem of ovarian tumorigenesis because they indicate that ovarian tumors may arise in an environment
190
W. U. GARDNER
that is not unlike that prevalent in certain parts of the estrous cycle; the greater effect is produced by the continuity of the gonadotropin to which the ovary is exposed rather than abnormally elevated levels of hormone. If such is true then mice of strains which survive for prolonged periods after ovarian regression and in which the adrenals do not assume a sex hormone function should acquire ovarian tumors. Inanition, hyperthyroidism, and hypothyroidism modify reproductive function. Hyperthyroid castrated mice bearing intrasplenic ovarian grafts and mice on two-thirds of the ad libitum food consumption had fewer ovarian tumors than did ad libitum or hypothyroid (thiouracil fed) mice (Table XI). Nutritional influences and other hormones than gonadotropic or sex hormones directly or indirectly modify experimental ovarian tumorigenesis (55).
TABLE XI Incidence of Ovarian Tumors in Intrasplenic Ovarian Grafts in Gonadectomized (CEJ 0 X CBA 3 ) Hybrid Mice under Different Experimental Conditions All fed a basic diet of Fox Chow (65) Tumors Condition
No. of Mice Total No.
Type
0.2 % desiccated thyroid added to diet
7
3
Diet ad libitum 0.2 % thiouracil added to diet 3 of ad libitum consumption # ad libitum for 7 months and then ad libitum
21 21 12
16 18
5
1 granulosa cell tumor 1 papillary cyst adenoma 1 tubular adenoma Granulosa cell or luteoma Granulosa cell and luteoma Granulosa cell and luteoma
9
7
Granulosa cell and luteoma
Ovarian tumors were observed in mice given roentgen irradiation (Table XII). These observations were made several years before ovarian tumors were noted during the experiments mentioned above. It was generally assumed that x-rays might produce in the ovaries changes that could be considered mutations much as mutations have been observed in cells of the germinal line subsequent to irradiation. Parkes and Brambel (57) reported that irradiated ovaries produced gonadal hormones in a somewhat cyclic manner even when all the ova were destroyed arid normal follicles failed to form, The hormonally active irradiated ovaries showed ingrowths of granulosa-like cells from the germinal epithelium. Subsequently detailed studies on the incidence and histogenesis of ovarian tumors in irradiated mice were made by Furth and his associates (58,59,60). The incidence of ovarian tumors is
191
HORMONES AND EXPERIMENTAL TUMORIGENESIS
high in mice that survive for prolonged periods and the tumors occur in mice of all strains that have been studied adequately (Table XII). The incidence tends to be higher when smaller amounts of irradiation are used or when strains of mice are used that are not too susceptible to leukemia subsequent to irradiation. TABLE XI1 Incidence of Ovarian Tumors in Mice Following Roentgen Irradiation
Investigator Furth and Furth (58) Kaplan (73) Geist, Gaines, and Pollack (65) Furth and Boon (59) Gardner (63)
Lick et al. (61)
No. of Mice
Treatment
and Strain
A.R.S. A
300-400 r 600 r 200 r 87 r* 175 r* 350 r*
No. of Tumors
775 99
66 10
6-24 1
38
(Rf X AK) 245
22 61 97 36
10-24 13-21 7-21 13-21
BC
45
24
Average 17
BC
45
29
Average 17
BC
42
-
-
Bagg Alb. Bagg Alb.
4 2
4 2
-
Bagg Alb. Bagg Alb.
15 24
24
0
-
(Rf X AK) 101
(Rf X AK) 195
+ +
280-380 r sesame oil 280-380 r testosterone propionate 280-380 r + estradiol benzoate 200 r to each ovary 200 r to 1 ovary and 1 ovary removed 200 r to 1 ovary 200 r whole body ~
~~
Age at Tumor (Month)
~
*Some mice received methylcholanthrene na well.
The hormonal mediation of ovarian tumors in irradiated mice is indicated by a number of experiments (Table XIII). Ovarian tumors do not occur more frequently or sooner in irradiated intrasplenic ovarian grafts than they do in nonirradiated grafts in gonadectomized mice. When one ovary was irradiated and one ovary was not irradiated, no ovarian tumors appeared, whereas tumors did appear when the nonirradiated ovary was removed (61). Furthermore irradiated ovaries transplanted intramuscularly into castrate nonirradiated hosts became tumorous but failed to become tumors in intact hosts (62,62a). These experiments demonstrated the failure of x-rays t o augment the ‘(hormonal-induction” of ovarian tumors and the capacity of intact ovaries or one intact ovary to prevent ovarian tumors. Irradiated mice given estradiol benzoate, 16.6 pg. weekly, did not have
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W. U. QARDNER
TABLE XI11 Effect of in Vitro Irradiation of Ovaries or Irradiation of Transplanted Ovaries or Hosts on Tumorigenesis Investigator Li et al. (62)
Kaplan (62a)
No. of No. of Age of Graft, Months Mice Tumors
Treatment
Strain
400 r to intrasplenic graft 200 r in vitro before grafting in spleen 300 r ovariectomired non-r ovary intramuscular Ovariectomized 300 r ovary intramuscular 300 r ovariectomized 300 r ovary intramuscular 9 300 r ovary intramuscular
Several
17
15
6-10
Several
5
6
5-10
L XA
31
1
LXA
26
19
L XA
28
17
LXA
30
0
+
+
+
+
ovarian tumors. This observation affords some evidence that the estrogenic hormones prevent the tumors. Ovarian tumors were not prevented in irradiated mice given 1.25 mg. of testosterone propionate weekly (63). In fact the incidence of ovarian tumors was actually higher than in placebo-treated irradiated controls. This amount of testosterone propionate is adequate to prevent ovarian tumors in mice bearing intrasplenic transplants and to reduce the incidence of mammary tumors in intact mice of the CIH strain and to reduce the incidence of lymphoid tumors in irradiated mice. The nontumorous ovaries of the irradiated testosterone propionate treated mice were, however, not unlike those of the nonhormone treated controls. As indicated by experiments involving parabiosis, pituitary gonadotropins are greatly elevated in irradiated mice. Testosterone propionate in doses such as were used in the experiments referred to above, prevented gonadal stimulation (64)as indicated by vaginal smears of the intact parabiont and by the weights of their genital tissues. The histogenesis of ovarian tumors in both x-rayed ovaries and in ovarian grafts in the spleens of castrated animals have been described in detail (35,38,65,66)and will be mentioned here only in so far as it may indicate the hormonal etiology of the tumors. In general both x-irradiation and intrasplenic transplantation of ovaries in castrated mice leads to a precocious loss of or decrease in number of ova and ovarian follicles. In the writer’s laboratory ova have never been observed in irradiated
HORMONES AND EXPERIMENTAL TUMORIOENESIS
193
mice that have had ovarian tumors, either in relation to the tumors or in the contralateral ovary when only one ovary was tumorous. Ova rarely exist in animals in which tumors have appeared in intrasplenic grafts. Ovaries of mice in which no or very few follicles or ova are present show hyperplasia of the surrounding epithelium-germinal epithelium. The epithelium grows into the ovarian stroma forming, in some, an extensive branching network of tubules. These ingrowths are formed in the relatively anovular ovaries of aged mice as well as in roentgenirradiated mice or mice with transplanted ovaries. In the latter circumstances they can be considered pretumorous in that they appear so consistently before tumors appear and because the tumors seem to arise in relation to them. At this time it seems that the tubular ingrowths appear in ovaries that are no longer producing significant amounts of estrogenic hormone. Irradiated mice given large amounts of estrogen do not show tubular ingrowths from the germinal epithelium, but do form anovular follicles. The action of pituitary hormones on ovogenesis as well as on the above described activities of the germinal epithelium is certainly not understood. The origin of granulosa cell tumors from the germinal ingrowths seems more readily associated with the abnormal levels of gonadotropin. Ova persist for longer periods in grafts made to nonportal areas or to portal areas that have become adherent to the body wall. The environment of grafts that secrete their hormones through the liver predisposes to precocious anovular states. The genesis of ovarian tumors in intrasplenic grafts in rats seems different than in mice. Luteomas apparently arising in corpora lutea appear after 150 days and grow progressively. Subsequently granulosacell-containing areas appear within and may replace the luteomas. I n general the tumor inducing environments are conducive of precocious senile changes. At times one wonders whether the hormonal environment is only indirectly responsible for these tumors-whether inherent potentialities are manifest because of precocious age changes. 8. Transplantability and Hormonal Functions of Ovarian Tumors
Many ovarian tumors arising in intrasplenic grafts or in irradiated ovaries are readily transplantable subcutaneously to intact or castrated male or female hosts (67,68,69,70), and occasionally metastasize to the liver and lungs (71). They are thus independent of the environment which gave origin to them. The incidence of successful transplants is not as high at the first transfer generation as subsequently. Some tumors grow more consistently when the hormone environment is modifled (72).
194
W. U. GARDNER
Evidence of estrogen, androgen, or progesterone production by the tumors exists, although some tumors exhibit no hormonal activity. Hypervolemia occurs in many tumor-bearing animals and cannot be associated with the tendency to produce known hormones (67,69,72). The administration of pituitary gonadotrophins should be tumorigenic if the above assumptions are correct. Several attempts have been made to induce ovarian tumors by such means (74,75). Because many preparations of gonadotropins have been antigenic and because of probable difficulties of getting proper levels of FSH and LH (luteinizing hormone) such experiments have been negative. Four small tumors were observed among twelve rats given daily a preparation of growth hormone for 500 to 700 days. Only one tumor occurred among the controls (76). Two tumors occurred among hypophysectomized rats, one of which received growth hormones for several hundred days (77). None of the above tumors was tested for malignancy. The largest attained a mass of only 336 mg. 3. Ovarian Tumorigenesis in Primates
Up to this time ovarian tumors have occurred under experimental conditions only in rodents. Intrasplenic ovarian grafts in ovariectomized monkeys have not become tumorous within two years; in fact the monkeys have shown regular menstrual cycles as revealed by vaginal bleeding and sex skin changes (78). There is no evidence that the liver of the rhesus monkey destroys estrogens (79,SO). At this time it is doubtful if the same experimental methods so successful in inducing ovarian tumors in rats or mice will be applicable to primates.
VI. TESTICULAR TUMORS Interstitial cell tumors of the testis have occurred in mice of some strains that have been exposed to estrogens for prolonged periods and more recently in rats bearing intrasplenic grafts of testes. The hormones of the pituitary have been implicated in the causal etiology of the tumors in both species. Estrogen-treated mice of most strains show marked testicular atrophy, both the spermatogenic epithelium and the interstitial tissue being decreased in amount (81,82). In some strains, however, the testes were either less markedly damaged or recover in part, after some initial regression (81,82,83). Mice of two inbred strains, A and C, have most consistently acquired such tumors after application of estrogens (Table XIV) although approximately 50% of the mice of the JK strain in one experiment had interstitial cell tumors. Several different estrogens were used in the experiments and with somewhat similar results in so far as can be
195
HORMONES AND EXPERIMENTAL TUMORIGENESIS
TABLE XIV Summary of Investigations on Testicular Tumors in Estrogen-Treated Mice ~
~
~~
No. No. Showing of Hyperplasia
Strain or Stock
Investigator Burrows (84) Gardner (81)
7 different A 5 different CaH, CBA, N, F 5 different
Hooker et al. (85) A Bonser and A Robson (86) R I11 Shimkin et al. (87) Hooker and Pfeiffer (83) Bonser (88) Gardner (89)
Bonser (90)
No. of Mice
162 15
29
S or E B T T
-
CBA C A
T S S
46 62 20
A A JK A CsH C121, CBA, N A
EB or S T T T T T T T T
96
n
IFS Gardner and Boddaert (91)
Estrogen Used*
(Cs7 X CBA)
TACE
23 61
TUor Duration mors Hypertrophy 5-9mo. 2.5-8mo. P9mo.
-
-
1
1-9mo. 2 20-69wk. 13 23 20-89 wk. - Slight hypertrophy 20-89 wk. - In t of mice 6-11 mo. 13 7mo. 2
17 14 16 9 39 20
6-14mo. 30-89wk. 8-16mo. 15-18mo. 9-22mo. 11-25mo. 20-99wk. 20-99 wk. 20-99wk.
39 8 7 7 1 0 8 3 4
92
8-26mo.
46
10 13
77 15
-
10
9 31 14
* 8,stiibeatrol; T, Triphenylethylene;EB, estradiol benzoate: TACE, Tri-para-aniaylchloroethylene. determined. Quantitative differences in estrogenic activity make such evaluations difficult. It is possible that triphenylethylene and its derivatives are more effective than the other estrogens (see below). The tendency for interstitial cell tumors in estrogen-treated mice is transmitted to hybrids derived by mating males or females of susceptible strains to mice of nonsusceptible strains and is not associated with the tendency to acquire mammary tumors or tumors of other endocrine glands (11,92). About 40% of the old untreated mice of one strain had either interstitial cell tumors or excessive interstitial tissue (93,94). Male mice of this strain uniquely acquire mammary tumors frequently indicating a probable high intrinsic production of estrogen. Such tumors occur rarely in male mice of all other strains. The humoral etiology of the tumors has been interpreted to be
196
W. U. GARDNER
pituitary gonadotropin, probably luteinizing hormone (LH) , or interstitial-cell-stimulating hormone (ICSH),the two being considered to be the same. Under certain conditions evidence of increased LH has been obtained subsequent to estrogen injections (95,96). Up to this time, however, critical experiments have not been undertaken to demonstrate the correctness of the assumed etiology. Attempts t o obtain interstitial cell tumors in mice by injection of gonadotropins have not been succesful. The strain-limited tendency for the appearance of testicular interstitial cell tumors in estrogen-treated mice may be due to (1) intrinsic differences in the testes or (2) of the pituitary gland, or (3) to differences in the metabolism of the hormones. The possibility exists that critical experiments may be designed to determine much more specifically what is transmitted from parent to offspring, but so far experiments of this type have not been reported. Some question now exists as to whether the strain differences are quantitative rather than qualitative. Hybrid X CBA) did not acquire testicular interstitial cell tumors when mice (CS, treated with estradiol benzoate (91). However, mice of the same stock did acquire such tumors when they were given tri-para-anisylchloroethylene (TACE) (91). Fifty per cent of the mice had such tumors. More recently interstitial cell tumors have been noted in many mice of the CS,strain that were similarly treated (97). Mice of this strain had pituitary tumors when treated with estradiol esters (98). TACE differs from some other estrogens in that it is stored in the body fat (99), and more estrogenic activity may be excreted than is injected (100). Further evidence of the involvement of the pituitary gland in interstitial cell tumors is indicated from their appearance in intrasplenic grafts of testes (Table XV). At least two tumorous intrasplenic testicular grafts were observed TABLE XV Testicular Tumors in Intrasplenic Grafts of Testes into Castrated Rats Age of Donor
No.
Age at Tumor No. of Host (months) Tumors _____~
~~~
Biskind and Biskind (101) Newborn 1 Newborn 25 Twombly e l d. (102) 60 50 Note: 83 had deteotsble nodules.
* Orohieotomised. t Overieotomised.
*
11
t
8-15 8-16
8
*
Type of Tumor
21 0 16 13
Interstitial cell Interstitial cell Interstitial cell
HORMONES AND EXPERIMENTAL TUMORIGENESIS
197
among castrated male rats that survived for eleven months, and other grafts were quite enlarged and contained abnormal tissues (101). In a more extensive experiment 100 gonadectomized male or female and twenty-five intact male rats received intrasplenic grafts of testes of newborn rats (102). Eighty-three animals had detacteble nodules of tissue in their spleens a t autopsy and twenty-nine of the gonadectomized rats had tumors, all interstitial tumors, one of which had extensive areas. of rhabdomyoblasts and other tissue elements and hence appeared teratomatous. Thirty-two testes grafted into the spleens of gonadectomized mice of the A strain failed to become tumorous within 275 days (102a). This experiment should be repeated; the animals should be followed for a longer period of time and animals of other strains should be used. The appearance of testicular tumors in the intrasplenic grafts could be explained on the same basis as ovarian tumors arising under comparable circumstances. If so, one must assume that the rat and mouse differ appreciably, either in the response of the pituitary gland t o castration or of the testis t o the gonadotropin produced. In the rat it seems that elevated levels of FSH were associated with both testicular and ovarian tumors, whereas in the mouse conditions assumed to reduce FSH (estrogen treatment) led t o testicular tumors. Attention must again be called to the experiments of Gans (53) who demonstrated that intrasplenic ovarian grafts in rats did prevent the usual increase in level of pituitary FSH. Whether testicular grafts would similarly prevent an elevation of FSH is unknown. The pituitary hormones must be involved in the origin of the testicular interstitial cell tumors, but the mechanisms are unknown. Until the proper mixtures of gonadotropin can be injected over prolonged periods the direct experiment will not be done. The testicular interstitial tumors in mice occasionally metastasize, the perirenal and lumbar nodes being most frequently involved. They are transplantable t o other mice of the same strain but usually require that the hosts receive estrogens for the first several transfer generations (86,103). The tumor tissue may persist without growth for several months when transplanted into untreated hosts and begin to grow rapidly later when estrogen is added. Once growth has started they grow progressively. The tumors produce androgenic effects in their hosts, sometimes even counteracting the effects of the injected estrogens. Histologically they consist of Leydig cells somewhat similar to those at several different stages of development (83).
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W. U. OARDNER
VII. ADRENAL TUMORS Two major types of tumor have appeared in the adrenal glands of animals that have been observed under experimental conditions ; pheochromocytomas and adrenal cortical adenomas or carcinomas. Five adrenal medullary tumors (pheochromocytomas) appeared in castrated and five in irradiated mice of different strains (104). One tumor was almost as large as the normal kidney. All the tumors arose in old mice, and none was observed in the control groups. Ten histologically similar tumors appeared in a group of fifteen rats treated daily with growth hormone for 350 to 485 days (105). Three of the tumors enlarged the adrenal glands and were apparent macroscopically. The humoral mechanism involved in the appearance of adrenal medullary tumors is vague although it is indicated, “that these changes are directly related to prolonged administration of preparations of growth hormone.” Adrenal cortical tumors have appeared in gonadectomized male guinea pigs (106) and in mice. Only in the latter species, however, have the observations been sufficient,ly extensive to merit much consideration here. One adrenal cortical carcinoma appeared spontaneously in a mouse of the C strain (107). Among intact and untreated mice of the NH strain, in at least one laboratory, adrenal adenomas and carcinomas occur quite frequently, thirteen adenomas and two carcinomas appearing among twenty-three mice (108). A few cortical adenomas appeared among mice of the Bagg Albino stock (109,110). The adrenal glands of both male and female mice of the dilute brown strain that were gonadectomized early in life frequently showed hyperplastic nodules of the cortical tissue and coincident hypertrophy of the mammary glands and other tissues that respond to estrogens (111,black and C6.r brown strains rarely 112,113). Castrated mice of the had nodular hyperplasia of their adrenal glands (114). Similarly castrated mice of the A and C3H strains rarely acquired adrenal carcinoma although cortical hyperplasia and adenomas were not uncommon (1 15). Mice of other strains have also been studied (Table XVI). Gonadectomized mice of the ce strain show an unusually high incidence of adrenal cortical tumors; among females the incidence is stated to be 100% after six months of age; among males the incidence is lower and they occur a t greater ages (116,117). Difficulty is encountered in comparing the incidence of adrenal tumors in mice of different strains and reported from different laboratories. In general two types of lesions have been described, nodular cortical hyperplasias and carcinomas and sometimes adenomas (Table XVI). The different lesions cannot be readily distinguished from all the reports.
HORMONES AND EXPERIMENTAL TUMORIGENESIS
199
Some tumors have been transplanted into intact and gonadectomized mice of the same strain and have grown progressively (118); they are not dependent growths. TABLE XVI Adrenal Changes in Gonadectomiaed Mice Strain
No. of Age Mice Castrated
Investigator
Woolley et al. (111,112,113) 217 1 day 15 43-65 days Gardner (120) Woolley et al. (116,128) Fekete and Little (129) Frantz et al. (109) Smith (115)
103 114 14 33
1-3 days 2 days Weaning 4 wk.
Smith (115)
33 4 wk.
Smith (115)
28 4 wk.
Frantz et al. (110) Dickie and Woolley Dickie and Woolley Dickie and Woolley Dickie and Woolley
(130) (130) (130) (130)
10 79 112 95 68
Weaning 1-3 days 1-3 days 1-3 days 1-3 days
Observations 179 cort. hyperplasias Cort. carcinomas hyperplasias 85 cort. carcinomas 109 cort. carcinomas
No carcinomas 9 cort. hyperplasia 4 carcinomas 29 cort. hyperplasia 8 carcinomas 13 cort. hyperplasia 10 tumors? 27 tumors? 93 tumors? 63 tumors? 45 tumors?
* Orchiectorniaed.
t Ovariectomized.
Most of the animals bearing adrenal tumors or nodular hyperplasias show evidence of abnormal hormonal stimulation. Some lesions produce estrogenic and others androgenic effects, as indicated by the condition of the responding tissues of their hosts (110,119,120) and by assay of the urine and feces (121). Adrenal lesions in mice of some strains are more frequently associated with evidence of androgen production, in others, with estrogen production (110). The appearance of hormone-producing adrenal lesions in mice of some strains following the cessation of ovarian activity or after gonadectomy can be interpreted t o indicate an abnormal adrenocorticotropic stimulation in the absence of gonadal hormones. Increased production of adrenocorticotropin in gonadectomized mice has not been demonstrated. Gonadectomized mice of the ce strain given stilbestrol did not have adrenal cortical tumors (122). The postcastration increase in gonadotropins would certainly be prevented by such treatment. The injection of estradiol, estriol, estrone, testosterone propionate, and testosterone dipropionate also prevented tumors whereas progesterone, cistestos-
200
W. U. GARDNER
terone, and desoxycorticosterone did not (123). The possibility that adrenocorticotropin might be tumorigenic was indicated in one experiment by Flaks (124). A highly malignant adrenal cortical tumor appeared in a castrated mouse of the Strong A strain that had received adrenocorticotropic extracts. As mentioned above, castrated mice of the A strain rarely had such tumors. A second tumor appeared in a stock mouse similarly treated. The strain differences in susceptibility to adrenal cortical tumors in mice permits another type of experimental approach t o the problem. The question might be asked whether the pituitary glands, the adrenal glands or the mechanisms for the inactivation of hormones differ among the several strains. Mice of the CsH strain (few adrenal lesions) were hybridized with mice of the ce strain (very susceptible to adrenal cancer) and it was found that the hybrids were also susceptible to adrenal tumors after gonadectomy. The tendency for adrenal cortical tumors was transmitted. When adrenals from mice of the CSH and ce strains were transplanted into gonadectomized-adrenalectomized hybrid mice (CsH X ce) only the adrenals from the ce donors became tumorous (125). This nice experiment demonstrates that the potentiality for tumors is within the adrenal glands and not in the environment because the adrenals of mice of both the CsH and the ce strains responded differently in the same environment. The same type of approach has revealed the differential susceptibility of pulmonary tissue to carcinogens; transplanted lung tissue from susceptible strains became tumorous in the hybrid while lung tissue derived from the resistant parent did not become tumorous (126,127). VIII. PITUITARY TUMORS Tumorigenesis of the pituitary gland has been studied primarily among the laboratory rodents. Among rats and mice of some strains small adenomatous areas occur frequently in the anterior lobe of the pituitary. Larger lesions occur most frequently in animals given estrogens, It is often difficult, as was true with the neoplastic adrenal lesions, to compare the reports from different laboratories because of differences in the methods of classification. In the writer’s opinion it is often diflicult to discern sharply between generalized chromophobe hyperplasias, which may be reversible, and chromophobe adenomas; the latter would presumably grow progressively. Arbitrary methods of classification have often been used such as weight or unusual localization of cells of the different types. I n general the tumors are not malignant and impair the well being of their hosts either because of interference with normal functions of adjacent structures or of endocrine function.
HORMONES AND EXPERIMENTAL TUMORIOENESIS
201
The pituitary glands of females are larger than those of males in most species of rodents studied in the laboratory and many investigators have reported that the pituitary hypertrophies in animals given female sex hormones (2,131,132). Furthermore, the pituitary gland of the intact female parabiont in union with a gonadectomized animal hypertrophies (Fig. 2), and other observations as well show that intrinsic hormones may cause either marked pituitary hypertrophy or tumors. Greene (133,134,135) noted that rabbits with an endocrine syndrome of hypergonadal nature had large pituitary glands and showed a generalized hypertrophy of the anterior lobe. A chromophobe adenoma was noted in one mouse with bilateral spontaneous granulosa cell tumors (136) and in rats bearing ovarian grafts (137,138). The incidence of pituitary tumors among untreated animals is quite low except in some strains of rats and mice that have been studied (130,139,140,141,142) so the association of pituitary tumors or hypertrophies with endocrine imbalances mentioned above may be of more significance than their infrequent occurrence would indicate. I n 1936, three groups of investigators reported the occurrence of chromophobe adenomas of the pituitary gland of rats and mice that had been subjected to prolonged estrogen treatment (Tables XVII, XVIII) TABLE XVII Pituitary Tumors among Estrogen-Treated Mice Investigator Cramer and Horning (145)
Burrows (167)
Gardner and Strong (98) Gardner (149) Gardner (150)
Strain
Number
Hormone Used
No. of Tumors
-
12 567 68 105 113
Estrin Estrone Estradiol benzoate Estradioi benzoate Estradiol benzoate
3 1 14 62 41
Stock Car (Cn X CBA) ((25, X CBA) and backcrosses
(143,144,145,146). Since then the observations have been repeatedly confirmed. Among both rats and mice strain difference in susceptibility to such tumors, which have usually been called chromophobe adenomas, have been described (98,147,148). Estrogen-treated mice of the Cay strain, in one laboratory, most consistently showed chromophobe adenomas; almost all the mice surviving for more than one year having such tumors (98). The tendency for these tumors was transmitted by both male and female mice to their first generation hybrids (149). Backcrosses of the F1 to the parental stocks indicated that thepredisposition for these tumors was transmitted genetically as a dominant (150).
202
W. U. GARDNER
TABLE XVIII Pituitary Tumors among Estrogen-Treated Rats Investigator Zondek (143) McEuen et al. (144) Wolfe and Wright (132) Zondek (168)
Strain
-
Hooded Deanesly (169) Noble et al. (170) Nelson (151,152) Selye (171) Segaloff and Dunning (148) Fischer A xC
No. of Rats
Hormone Used
5 11 37 41
Estradiol Estrone Estrin Estradiol benzoate Estradiol Estrone Stilbestrol Estradiol (Estradiol) (Stilbestrol) (Estrone)
32 15 28 10 69 21
No. of Tumors ? 5
9 (1)
22
3 ? 9 ? Extensive hypertrophy Moderate hypertrophy
Among rats the pituitary tumors have been said to regress following cessation of estrogen treatment (151,152),but evidence of regression has not been observed in mice (150). The tumors of both rats (153,154)and mice (155) have been transplanted and have grown progressively. Usually they are not completely autonomous and grow only in specific environments and after prolonged “dormant” periods. I n mice, when transplanted subcutaneously, the tumors have grown only in estrogentreated animals and have not become apparent until after ten or more months, a t which time tumors are usually present in the host’s pituitary gland (155). One group of investigators have designated the spontaneous tumors appearing in the mouse’s pituitary as basophilic tumors, in part because of the negative Golgi configuration, and have associated adrenal hyperplasia with their appearance (130). For the greater part these tumors were small, rarely enlarging the gland. Androgens prevent the appearance of tumors in rats given estrogens (148,156). Progesterone has been stated to both augment and inhibit the tumorigenic action of estrogens (157,158). Undoubtedly the doses are important. More recently pituitary tumors have been described in mice that have been made thyroid deficient by the injection of large doses of radioactive iodine (159). Although mice of four different strains were studied pituitary chromophobe adenomas were frequent only among mice of the Cb7strain, a strain particularly susceptible to such tumors subsequent to estrogen injection. Similar lesions were found in another laboratory in mice of the Ca7 strain following the injection of F. The first tumors
HORMONES AND EXPERIMENTAL TUMORIQENESIS
203
appeared ten months or more after treatment was started. All mice surviving fifteen months or more had tumors. The tumors grew following transplantation only in Ilalpretreated hosts (160). A benign hypertrophy of the pituitary glands (glands weighing 14.9 mg.) of Swiss mice given when 6 weeks old was noted when the mice were 8 to 11 months old. Unfortunately they were not followed longer because such lesions are probably pretumorous. Mice similarly treated but given also desiccated thyroid had no pituitary enlargement (161). Rats given comparable amounts of Ilal did not show either pituitary hypertrophy or tumors within eight months (162). Adenomatas of the intermediate lobe have been noted in two estrogentreated rats (144,163) and in hamsters (164,165). I n hamsters these tumors were locally invasive and attained relatively large size. Abnormal nodules in the pituitary glands occurred in eleven rats treated with growth hormone and in only nine of fifteen controls. The nodules (adenomata) were either single or multiple and composed of cells of either one or more types (166). Explanations of the rather well-substantiated observation on the appearance of pituitary tumors in estrogen-treated and thyroid-deficient animals are inadequate at this time. Certainly their appearance is species limited. Dogs, rabbits, and monkeys given estrogens for prolonged periods have not had such tumors nor have rats or mice of some strains, as mentioned above. Species or strain differences must exist in either (1) the responses of the pituitary gland, (2) the metabolism of the hormone, if we assume a metabolic product to elicit the abnormal response, or (3) of ancillary responses of other tissues and organs which are directly responsible for the abnormal hypophyseal response. Little is known of these possibilities a t this time. Furthermore, thyroid deficiency produced by large doses of P a l has given much greater hyperplastic responses of the pituitary than has thyroidectomy or treatment with antithyroid chemicals if one, for the moment, would not consider possible strain or species specificities; animals of the same strains have not been used in all experiments. All methods of thyroid deficiency do cause a degranulation of and reduction of number of acidophilic cells and changes in some basophilic cells. The possibility exists that the experimental conditions predisposing to pituitary tumors in suitable animals merely induce precociously nonspecific age changes that are tumorigenic. The pituitary glands apparently attempt to readjust to the altered environment produced by changes in concentration of hormones of their dependent organs. After prolonged attempts a t readjustment abnormal hyperplasia and neoplasia appear. The above attempt to explain the etiology of pituitary tumors
204
W. U. QARDNER
is certainly inadequate. Methods are available for further elucidation of the problem, but the experiments are tedious and time consuming.
IX. LYMPHOID TUMORS Lymphoid tumors occur more frequently in mice of some strains that have been exposed to high doses of x-rays, to carcinogenic hydrocarbons, or to estrogens than in untreated controls. Mice of the different strains may show greater increases in the incidence of leukemia after any one or two of the inciting treatments, but not necessarily after all of them. Furthermore, combinations of treatments may have an augmenting effect. The lymphomagenic action of estrogens is inhibited by testosterone propionate and more recently it has been shown that testosterone propionate similarly inhibits the lymphomagenic action of x-rays. Cortisone may have a similar effect. All aspects of the experimental work have been reviewed recently (172). Lymphoid tumors in estrogen-treated mice (Table XIX), apparently arising in the thymus, were first reported by Lacassagne (173). Similar tumors appeared among estrogen-treated mice of other strains (174). In mice of the CaH strain the incidence was increased about thirty times, 15 % of the estrogen-treated mice acquiring lymphoid tumors and about 0.5% of the controls (175). Furthermore this study revealed a high incidence of lymphomas among those mice of certain strains that received estrogens for only ten weeks or more. The lymphomas appeared several months after the cessation of hormone treatment in these instances. Mice given testosterone propionate in conjunction with estrogens had no more lymphoid tumors than the controls (176). Several different estrogens (estradiol, estradiol benzoate, estradiol dipropionate, estrone, equilin benzoate and stilbestrol) were effective. Estrogens have been administered in solution in oil (173,174,175,176,177,178,179,180,181,182) as pellets of pure hormone, or as pellets of hormone and cholesterol (87,176,179,183), or percutaneously in chloroform (185) with similar results. The lymphoid tumors in the estrogen-treated mice occur at earlier ages than in the controls. The tumors usually arose in the thymus and all the lymphoid tissues were often involved, probably secondarily (176). They are transplantable although in one laboratory attempts a t transplantation failed (184). The transplants grew either as localized lymphosarcoma, some of which later spread to involve remote tissues, or they spread throughout the body without appreciable local growth. Estrogens cause an involution of the thymus at least in intact animals. If estrogen treatment is continued more than ten weeks in mice, however,
205
HORMONES AND EXPERIMENTAL TUMORIGIENESIB
TABLE XIX Lymphoid Tumors among Estrogen-Treated Mice
Investigator
No. of Strains Used Mice
No. or % Lymphoid Tumors
Gerdner (174) Laaassagne (173)
A, CIH, CBA RI, 30, 39, 17
111 1
4 14
Gerdner st al. (176) Shimkin et 02. (87)
CaH C
136 61
22 7
Biechoff et al. (177,178)
Marah-Buffalo
192 &50%
CIH
97 747
2 108
CBA PM A Csr JK CllI
445 143 94 170 64 136 1799 1463
67 22 3 3 3 8 216 166
Shimkin and Wyman (183)
Gardner et al. (176)
CcH
Total Gardner and Dougherty 16 hybrid (179) groups Silberberg and Silberdbe berg (181) dba dba t dba d Dmochowski and Horn- H t ing (184) H d H X R t
*
Hd
Murphy and Sturm (180) RIL d Silberberg and giberCaH Q berg (182) CaH 0 CaH d CaH t Gardner and Boddaert (91) (CII X CBA)
Estrogen Used Several Eetrone bens. or EB. pit. Several Stilbestrol
+
depend- Eetrone or ing on group estriol
67 60 73 68 97 100 49 60 116 19 19 14 34
aa % 40 % 61 % 45 % 38 6 32 9 71.6% 4 6 1 2
92
13
Stilbestrol Several estrogens in different doses
Lymphoid Tumors in Controls % None mentioned None in 5 yr. 0.5
None until 24 rno old
.
5-18
0.6 1
3
0 0
5 3 0 1.2%
Several estrogens
Estradiol benzoate
4%
2%
0
Stilbestrol Estradiol henz.
TACE
42 % 0.7%
-
* Overiectomized. t Orohiectomized.
the thymus regenerates appreciably. The discontinuance of estrogens after this time does not reduce the incidence of tumors. If the injection of extrinsic androgens and estrogens effects the appearance of lymphoid tumors, one might ask whether or not a sex difference in lymphoma exists among untreated mice. Among mice of some strains [AK (185); RIL (180,186); dba, CBA and F (188); and C58 (189)] the incidence of lymphomas was higher in females than in males, although in some strains the differences were not great. The injection of testosterone propionate reduced the incidence of lymphomas in the high-leukemia
206
W. U. GARDNER
RIL strain. Castrated males usually had a higher incidence of lymphomas than did intact males (185,186,189). The incidence of lymphomas among irradiated female mice was almost always higher than among males (58,63,73,18SJ190,191). In some strains the differences were again quite small but in others the female mice acquired tumors two to three times as frequently as males. The great range of doses of x-rays given and difference in methods of application preclude comparisons of the figures obtained on mice of the same strain in different laboratories. Also the number of animals was not large in some experiments. The injection of extrinsic estrogens decreased the incidence of lymphomas in irradiated males, and testosterone propionate reduced the incidence in irradiated females (Table XX). Extrinsic androgens, if the treatment is started at the time of irradiation, inhibit the lymphomagenic action of x-rays (63,192,193). TABLE XX Influence of Testosterone Propionate (1.25 mg. weekly) and Estradiol Benzoate (16.6 pg. weekly) on the Incidence of Lymphomas in Irradiated Mice (380-280 r in one dose) of the BC Strain (63,192)
No. of Mice
Sex
Treatment
39 44 40 45 45 42
8
Sesame oil Testosterone propionate Estradiol benzoate Sesame oil Testosterone propionate Estradiol benzoate
3 3 9
0
9
No. with Lymphoid Tumors
5 4 28 21 9 23
Several investigators have reported the incidence of lymphomas among male and female mice treated with carcinogenic chemicals such as dibenzanthracene or methylcholanthrene (188,194,195,196). The incidence of lymphomas has generally been slighly higher in the females. Lymphoid tumors also occur in estrogen-treated rats (197), but it is questionable whether the incidence is higher than among the controls. The tumors usually involve the mesenteric nodes. Five of fifteen rats given six injections of purified growth hormone weekly for periods up to 685 days showed lymphoid invasion of the lungs at autopsy that were diagnosed as lymphosarcoma (198). All rats so treated showed some peribronchial lymphoid hyerplasia; a similar condition of the lungs was observed in some of the controls. These rats showed lesions of the adrenal and pituitary glands referred to earlier (105,199). Attempts to find a common mechanism for the lymphomagenic
HORMONES AND EXPERIMENTAL TUMORIGENESIS
207
action of estrogens and x-rays implicated the adrenal glands (176), although no direct experiments were available a t that time. I n animals with intact adrenals both x-rays and estrogens are followed by regression of the thymus and probably other lymphoid tissue. It was thought that both x-rays and estrogens acted in part on the lymphoid tissue through stimulation of the adrenal cortex. A decrease of successful “takes” of transplanted lymphoid tumors occurred in rats given adrenal cortical extracts (200,201) and adrenalectomy increased the number of “takes” of transplanted lymphomas (202) much as x-rays do. Adrenalectomized mice of the c68 strain showed an increased incidence of lymphoid tumors (189) and adrenalectomized mice of the c67 strain that were irradiated also showed a higher incidence of mediastinal lymphomas than did the irradiated intact controls (203). Irradiated mice given cortisone had fewer lymphomas. The lymphomagenic action of estrogens is not inhibited by cortisone (204). Experiments in which the corticoids have been used are a t present few in number. The preliminary experiments make the earlier hypothesis of the intermediary action of the adrenal cortex in lymphomagenesis following x-irradiation or estrogen treatment less probable. Prolonged exposure to estrogens may induce adrenal deficiency.
X. UTERINETUMORS Some of the earlier experiments implicated estrogens in carcinogenesis or in abnormal hyperplasia of the uterine cervix. Abnormal epithelial growths of the uterine cervix occurred in estrogen-treated monkeys, and in some animals in which the cervices were traumatized as well the lesions suggested malignancy (205). Further study, however, revealed that although metaplasia of the cervical epithelium did occur in monkeys given estrogens the lesions were reversible following cessation of treatment and when progesterone was injected (206), and that they did not become invasive or malignant even when estrogens were given for very long periods (207,208). Uterine or uterine cervical cancers occur rarely in experimental animals so that opportunities to study their association to possible abnormal hormonal states have been infrequent. Rabbits more frequently have uterine tumors than neoplasia a t other sites (209,210). Among rabbits of some stocks approximately 75% of the females from 5 to 6 years of age had uterine adenomas (133). Many of these tumors later metastasized and killed their hosts (211). The cancers occurred in animals that had had symptoms similar to toxemia of pregnancy, and adrenal and pituitary lesions indicative of an abnormal endocrine condition (134). Not only were the lesions associated with evidences of a
208
W. U. OARDNER
polyglandular dysfunction, but the cancers appeared in uteri that first showed generalized endometrial hyperplasia, then localized dependent neoplasia, and finally autonomous neoplasia (211). Furthermore, they appeared in rabbits of stocks that also showed high incidences of mammary cancer and abnormal mammary hyperplasia. Tumors of the uterine cervix were not noted, but three rabbits showing similar syndromes had epidermoid carcinomas of the vaginal wall apparently arising at the squamomucosal junction (212). Epithelial tumors of the uteri of untreated mice are extremely rare. Woglom (213) described one undifferentiated carcinoma of the uterus of one mouse, Thirteen of fifty-six untreated mice of one stock died with uterine cervical or uterine tumors (214) of which five were epidermoid carcinomas, seven undifferentiated cell neoplasia and one a spindlecelled sarcoma, Although objective evidence of a humoral etiology was lacking, these mice were from a stock that showed a high incidence of sterility and several females had imperforate vaginas. Untreated mice of other strains in the same laboratory had no epithelial tumors of the uteri or cervix. Uterine sarcomas occurred infrequently in untreated mice. Abnormal hyperplasias of the uteri have been repeatedly observed in guinea pigs that have had a considerable proportion of their ovarian tissue removed (6,215,216,217) or that had their ovaries irradiated (218). The animals showing the lesions were the ones that presented evidence of prolonged exposure t o intrinsic estrogens. Similar procedures did not induce abnormal growths of the uteri in monkeys (219,220,221). Four liomyomas (222) and one adenocarcinoma (223) appeared among rats that showed continuous or greatly prolonged estrous cycles subsequent to experimental imbalances induced by a method referred to previously (12). Other methods of inducing abnormal production of intrinsic hormones have been associated with evidences of abnormal uterine hyperplasia but rarely of malignancy in the laboratory animals. Abnormal growths or cancers have been observed in animals subjected t o extrinsic estrogens. These lesions have ranged from liomyomatous growths to uterine cervical cancer. Lipschutz and his associates have studied fibromuscular growths, arising from the uterine and also other peritoneal surfaces, in hundreds of guinea pigs given different estrogens by different methods. (See reference 6 for extensive list of references.) Depending upon the effective amounts of estrogen administered, the tumors appeared in from six weeks to several months, first appearing as serosal thickenings (tumoral seeds) and then growing t o large siae; some showing evidence of local invasiveness. The tumors invariably regressed after cessation of treatment. In addition t o the
HORMONES AND EXPERIMENTAL TUMORIQENESIB
209
parametrium and uterus the gastroepiploic area was an especially preferred site for these tumors. The term “tumorigenesis” was suggested rather than carcinogenesis because these lesions were benign. Animals given testosterone or its esters, or progesterone concurrently with tumorigenic doses of estrogens did not have tumors, and these substances were called antitumorigeaic. The tumors occurred later in intact females than in ovariectomized animals indicating that the intact gonads had some inhibiting effect on the extrinsic estrogens. The peritoneal areas of estrogen-treated males also showed the tumors. The earliest recognizable lesions were small serosal concentrations of macrophages. Hyperplastic fibroblasts appeared in relation to these areas and formed the greater part of the larger tumors. Similar fibromas, but limited to the uteri of estrogen-treated guinea pigs, had been described by others (224,225,226). Tumors of this type have occurred only in guinea pigs. Estrogen-treated rats (197) and rabbits (227) have infrequently had uterine or uterine cervical cancers. Several neoplasias have been observed in untreated animals of these species (228). Cancerous or precancerous invasive lesions of the uterine cervix that have appeared in estrogen-treated mice have been described by at least four different groups of investigators (Table XXI). Mice of all the strains that have been adequately studied and that have survived for prolonged periods, usually more than one year, have had uterine cervical carcinomas (229). Little evidence exists that mice of any one strain are more susceptible than animals of another strain. The fact that uterine cervical cancers occur, although infrequently, in estrogen-treated mice, and so rarely in untreated mice, indicates a causal association. The uteri and vaginas of estrogen-treated mice remain in a hypertrophic state for several months. Then, even when the hormones are still being administered some regression occurs. The vaginal smear no longer contains only cornified cells. The cervical mucosa becomes less cellular and sometimes almost myxomatous, and the basement membrane thickens. The mucosal connective tissue of the uteri becomes hyaline. Certain tissues of the female genital tract are apparently no longer able to respond normally to estrogens over prolonged periods. The capacity to resist infection is reduced and septic pyometra occurs in many mice. The columnar epithelium of the uterine cornua often undergoes squamous metaplasia or is replaced by an extension of the squamous cervical epithelium. The extent to which infection may be the cause of the epithelial metaplasias and connective tissue changes is unknown. Even if pyometra is not evident it is quite probable that the uteri of all estrogentreated mice have been septic at one time (230). Infection apparently
210
W. U. GARDNER
TABLE XXI Uterine Cerival and Uterine Tumors, or Pretumorous Lesions among Mice Given Estrogens or Estrogens Plus Other Substances
Investigator Lacassagne (235) Loeb et al. (236)
Strain
No. Hormones Duraof No. of or tion, TuMice Chemicals* Weeks mors
Several 40 Old Buffalo 1
11-29 104
Ee E
Perry (237)
-
27
E + dibenz.
Perry et al. (238)
-
75
E
Several
128
Es
Suntzeff st al. (239)
<28
+ dibenz. 26-40 < 106
Description
1 Epidermoid 1 Epidermoid cancerlike 3 Epidermoid cancerlike 3 Epidermoid cancerlike 26 Epidermoid cancerlike 19 1 carcinoma; 18 invasive lesions 3 Carcinoma 23 Invasive lesions
Several 100+ E B 38-52 Gardner et al. (240) Loeb et al. (241) Several 324 Es 4-80 Gardner and Allen 8 different 134 Es 55 (229) Es+ t.p. Es p. progesterone Allen and Gardner CC hybrids 44 E B 52+ 25 Carcinoma and invasive lesions (242) 36 Fibromata fibroMiller and Pybus 3 different 151 Es (243) sarcomata Crossen and Loeb CBA Invasive (244) C67 16 Ee 9 Precancer Pan and Gardner BC hybrids 35 EB or S Cancer (245) EB t.p. 29+ 13 Precancer
+
+
*
~~~~
E, estrone; dibenz., 3-4 dibenzanthracene; EB, eatradiol benzoate; EB,estrogen; t.p., testosterone propionate; 8, stilbestrol.
occurs through the cervix rather than through the blood. The usual regressive epithelial and mucosal changes were not observed in the transplanted and aseptic genital tissues of estrogen-treated mice (231). The somewhat abnormal distension of these grafts, however, permitted only limited interpretations from them. Whatever the real cause of the regressive changes they generally preceded the early evidences of neoplasia. At local areas, usually a t the distal cervical canal or outer surface of the cervix, small infiltrative epithelial growths occurred. Larger growths of the same general character were noted in other animals, growths that often could not be localized as to origin. I n other animals the epidermoid invasion replaced or occupied the entire cervical and
HORMONES AND EXPERIMENTAL TUMORIGENESIS
21 1
vaginal region; sometimes metastatic growths occurred. Several carcinomas grew progressively when transplanted into other hosts. Whether the small infiltrative lesions are neoplastic is not known but the nice sequences of lesions would indicate that they are. It is not impossible, however, that the same sequence of abnormal changes occurs in the genesis of these tumors in mice as in the rabbit (211). All the estrogens seem to be similarly tumorigenic. Testosterone propionate, when given concurrently, did not prevent the cervical lesions or cancers (229). In fact the incidence was somewhat higher because of the longer survival of these animals. The effects of progesterone have not been studied adequately. The cervix of the mouse is susceptible to cancer as indicated by effects of carcinogenic hydrocarbons. The aseptic cervices of young mice, when placed in contact with carcinogen and transplanted subcutaneously gave origin to epidermoid carcinomas (232). The intravaginal instillation of carcinogens in different solvents resulted in none or few uterine cervical or vaginal carcinomas (233,234). Two carcinomas appeared in mice in which estrogens were instilled intravaginally for prolonged periods (234). The total data available on the natural history of uterine cancers in rabbits and experimental uterine cervical carcinogenesis in mice affords appreciable evidence of the carcinogenic quality of environments high in estrogens. The necessity for prolonged exposure to the abnormal environment indicates a relatively great resistance to tumorigenesis at these sites in most of the animals that have been studied and certainly increases the possibility that the hormones. act indirectly as carcinogenic agents.
GLANDS XI. MAMMARY The role of hormones in mammary tumorigenesis has been investigated and reviewed most extensively (8,9). The superficial location and ease of identification of mammary cancer in laboratory animals must have abetted early investigations. The contributions of geneticists have provided abundant material for investigation on many aspects of the disease. Among the laboratory animals mammary cancers occur in mice, rats, and rabbits of some strains or stocks. Dogs not infrequently have mammary cancer (246). Guinea pigs, hamsters, and monkeys rarely or never have mammary cancer. The mammary glands are “end organs” of endocrine reactions; unlike the pituitary or adrenal glands, or the gonads they are not themselves involved in the initiation of changes in other tissues or organs except in a general way during lactation when their activity may necessitate exten-
212
W. U. GARDNER
sive metabolic demands. Under such circumstances lactation may be associated with such evidence of endocrine imbalances as prolonged anestrum or delayed implantation of fertile ova. Furthermore, the mammary glands, as modified skin glands, have acquired their function of postnatal nutrition of the young relatively late phylogenetically. They enjoy an endocrine control mechanism that is unusually complicated for simple end organs. Hormones of the ovary, anterior and posterior pituitary, all seem directly involved in either their growth or function (247,248,249). Species differences in mammary responses are probably greater than those of any other end organ. Among the laboratory animals mammary cancers are almost entirely found in females. This would implicate the ovaries and their hormones. Among mice of some strains they occur more frequently in animals that have been pregnant. Furthermore; removal of the ovaries reduces the incidence of or prevents mammary cancer in mice (exceptions to this have been noted, pages 198-200). Mammary tumors, however, did not occur in some of the earlier experiments in which ovaries were transplanted into males (250,251), but more recently the transplantation of ovaries into gonadectomieed males resulted in mammary cancer (252,253,254,255,256,257,258,259). In fact the incidence of mammary cancer in gonadectomized males of the A strain bearing subcutaneous ovarian grafts has exceeded that of virgin females of this strain but has not equalled that of multiparous females (Table XXII) (258). When mammary cancers have occurred in male mice (strain H of Athias, 93,94) TABLE XXII Mammary Tumors in Castrated Male Mice Bearing Ovarian Grafts Investigator Murray (252,253)
Strain dba dba
deJongh and Korteweg (269) 1 Loeb et al. (254) A, CaH, dba, etc. Huseby and Bittner (255) A X CaH A X CsH intact Silberberg and Silberberg A (256,257) A A A Huseby and Bittner (258) A
* Pituitary glands elso implanted.
No. of No. of Age at Transplantation Mice Tumors
4-6 wk. Controls to above 1 1
4-6 wk. 4-6 wk. 1 mo. 1 mo.* 7 mo. 7 mo.* 4 6 wk.
210 241
16
?
31 39 27 26 28 29 38
32
0 9 1
27 1
1 8
-
16
HORMONES AND EXPERIMENTAL TUMORIGENESIS
213
they were associated with evidences of elevated intrinsic estrogens. Ovaries transplanted into the subcutaneous tissues of intact males did not induce many tumors or mammary growth (255) although when transplanted into the testis mammary growth occurred (260). Rats of one strain frequently showed an endocrine imbalance indicating hyposecretion of the pituitary as indicated by frequent failure of ovulation, failure of luteinization, long estrous periods, decreased fertility, and reduced body growth (140,261,262,263). Mammary fibroadenomas occurred among those rats of this strain that showed the most exaggerated imbalances. Among rabbits of some stocks mammary cancers also occur frequently. These rabbits also show symptoms of an endocrine imbalance (135,264); multiple tumors of the genital tissues were not infrequent. Male mice, unlike males of some other species have very small mammary glands when fed on most diets (265). The addition of desiccated thyroid t o the diet, however, has resulted in some mammary growth (266). Because breeding female mice have a high incidence of mammary cancer from those strains in which mammary tumors have arisen in castrated males bearing ovarian grafts these experiments permit only the conclusion that mammary glands of some size are necessary for mammary cancer, that the male’s tissues are susceptible to cancer when properly stimulated and that one ovary in the castrate male stimulates mammary growth. The presence of intact testes prevented mammary growth and mammary tumors in hybrid males bearing ovarian grafts (255). Mammary cancer has been associated with abnormal endocrine states in rats and rabbits as noted above. Among mice, however, evidence of abnormal or unusual ovarian, adrenal or pituitary structure or function have been described but generally could not be correlated with tendencies to have mammary cancer. Several investigators have reported differences that existed between one or more strains that were susceptible to mammary cancer and one or more that were not susceptible. When animals of other strains were added, however, these correlations usually were weakened or no longer existed. For example, some studies have indicated a correlation between the incidence of mammary tumors and the length and regularity of the estrous cycles of strains susceptible and resistant to mammary cancer (267,268). When an additional strain was studied the correlation no longer existed (269). Subsequently more extensive studies, although they confirmed the existence of strain differences in certain characteristics of the estrous cycle, demonstrated no correlat,ion of any of these with the tendency to have mammary cancer (270,271,272,273,274,275,276). Strain differences in ovarian structure and the sequences of age changes have been described and associated
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W. U. GARDNER
with the tendency to have or not have cancer (277), but again the association has not been consistent when more extensive studies have been made (278). Among mice the strain differences in the mammary glands alone can be quite consistently correlated with the tendency to have mammary cancer (277,279,280,281). These differences become apparent sometime after sexual maturity and may well be early stages of an abnormal process culminating in mammary cancer. The glands of female mice of those strains susceptible to mammary cancer almost always show localized nodules of mammary tissue in glands that may otherwise be partially atrophic (280). The nodules differ greatly in structure, some being histologically similar to small lobules of normal growing mammary gland or lactating gland, some are abnormally hyperplastic but well organized, some are adenomatous and some seem to be regressing. Often nodules of several types are found in glands of the same animal. Mammary adenocarcinoma generally arise in these nodules. It seems quite probable that these localized abnormal areas-some obviously abnormal only in that they exist in otherwise atrophic or relatively atrophic glands-are attributable to the same direct causes as the mammary cancers. When present they have some autonomy; some will persist or grow after hypophysectomy (282) whereas hypophysectomy at early ages prevents mammary cancer (283). Some investigators have described strain differences in the “normal” structure of the mammary glands and they undoubtedly occur (284,285) but their associations with the tendency t o have mammary cancer is controversial (280,286). The reduction of ovarian function, whether by ovariectomy (251) or caloric restriction, reduced the incidence of mammary tumors (287). Inanition even reduced the evidence of estrogen production by hyperplastic adrenals of ovariectomized mice of the C3H strain (288). Inanition decreased the response of the mammary glands of mice to extrinsic estrogens (288a). Many comparisons have been made between mice of a mammary tumor susceptible and nonsusceptible strain. When differences exist they are of interest because they do demonstrate strain differences but they do not necessarily demonstrate correlations with a tendency for mammary cancer. For example, mammary-tumor-susceptible mice of the CsH strain retained 1‘01 in such a manner as to indicate a thyroxine content of 1.5 times greater than in the bodies of mice of the Cs7strain (289). Several other strains must be similarly studied before such observation can be correlated with a predisposition to cancer. The rather large number of experiments on the influence of extrinsic estrogens on mammary tumorigenesis in mice have been reviewed
HORMONES AND EXPERIMENTAL TUMORIGENESIS
215
repeatedly. The original experiments of Lacassagne (290) have been repeatedly confirmed. This aspect of the problem will not be reiterated here. In general, however, the administration of suitable amounts of all the estrogenic chemicals have been followed by mammary cancers in mice of suitable strains (291), and when the mammary tumor virus or agent is present (292,293). The incidence may be higher in the estrogentreated males of some strains than in the multiparous females of the same strain, but in general this is not true. Mice given very large doses of estrogen show less extensive mammary growth and fewer tumors than those given smaller amounts (15). The morphological changes in the mammary glands generally parallel those observed in multiparous females except that more cystic distension and more numerous hyperplastic nodules appear. More estrogen is required for mammary cancer than is necessary for the mere growth of the mammary duct system. In the writer’s opinion estrogens do no more than do the intrinsic hormones of multiparous females in their contribution to mammary carcinogenesis in the mouse. Both male and female rats from some stocks or strains given estrogens in large amounts acquire mammary cancers, either adenocarcinomas, squamous carcinomas, carcinoma simplex, or mixed tumors (Table XXIII). I n general the larger the amount of hormone given the sooner the tumors appear. Some of the tumors have metastasized to regional lymph nodes and the lungs (152,295) and are often multiple. Genetic or parentally transmitted tendencies for mammary tumors in rats are also revealed. A mammary tumor virus or agent has not been demonstrated however. Some of the earlier experiments revealed no tumorigenic action of estrogens in rats, but inadequate amounts were probably used. The very low incidence of spontaneous mammary cancers in untreated rats and their appearance in large numbers when large amounts of estrogens were injected, or smaller amounts absorbed from pellets, affords rather convincing evidence of the carcinogenic effect of estrogens. The incidence of mammary tumors in castrated estrogen-treated male mice of the CIH strain (299) and A strain was greater than in intact males similarly treated (300). These experiments indicate that the intact testes may produce enough androgen to oppose mammary tumorigenesis. Both experiments are somewhat inconclusive, however, in that in the first estrogen injections were stopped after five months, and in the second, animals of the A strain were used which are particularly susceptible to testicular interstitial cell tumors. Female mice given testosterone propionate in adequate amounts and beginning when 3 to 6 months of age had fewer tumors than did untreated controls (301,302,303,304). Because the amounts of androgen used
216
W. U. QARDNER TABLE XXIII Mammary Cancers in Estrogen-Treated Rats Investigator
Geschickter (294)
Stock Wistar Wistar
Wistar McEuen (197) Hooded Hooded Noble et al. (170) Geschickterand Byrnes Albino (295)
Eisen (296) Chamorro (297) Nelson (152) Dunning el al. (147)
Dunning et al. (298)
Sherman
Estrogen Used
Amt. 50-200 pg. daily Pellets 2.57.5 mg.
Estrone Estrone Estrone Estrone Estrone Several
No. of No. of Rats Cancers 12 11
12 8
Pellets Variable
38 34 49 555
22 2 28 202
1-20 mg. total
134
2
10
6
131 29 58 43
36 10 22 0
52
6
Irregular
80-200 pg. daily
Fischer
Estradiol dipropionate Estradiol benzoate Stilbestrol Stilbestrol Stilbestrol Stilbestrol
Fischer
+
weekly 50 daily Pellets Pellets Pellets 15-25 mg. Pellets
+
Pellets Pellets
59 58
0 39
+
Pellets Pellets
36 58
0 0
Pellets 4-6 mg.
67
58
-
Stilbestrol cholesterol AXC Stilbestrol AXC Stilbestrol cholesterol Copenhagen Stilbestrol Copenhagen Stilbestrol cholesterol AX C Stilbestrol
50-100 pg. 2 X
would impair ovarian function, however, mice castrated at the same ages should have been used as controls. Other investigators injected both testosterone acetate or propionate and estrogens simultaneously (305,306,307,308,309). Under such conditions testosterone reduced the number of mammary tumors. The most extensive study showed some tendency for fewer mammary tumors to appear when the amounts of androgen were largest in relation to the amount of estrogen (Table XXIV). The development of the mammary glands was also impaired and the number of hyperplastic nodules was reduced. The injection of progesterone with estrogen has given variable results; in some experiments the incidence of mammary tumors was increased (310), in others decreased (311), and in others had little or no effect (312). In all instances it is very probable that the amounts of hormone given were too small to be highly effective. From 1 to 2 mg, of proges-
217
HORMONE8 AND EXPERIMENTAL TUMORIGENESIS
TABLE XXIV Incidence of Mammary and Other Tumors among Mice of CsH Strain Which Received Both Estrogen and Androgen and Estrogens Alone Amount of Number Amount of Testosterone of Estrogen+ Propionate Mice pg. Weekly mg. Weekly 12 24 12 20 35 18 5 26 22 6
16.6 16.6 33.3 16.6 16.6 16.6 33.3 ES pellet 16.6 33.3
180 (total) 118 as above
Mammary Age at Age at Tumors Death Death No. and Range Av. Av. Age
Sex
Ages Started Range
0.625 1. 1. (12)* 1. 1.25 (lO)t1.25 1.25 1.25 2.5 2.
83 49 173 7 9 1 8 11 9 203 359 183 4 3 19 10 3 16 9 6 8 160 4 3 29
36-63 31-72 53-58 27-86 30-79 3444 54 30-52 24-114 29-31
177-576 449 134-733 376 205-479 389 156-652 440 221-659 429 241-535 445 181-234 209 98-258 183-553 373 176-572 409
1 (239) 2 (465) 1 (572)
none
9 7 3 21 9
27-97
131-569
407 356
17 (424) 51 (325)
1 (323)
4 (384) 5 (443) 2 (478) 1 (440)
* Estradiol benzoate or dipropionate unless specified; ES, estrone. t (12)
and (10). injection given weekly for twelve or ten weeks and then stopped.
terone are required daily to maintain pregnancy in mice that have been ovariectomized (313). When pellets of progesterone were given in such a manner as to assure the average daily absorption of 0.5 mg. the incidence of mammary tumors was increased, and the age a t which tumors appeared was decreased (Trentin, unpublished). Foulds (314) has described the repeated reappearance of mammary tumors in mice during pregnancy with regression between pregnancies. Eventually many of these tumors became autonomous and grew progressively. Progesterone levels are high during pregnancy (315), and this might have been the cause for the repeated appearance of the dependent mammary tumors. An “inherited hormonal influence” has been described recently t o account for the lower incidence (3.9%) of mammary tumors in virgin mice of the A strain as compared with multipara (86.7%) of the same strain (316). I n mice of the CaH strain the incidence of mammary tumors in virgins was 63% and in multipara 95.1%. Other investigators have reported less divergent incidences in virgin and multiparous mice of the CsH strain (317). In general the incidence was higher in the F1 and Fz in both the multiparous and virgin animals than in either parental stock. This difference is probably not due to a difference in the milk transmitted virus (291). Neither the estrous cycles of mice of the A and CIH strain (275) nor the estrous cycles of ovariectomized F1 mice
2 18
W. U. GARDNER
bearing transplanted ovaries of either A or C3H mice were significantly different. The “inherited hormonal influence” is a somewhat vague entity and is manifest only in virgin mice of some strains. The term probably could be applied to strain differences in the tendencies to have pituitary, or adrenal, or testicular tumors for example. Some preliminary investigations have indicated that mice with the mammary tumor virus excrete less 17-ketosteroids in their feces than do mice without the virus (318,319). High levels of ketosteroids might indicate high levels of androgenic mubstance and hence promote an endocrine environment not permitting mammary tumors. If so, it is not revealed by differences in the vaginal responses to estrogens (20,21,276,320). It would certainly be extremely interesting if the mammary tumor virus did modify the metabolism of hormones or their production whether or not this might have any effect on mammary tumorigenesis. If mammary tumorigenesis were controlled through such a mechanism, one might expect that mammary tumors could be more readily induced in the absence of the virus by injection of estrogenic hormones. That the absence of the agent can be compensated for is indicated by some experiments indicating that carcinogenic hydrocarbons may induce mammary cancer (321,322). In general estrogens are less effective in inducing mammary cancer in mice lacking the mammary tumor virus than are carcinogenic hydrocarbons. Mammary tumors although of somewhat different types may occur in mice subjected to intensive breeding but presumably without the milk agent (149). Approximately 40% of “force bred” female mice born of low-tumor mothers (Cb,) had mammary tumors. Thirty-eight per cent of 188 intensively bred, agent-free mice of the C3H strain acquired mammary tumors (323). Irradiated C3Hb mice without mammary tumor agent (0.11 to 8.8 r daily during eight hours) also showed an increased incidence of mammary carcinomas but surprisingly a high incidence of mammary sarcoma (324). Most of these mice had ovarian tumors as well. Furthermore, it is well known that carcinogenic hydrocarbons increase the incidence of mammary tumors in agent-free mice (see 325 for review). The carcinogenic hydrocarbons are however effective only in females in which mammary growth is induced by intrinsic hormones (326) or when extrinsic hormones are added. More tumors tend to show squamous metaplasia (321,322). These tumors arising in carcinogentreated mice without the mammary tumor virus did not contain the virus (327). This indicates that in mice mammary tumors may have divergent causes. I n one instance estrogenic hormones, plus a suitable genetic makeup, plus the mammary tumor virus are needed for mammary cancer, in the other, carcinogenic hydrocarbons, plus intrinsic estrogens.
HORMONES AND EXPERIMENTAL TUMORIGENESIS
219
What part do hormones play in mammary cancer? At the very least they are necessary for some development of the mammary glands; the “substrate” in which tumors can develop. Furthermore it seems that hormones must be necessary for the appearance and early progressive growth of many of the premalignant hyperplastic nodules (282,328) in which mammary cancers arise frequently in mice with the mammary tumor virus. Furthermore, some of the small “virus induced” nodules may require estrogens for their continued growth, in other words, be hormonally dependent. Indeed some mammary tumors in mice grew only when transplanted first into female or estrogen-treated male mice (329). The progressive growth of these dependent tumors depended more upon the proper hormonal environment than the presence of the mammary tumor virus. The possibility exists that a proper hormonal environment is necessary, not only for early mammary growth, but for growth of some abnormal hyperplasias or dependent neoplasias, although these are caused by the mammary tumor virus or carcinogens. The assumption of independent growth-cancer-has really not been proved to be caused by either the mammary tumor inciting virus or hormones. Testosterone propionate seems to inhibit not the virus but the hormonal action of estrogens on the glands and dependent localized hyperplasias. XII. HORMONES IN RELATION TO TUMORS OF SECONDARY SEX ORGANSOF MALES
THE
Males of several species have been carefully examined after prolonged exposure to estrogens. The secretory epithelium of the prostate, seminal vesicles, and of other adjunctive glands that occur in animals of some species regresses when the amounts of estrogen are sufficient to impair the hormonal activity of the testes. The fibromuscular tissues hypertrophy relatively. The glandular epithelium of some of the glands shows squamous metaplasia; the coagulating glands of mice often become filled with desquamated cornified cells and abscessed. Metaplasia of the urethral epithelium also occurs, a t least in mice (330) and monkeys (331). Male dogs and mice often show marked distension of the urinary bladder due to urethral occlusion a t the proximal part of the cavernous urethra. The above abnormal responses have not led to malignant growths and, because they have been adequately considered in earlier reviews (2,9) will not be cited here. Tumors of the male’s accessory genital tissues are rare in most species except in man and the dog. In dogs prostatic hypertrophy is often associated with an endocrine disturbance indicative of abnormal steroid metabolism (332,333,334) but the attempts made to reproduce other than hypertrophy and metaplasia have been futile to date. It is probably
220
W. U. GARDNER
significant that neoplasia occurs so rarely in these tissues of other laboratory animals and that they have not been induced by hormones. The potentialities for such a response must be very slight. Nevertheless, rats of several strains have acquired squamous carcinomas of the prostate when carcinogenic hydrocarbons were applied directly (335,336). Malignant epidermoid and glandular carcinomaa have arisen in transplanted prostatic tissues of mice that were in contact with carcinogens (337,338,339). The glandular tumors usually grew subsequent to transplantation only in mice given testosterone propionate and underwent squamous metaplasia when stilbestrol was injected. The prostatic tissue of rats and mice is therefore not refractory to malignant changes. Although hormones may be associated with prostatic cancer in man they have not been so determined in other animals.
XIII. OTHER TISSUESOR ORGANSIN WHICHSEX OR SEX HORMONES MODIFY THE APPEARANCE OF TUMORS 1. Liver
Hepatomas occur in mice of some strains (340). Several investigators have noted the more frequent occurrence of hepatomas in male than female mice and the tendency for castration to reduce the incidence of tumors among the males (Table XXV). The incidence of such tumors TABLE XXV Influence of Sex on Appearance of Hepatomas in Mice
Investigator Gorer (341). Pybus and Miller (342)
Strain
Sex
CBA
d 0 d 0 d
CBA CBA
Andervont and McEleney Ca€I (317) Burns and Schenken (344) CsH Burns and Schenken (345) CaH
Andervont (346)
CsH
CBA
0
d 0
3 0
d
0 Orchiectomized 3 0
Orchiectomized
No.
Age Months
No. or %with Hepatomas
39 >14 14 39 >14 4 285 Life span 116 (28.6mo.) 229 Life span 61 (30.1mo.) 320 >12 26.8% (15.3mo.) 9.9% (17.5mo.) 141 >12 38 16 (15.3mo.) 46 0 (12.9mo.) 60 16 (15.5 mo.) 47 -(14 mo.) 426 >15 144 541 >15 51 183 >15 21 80 92 32
>15 >15 >15
23 4 8
HORMONES AND EXPERIMENTAL TUMORIGENESIS
221
is generally lower in castrated than in intact males. The effect of extrinsic hormones on the incidence of hepatomas is less compatible with the above interpretation. The incidence of hepatomas was increased in estrogen-treated male mice of the CaH strain and testosterone propionate failed to increase the incidence in females (347). 2. Bone
The sex hormones have been associated with dimorphism of the skeleton for many years (348) and certainly produce local or general skeletal responses in animals. Osteogenic tumors occurred in female mice of one strain (77.3 '3%) more frequently than in males [29.6% (349,350,351)l. The tumors arose from different parts of the skeletal periosteum and were often multiple, indicating multifocal origins and possible general humoral influences in their origin. Strain differences in the response of osseous tissues of mice do exist, the strain susceptible to estrogenic tumors showing a less organized osteoblastic response (349,352).
XIV. URINARY TRACT Sex differences exist in the kidneys of mice (353,354), the lining of Bowman's capsule being composed of cuboidal or columnar cells more frequently in males. Testosterone propionate accentuates the sex difference (354). After sexual maturity the levels of renal glucuronidase (355) and distribution of alkaline phosphatase differ in mature animals of the two sexes (356). Male hamsters given stilbestrol, either as pellets or injected in solvents, show tumorous growths after 200 days or more (164,357,358). The tumors apparently arose both in the renal pelvis and the cortex, often from multiple foci. They attained sizes almost as great as the normal kidney, and were histologically malignant although metastases were never observed. Female hamsters, similarly treated, did not have such tumors. Cancer of the bladder and papillomas of the bladder occurred in rats bearing subcutaneous pellets of stilbestrol or stilbestrol-cholesterol(l47). These tumors occurred most frequently among rats of one strain and were always associated with urinary calculi. Calculi also occur in the bladders of estrogen-treated mice but neoplasia has not been reported.
XV. GENERALDISCUSSION Although repeated attempts have been made to associate the abnormal metabolism and excretion of hormones, especially the steroid hormones, with the appearance of cancer in man, few such attempts have been made in experimental animals. The small size of most of these animals adds to the complexity of the problem. Small amounts of most hormones are
222
W. U. GARDNER
produced and the technics available for quantitative determination of the small amounts of hormones or metabolites are scarcely adequate. Nevertheless much has been learned of the internal environment by the observation of the animal’s own end organs. To be sure, these observations are not always specific or uninfluenced by associated supplementary or opposing substances. In some instances it has been possible to associate tumors with evidences of abnormal endocrine environments. In other instances it has been possible to modify the intrinsic production of hormones so that abnormal hormonal environments are produced and so that tumors appear. Other tumors, for example, mammary tumors in mice, develop in hormonal environments that do not seem to deviate from those compatible with normal reproductive functions. The use of hormones tagged with radioactive atoms may help in determining whether the hormone itself or one of its metabolic products is carcinogenic. Neither, however, may be carcinogenic. Hormones may incite the formation of carcinogenic substances or changes in “end organs,” and there is certainly no indication as to what these may be; they may be quite different from the hormone. If so it is quite probable that endocrine stimulation may merely accelerate the appearances of these substances or changes, that would normally appear with advancing age should an animal live long enough. Superficial association of hormones, predominantly growth or hyperplasia inducing hormones, with tumors of certain organs or tissues, has been demonstrated repeatedly. In general it is the more highly differentiated tissues or organs that are affected; organs that respond in a cyclic fashion to cyclic changes in the hormonal environment. The numerous instances in which other carcinogenic stimuli can induce similar neoplastic responses predisposes to the hypothesis that hormones may act indirectly; some common direct mechanisms being involved. The changes induced by prolonged hormonal stimulation that leads to cancer may be nonspecific. The sequences of premalignant responses of tissues are in general quite similar in the different tissues or organs that become cancerous when subjected to abnormal hormonal environments. First growth is produced and maintained for variable periods; then the growth response ceases and the organs may actually regress. At this time localized hyperplastic foci appear. These early localized growths seem to be a t first dependent upon special hormonal environments for their persistence and growth but subsequently either become, or give origin to cells that do become, autonomous. The sequence of stages or steps are often sufficiently long to be evident in many of the neoplastic responses to abnormal hormonal environments.
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223
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35. Biskind, G. R., and Biskind, M. S. 1949. Am. J . Clin. Path. 19,501-21. 36. Biskind, M. S., and Biskind, G. S. 1944. Proc. SOC.Exptl. Bibl. Med. 66, 176-79. 37. Peckham, B. M., Greene, R. R., and Jefferies, M. E. 1948. Science 107, 319-20. 38. Li, M.H., and Gardner, W. U. 1947. Cancer Research 7, 549-66. 39. Furth, J., and Sobel, H. 1947. J . Natl. Cancer Inst. 8, 7-16. 40. Li, M. H., and Gardner, W. U. 1949. Cancer Research 9,35-41. 41. Li, M. H.,and Gardner, W. U. 1950. Cancer Research 10, 162-65. 42. Klein, M. 1952. J . Natl. Cancer Inst. 12, 1003-1010. 43. Frantr, M., Kirschbaum, A,, and Casas, C. 1947. Proc. SOC.Exptl. Biol. Med. 66, 645-46. 44. Rush, B. F. 1950. Proc. SOC.Exptl. Biol. Med. 74,712-14. 45. Twombly, G.H., and Taylor, H. C. 1942. Cancer Research 2, 811-17. 46. Miller, 0.J., and Pfeiffer, C. A. 1950. PTOC. SOC. Exptl. Biol. Med. 76, 178-81. 47. Boddaert, J. Unpublished. 48. Biddulph, C., Meyer, R. K. and Gumbreck, L. G. 1940. Endocrinology 26, 280-84. 49. Bymes, W.W.,and Meyer, R. K. 1951. Endocrinology 48, 133-36. 50. Bunster, E., and Meyer, R. K. 1938. Endocrinology 23, 496-500. 51. Greep, R. 0. 1940. Proc. SOC.Exptl. Biol. Med. 44,214-17. 52. Biddulph, C., and MeyerkR. K. 1946. Proc. SOC.Exptl. Biol. Med. 63, 9295. 53. Gans, P. 1950. A d a Physiol. Pharmacol. Neerland. 1, 229-87. 54. Gaarenstroom, J. H., deJongh, 5. E., and Paesi, F. J. A. 1950. Actu Physiol. Pharmacol. Neerland. 1, 90-98. 55. Miller, 0.J., and Gardner, W. U. Unpublished. 56. Jungck, E. C., Heller, C. G., and Nelson, W. 0. 1947. Proc. SOC.Exptl. Biol. Med. 66,148-52. 57. Brambel, F. W.R., and Parkes, H. S. 1927. PTOC.Roy. SOC.(London) 101, 316-28. 58. Furth, J., and Furth, 0. B. 1936. Am. J . Cancer 28, 54-65. 59. Furth, J., and Boon, M.C. 1947. Cancer Research 7, 241-45. 60. Furth, J., and Butterworth, J. S. 1936. Am. J . Cancer 28, 66-95. 61. Lick, L.,Kirschbaum, A., and Mixer, H. 1949. Cancer Research 9,532-36. 62. Li, M. H., Gardner, W. U., and Kaplan, H. S. 1947. J . Nutl. Cancer Inst. 8, 91-98. 62a. Kaplan, H. S. 1950. J. Natl. Cancer Inst. 11, 125-132. 63. Gardner, W.U. 1950. Proc. SOC.Exptl. Biol Med. 76, 434-36. 64. Chang, C. H.,and Van Eck, G. 1952. Cancer Research 12, 254 (ribs.). 65. Geist, 5. H., Gaines, J. A., and Pollack, A. D. 1939. Am. J . Obstet. Gynecol. 38, 786-97. 66. Butterworth, J. S. 1937. Am. J . Cancer 31, 85-99. 67. Furth, J., and Boon, M. C. 1945. PTOC.SOC.Ezptl. Biol. Med. 68, 112-14. 68. Furth, J. 1946. Proc. SOC.Exptl. Biol. Med. 61, 212-14. 69. Furth, J., and Sobel, H. 1946. J . Natl. Cancer Inst. 7, 103-13. 70. Furth, J., and Sobel, H. 1947. Cancer Research 7, 246-62. 71. Li, M. H. 1948. Am. J . Obstet. Gynecol. 66, 316-20. 72. Cliffton, E. E., and Wolstenholme, J. T. 1949. Cancer Research 9, 331-35. 73. Kaplan, H. 5. 1947. Cancer Research 7, 141-47. 74. Pfeiffer, C. A.,and Hooker, C. W. 1942. Anat. Record 84,311-29.
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Heiman, J. 1945. Cancer Research 6, 426-30. Burrows, H., and Hoch-Ligeti, C. 1946. Cancer Research 6, 60849. Crossett, A. D., Jr. 1950. Thesis, Yale University School of Medicine. Foulds, L. 1949. Brit. J. Cancer 3, 345-75. Forbes, T. R. 1948. Anat. Record 100, 29-30. Bittner, J. J., and Huseby, R. A. 1946. Cancer Research 6 , 235-39. Andervont, H. B., and McEleney, W. J. 1941. J . Natt. Cancer Inst. 1, 73744. Bittner, J. J. 1948. Cancer Research 8, 625-39. Samuels, L. T., Bittner, J. J., and Samuels, B. K. 1947. Cancer Research 7,722. Armstrong, E. C. 1948. Brit. J. Cancer 2, 59-69. Strong, L. C., and Williams, W. L. 1941. Cancer Research 1, 886-90. Kirschbaum, A., Williams, W. L., and Bittner, J. J. 1946. Cancer Research 6, 354-62. Heston, W. E., Deringer, M. K., Dunn, T. B., and Levillain, W. D. 1950. J. Natl. Cancer Inst. 10, 1139-51. Lorenr, E., Eschenbrenner, A. B., Heston, W. E., and Uphoff, D. 1950. J . Natl. Cancer Inst. 10, 947-61. Dmochowski, L., and Orr, J. W. 1949. Brit. J. Cancer 3, 376-84. Strong, L. C., and Smith, G. M. 1939. Yale 1.Biol. and Med. 11, 589-92. Dmochowski, L., and Orr, L. 1947. Brit. J . Cancer 3, 520-25. Pullinger, B. 0. 1947. Brit. J. Cancer 1, 177-91. Foulds, L. 1949. Brit. J. Cancer 3, 24046. Burrows, H. 1935. Am. J. Cancer 23, 490-512. van Wagenen, G. 1935. Anat. Record 63, 387403. Greulich, W. W., and Burford, T. H. 1936. Am. J. Cancer 28, 496-511. Huggins, C., and Moulder, P. V. 1945. Cancer Research 6, 510-14. Zuckerman, S., and McKeown, T. 1938. J. Path. Bact. 46, 7-19. Dunning, W. F., Curtis, M. R., and Segaloff, A. 1946. Cancer Research 6, 256-62. Moore, R. A., and Melckionna, R. H. 1937. Am. J . Cancer 30, 731-41. Homing, E. S. 1946. Lancet 2, 829-32. Homing, E. S. 1949. Brit. J. Cancer 3, 211-30. Homing, E. S., and Dmochowski, L. 1947. Brit. J . Cancer 1, 59-63. Strong, L. C., and Smith, G. M. 1936. Am. J . Cancer 27, 279-84. Gorcr, P. A. 1940. 17th Ann. Rept. Brit. Emp. Cancer Camp., p. 232. Pybus, F. C., and Miller, E. W. 1942. 19th Ann. Rept. Brit. Emp. Cancer Camp., p. 42. h d e r v o n t , H. B. 1939. U.S. Pub. Health Rept. 54, 1158-69. Burns, E. L., Schenken, J. R. 1940. Am. J . Cancer 39, 25-35. Burns, E. L. 1943. Cancer Research 3, 691-92. Andervont, H. B. 1950. J . Natl. Cancer Inst. 11, 581-92. Schenken, J. R., and Burns, E. L. 1943. Cancer Research 3, 693-96. Gardner, W. U., and Pfeiffer, C. A. 1938. Proc. SOC.Exptl. Biol. Med. 37, 678-79. Miller, E. W., Orr, J. W., and Pybus, F. C. 1943. J . Path. Bact. 66, 137-50. Pybus, F. C., and Miller, E. W. 1938. Am. J . Cancer 34, 248-51. Pybus, F. C., and Miller, E. W. 1940. Am. J. Cancer 40, 47-61. Gardner, W. U. 1944. Report a t 6th meeting of Conference on Metabolic Aspects of Convalescence Including Bone and Wound Healing. Josiah Macy Jr. Pub., pp. 84-88.
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353. Crabtree, C. E. 1941. Endocrinology 29, 197-203. 364. Pfeiffer, C. A,, Emmel, V. M., and Gardner, W. U. 1940. Yale J . BioZ. and Med. 12,493-501. 366. Morrow, A. G., Carroll, D. M., and Greenspan, E. M. 1951. J . Natl. Cancer Inet. 11, 663-69. 356. Dunn, J. B. 1948. Am. J . Path. 24, 719-20. 357. Matthews, V. S., Kirkman, H., and Bacon, R. L. 1947. Proc. Soc. Exptl. Biol. Med. 66, 195-96. 358. Kirkman, H., and Bacon, R. L. 1950. Cancer Research 10, 122-24. 359. Schenken, J. R., Burns, L. E., and McCord, W. M. 1942. Endocrinology SO, 344-52.
Properties of the Agent of Rous No. 1 Sarcoma R. J. C. HARRIS* Chester Beatty Research Institute, Royal Cancer Hospital, London, England CONTENTS
Page I. Introduction,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Agent and Host ................................... 1. Localization and Distribution of Agent.. . . . . . . . . . . . . . . . . . . . . . . . 235 2. Adaptation and Variation of Agent.. . . . . . . . . . . . . . 3. Inhibition of Growth of Rous No. 1 Sarcoma i n uiuo . . . . . . . . . . . 240 4. Assay of Rous No. 1 Agent.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241 111. Agent and Malignant Cell.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 1. Histogenesis of the Tumor.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 2. Electron Microscopy of Chicken Tumor Cells.. . . . . . . . . . . . . . . . . . . . . . . 245 3. Agent and Cell Metabolism.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 IV. Isolation and Properties of Rous No. 1 Agent,. . . . . . . . . . . . . . . . . . . . . . . . . 250 ............................................... 250 2. Size of Agent.. .:. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 3. Lipid Content of Agent.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 4. Stability of Agent.. . . . . . . . . . . . . . . . . . . . . ...................... 257 A. pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 B. Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258 C. Chemical Inactivation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258 D. Enzymic Inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258 E. Irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 F. Freezing and Freeze-Drying ........ ........................ 259 5. Immunological Properties of Rous Agent. .......... V. Relationship of Rous Agent to Fowl Tumors and Leucoses.. . . . . . . . . . . . . . 261 1. Carcinogen-Induced Fowl Tu ................. 2. Fowl Leucoses.. . . . . . . . . . . . . ................................. 262 VI. Origin of Rous Agent.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264 VII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. ............................................
...
I. INTRODUCTION The filterable fowl tumors were last reviewed by Foulds in 1934, but no recent review has been devoted entirely to the properties of the filterable agent of the Rous No. 1 sarcoma. It is by no means generally agreed that the actual Rous agent has ever been prepared. Beard (1948) stated that it is improbable that the observations on the particles isolated
* Junior Research Fellow, British Empire Cancer Campaign. 233
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R. J. C. HARRIB
by Claude and others have any bearing on the properties of the Rous sarcoma virus, because studies of the sedimentation velocities of the preparations have given no evidence of homogeneity. Viruses which have been obtained in states of significant “purity” have revealed, according to Beard, (1) characteristic forms in electron micrographs, present in repeated examinations, in amounts greatly predominant over other particles; (2) single boundaries in the ultracentrifuge compatible with the variation in size found in electron micrographs; (3) single boundaries in the Tiselius electrophoresis apparatus; (4) uniformity of chemical constitution. Judged by these criteria it must be admitted that Rous agent preparations hitherto described are not composed of “pure ” or “homogeneous” agent. It is possible, however, that Beard’s criteria are inadequate. It is immediately apparent that biological criteria, such as infectivity, are omitted. Criteria (1) and (2) depend upon the results of electron microscopy, but the selection and preparation of specimens for examination and the evaluation of the results are all highly subjective procedures. How many specimens require t o be examined for example, for the assessment of the homogeneity of a virus? Moreover, “virus-like” particles appear to be derivable from almost all mammalian tissues and, in those malignant tissues studied by De Witt Fox (1951), are held t o have a “morphology characteristic of the tissue.” The paper quoted, and the electron micrographs recently published by Gregory (1948, 1951) lend excellent support for ROUS’contention that “the tumor problem is the last stronghold of metaphysics in medicine.” Fox’s estimate of the number of samples which require to be examined for the determination of homogeneity and also of the subjective but otherwise highly satisfactory (!) nature of the statistical results, are illustrated in this extract from the table: ’
Tissue
Rous sarcoma Chick embryo
No. of Specimens Examined 2 3
No. in Which “Bodies” Were Present 2
1 (?I
No. in Which “ Bodies ” Were Absent
%
0
100.00
2
33.33
So far as criteria (3) and (4) are concerned it should be remembered that serological differences in proteins can exist that are not, a t present, perceptible by electrophoretic measurements and that “uniformity of chemical composition ” is meaningless when applied t o particles with weights 1 X lo8 greater than that of the hydrogen atom.
AGENT OF ROUS NO.
1
SARCOMA
235
We need not, therefore, for these reasons be deterred from a consideration of the properties of preparations of Rous No. 1 sarcoma agent. Preoccupation with the chemical and physical properties of isolated viruses has been the inevitable result of a situation in which the extracellular infective phase of a virus was the only experimentally accessible one. Attention is now being turned increasingly to the more fundamental problems of the modes of intervention of the infecting virus in the metabolism of the cell and the nature of the cell’s response, involving as it does, the replication of the infecting particle. In the consideration of the avian tumor viruses this approach, hitherto entirely neglected, is of overriding importance since these viruses have a primary action on the infected cell resulting in the production of a malignant cell and are, in fact, the only carcinogenic agents which are known to produce tumors by direct action (Rous, 1943). The knowledge, therefore, of the nature of the cellular metabolic changes accompanying infection may provide valuable data towards the elucidation of the more general problem of the relationship of the normal to the malignant cell, whether the change from normality to malignancy has been brought about directly as in the filterable tumors, or indirectly, as in the carcinogen-induced tumors. 11. AGENTAND HOST 1 . Localization and Distribution of Agent
Rous et al. (1912) observed that the tumor-producing efficiency of a filtrate of Rous No. 1 sarcoma could be augmented by the prior or simultaneous injection of diatomaceous earth and further, that although intravenous injection of filtrates rarely produced tumors (but see DuranReynals, 1940b), in a number of cases tumors were found in the ovary of laying hens, where injury and proliferation occur daily. These observations were confirmed by Pentimalli (1924). The agent was injected intravenously and localized where tissue was injured by the thermocautery. Mackenzie and Sturm (1928) produced a similar localization a t the site of injection of such substances as Scharlach R, tar and chick embryo hash. The degree of localization depended upon the stage of tissue reaction to the substances used. The earlier stages of the reaction localized more regularly than later stages. After intravenous injection demonstrable agent disappears from the blood stream with great rapidity (Doerr et al., 1932; Sittenfield et al., 1932). Sittenfield and his associates claimed, however, that agent could be obtained from the blood by precipitation at pH 4.0 and elution a t pH 8.0 up to eight days after the intravenous injection of 10 to 20 m.i.d. There is no evidence that the agent can multiply in the blood stream, and it therefore appears that the
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agent, present initially in a free state, rapidly becomes adsorbed t o blood elements. In birds with well-developed tumors the blood becomes infective (Fraenkel, 1927; Ragnotti, 1929; Doerr et al., 1932). Ragnotti concluded that the agent was adsorbed to the leucocytes, although both plasma and erythrocytes were also infective. Pentimalli (1935) claimed that chick erythrocytes adsorbed the agent in vitro, adsorption being favored by low temperatures. In our hands, attempts to adsorb the agent on to chick erythrocyte “ghosts” have been unsuccessful. Shrigley (1946) grew the sarcoma by intraocular transplantation in guinea pigs. A spread of the agent or of tumor cells was observed and, although lungs and blood gave negative results, the ground spleens of the pigs were found to be tumor-producing between one day and eight days after transplantation. It would be of interest t o know whether this infectivity is attributable to free agent since Duran-Reynals and Murphy (1929) found that ground muscle from rabbits and pigeons, unlike ground muscle from susceptible chickens, did not adsorb the agent. It is generally agreed that the agent liberated from an actively growing Rous tumor can exist in most tissues of the bird without producing tumors (Fraenkel, 1927; Mellanby, 193810). In fowls which had borne Rous tumors for less than twenty-eight days, both spleen cells and extracts of spleen transmitted the tumor (Carr, 1944a). However, quantitative experiments showed that the maximum amount of agent present was only 20 m.i.d./g., whereas the tumor tissue itself contained lo6to lo7 m.i.d./g. The disseminated agent in the whole bird amounted to less than the agent content of 1 mg. of tumor, and it is reasonable to accept Carr’s suggestion that the whole of this amount may be referred to that contained in the blood and phagocytic cells present in the tissues. Mellanby (1938a) also claimed that, in a bird bearing both a Rous sarcoma and a chemically induced tumor, the cells of the nonfilterable tumor were invaded by agent from the filterable tumor. Transplantation of the cells of such a nonfilterable tumor into a fresh host produced a typical nonfilterable tumor, whereas injection of a cell-free filtrate from the same tumor produced, if a tumor were produced a t all, a typical, filterable, Rous sarcoma. Carr (1945a) found, as before, that the amount of tumor agent present in the chemically induced tumor under these conditions was minimal, in fact, the amount which might be expected to be present in the blood contained in the tumor. There was no indication of attraction between the tumor agent and either the intact or the damaged nonfilterable tumor cell. Mellanby (19384 had observed that injection of Rous agent directly into the nonfilterable tumor did not cause regression of the established tumor but that islands of Rous sarcoma were produced in it. It is scarcely conceivable that the infected cells
AGENT OF ROUS NO.
1
SARCOMA
237
were those of the nonfilterable tumor and probable, therefore, that the Rous tumor arose by localization of the agent, in the usual way, a t the site of injury. There is considerable evidence that, under certain conditions, the agent may survive in fowls for long periods of time. Carr (1942b) found that injection of 20-methylcholanthrene into fowls carrying a Rous sarcoma produced a violent local reaction a t the site of injection. The established tumors regressed, but new tumors arose some three weeks later at the site of injection of the hydrocarbon. These, however, were filterable and similar to the original Rous sarcoma. Furthermore, the small tumors that were produced by injection of agent into Carr”s nonsusceptible (“N-S ”) strain of fowls (Carr, 1942a) usually regressed within twenty-eight days, but later, and in one case, one year later, tumors appeared again at the same site. The reason for such recurrence appeared to be a decrease, with age, of resistance to the tumor, but the implication is that either tumor cells, or the tumor agent, presumably in an intracellular state, had been immobilized and preserved in the fowl. These lines of evidence thus indicate that the Rous agent may be widely distributed in small amounts in the tissues of fowls bearing an actively growing young tumor. This is probably merely a reflection of its presence in the blood stream, from which under normal conditions i t is rarely localized, although tumors may develop a t the site of an injury. 9. Adaptation and Variation of Agent
Rous and Murphy (1913) found that, after several transplantations, variations in chicken tumor 1 began t o occur, exemplified by hemorrhagic growths, the appearance of spherical rather than spindle cells and of giant cell forms. Some of these variations were considered to be the expression of changes in the growth’s causative agent but it has lately appeared (Milford and Duran-Reynals, 1943) that the host’s natural resistance may be equally, if not more, important in determining the nature of the lesion. In chickens injected intravenously with doses of Rous No. 1 (or Fujinami myxosarcoma) agent proportional to body weight, notably-different types of lesion were obtained according to the age of the bird (Duran-Reynals, 1940b). In the young chick, devoid of virus-neutralizing antibody, a “hemorrhagic ” disease was produced characterized by the development of lesions confined to the viscera and consisting of blood blebs and extravasations of blood (cf. Sugiura, 1926). I n pullets, gross tumor nodules were found, together with “hemorrhagic ” lesions. In adult birds tumors were usually produced at the site of injection. The route of injection is of great importance since, in a large number of experiments involving intramuscular injection of the agent
238
R. J. C. HARRIS
into 2-day-old chicks, this “hemorrhagic” disease has not been observed (Carr and Harris, 1951a). Injection of agent intravenously or intracoelomically into the chick embryo elicited no tumors but produced “hemorrhagic )’lesions only (Milford and Duran-Reynals, 1943). However, intravenous injection of extracts of these lesions into pullets or adult chickens gave true tumors. It appears, therefore, that neoplasia in the fowl, following the intravenous injection of tumor agent, appears when the host is endowed with a certain resistance against the agent. In chicks with a low resistance or none, the necrotic, hemorrhagic lesions appear. Variation of the agent has been obtained most consistently after passage to another host followed by a return to fowls. Ducklings may be infected with Rous agent at an age of one day provided that large amounts of the agent are given intravenously (Duran-Reynals, 1941). The ducklings showed a detectable hourly increase in resistance and the lesions produced could be divided into two stages (Duran-Reynals, 1942) (1) “immediate,” which developed within thirty days and resembled Rous No. 1 sarcoma and (2) “late,” which developed after several months (three different sarcomata and one lymphoblastoma in different locations). Extracts from type (1) lesions produced Rous No. 1 sarcomata in chickens and were notably ineffective in ducks, i.e., the agent had not undergone variation. Extracts from type (2) lesions, however, had no action on adult chickens, but produced a generalized disease in ducks characterized by the induction of periosteal sarcomata. The agent was, therefore, a variant. The variation of “chicken” agent to “duck” agent was, moreover, reversible since extracts of two of the type (2) lesions injected into chicks produced again “immediate” and “late” lesions: (a) the “immediate,” which developed within one hundred days, were spindle-celled sarcomata arising from the periosteum, and (b) the “late,” which developed after some months, were leucoses and osteopetroses. Type (b) lesions were transmissible only to chicks and to a few adult birds producing, as might be expected, leucosis and osteopetrosis. Three further duck variants have been studied (Duran-Reynals, 1947a). These did not lose their affinity for chickens, but the tissue specificity of the agents was different. After a passage in adult chickens, one variant lost its acquired duck affinity and reverted completely t o a chicken-type tumor. These duck variants could be passed to young pigeons by cell graft into the pectoral muscle. Local growths, which did not generalize were produced by two of these but a third induced, even in l-year-old pigeons, large local growths which often metastasized (Duran-Reynals, 1947b).
AGENT O F ROUS NO.
1
SARCOMA
239
These variations of the agent apparently involved considerable change in the antigenic structure since the natural immune bodies of aging chickens neutralized both chicken and duck agents with respect to the infection of both chickens and ducks whereas the natural immune bodies of aging ducks neutralized only the duck agent with respect to the infection of ducks (Duran-Reynals and King, 1947). An analogous series of variations has been found with turkeys and with guinea fowls (Duran-Reynals, 1943). Within ten weeks of hatching (five weeks for guinea fowls) 80% of young turkeys or guinea fowls given Rous agent intravenously, developed tumors. Cell grafts were passed to a few adult pheasants, but both newly hatched and older pigeons were wholly resistant t o both cells and agent. In both turkeys and guinea fowls the tumors were viscous, very collagenous and similar t o the duck variants in that analogous periosteal and endosteal tumors developed. The agent isolated from these tumors would regularly reinfect adult chickens but gave evidence of variation in that it showed the same tissue affinities in chickens as in turkeys and guinea fowl. There are at least two possible processes at work here. First, a variation in the antigenic structure of the virus and/or, secondly, a variation in the type of lesion produced in the host. It is probable that the second process could be a result of selection, i.e., that the tumor agent population was already heterogeneous with respect to, for example, tissue affinity, and that a changed host environment had exerted a selective action (cf. Findlay, 1936). Thus Shrigley et al. (1945) and Shrigley (1947) found that passage of the tumor in the anterior chamber of the guinea pig eye followed by return t o chicks increased the incidence of periosteal and hemorrhagic lesions. Bone lesions were, however, found in the control birds, which would suggest that residence in the guinea pig eye had merely accentuated a normally occurring virus tissue-heterospecificity . Pikovski and Doljanski (1946) have also claimed that intravenous injection into chicks of agent derived from cultures of Rous sarcoma cells gave osteoid sarcomata. There is some evidence too, that the nature of the agent derivable from the Rous No. 1 sarcoma may vary with the age of the tumor-bearing host. Duran-Reynals (1946a) showed that, adaptation of the agent to other species is easier when the tumor from which the agent was prepared had been grown in adult chickens, several months old. Adaptation was never successfully accomplished when the tumor had been grown in chicks and rarely when in older birds. The results of the study which Duran-Reynals (1946b) and Duran-Reynals and Shrigley (1946) made of some fifty spontaneous chicken tumors are in agreement with these views. Of seven sarcomata occurring in fowls aged 5 t o 10 months, four were
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R. J. C. HARRIS
transplantable indefinitely and were transmissible by cell-free extracts, whereas in the age group 3 weeks to 18 weeks, only three possibly transplantable sarcomata were found and, in the age group 12 to 18 months, one sarcoma out of four was found to be transplantable indefinitely and transmissible by agent. An argument which has often been raised against the hypothesis of a virus origin for canuer is that a great many different viruses would be required. The great variety of stable lines of tumors and of leucoses which may be produced by variation of the Rous agent, constitute a sufficient answer to such an objection. 3. Inhibition of Growth of Rous No. 1 Sarcoma in vivo
An enormous number of different substances has been tested in attempts t o inhibit the growth of the filterable tumors i n vivo. Peyron et al. (1937); for example, found that colchicine inhibited the Shope virus, but neither colchicine nor 2,4-dinitrophenol had any effect on the growth of Rous No. 1 sarcoma (Carr, 194213). Carr also found that injection of 20-methylcholanthrene into fowls with established Rous No. 1 tumors resulted in a violent local reaction and regression of the tumor. However, some weeks later Rous type tumors appeared a t the site of injection of the carcinogen. The importance of this finding in discussion of the localization of disseminated tumor agent a t the site of an injury has already been mentioned but another equally important consideration is the possibility that filterable fowl tumors claimed to have been produced by injection of tar or carcinogens (McIntosh, 1933) may have arisen by localization of a latent preexisting virus in the fowl. Bryan, Riley, and Calnan (1947) did not observe any effect on the latent period of tumors (induced by subcutaneous injection of the agent) by intraperitoneal administration of urethane. Little et al. (19484 concluded from a nutritional study of young chicks that folic acid was the only dietary factor required to the extent that tumor response was prevented by its absence from the diet and stimulated by its addition to the standard diet. A similar conclusion about folic acid was reached by Woll (1948). Moreover, folic acid antagonists such as 4-aminopteroylaspartic pcid, 4-aminopteroyl-d(-)glutamic acid and 4-amino-N-methyl folic acid actively inhibited the growth of the tumor when they were fed at high concentrations (Little et al., 1948b). Folic acid deficiency could be overcome completely by injection of a liver extract, and partially by administration of vitamin B12 (Oleson and Little, 1949) and it was concluded, therefore, that not only folic acid but also vitamin Blz has a role in the growth of Rous No. 1sarcoma. There is some doubt as to whether chicks deprived of dietary folic acid were, in
AGENT OF ROUB NO.
1 BARCOMA
241
fact, folic-acid free. Engelbreth-Holm (1951) has repeated Little’s work and found that, on a folic acid-free diet, day-old chicks ceased to grow at 16 days and died between 24 and 28 days. Compared with the controls (day-old chicks forced to the same growth rate by food restriction) the “takes” (90-1000/0) of cell transplants of the tumor were unaffected, although “takes” from agent inoculation were somewhat less. The resulting tumors were always smaller in the folic acid-deprived birds but appeared to contain as much agent per gram of tumor as the controls. In fact, the folic acid-deprived birds were not folic acid “free” since their organs contained about one-third of the amount of folic acid in the similar organs of chicks on a standard diet. The effect of vitamin BH deprivation was similar but less pronounced. Engelbreth-Holm has suggested, therefore, that folic acid deprivation has no specific action on the growth of Rous No. 1 sarcoma but it prolongs the latent period of tumor induction.
4. Assay of Rous No. 1 Agent It would not be appropriate to close this section of the review without mentioning the methods available for assay of the agent. In very many of the papers, especially those dealing with the preparation and properties of the agent, the effects of procedures, such as delipidation, have been presented in qualitative terms as “survival” or “nonsurvival ” of infectivity. The various methods which have been used for the determination of the tumor-producing activity of Rous agent preparations fall essentially into two groups. In the first group, serial dilutions are injected into susceptible fowls. The highest dilution a t which a tumor is produced gives a “minimal infective dose” (m.i.d.). In the second group the agent suspension of unknown titer is injected into a fowl together with a standard agent suspension of known titer. The titers are compared either by the relative sizes of the tumors ultimately produced or by the time elapsing (“latent period”) before detectable tumors arise (Bryan, 1946a,b). So far as the latter methods are concerned there appears to be no a priori reason for assuming that a constant reference standard can be obtained or that the subsequent comparison would be unaffected by such factors as the season of the year or the nature of the medium in which the agent was suspended. A further #importantconsideration is the age of the bird which is used. Carrel (1925a) found that younger birds were more susceptible than older, and Sugiura (1926) stressed the advantages of using chicks less than 8 days old. Riley et al. (1946), however, claimed that there were no significant differences in susceptibility
242
R. J. C. HARRIS
in fowls of between 3 and 10 weeks old and that 4-day-old chicks were less susceptible than those which were 4 weeks old. This is contrary to the findings of others who have worked with birds of a known degree of natural resistance to the tumor (Carr, 1943). Moreover, older birds frequently show a variable and unpredictable resistance to the agent (cf. Bryan, 1946a) in that they may not react to 1000 times the dose that will produce tumors in susceptible birds. The titer end point being dependent, as may be shown, upon a Poisson distribution of particles, then a large number of fowls would be required for an accurate assay. For these reasons Carr and Harris (1951a) used 1-day-old chicks for assay purposes. Large numbers may be employed; but since metastasis to a site of injury produced by a noninfective inoculation may occur (Carr, 1943), only one test injection in each chick is possible. Table I TABLE I Assay of Roue No. 1 Agent in Day-Old Chicks Titer
gives examples of the assays obtained. In the table, the numerator of each fraction refers to the number of tumors obtained and the denominator to the number of chicks used for each dilution. All titers were referred back to the wet weight of tumor tissue used initially. Thus is equivalent to lo6 m.i.d. per gram original tumor. The 1X m.i.d. of an agent preparation assayed on day-old chicks was, on average, found to be one-tenth to one-fiftieth of that obtained with the use of 6-week-old birds of the same strain. Riley (1950~)has claimed that the minimum number of agent particles required for infection lies between 1 and 100. Since however, a Poisson distribution of tumors was not obtained, the tumor score per forty sites inoculated a t 10-7 being 5; 1and 10-lo, 3, the actual titration 3; end point is obscure. Claude and Rothen (1940) recorded the weight of g. (=2,000 the m.i.d. of their highly purified material as 4.0 X particles). It is conceivable, of course, that, effectively one agent particle may produce a tumor, and such a result has been derived from statistical analysis of the figures recorded by Carr and Harris (195la).
AGENT OF ROUS NO.
1 SARCOMA
243
111. AGENTAND MALIGNANT CELL
I. Histogenesis of the Tumor Rous No. 1 sarcoma appears to be composed of cells of a t least two types, fibroblast and ameboid (the latter variously described as monocyte, histiocyte, or macrophage). Both of these cell types, if, indeed they can thus be differentiated, have been claimed as the malignant cell. Carrel (1924a,b) obtained “pure” cultures of each cell type. Cultures of fibroblasts less than 7 days old produced tumors upon inoculation into susceptible fowls but older cultures did not. Monocyte cultures, 14 to 33 days old, also transmitted the tumor. Carrel (1924c, 1925b) held that blood macrophage cultures could be infected with the agent and transformed into malignant cells, whereas attempts thus to transform fibroblast cultures were unsuccessful. However, Fischer (1924) and, later, Carrel and Eberling (1926) and Fischer and Laser (1927) observed that fibroblasts appeared in cultures of monocytes derived from the tumor, a process which Carrel and Ebeling regarded as the tendency of a cell type susceptible to the agent to become transformed into an immune cell. The alternative view that the tumor arose by infection of fibroblasts was defended by Mottram (1928) and by Llombart (1935), who believed that the infected fibroblasts changed into malignant fibroblasts. On evidence obtained from vital staining Haddow (1933) considered that the free histiocytes took up the agent and underwent development: infected free histiocyte + histioblast --+ malignant fibroblast. Ludford (1937), unlike Carrel, claimed under favorable conditions to have infected fibroblasts in cultures of chick pectoral and heart muscle with the agents of either Rous No. 1 sarcoma or Fujinami myxosarcoma. The possibility that the tumor was produced by inoculation of free agent in the culture fluid was excluded by treatment of the culture with immune serum before inoculation. However, Carrel (1926) had already demonstrated that the agent would survive for several weeks in association with fibroblasts, although it disappeared rapidly from the culture medium. This suggests that Ludford’s fibroblasts may have “carried” the agent without having become malignant. Fixation of the agent by ground chick muscle has been described by Duran-Reynals and Murphy (1929) and Carrel also found that the fluid (1:3: 1 plasma-Tyrode-embryo extract) medium for the culture of leucocytes (from buffy coat or spleen) had a preservative action on the agent, which suggests more probably that, unlike fibroblasts, the leucocytes had no capacity for “fixing” the agent which thus remained free in the medium; Ludford found also that buffy coat cultures
244
R. f . C. HARRIS
could not be infected and, moreover, that there was no evidence to show that fibroblasts could be produced from leucocytes. The evidence from tissue culture studies is therefore equivocal, and it is doubtful even whether it would be true to say, in default of other evidence, that both cell “types” can “carry” the agent (Doljanski and Tenenbaum, 1941, 1942). These divergent views would, however, be reconciled, if it could be shown, unequivocally, that fibroblasts and macrophages are merely functional variants of a single cell type. Thus Parker (1932) has claimed that a long-established strain of chick fibroblasts could be transformed into macrophages by changing the nutritional medium of the culture from one containing embryo extract to one containing plasma alone. (It had been shown by Carrel (1924a,b) that macrophage growth was suppressed in a medium heavily fortified with embryo extract.) Conversely, Moen (1935) claimed to have produced a permanent culture of fibroblasts from a single macrophage. There may exist some sort of symbiotic relationship between macrophages and fibroblasts in the Rous tumor. de Bruyn (1949) and de Bruyn et al. (1949) have studied a transplantable mouse lymphosarcoma which, again in tissue culture, revealed two main types of cell, lymphoblast-like and fibroblast. In culture media containing embryo extract the growth of the lymphoblasts could be quickly suppressed. The lymphoblasts were, however, the true malignant cells, but for successful continuous culture low concentrations of embryo extract were required and, in addition, active mesenchymal cells had to be present. A strain of fibroblast-free lymphoblasts was obtained, but, after independent growth for 157 days these appeared to have lost their ability to produce a tumor. de Bruyn et al. concluded that the evidence suggested a symbiosis between lymphoblasts and fibroblasts. Doljanski, Hoffman, and Tenenbaum (1944) observed that extracts of chicken tumor I stimulate the in vitro growth of chick heart fibroblasts to a marked degree, and the very interesting suggestion has been made (Waymouth, 1952) that cytoplasmic particulate material liberated by the agent-producing cells of the tumor may act as growth stimulants (in a manner similar t o that of embryo extract) for the associated fibroblasts, thus preserving an overall balance of cell “types” in the tumor. Moreover, Fischer and Laser (1927) observed that both the macrophages and the fibroblasts from the tumor were phagocytic although normal fibroblasts were not. It has been concluded already that the localization of the agent from the blood stream, by injury or by irritation, could be ascribed t o a mobilization of monocytes (see also Levine, 1939). The next stages, however, remain obscure, especially since all the filterable fowl tumors
AGENT OF ROUS NO. 1 SARCOMA
245
undoubtedly arise from one or other of a group of closely related cells which are highly variable and probably interconvertible. 2. Electron Microscopy of Chicken Tumor Cells
The problem of the type of cell with which agent replication is associated in the tumor might be solved if virus could be unequivocally detected microscopically. In 1947, Claude, Porter and Pickels published electron micrographs of portions of the peripheral cytoplasm of cells derived from explants of a 5-day-old Rous No. 1sarcoma from the chorioallantoic membrane, and from explants of a 14-day-old tumor from a pullet. Small, osmiophilic bodies of the same size as the agent (70-100 mp) were observed. In chicken tumor 10 these small bodies occurred in aggregates of large numbers but in chicken tumor 1 (Rous No. 1sarcoma) cells they were dispersed singly or in pairs. No such particles were seen in normal fibroblasts or macrophages (from buffy coat), and they had the appearance of entities distinguishable from normal cell constituents by their regular granular shape, their relatively uniform size, and their greater density in osmium-fixed preparations. The microsomes of normal cells, which are in the same size range as the agent particles appear as vesicles in electron micrographs since they swell considerably in water after fixation with osmic acid (Claude, 1947-8). A similar claim has been put forward by Oberling et al. (1950), but the density of these particles was found to be intermediate between that of mitochondria and of microsomes. The particles were found typically in only a small percentage of the Rous cells examined and this was considered to be the result of the virus being too closely associated with the dense nucleus. However, the identity of these particles must remain in doubt since the tumor from which cells were examined was not filterable “pendant toute la d u d e de nos investigations.” A very large number of cells from primary cultures of Rous No. 1 sarcoma derived from actively growing young tumors has been examined under various fixation conditions by my colleagues F. W. Cuckow and C. JVaymouth. Osmiophilic particles in a size range of from 70 to 500 mp have been observed. While a few scattered particles of the same size as the agent may be seen, no groups of particles of uniform size similar t o those obtained by Claude et al. have ever been observed. 3. Agent and Cell Metabolism
It may be assumed that the Rous No. 1 agent infects a cell of the monocyte or related type and converts it into a malignant cell. In the cell division which follows, the agent, which also undergoes multiplication, must be distributed among some, if not all, of the progeny of the
246
R. J. C. HARRIS
first infected cell and probably multiplies yet again within some, if not all, of them. Carr (1947) was the first to point out that the amount of agent found experimentally in the avian filterable tumors was entirely inadequate to support the usual theories. Carr calculated that there is one sarcoma cell in every (10 p ) s of tissue and if there is one agent particle per cell, then it follows that there must be about lo9 particles per gram of tumor. The best experimental recovery so far obtained gave a figure of 3.75 X lo7infective doses per gram (Claude and Rothen, 1940). The amount of agent isolated, therefore, was a t the most less than 5% of the anticipated amount, and Carr suggested a number of possible explanations for this discrepancy. First, that the agent is not there, i.e., statistically only one cell in every twenty contains an agent particle, and it would follow from this, if one accepts the evidence of published electron micrographs of avian tumor cells, that far fewer cells are actually producing agent, and that the remainder are either nonmalignant, or malignant, but devoid of agent, in which case it should be possible to obtain a nonfilterable Rous tumor by selecting such malignant cells (cf. the view of Peacock (1946) that the malignancy of the filterable fowl tumors is not irreversible but depends upon the continued existence of the agent which, as it were, “goads the cells on to useless multiplication”). Some support for Carr’s views may perhaps have been provided by Fraenkel’s (1927) observation that for the passage of the Rous No. 1 tumor, an inoculation of 50,000 cells never produced a tumor, and 500,000 cells, not invariably. If far less than one in twenty of the former number of cells were capable of transmitting the tumor, it is not difficult to see why these figures should apparently be so large. Carr also considered and rejected explanations based on errors due to the method of titrating the agent or to its incomplete extraction from the cells. A fourth possibility is that not all the particulate material produced by the cells is infective, although this associated product of the tumor would appear to be physically, chemically, and immunologically indistinguishable from the infective particles and present in far greater quantity. This possibility would in particular appear to be worth investigation since there is immunological evidence (see Sec. V, 1) that a similar type of noninfective particle is present in some nonfilterable avian tumors (Andrewes, 1936; Foulds, 1937; Gottschalk, 1943). The sequel to the successful infection of a susceptible cell with any virus is the appearance of a new unit system, the virus-infected cell (Luria, 1950), which contains the existing enzymatic machinery of the host cell and, together with it, a new pattern derived from the virus and directing, among other things, the synthesis of new virus material. So far as the avian virus-induced tumors are concerned, a further metabolic ’
AGENT OF ROUS NO.
1 SARCOMA
247
pattern emerges such that the new cell is not only (and probably, not primarily) an agent-synthesizing system but a malignant cell. Whereas, in the unit system “phage-bacterium” it may be permissible to regard the phage as imposing an overriding metabolic pattern upon the bacterium, so that, for example, phage-infected B. coli are unable to form adaptive enzymes (Monod and Wollman, 1947), in the virus-induced avian malignant cell part, a t least, of the normal metabolic pattern would appear to be retained, notably that part which is concerned with the synthesis of new proteins etc. for cell growth. The channels into which protein synthesis are directed in these malignant cells, therefore, end not only in protein for agent replication but also in the production of normal proteins. It is probable, however, that the agent protein is formed a t the expense of normal protein (cf. Bawden, 1951). The effect of the tumor agent on the protein-synthesizing system of the infected cell may be summarized : (1) The rate of production of normal cell proteins (e.g., structural proteins) is increased. (2) The infecting agent is replicated. (3) A variety of other noninfective products that resemble the infecting agent to a more or to a less extent are, no doubt, also synthesized. The first effect is of the utmost importance in the consideration of the way in which a normal cell can be converted into a malignant cell. The product of the second effect has received the major part of the attention devoted to the study of the fowl tumors in the last forty years. The third effect, which has not received any attention, may provide the data with which to attack the problem of the relationship between the filterable and the nonfilterable fowl tumors. It is probable that the chemically induced fowl tumors also have an aberrant protein synthesis, covering effects 1 and 3, of which the end products are tumor specific, but not infective, particles. It is also possible on these grounds to explain the fact that the presence of agent is not demonstrable in some Rous No. 1 tumors (Gye and Andrewes, 1926; Carr 1942a, 1944b) and also the low agent yield (Carr, 1947), the explanation being that in some circumstances the malignant cells produce only a few infective specific particles. The condition in which this occurs may depend, for example, on the degree of host resistance or on the rate of growth of the sarcoma. A situation, capable of a similar explanation, probably obtains with the Shope papilloma virus. The virus may be recovered in large amounts from natural and experimental papillomata of the wild, cottontail rabbit, but it is only occasionally demonstrable in small amounts in extracts of papillomata from domestic rabbits and not a t all from the resulting carcinomata (Shope, 1934; Kidd, 1938a,b; 1939; Friedewald and Kidd, 1944). Indirect evidence, obtained by serological and immunological methods indicated that the virus, in “masked ” or altered form, persisted
248
R. J. C. HARRIS
in the cells of two transplanted carcinomata (Vl, V2) which had originated from virus-induced papillomata, and that this “virus” increased in amount as the tumor cells proliferated (Kidd and ROUS,1940; Kidd, 1942). However, after twenty-five transplant generations this “virus ” disappeared from the tumor which then became free from papilloma virus antigen (Smith, Kidd, and ROUS,1947). Kidd (1946) concluded it possible that a constituent of the tumor cells, other than the virus, was responsible for the continuing malignancy, and Syverton et al. (1950), that it was improbable that the papilloma virus formed an essential part of the ultimate tumor. There are alternative explanations, namely (1) that during continuous passage the malignant cells had undergone selection such that those which had infective virus, masked “virus,” or virus antigen as one of the end products of the cell’s protein synthesis were gradually eliminated in favor of a cell lineage which produced none of these, or (2) that all the malignant cells had undergone, with successive transplantation, a metabolic change. The most direct evidence that products, resembling but other than, infective particles may be produced by virus-infected cells, has been given by Hoyle (1948). When embryonated eggs were inoculated with very large doses of influenza A virus, a very rapid production of a soluble antigen took place, indicating a rapid intracellular replication of virus antigen, but the resulting yield of infective, extracellular virus was much reduced, and in some cases, negligible. The soluble antigen was described by Hoyle (1945) as a component of infective influenza virus, from which it could be liberated by ultrasonic disruption (Wiener, Henle, and Henle, 1946). The soluble antigen had the same physicochemical properties as the virus but was noninfective. Hoyle also found that the soluble antigens of all strains of influenza virus A appeared to be serologically identical. These data lead him to the conclusion that, under certain circumstances, the infected cells were unable to produce the complete extracellular infective form of the virus since, of 3600 units of complement-fixing antigen produced after thirty-six hours incubation only 600 units represented soluble antigen associated with infective particles and the rest was in the form of free soluble antigen. Gard and von Magnus (1946) also observed a decrease in the infectivity of an influenza virus preparation during egg passage. The decrease was associated with an increase at the expense of the infective particle (sedimentation size, 700 S) of a noninfective particle of sedimentation size 500 S, which may be related to the soluble antigen. Infective influenza A virus, therefore, appears t o be composed of a soluble antigen portion plus strain-specific antigens and an enzymic mechanism enabling it to gain access to the cell. It would be foolish to
AGENT OF ROUS-NO. 1 SARCOMA
249
force the avian tumor agents into the same mold, but the implications for future work in this field are worth noting. The soluble antigen of influenza virus would have as its analogue in the tumor agents the small noninfective antigens common to both filterable and nonfilterable fowl tumors (Foulds and Dmochowski, 1939). Both the Rous No. 1 agent and influenza A virus (PR 8 strain) are also known t o carry an antigen characteristic of particulate material from the normal host cell (Amies and Carr, 1939; Knight, 1946b) and the slightly smaller noninfective influenza virus particle of Gard and von Magnus may have its tumor counterpart both in the noninfective particulate material which may be isolated from nonfilterable fowl tumors, for example the carcinogeninduced G.R.C.H. 15 sarcoma (Carr and Harris, unpublished) and in the large amounts of chemically, physically, and immunologically similar particles which appear to accompany the isolated Rous agent (Carr, 1947). The antigenically related cytoplasmic particles from the nonfilterable avian tumors would thus resemble a Rous agent stripped of its capacity for assuming an extracellular infective phase. Further, the variations which the avian tumor agents so readily undergo become explicable in terms of association with the primary virus antigen of different host antigens and the superimposition of a suitable cell-invasive mechanism. Another approach t o the problem of the relationship of the virus to the malignant cell lies in the elucidation of the directions in which the cell’s metabolism and enzyme “spectrum” have been changed; Lewis et al. (1931) and Barron (1932) claimed that Rous sarcoma tissue did not contain succinic dehydrogenase. Reinvestigation by Schneider and Potter (1943), however, showed that the tumor content of both cytochrome oxidase and succinic dehydrogenase was of the same order as for a number of other non-virusinduced mammalian tumors but much less than for normal tissues. In rat liver, these two enzymes are largely associated with mitochondrial particles of a size range from 0.5 p to 2.0 p (Hogeboom et al., 1946; Schneider et al., 1948), and Schneider (1946) has found that less mitochondrial material, could in fact, be isolated from rat hepatoma with its lowered enzyme content. Moreover, apart from the quantitative differences, there appeared t o be a different pattern of enzyme distribution in the hepatoma cells. Thus, in normal liver, 50% of the adenosine triphosphatase was associated with the mitochondrial fraction, whereas in the hepatoma only 12% was so associated, although the total quantity remained the same. The Rous agent is within, or associated with the microsome size range of 50-150 mp, and the enzymic properties of the microsomes are less well known. In mouse liver, esterase (Omachi, Barnum, and Glick, 1948) and
250
R. J. C. HARRIS
thrombokinase (Jeener, 1948) are associated with these small granules, and in rat liver, this fraction has the highest activity (per mg. N) of DPN-cytochrome C reductase. Changes in the amounts of enzymes after virus infection have been found for a number of tissues but these changes have not yet been associated with changes in cell fractions such as mitochondria and microsomes (Racker and Krimsky, 1948; Krimsky and Racker, 1949; Bauer, 1947, 1948) although Claude (1947-8) has discussed in general terms the possible function of the microsomes in anaerobic glycolysis.
IV. ISOLATION AND PROPERTIES OF Rous No. 1 AGENT 1. Isolation It was early shown (Murphy et al., 1928) that the infective principle of Rous filtrates was associated with a nucleoprotein fraction that could be precipitated in active form by half-saturation of the filtrate with ammonium sulfate (Sugiura and Benedict, 1927 ;Lewis and Mendelsohn, 1930) or by adjustment of the pH to 4.0 followed by extraction of the precipitate at pH 8.0 (Sittenfield and Johnson, 1930). Claims were subsequently made that protein-free filtrates were infective (Lewis, 1931 ; Lewis and Mendelsohn, 1931), but these claims rested on inadequate analytical evidence. Pirie (1931) first used an adsorption procedure for concentration of the agent. Chemical, as distinct from physical procedures, for agent concentration were reinvestigated by Shemin, Sproul, and Jobling (1940), and by Shemin and Sproul (1942), who found that the agent was precipitated from polysaccharide-free filtrate by papain or by calf thymus histone. Such a phenomenon had previously been described by Stern and DuranReynals (1939) with protamine and a similar procedure was used by Chambers and Henle (1941) for the concentration of influenza A virus. The papain-Rous agent complex was a t least as active as the original filtrate and could be decomposed electrophoretically. The liberated agent was apparently electrophoretically homogeneous at pH 7.4, but contained less nucleic acid purine than Claude’s centrifugally concentrated material. With an optimum amount of adsorbent alumina gel, it was possible to recover the agent from the adsorbate by elution at pH 9.2. Similar chemical procedures were employed by Murphy et al. (1932) and Claude (1934, 1935a,b). Nonviral proteins were removed by adsorption t o alumina gel, the viscous polysaccharide was precipitated by gelatin, and salts were finally removed by dialysis. A thirty-fold concentration was thus achieved, and 93 % of the total solids were removed (cf. Dmochowski, 1948a).
AGENT O F ROUS NO.
1 SARCOMA
251
A variant of the early gross adsorption procedures has recently been described by Riley (1948, 1950a,b), who claimed that the agent may be adsorbed t o diatomaceous earth (Celite 501 or 503) from suspension in physiological saline and increasingly eluted therefrom with salt solutions of decreasing molarity. The early experiments showed that small concentrations could be achieved and that there was an equally small gain in purity as measured by the ratio of the potency (per cent activity of a “standard ” reference material, usually partially purified starting material) to nitrogen content. Subsequently Riley prepared agent concentrates by adsorption from physiological saline in the top few millimeters of a column of Celite. The column was then washed with physiological saline, extruded, an arbitrary top zone was removed, and the agent eluted with water. A typical result gave, in the top 5-mm. zone of such a “chromatogram,” 3.9% total N and 79.5% total agent. Electron microscopy of such eluates showed that the size range of the particles present was 20-70 mp with a maximum distribution a t 40 mp. Riley suggested that the agent, like other small proteins, was “ saltedout” when the salt concentration was increased in the presence of a suitable adsorbent and desorbed as the salt concentration was decreased. There is, however, some evidence for an alternative explanation. Oberling, Guerin, and Guerin (1946) studied a spontaneous fowl sarcoma which was cell-transmissible for sixty-three passages and extracts of which in physiological saline gave uniformly negative filtrates. Subsequently, however, it was found that extracts in distilled water gave successful filtrates and Oberling et al. suggested that there was a decrease in effective virus volume in water extracts. Moreover, Taylor, Sharp, Beard, and Beard (194213) isolated particles from chick embryos extracted with water which were considerably smaller (23 mp) than the 50- to 100-mp particles isolated by Claude (1938, 1940) using dilute salt solutions as the extraction medium. The virus of rabbit papilloma precipitates when the salt molarity of the suspending medium is reduced to less than 0.05M and all three strains of influenza virus are agglutinated in distilled water (Sharp and Taylor et al., 1945). The PR 8 strain of influenza virus A is less infective in distilled water than in phosphate, veronal, or borate buffers, but the effect is reversible, and full infectivity is recovered on addition of 0.1M phosphate (Knight, 1944). The effect of salt concentration on the agent must therefore be more clearly elucidated before Riley’s explanation of the “chromatography” can be accepted. If a decrease in effective virus volume (and it is significant that electron micrographs were stated to show particles of average
252
R. J. C. HARRIS
size 40 mp.) with decreasing salt molarity is the reason for this “chromatographic” behavior, then it would be important to know whether this is reversible or whether inactive material has been removed from the agent. McIntosh (1935) demonstrated that the agent could be deposited by centrifugation in a “spinning-top ” centrifuge. By fractional centrifugation agent concentrates were prepared by Amies (1937), Amies and Carr (1939), Claude (1937,1938, 1939a, 1940), Claude and Rothen (1940), Pollard (1938, 1939), Stern and Duran-Reynals (1939), McIntosh and Selbie (1940), Dmochowski (1948b), and Carr and Harris (1951a). Some of the results are shown in Table 11. TABLE I1 Concentration of Rous No. 1 Agent by Fractional Centrifugation Experimental Data
Yield per Gram Tumor
2 . 2 mg. Yield enhanced by prior freezing of the tumor to -80%. Frosen and thawed many times after grinding with nand in physiological saline. Deposition of agent at pH 6.0, after clarification of initial extract with ’ Sharplea centrifuge. Nonvirus protein digested a t pH 9.0 with 0.1 % Merok “panoreatin absolute.” 0.2-0.3 mg. Deposition at pH 6.0. Nonvirus protein digMted with trypain. 1 . 3 mg. Enzymatic degradation of the polysaccharide, removal of nonviral protein by hydrolysis with cryatalline trypein.
Infectivity m.i.d.
”Purity”
Free from deoxypen- 5 . 2 X lO-’*g. 4 . 0 X 1O-lBg. tosenucleio acid Final produot free 5000 m.i.d./g. original tumor from fowl protein Final product free 80,000 m.i.d./g. from fowl protein original tumor
Final “concentrate” contained 2% of N of initial extract; free from deoxypentosenucleio acid
1
x
1o-og.
1 x 10-1yg.
Reference
Claude (1938) Claude and Rothen (1040) Amies (1037)
Amies and Carr (1939)
Pollard (1038, 1939) Carr and Harris (1951a)
Carr and Harris (1951a) have investigated a number of methods for the preparation of agent concentrates, via., precipitation from crude extracts with papain, by adjustment of extracts to pH 5.0 (Amies and Carr, 1939; Pollard, 1939) followed by elution of the agent a t pH 8.0; or by addition to such extracts of methanol in the cold, a method which has been used by Cox et al. (1947) and by Moyer et al. (1950) for the preparation of influenza virus, by Wagner et al. (1948) for psittacosis virus, and by Brumfield et al. (1948) for mouse poliomyelitis. Carr and
AQENT OF ROUS NO.
1 SARCOMA
253
Harris concluded that fractional centrifugation together with the appropriate enzymatic degradation of polysaccharide and nonagent protein was the best method for preparing adequate quantities of agent concentrate. Pirie (1940) has dealt exhaustively with the criteria of purity used in the study of large molecules of biological origin. It seems clear, however, that animal viruses cannot be included in this category. Studies on many different preparations of rabbit papilloma virus showed, for example, that there were considerable variations, far outside the limits of experimental error, in the sedimentation rates of virus obtained from different batches of warts (Neurath et al., 1941). This was taken to indicate that there were naturat variations in the size, shape, and density of the virus particles from different sources. A similar size distribution has been shown for influenza virus A (PR 8 strain) and B (Lee strain) and for swine influenza virus (Sharp et al., 1944) where there was in each case a k 5 0 % distribution about the mean. It has also been shown for the PR 8 strain of influenza A that the chemical composition of the virus is not independent of the host. Isolated from mouse lung, the virus was found to contain 38% lipid, from chick embryo allantoic fluid the lipid content was 26.8% (Knight, 1946a). Moreover, preparations of many viruses appear to be contaminated with particulate matter normally present in the host’s cells. Thus Claude (1938, 1940) was able to isolate from normal 8-day-old chicken embryos a particulate fraction which sedimented in exactly the same way as the Rous agent. The particles appeared t o be of the same size, shape, and density and gave similar chemical analysis (see Table 111). However, Taylor el al. (194213) found that aqueous, as distinct from dilute buffer extracts, of 11- 12-day-old chick embryos gave particles of size, 15 to 40 mp (Sharp et al., 1943), although the chemical properties were similar (Table 11) to those prepared by Claude. Taylor et at. claimed that these 23-mp particles were unstable in slightly alkaline and disintegrated into smaller buffer solutions, even as dilute as 0.005M, fragments. A smaller particle may also be isolated from the chorioallantoic fluid of the developing chick embryo. Chambers and Henle (1943) and Chambers et al. (1943) found a particle of estimated size 10 to 12.5 mp in the extraembryonic fluid of eggs inoculated with influenza virus. This was considered to be the infective agent, but Stanley (1944) showed that the supposed virus activity of this fraction was a result of contamination with a small proportion of a much larger particle. He also showed that a well-defined component similar in size to that isolated by Chambers et al. was invariably present in high concentration in the allantoic fluid of chick embryos inoculated with influenza virus (F 12 strain). The
254
R. J. C. HARRIS
larger virus particle (600 S) was accompanied by some 50 % of the smaller (30 S). Normal allantoic fluid was also found to contain characteristic components with a strong serological relationship to influenza virus (Knight, 1944). The major component had a sedimentation constant of 170 S giving a diameter, confirmed in the electron microscope, of 40 mp. Analytically, this particle was identical with influenza virus (Table 111). TABLE I11 Comparative Chemical Composition of the &us Agent
Particle
Whole Partiole Lipid Nonlipid Total, Total, D.n.a. P.n.a. N, % P, % % Phospholipid, % % % %
Chiok embryo component Chiok embryo component Rous agent
9.5
2.3 35
66.5
-
8.22 7.2
49 2.1 51 1.56 43.5 Lipid P, 1.26% 58.5
-
Rous agent Roue agent
8.5-9.0 1.5 35 7.5 1.22 47
Equine enoephalomyelitis (Eastern strain) Component of allantoio fluid Intluenaa A (PR 8)
7.7
V
h
Rabbit papilloma virue
2.2 54.1
10.0
1.0 25
10.0 15.0
0.97 23.4 0.94 1.5
23.4
Lipid P, 2.4% Lipid P,0.5% 35.0
-
10.6
Taylor et al. (1942b)
7.5-8.5 Claude (1940) P, 1.69% Shemin & Sproul (1942) P, 1 . O 76 Claude (1939a) P, 1.857% Harris L Carr (unpublished) 4.4 Taylor et al. (1943)
65 53
-
53.0
-
75
-
-
77.5 98.5
1.5 8.7
-
-
Reference
-
Knight (1944) Taylor (1944) Taylor el al. (1942s)
This normal material was associated with the 80- t o 120-mp (600 S) particles with which influenza virus activity is also associated. The isolated virus of F 12 strain may contain as much as 30 % normal material and PR 8 and Lee strains, 50%. The relationship of such particles to soluble antigen remains to be determined. In view of these facts, it is obvious that the usual chemical and biochemical criteria of ‘(purity” are inapplicable to the animal viruses, the particles of which are probably no more identical with one another than are individual rats. It may seem unnecessary therefore to spend any time on the properties of the Rous agent concentrates which have, so far, been prepared, but there is a general agreement and it would appear t o be worth while to compare the properties of agents isolated by different procedures and of the particles derived from normal cells and from tissues infected with different viruses, albeit that the latter are just as poorly “defined” as the Rous agent.
AGENT OF ROUB NO.
1
SARCOMA
255
There are several points of interest, first, the amount of lipid found by different workers is variable-from 35 to 47%. Pollard’s (1939) best preparation (m.i.d. 1 X lomB g.) contained 53% lipid. It is evident that where a trypsin digestion has been used in the preparation of the agent, the product invariably has a high lipid content (Pollard, 1939; Carr and Harris, 1951a). This suggests either that free lipid liberated during hydrolysis may become associated with the agent or that tryptic digestion removes a diluting protein. Secondly, viruses which contain only, or largely, deoxypentosenucleic acid appear to contain much less lipid than others. TZ bacteriophage (Taylor, 1946), vaccinia (Hoagland et al., 1940) and rabbit papilloma (Taylor et al., 1942a) contain respectively 1.8, 5.7, and 1.5% lipid and 97%, loo%, and 100% of the total nucleic acid as deoxypentosenucleic acid. The only well-characterized virus which contains pentosenucleic acid alone is Eastern strain equine encephalomyelitis (Taylor et al., 1943) which has 54.101, lipid.
2. Size of Agent The four methods which have been applied to this problem are (a) ultrafiltration, (b) analytical centrifugation, (c) electron microscopy, and (d) X-irradiation. Before the adoption of Elford’s “ Gradocol ” type membranes, filtration experiments had given values for the size of the agent varying from 10 mp (Fraenkel, 1929) to 200 mp (Teutschlaender, 1923). Elford and Andrewes (1935) obtained a value of c 100 mp which was confirmed by Yaoi and Nakahara (1935). By analytical ultracentrifugation Stern and Duran-Reynals (1939) found that activity was associated with a particle of average sedimentation constant sz055O X 10-la cm. dynes-’ sec.-l corresponding t o a sphere of diameter 70 mp. The material was of low homogeneity and stated to contain deoxypentosenucleic acid, and the value 76 mp obtained in 1937 by McIntosh and Selbie is almost certainly better. By direct observation of particulate material in the cytoplasm of tumor cells, Claude, Porter, and Pickels (1947) estimated a size of 67 to 80 mp for the agent of Rous No. 1 sarcoma and 60 to 70 mp for that of chicken tumor No. 10. Oberling et al. (1950), however, claimed to have observed larger (70-100-mg) particles. Bryan, Lorenz, and Moloney (1950) investigated the action of X-rays on different types of impure and partially purified suspensions of the agent t o determine whether the dose-survival curves differed appreciably. The most ‘(pure” preparation used required 43,700 r for 37% survival (as judged by measurement of “potency” of the preparation compared with the unirradiated control). Assuming that the particle is spherical,
256
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C. HARRIS
has a diameter of 70 mp, a density of 1.3, and is inactivated by a “directhit” mechanism, 18,000 r should have sufficed. This discrepancy disappears if the true agent size (or, correspondingly, the diameter of the radiation-sensitive portion) is 49 mp. Bryan et at. used 2% normal rabbit serum as a suspending medium in order to rule out any possibility of indirect action of the radiation (cf. Friedewdd and Anderson, 1941), but apparently did not attempt to measure the 37% survival dose of radiation for differing virus concentrations in the same concentration of serum. If the inactivating dose decreases for decreasing virus concentration then some indirect effect is still operating. The results, in fact, suggest this. It is doubtful, however, whether this method, even under more carefully controlled conditions, should be used t o estimate agent volume, although it has the advantage of trying to do so in suspension. It appears, therefore, that agent activity has usually been found t o be associated with particles of 70 to 100 mp diameter. It may be anticipated, however, (a) that it may yet be possible to remove contaminating lipids and (b) that the agent itself may show a size variation of the same order ( I t 50%) as other animal viruses, such as influenza (Sharp et al., 1944). 3. Lipid Content of Agent
A number of reports have appeared claiming that lipid extracts from wet or dried tumor tissue were capable of transmitting the tumor (Jobling, Sproul, and Stevens, 1937; Sproul, Stevens, and Jobling, 1937). These claims could not be confirmed by Levine and Baumann (1937), by Pollard and Amies (1937), or- by Fraenkel and Mawson (1937), and it was generally concluded that contamination of the extracts with proteincontaining agent had occurred, despite the claims of Sproul, Stevens, and Jobling that their extracts were chemically and immunologically proteinfree. It is of particular interest that these authors found that the activity of the lipid extract could be augmented by addition of saline extracts of normal fowl muscle, fowl protein, casein, gelatin, and gum acacia, or, in fact, by any addendum which might be expected to protect a very dilute agent or protein suspension against inactivation (cf. Adams, 1948). It has been shown, without doubt, however, that a considerable proportion of the lipid may be removed from dried Rous tumor tissue without inactivating the residue (Hawkins, 1928-9; Fraenkel and Mawson, 1937; Gye and Purdy, 1931; Claude, 1935b; Pollard and Amies, 1937; Pollard, 1938). Pollard found that tumor desiccates could be extracted with anhydrous solvents such as ethanol, ethyl ether, benzene,
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petroleum ether, or chloroform at a low temperature (20-35OC.) in nitrogen at reduced pressure. Such an extracted desiccate gave an active agent preparation (m.i.d. 2-5 X lo-* g.) containing some 6 % lipid instead of the usual 40-50%. The possibility remains that such a preparation contains a small proportion of active lipid-containing material together with a large proportion of lipid-free, inactive contaminating nucleoprotein. The evidence for this derives chiefly from the fact that efforts to delipidate the separated agent have been almost completely unsuccessful (Pollard, 1939; Harris and Carr, 1950).
4. Stability of Agent A. p H . Lewis and Michaelis (1928) found that the agent retained its infectivity in suspensions buffered in the range pH 4.0 to 12.0, but not a t pH < 3.0 or > 12.5. Bryan, Maver et al. (1949,1950), using a partially purified suspension and phosphate buffers of 0.05M final concentration, obtained maximum potency a t pH 7.0 to 7.5, but infectivity was retained between pH 5.0 and 8.5. So far as the physical stability is concerned, the purified agent forms a stable colloidal suspension between pH 7.0 and 11.0. Between pH 11.5 and pH 13, the initially opalescent suspension becomes transparent. Aggregation of the particles begins a t pH 6.6 and the agent has a minimum solubility at pH 3.5. Below pH 2.4, the opalescent solution again clears and the suspension is almost transparent at pH 1.0 (Claude and Rothen, 1940). A nonsedimentable fraction was split off below pH 2.0 and above pH 12 and spectrophotometric evidence suggests that this was nucleic acid. The fission of nucleic acid from the agent is probably not complete, since the residue, after precipitation of the agent from purified suspensions with 5% trichloracetic acid and removal of the lipids, contains only 0.2%phosphorus, whereas the residue prepared by delipidation of dialyzed and frozen-dried agent suspensions ’ phosphorus (Harris, unpublished). There is, therefore, contains 1.85% a very wide pH range within which the Rous agent is stable both as an infective unit and as a particle. Comparison with the viruses of rabbit papilloma (low lipid, deoxypentosenucleic acid) and Eastern strain equine encephalomyelitis (high lipid, pentosenucleic acid) shows that the latter, as might be expected, closely resembles the Rous agent. The infectivity range extends from pH 6.5 to 9.0 (Finkelstein et al., 1938), and the range of physical stability from pH 6.5 to 10.0, with agglutination beginning at pH 5.8 (Taylor et al., 1940). The encephalomyelitis virus may, indeed, have a very close physical and chemical resemblance to the Rous agent, since, in its preparation from infected embryos it may be accompanied by normal
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chick embryo component, which, however, was stated by Taylor et a2. (194213) to be unstable at pH 8.5. Rabbit papilloma virus has a remarkable range of physical stability (pH 2.74.0 and 5.8-9.7) ;between p H 4.0 and 5.8 the virus is aggregated but redisperses as the pH is raised or lowered. Below pH 2.8 (2.4 for the Rous agent) disintegration occurs and a second component may be detected (Beard and Wyckoff, 1938). B. Oxidation. Like many viruses, as purification advances the Rous agent becomes progressively more unstable, particularly in dilute suspensions. Mueller (1928) found that the rapid autoinactivation a t 37" of Berkefeld filtrates of the tumor was due largely to oxidation and could be prevented by addition of cysteine at pH 7.4 (v.a., Loewenthal, 1931). Under anaerobic conditions sodium hydrosulfite also has a stabilizing action (Pirie, 1931; Pine and Holmes, 1931) but under aerobic conditions both destroy the agent rapidly. Cyanide also has been used t o inhibit the oxidative enzyme systems of tumor extracts (Gye and Purdy, 1930). A similar effect of cysteine has been observed on equine encephalomyelitis virus (Bang and Herriott, 1943) which is also relatively unstable after purification (Wyckoff, 1937; Taylor et al., 1943). The fowl leucosis agent is sensitive to oxidation, but the agenKthus attenuated may be reactivated by reducing agents (Engelbreth-Holm and Frederiksen, 1938). No such reactivation could be shown for attenuated Rous agent (Pirie and Holmes, 1931). C. Chemical Inactivation. A vast range of salts, dyes, and colloids have been tested under many different conditions against relatively crude agent suspensions. Carr (1942b) observed no action of colchicine on suspensions of purified agent (cf. Sec. 11, 3) but, in the presence of glucose, notatin (which liberates hydrogen peroxide) had a marked inactivating action (Carr, 1945b). Pollard (1939) claimed that the observed decrease in infectivity after treatment with formaldehyde could be correlated with a decrease in the number of free amino groups. The great difficulty with this sort of experiment, however, is to distinguish partial saturation of active agent groups (cf. partial removal of the agent's lipid) from the complete saturation of all the groups of some of the agent. D. Enzymic Inactivation. Treatment of cell-free extracts of tumor tissue for twenty-four hours a t 4.5OC. a t optimum pH with castor bean lipase, soybean urease or an amylase had no action on infectivity (Sugiura, 1932). The effects of proteolytic enzymes have depended upon the purity of the enzyme preparations. Thus in alkaline media, commercial trypsin products inactivate the agent (Baker and McIntosh, 1927; Sugiura, 1932). In acid media, however, treatment with the same enzyme augments infectivity (Baker and McIntosh, 1927; Selbie and
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McIntosh, 1939). These results suggest that some “inhibitor” is removed. Pirie (1935) observed that the inactivation of the agents of both Rous No. 1 and Fujinami tumors with pancreatic extracts was not a result of degradation by either proteases or pancreatic lipase. The viricidal component of the extract had the properties of an unstable fatty acid and the higher unsaturated fatty acids were indeed found to inactivate the agents, possibly by physical disruption of the particles. These results were confirmed by Helmer (1936), who isolated an unsaturated viricidal fatty acid fraction from frozen pig pancreas and showed that it resembled oleic acid and that it was inactive after hydrogenation (cf. Begg and Aitken, 1932). Pirie (1935) showed that crystalline trypsin and chymotrypsin had only slight inactivating action on the agent, and tryptic digestion of agent concentrates has since been used as an aid in purification by Pollard (1939), Amies and Carr (1939), and Carr and Harris (1951a). Carr and Harris showed that, after the first stage of fractional centrifugation, 60% of the total protein of the agent concentrate became nonsedimentable after short digestion a t pH 7.5 to 8.0 with crystalline trypsin and that the infectivity of the agent was unimpaired. Hoffman, Parker, and Walker (1931) claimed that treatment of a cell-free filtrate of the tumor with rabbit testis extract before injection stimulated the growth of the tumor. The result could not be confirmed by Sturm and Duran-Reynals (1932) or by Duran-Reynals and Claude (1934), and it has since become evident that neither hyaluronidase (from rat or bull testes) nor azoprotein solutions, which have a similar action (Claude, 1939b) either activate or inactivate the agent. Its use in the early stages of the preparation of the purified agent considerably simplified the procedure (Carr and Harris, 1951a) and avoids such unnecessary waste as the rejection of the first tumor extract (Bryan et al., 1947). E. Irradiation. It was early realized that the agent in cell-free extracts of tumor tissue was much more resistant to inactivation by X- and /%irradiation than the cells of rat and mouse tumors but less resistant than enzymes (Russ and Scott, 1926; Lacassagne, Levaditi, and Galloway, 1927; and Baker, 1935). Claude and Rothen (1940) irradiated a suspension of highly purified agent with ultraviolet light for from five minutes to five hours. After thirty minutes, the degree of absorption at 2400 d and 2700 d had markedly decreased, and the suspension was noninfective. F. Freezing and Freeze-Drying. It has long been known that the filterable fowl tumors may be preserved in the frozen state for long periods and may even withstand many cycles of alternate freezing and
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thawing without loss of their ability to transmit the tumor. It remains to be determined whether this resistance to freezing depends only on the resistance of the agent to freezing or whether whole cells may, in fact, survive. The agent in filtrates may survive as many as sixty cycles of rapid freezing and thawing without inactivation (Miller and Eggers, 1935), but more highly purified preparations of the agent are almost completely inactivated by one cycle (e.g., Pollard, 1938). The Rous No. 1 sarcoma is also transmissible by dried tumor tissue, and it has been presumed, that, under such conditions, the agent alone survives (Rous, 1911; Rous and Murphy, 1914; Hoffstadt and Tripi, 1946). Knox (1939) dried Berkefeld filtrates of both Rous and Fujinami sarcomata but with large attendant loss of infectivity. Dmochowski (1948~)claimed that, although aqueous agent suspensions could not be satisfactorily frozen-dried (cf. Pollard, 1938) the addition of 0.001 % HCN or of a drop of fowl or rabbit serum to the suspension gave 60 to 80% preservation. A detailed study of the preservation of the Rous agent by freeze-drying has been made by Carr and Harris (Harris, 1951; Carr and Harris, 1951b). A large number of different suspending media was tested, and optimum results (i.e., 100% recovery of infectivity after rehydration) with highly purified agent suspensions were obtained with media containing broth. 6. Immunological Properties of Rous Agent
Experiments with highly purified Rous No. 1 agent suspensions support the view that the agent has a t least two antigenic components, corresponding to two antibodies, one of which (antiagent) is present in Rous-immune fowl serum and the other in antifowl rabbit serum. Amies and Carr (1939) considered that both the antigens were part of the agent. Foulds and Dmochowski (1939) found by complement fixation tests that Rous No. 1 sarcoma and the carcinogen-induced sarcoma (RFD 2) had two common antigens, which by the methods used were not demonstrated in normal fowl tissues. One of these antigens passed an 800-mp Gradocol membrane but not a 140-mp one, and was therefore comparable in size with the Rous agent, whereas the other passed the 140-mp filter and was thus considerably smaller. Kabat and Furth (1940) found no demonstrable immunological differences between particulate products from S1 sarcoma-leukosis and from normal spleen extracts, which was held to suggest that the tumor preparations were very heavily contaminated with normal cell components. However, Gottschalk (1943) found that rabbit antisera to normal fowl tissue had no neutralizing action against the agent from his 513 fowl tumor (although the agent was precipitated), which was inter-
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preted to mean that the agent of 513 does not contain normal fowl antigen. If this observation is confirmed, it may be important since the rabbit papilloma virus is almost lipid-free and appears to have only one antigen whereas the majority of other animal viruses evoke at least two antibodies (Lennette, 1943). Knight (1946b) has shown by precipitation tests that the most highly purified preparations of Lee and PR 8 strain of influenza virus obtained from the allantoic fluid of infected embryos contain an antigen characteristic of normal allantoic fluid, and, similarly, that purified PR 8 virus from mouse lung contains an antigen characteristic of normal mouse lung but the influenza virus, unlike the Rous agent but like the S13 agent, is not neutralized by antisera t o the respective normal host antigen.
V. RELATIONSHIP OF Rous AGENT TO FOWL TUMORS AND LEUCOSES 1. Carcinogen-Induced Fowl Tumors Carrel was the first to claim that tumors, induced in the pectoral muscle of fowls by injection of a mixture of embryo pulp and a little arsenious acid or indole, were filterable. The subsequent literature has contained both claims and counter-claims. Murphy and Landsteiner (1925) and Murphy and Sturm (1941) concluded that, for fifty-two induced fowl sarcomata, of which twelve were transplantable, there was no evidence of a transmissible agent. In 1946 Peacock presented a comparative study of fifty-five chemically induced fowl sarcomata and three filterable sarcomata (Rous No. 1, Fujinami and McIntosh No. 5). Thirteen of the induced tumors were transplanted once or more, and none of them could be transmitted by cell-free extracts. After careful assessment of claims that some induced tumors were transmissible by filtrates (Des Ligneris, 1935, 1936; McIntosh and Selbie, 1939) Peacock concluded that only one (McIntosh and Selbie’s No. 9) was an apparently uncomplicated filterable spindle-cell sarcoma directly induced by the injection of tar. Since 1946, a fresh claim has been put forward by Oberling et al. (1946) and by Oberling and Guerin (1950) that a tumor induced by 20-methylcholanthrene was filterable between the fifth and sixth passages but not subsequently. These authors claimed that the possibility of contamination with Roue No. 1 tumor was negligible because none was present in the laboratory when the filterability was discovered. However, it was found that chickens resistant to their virus sarcoma (T 25) showed resistance to grafts of the new tumor and it seems possible, therefore, that contamination with the agent of T 25 could have occurred. There appears to be no doubt that some of the tar tumors are antigenically related to the Rous No. 1 or other filterable tumor agents.
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Thus Andrewes (1936) found that pheasants inoculated with a nonfilterable tar-induced fowl tumor developed neutralizing antibodies to Rous sarcoma I. These were absent from normal pheasant sera and were not induced by immunizing pheasants with normal fowl embryo. Andrewes concluded that the tar sarcoma contained a “virus,” although it could not be directly demonstrated by filtration or desiccation. Amies, Carr, and Purdy (1939) prepared a particulate agent from Des Ligneris’ tumor (Des Ligneris, 1936). Immunologically the agent was distinct from the Rous No. 1agent but closely resembled that from the Fujinami myxosarcoma, but neutralization tests with rabbit antisera to tumor agent suspensions from Rous No. 1 and the Des Ligneris tumor showed that each tumor agent was inhibited by the homologous and, t o a lesser degree, by the heterologous serum. Gottschalk (1943, 1948) found similarly that his nonfilterable induced tumor (516) contained an antigen, related to or identical with an antigen from the filterable sarcoma, 513. Andrewes (1931, 1933), Foulds (1937) and Dmochowski and Knox (1939) showed that all fowl tumor viruses which have been studied have some degree of antigenic relationship and further that both filterable and nonfilterable tumors have common antigen factors. It is reasonable to conclude that the nonfilterable tumors contain cytoplasmic particles which closely resemble the agents from filterable tumors and to infer that the nonfilterable tumor cells are unable to “complete” a virus that is infective in the extracellular phase, possibly because the “pattern” for the replication of such a “complete” virus has not been provided to the cell. Should it be possible to discover wherein the difference between infective and noninfective particle lies, then it might be equally possible to force the carcinogen-induced tumor cell to produce transmissible agent as the result of an “agent” mutation induced by means of the appropriate substrate. 2. Fowl Leucoses
The transmissible fowl leucoses have a number of features in common with the filterable tumors and are transmissible by cell-free agents with similar properties. Moreover tumor-like proliferations are frequently associated with leucoses (Furth, 1933, 1934; Oberling and Guerin, 1933a,b, 1934). Unlike the Rous No. 1 sarcoma, however, these sarcomata and endotheliomata are not transmissible in series. Moreover, it has been stated (Pikovski et al., 1947; Pikovski and Doljanski, 1950) that cell-free leucotic material never produces a tumor at the site of injection and that the cell-free agents of the t‘pure” leucosis strain apparently have an affinity for primitive blood cells only [cf. the observation on avian
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lymphoid tumors by Burrnester, Prickett, and Belding (1946) and Burmester and Cottral (1947)l. The cell-free agent of S1 leucosis was isolated and studied by Kabat and Furth (1940). Stern and Kirschbaum (1939) and Stern et a,?. (1940) found that partially purified preparations of the agent of S 1 were even more labile than the Rous No. 1 agent and material prepared by fractional centrifugation underwent considerable aggregation. The agent of Furth and Kabat’s S13 strain was isolated by Gottschalk (1946), and the final preparation had a m.i.d. of 8.6 X g. N. Yet another particulate component, associated with avian erythromyeloblastic leucosis was isolated from the plasma of infected fowls (Beard et al., 1950). The component had a sedimentation size of 630 S but was not homogeneous. Electron micrographs showed spheres in the size range 60-100 mp, some of which had “tails” 100 to 200 mp long. These particles were not found in normal fowl plasma, but their relationship to the disease has not yet been defined. The plasma from which these particles were isolated contained a viscous mucoprotein; it is probable that the “tails ” are artefacts, and it would be interesting to know whether they disappear after treatment with a mucinase. Rothe-Meyer and Engelbreth-Holm (1933) considered that avian leucoses were neoplasms of undifferentiated mesenchyme which, normally, produces either connective tissue cells, endothelial cells, or erythrocytes. The particular manifestations of the disease would then be dependent upon the route of inoculation of the infective material. Foulds (1934) regarded the greater variety of manifestations of the leucoses compared with the filterable tumors as more probably due to the greater susceptibility of the cells to external influences rather than to conversion of different cell types or t o changes in the cytotropism of the agent itself. Uhl et al. (1936) assumed that each infective agent could induce tumors not only in the same kind of cells as the ones from which it originated but also t o a lesser degree, in genetically related cells whether precursors or descendants. It is possible, however, that selection from an infective agent which is not homogeneous with respect to cytotropism could equally well explain these results, for Oberling and Guerin (1933a,b, 1934) found that prolonged storage of leucotic material either in glycerol or in the ice chest, gave a “leucosis” virus with an increased tendency to produce sarcomata, although on further passage all the sarcomata eventually gave rise to leucoses. Findlay (1936) postulated that prolonged passage of a leucosis virus in tissue cultures of fibroblasts might result in a virus capable of producing only sarcomata. The most important conclusions which may be reached from consideration of the relationship of these avian leucoses and their agents
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to the Rous No. 1 agent are: first, the impossibility of reaching an unequivocal decision on the question of the filterability of the carcinogeninduced tumors since it is not sufficient that the fowls in question should be demonstrably free from Rous No. 1 agent alone; secondly, a further emphasis on the need for a complete comparative study of the products of the cells of nonfilterable tumors, sarcomata, and leucoses. VI. ORIQIN OF Rous AGENT There has been much discussion about the extrinsic or intrinsic origin of the Rous agent. The original tumor arose spontaneously in 1910 and has not been contagious, although under the right conditions (e.g., the direct contact of virus from one bird with a wound in a second), infection would, no doubt, occur. This,together with the fact that the agent has a t least two antigens, one of which is related to normal host cell protein has led recent authors to describe the agent as (‘a homologue of protoplasmic organizers determining the malignant transformation in nonfilterable tumors” (Gottschalk, 1948) or, more simply, as a “mutant plasmagene” (Haddow, 1944, 1947; Darlington, 1948). Haddow (1947) has suggested that the result of the penetration of the agent into the cytoplasm of a susceptible normal cell (that is, a cell containing a “required” gene) is the continued production of agent and the development of malignancy, both being determined by the combined presence of gene and agent. Darlington postulates different types of cancer-producing agents with the properties of plasmagenes, proviruses and true viruses. Most mammalian tumors, transmissible by intact cells are classified only in the first group. The avian tumor agents are stated to fall.into the provirus group, proviruses being those (‘self-propagating proteins in the cells of plants and animals which are normally transmitted by heredity, but have the properties necessary for natural infection if an infective agent or vector were to come along.” It is postulated that the proviruses have arisen intrinsically by the “mutation ” of existing cytoplasmic selfpropagating particles. The “spontaneous” tumor in the fowl, therefore, is believed t o be the expression of a plasmagene mutation, since, according to Darlington, no such mutation can express itself in a mature cell except by renewing the growth which has ceased. But, as Andrewes (1939-40), Duran-Reynals (1940a), and others have shown, most normal fowls, as they grow older develop neutralizing antibodies active against the Rous agent. This suggests that many tumor-free birds, i.e., birds lacking the L( expression” of the supposed plasmagene mutation, are either carrying or have had contact with the Rous or related virus. A mutation of a cytoplasmic determinant must, on Darlington’s hypothesis, have occurred, therefore, without causing resumption of growth.
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It must be concluded that the origin of the Rous agent is as obscure as the origin of any other virus and that attempts to solve the pr6blem in terms of the mutation of yet another particle of unknown properties and origin are of little help. VII. CONCLUSION In the last decade considerable advances have been made in the analysis of the functional organization of cells. What is now needed is an understanding of the behavior of viruses in relation to this organization. How do they invade the host’s cells, how are they replicated, how do they intervene in the normal functioning of cytoplasm and nucleus? These are basic problems, but there can be no doubt that the progress made toward their solution, whether in the field of plant, animal or bacterial viruses will be of the greatest importance to those who are investigating the virus-induced tumors. Moreover, the interest of the virus-induced tumors for the study of malignant growth does not lie in the naive assumption that all tumors are the result of a virus infection but in the conviction that knowledge of the mode of intervention of a tumor virus in the normal organization of the infected cell will help to elucidate the nature of the change from the normal to the malignant cell. REFERENCES Adams, M. H. 1948. J . Gen. Physiol. 31, 417-31. Amies, C. R. 1937. J . Path. B a d . 44, 14146. Amies, C.R.,and Carr, J. G. 1939. J . Path. Bact. 49,497-513. Amies, C. R., Carr, J. G., and Purdy, W. J. 1939. Am. J . Cancer 36,72-79. Andrewes, C. H. 1931. J. Path. Bact. 34,91-107. Andrewes, C. H. 1933. J . Path. Bact. 37,2744. Andrewes, C.H. 1936. J . Path. Bact. 43, 23-33. Andrewes, C. H. 193940. Proc. Roy. SOC.Med. 33, 75-86. Baker, S.L. 1935. Brit. J . Exptl. Path. 18, 148-55. Baker, S. L.,and McIntosh, J. 1927. Brit. J . Exptl. Path. 8,257-65. Bang, F. B., and Herriott, R. M. 1943. Proc. SOC.Exptl. Biol. Med. 62, 177-80. Barron, E. S. G . 1932. J . Exptl. Med. 66, 829-35. Bauer, D.J. 1947. Brit. J . Exptl. Path. 28, 440-46. Bauer, D.J. 1948. Nature 161, 852. Bawden, F. C. 1951. Science Progress 39, 1-12. Beard, D.,Eokert, E. A., Csaky, T. Z., Sharp, D. G., and Beard, J. W. 1950. Proc. Soc. Exptl. Biol. Med. 76, 533-36. Beard, J. W. 1948. J . Immunol. 68,49-108. Beard, J. W., and Wyckoff, R. W. G. 1938. J . Biol. Chem. 123,461-70. Begg, A. M., and Aitken, H. A. A. 1932. Brit. J . Exptl. Path. 13,479-88. Brumfield, H. P., Stulberg, C. R., and Halvorson, H. 0. 1948. Proc. Soc. Exptl. Biol. Med. 68, 410-13. de Bruyn, W. M. 1949. Bijdragen tot de Dierkunde 28, 77-85.
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de Bruyn, W. M., Korteweg, R., and Kits van Waveren, E. 1949. Cancer Rqsearch 9, 282-93. Bryan, W. R. 1946a. J. Natl. Cancer Inat. 8,22537. Bryan, W. R. 1946b. J. Natl. Cancer Znat. 8, 373-77. Bryan, W. R., Lorenz, E., and Moloney, J. B. 1950. J. Natl. Cancer Znst. 10, 1215-37. Bryan, W. R., Maver, M. E., Moloney, J. B., Thomas, M. A., and Sells, M. T. 1949. J. Natl. Cancer Znst. 10, 647-56. Bryan, W. R., Maver, M. E., Moloney, J. B., Wood, M. T., and White, C. C. 1950. J . Natl. Cancer Insl. 11, 269-77. Bryan, W. R., Riley, V. T., and Calnan, D. 1947. J. Natl. Cancer Znst. 7, 203-06. Bryan, W. R., Riley, V. T., Deihl, D. G., and Voorhees, V. 1947. J. Natl. Cancer Znst. 7, 447-53. Burmester, B. R., and Cottral, G. E. 1947. Cancer Research 7, 669-75. Burmester, B. R., Prickett, C. O., and Belding, T. C. 1946. Cancer Research 6, 189-96. Carr, J. G. 1942a. Brit. J. Ezptl. Path. 28, 206-13. Carr, J. 0. 1942b. Brit. J. Exptl. Path. 29, 221-28. Carr, J. G. 1943. Brit. J. Ezptl. Path. 24, 127-32. Carr, J. G. 1944a. Proc. Roy. SOC.Edinburgh 62B,51-53. Cam, J. G. 1944b. Brit. J. Exptl. Path. 26, 56-62. Carr, J. G. 1945a. Brit. J. Exptl. Path. 27, 1-3. Carr, J. G. 1945b. Nature 166, 202. Carr, J. G. 1947. Proc. Roy. SOC.Edinburgh 82B, 243-247. Carr, J. G., and Harris, R. J. C. 1951a. Brit. J . Cancer 6, 83-94. Carr, J. G., and Harris, R. J. C. 1951b. Brit. J . Cancer 6, 95-105. Carrel, A. 1924a. Compt. rend. soc. biol. 90, 1380-82. Carrel, A. 1924b. Compt. rend. soc. biol. 91, 1067-69. Carrel, A. 1924c. Compt. rend. soc. biol. 91, 1069-71. Carrel, A. 1925a. Compt. rend. soc. biol. 99, 85-87. Carrel, A. 1925b. J. Am. Med. Assoc. 84, 157-58. Carrel, A. 1926. J. Ezptl. Med. 49, 647-68. Carrel, A., and Ebeling, A. H. 1926. J. Ezptl. Med. 43, 461-68. Chambers, L. A., and Henle, W. 1941. Proc. SOC.Ezptl. Biol. Med. 48, 481-83. Chambers, L. A., and Henle, W. 1943. J. Exptl. Med. 77,251-63. Chamben, L. A., Henle, W., Lauffer, M. A., and Anderson, T. F. 1943. J. Exptl. Med. 77, 265-75. Claude, A. 1934. Am. J. Cancer 22,586-89. Claude, A. 1935a. J. Exptl. Med. 61, 27-40. Claude, A. 1935b. J. Ezptl. Med. 81, 41-57. Claude, A. 1937. J. Exptl. Med. 88, 59-72. Claude, A. 1938. Science 87, 467-68. Claude, A. 1939a. Science 90, 213-14. Claude, A. 1939b. J. Exptl. Med. 69,641-48. Claude, A. 1940. Science 91, 77-78. Claude, A. 1947-8. Harvey Lectures Set. 43, 121-64. Claude, A., Porter, K. R., and Pickela, E. G. 1947. Cancer Research 7, 421-30. Claude, A,, and Rothen, A. 1940. J. Ezptl. Med. 71, 619-33. Cox, H. R., van der Scheer, J., Aiston, S., and Bohnel, E. 1947. J. Immunol. 66, 149-66. Darlington, C. D. 1948. Brit, J . Cancer 2, 118-26.
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Applications of Radioisotopes to Studies of Carcinogenesis and Tumor Metabolism CHARLES HEIDELBERGER The McArdle Memorial Laboratory, The Medical School for Cancer Research, University of Wisconsin, Madison, Wisconsin CONTENTS
I. Introduction 1. General 2. Scope of the Review 3. Tracer Methodology A. Unique Applications B. Tracer Requirements C. Purity of Samples D. Isotope Dilution and Carrier Technics 11. Metabolism of Carcinogenic Hydrocarbons 1. Introduction 2. Distribution of Radioactivity 3. Chemical Fractionation of Metabolites 4. Rates of Elimination 5. Identification of Metabolites 6. Interaction with Tissue Components 111. Other Carcinogenic Compounds 1. Carcinogenic Azo Dyes 2. Acetylaminofluorene and Derivatives 3. Carcinogenic Beneacridines IV. Oxidative Metabolism of Tumors 1. Deficiency in Tumors? 2. Krebs Cycle in Tumors 3. Present Status V. Incorporation of Amino Acids into Tumor Proteins 1. Introduction 2. I n vivo Studies 3. In vitro Studies VI. Nucleic Acids 1. Introduction 2. Radioactive Phosphorus Studies 3. Nucleic Acid Purines 4. Nucleic Acid Pyrimidines VII. Miscellaneous Compounds 1. Iodinated Polysaccharide from Serratia marcesnes 2. A Radioactive Oxazine Dye 3. Stilbamidine 273
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4. Triphenylbromoethylene.. .......................................
6. Diethylstilbestrol. . . .
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VIII. Conclusion.. . . . . . . . . . . . . . . . . . . . . 333 References.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
I. INTRODUCTION 1. General
The extraordinary proliferation of biochemistry within the last two decades has undoubtedly been due to a very large extent to the introduction of the use of isotopic tracers. The application and exploitation of new technics made possible by this new methodology has profoundly revolutionized our concepts, not only of biochemistry but also of biology itself. Since the problem of cancer is a biological one, which may be described in many aspects in biochemical terms, it is only natural that the impact of the use of isotopes has been felt within the realm of oncology. However, just as biochemistry has only recently become admitted as a fairly respectable member of the family of oncological sciences, even so, it is only within the last five years that the tracer methodology has been applied extensively to problems of cancer biochemistry. Nevertheless, its use has increased rapidly during this period. A t the 1945 annual scientific meeting of the American Association for Cancer Research of the 26 papers presented, none dealt with the use of isotopic tracers; a t the 1951 meeting of the same society, 18 of the 174 papers involved the use of this technic. This enormous increase in the use of isotopes has been brought about largely as a result of the farsighted and successful efforts of the United States Atomic Energy Commission in providing a wide variety of radioactive isotopes to qualified scientists and in conducting and encouraging training programs through which scientists may learn the safe handling and accurate measurement of radioactive materials. Another factor that has greatly increased the availability of this technic has been the commercial production of satisfactory instruments for isotopic measurements at reasonable costs. In 1949 Calvin, Heidelberger, et al. (1949), stated: (‘It, is our conviction that isotopic . . . tracers will very soon find its place as a tool in many kinds of scientific laboratories. Its use on a routine basis for the investigation of physical, chemical, and biochemical transformations is comparable with that of the microscope, spectroscope, or x-ray analyses in their respective spheres.” This reviewer has also stated (Heidelberger, 1951), (IWith this everincreasing use of isotopes, the tracer technic is no longer an end in itself,
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but is assuming its proper perspective as a useful tool in research. . . . Concomitantly with this progressively larger scope of isotope researches it would appear desirable to shift the emphasis in review articles away from the tool and back to the subject matter itself. In any event, because the literature on the use of isotopes in biochemistry is increasing exponentially, such reviews will have to deal with a more and more restricted subject matter.” While still in full agreement with the foregoing statement, this reviewer will nevertheless have the temerity t o undertake the present chapter because of the restricted subject matter and the relatively small number of “applications of radioisotopes to studies of carcinogenesis and tumor metabolism’’ at the time of this writing. Cancer is primarily a problem in growth. As Greenstein has written (1947), “ Cancerous growth capacity is apparently independent of the organism and hence, the growth capacity of the tumor is a nearly unique property of the tumor itself. This property of autonomous growth is the most striking characteristic of tumors as a class and within the factors responsible for this property lies the secret of the control of this growth.” It would be very attractive indeed, to include all aspects of growth within the framework of this review, but it would obviously be beyond the ability of this writer and the scope of this review to be so all inclusive. Therefore, a t the risk of omitting many important aspects of growth and development of normal tissues, the subject matter will arbitrarily be limited to that included in the title. Few or no references will be made to contributions outside of the domain of research actually carried out in the production and investigation of tumors. An attempt will be made a t a critical evaluation of the literature until September 1951, and in a few cases of which the author is directly aware more recent reports will be included. Since the tracer technic has been used for a variety of researches, this review will necessarily be a reflection of the present state of cancer biochemistry; some attention will be given to pointing out significant trends in various fields.
2. Scope of the Review Many important applications of isotopes in the field of cancer treatment and research are beyond the scope of the present discussion. Fortunately, several excellent reviews on specific aspects of the problem are available. A Manual of Artificial Radioisotope Therapy by P. F. Hahn (1951) authoritatively summarizes therapeutic uses, and diagnostic applications have been discussed by Quimby (1951). Brues (1951) has summarized the work of his group and others on the carcinogenic effects of radiation, and Pressman (1951) has reviewed the use of isotopes in
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studying the immunology of cancer; these fields will also be arbitrarily excluded from this chapter. The wealth of information that is available on the production of isotopes and their measurement will not be covered in this review. It is to be assumed that the reader has some familiarity with the subject. Textbooks which are of perhaps most use to those interested in biological problems have been written by Kamen (1951), Hevesy (1948a), and Calvin, Heidelberger, Reid, Tolbert, and Yankwich (1949). The pioneer experiments with tracers were carried out by Hevesy in 1923, when he studied the transport of lead in plants, using a naturally occurring radioisotope lead-212 (thorium B). A decade later, following the concentration of deuterium oxide by Urey, a series of investigations by Schoenheimer and Rittenberg, using stable isotopes, was begun. These studies, which have admirably been summarised by Schoenheimer (1946), resulted in one of the most important concepts of modern biochemistry, ‘(the dynamic state of body constituents” and the concept of the metabolic pool. Up to this time there had been a rigorous separation of exogenous and endogenous metabolism. The adult living organism was considered as analogous to a steam engine, undergoing frequent repairs, whose fuel supply provided the necessary energy, but contributed only a small amount of soot to the actual structural body of the machine. I n the dynamic state, which could only have been discovered through the use of isotopic tracers, the following analogy has been cited by Schoenheimer (1946). “ A military regiment resembles a living adult organism in more than one respect. Its size fluctuates only within narrow limits, and it has a well-defined, highly organized structure. On the other hand, the individuals of which it is composed are continually changing. Men join up, are transferred from post to post, are promoted and broken, and ultimately leave after varying lengths of service. The incoming and outgoing streams of men are numerically equal, but they differ in composition. The recruits may be likened to the diet; the retirement and death correspond to excretion.” 3. Tracer Methodology
A. Unique Applications. The following will enumerate some of the unique applications of tracers to the field of biochemistry, and many examples.of each of these points will be found in the main body of this review: (1) The discovery of new metabolic processes and the identification of intermediates in the process. (2) Quantitative studies of metabolism and physiology in vivo.
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(3) Experiments on a very small scale, below the sensitivity of most conventional methods of analysis. (4) Measurements on equilibria in the steady state. (5) Measurements on the kinetics of metabolic reactions. In essence, the tracer method consists in the synthesis of labeled compounds, administration to the test system, and isolation of other compounds and assay of their tracer content. While this sequence of events appears to be quite simple, a number of requirements must be met in each situation before an experiment can be interpreted with any degree of probability. B. Tracer Requirements. (1) The isotope must behave chemically and biologically in a fashion identical to that of the natural element. While this requirement is ordinarily met, there are some cases known in which the tracer isotope reacts chemically and biologically a t a different rate from that of the natural isotope. This is particularly true of low atomic weight isotopes, such as tritium and deuterium, whose masses differ by large ratios from that of hydrogen, and significant errors may thus be introduced into studies of rates or equilibria. (2) The isotope must be present in sufficiently high concentration to withstand the dilution that is encountered in the experiment. This dilution in large animals might well amount t o several million-fold, and under these conditions radioactive isotopes of sufficiently high specific activity can be used, whereas stable isotopes could not be detected. This makes it possible to administer minute doses of radioactive tracer substances that will not upset the physiological state of the test animal, while with stable isotopes it is all too frequently necessary to flood the animal with doses that are enormous relative to the normal body pool. (3) The label must be retained throughout the experiment. If the label is introduced into a chemically reactive part of a molecule it may frequently be lost through exchange reactions. In other cases metabolic transformations may cause the loss of the tracer atom, which might have been retained were it in a different location within the molecule. (4) There must be no biological effect caused by the toxicity of the tracer itself. The bioIogica1 effects of radiation are an ever present consideration in work with radioactive isotopes. The specific activity of the tracer should be calculated in such a way as t o minimize this hazard in whatever biological system is under investigation. A true tracer should never be an “influencer.” ( 5 ) The half-life of the radioactive isotope should be long enough for the experiment. It would clearly be impossible to investigate the induction of tumors, a process which often takes six months, with a carcinogen labeled with carbon-11, which has a half-life of 21 minutes. The 5300-
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year half-life of carbon-14 not only makes the experiment feasible but also gives the investigator ample leisure to ponder the significance of the results. (6) The labeled atom should be an isotope of the element of actual interest. There is an unfortunate case in which a tremendous amount of effort was expended in studying the distribution of an iodinated steroid, labeled with radioactive iodine, in spite of the fact that this particular compound was devoid of biological activity. It is difficult to imagine any way in which the data derived from those careful experiments can be extrapolated to the hormone itself. It is to be hoped that as thorough a study will be carried on the natural steroid, when the CI4-labeled compound becomes available. C. Purity of Samples. I n tracer work it is customary to isolate the product under investigation and determine its isotopic content, but before a valid interpretation can be made the compound must be scrupulously purified. This is necessary particularly when dealing with radioactive isotopes, because a contamination of a sample with a minute quantity of a highly radioactive impurity could vitiate the entire experiment. Because of this fact, ordinary criteria of purity such as melting point, carbon and hydrogen analysis, and spectrum are inadequate. Thus, new criteria of purity must be devised and exceptionally rigorous purification procedures must be employed. In general, conventional methods of purification such as recrystallization or distillation do not suffice to give samples of adequate purity. It is always desirable to design the isolation method so as to be as specific as possible for the compound in question, and in addition to the usual means of purification, chromatographic and liquid-liquid separations should be used t o ensure separation of closely related compounds. Thus, it is only by a combination of several types of purification procedures that one can be assured of having achieved isotopic purity. The reader, when perusing the literature, should critically examine the methods that have been used for isolations in order to evaluate for himself the validity of the conclusions that have been drawn from the experiment. Unfortunately, it is all too frequent that investigators give insufficient attention to this matter. D. Isotope Dilution and Carrier Technics. Two technics have been developed for tracer work that are particularly useful in metabolic studies; these are the carrier and isotope dilution methods. Although these terms are often used interchangeably, the two processes are actually completely different. The isotope dilution method was first conceived and applied by Rittenberg and Foster (1940). It consists in the addition of an isotopically labeled compound to an unlabeled system, isolation, and purification of the product. The isotopic content of the compound
RADIOISOTOPES APPLIED TO TUMOR STUDIES
279
is then measured, and the dilution of isotope represents a quantitative measure of the amount of unlabeled compound present in the system. This principle has been extended to permit the analysis of multi-component systems (see Calvin, Heidelberger et aZ., 1949, Appendix 1). The carrier technic consists in the addition of a nonlabeled compound to an isotopic system followed by isolation and purification of the product. The amount of label present in the carrier compound is a quantitative measure of the amount of that labeled compound present in the system. By the addition of nonradioactive carriers it is often possible to carry out extensive purification and chemical degradations on what was originally a very minute quantity of compound. The measurement of kinetics and equilibria in living animals can often be accomplished only by means of tracer technics. These studies ordinarily measure the “turnover” of a metabolite or of a precursorproduct relationship and involve measurements of isotope content a t a number of times after administration of the labeled compound. The theory of these concepts is developed in an important paper by Zilversmit et al. (1943). There has been an unfortunate tendency in the recent literature to refer incorrectly to turnover and renewal rates based on measurements at only one or two times.
11. METABOLISM OF CARCINOGENIC HYDROCARBONS 1. Introduction
The contribution of chemical carcinogenesis to the field of oncology is one of considerable significance. It is a valuable tool for the production a t will of a variety of tumors in experimental animals. However, of perhaps greater importance is the use of chemical carcinogens to provide a means of studying the mechanism or mechanisms of tumor production. It is a rather unique situation when a pure chemical compound induces a profound series of chemical and biological changes that result in cancer, and thus it provides a challenging opportunity to study the phenomenon in biochemical terms. If it were possible to discover any metabolic sequence of reactions that was specifically affected by the carcinogen, then progress toward the prophylaxis and treatment of endogenous cancer would be materially advanced. Yet in spite of the tremendous effort that has been expended in the synthesis and biological testing of thousands of polynuclear hydrocarbons, we are still pitifully far from a knowledge of their mode of action. In an attempt to arrive at an understanding of the mechanism of action of carcinogenic hydrocarbons the Pullmans (1946) have calculated the pi electron density of the phenanthrene double bond or “ K ” region
280
CHARLES HEIDELBERGER
of these substances and were able to obtain a fairly satisfactory correlation between this quantity and the carcinogenic potency of the compounds. Several studies have been carried out in England (Cook and Schoental, 1948; Badger, 1949; Boyland, 1950) in which some correlation has been obtained between carcinogenicity and the ease of addition of various reagents to the phenanthrene double bonds of carcinogenic hydrocarbons. However, there is a large biological and biochemical gap, as yet unfilled, between pi electrons and the malignant cell. It is to be hoped that this gap can be bridged by a more detailed study of the metabolism of carcinogens and their chemical interaction with tissue constituents. The use of isotopically labeled carcinogens provides an important tool for the attack on this problem, and will be discussed in some detail. A series of authoritative reviews on chemical carcinogenesis by Haddow and others has appeared in the British Medical Bulletin (1947), and Rusch and LePage (1948) and Boyland (1949) have reviewed other recent aspects of this field. At the present time, the synthesis of three C14-labeled carcinogenic hydrocarbons have been reported. Heidelberger et al. (1947) prepared 1,2,5,6-dibenzanthracene-9,10-C14 (I), Dauben (1948) made 20-methylcholanthrene-1 1-C14 (11), and Heidelberger and Rieke (1951) have synthesized 3,4-benzpyrene-5-C14 (111). These compounds will be considered as a group in the subsequent discussion, which will be concerned with information that could only be obtained by means of tracer technics.
I
I1
I11
2. Distribution of Radioactivity
To gain information on the metabolism of carcinogenic hydrocarbons it is important to determine quantitatively the distribution of the compound following administration to the test animal. Before the availability of labeled carcinogens, advantage was taken of the characteristic ultraviolet absorption spectra and fluorescence in studies of metabolism. However, even under the most carefully controlled conditions, only a
RADIOISOTOPES A P PL I l D TO TUMOR STUDIES
281
small percentage of the administered dose could be accounted for (Jones et al., 1944). Therefore, it seemed important to initiate the metabolic studies with labeled hydrocarbons by a careful quantitative study of the distribution in tissues. Heidelberger and Jones (1948) measured the distribution of radioactivity following administration of 1,2,5,6-dibensanthracene-9, 1O-Cl4 to mice. Essentially quantitative recoveries of C l 4 were obtained following administration of the compound by intravenous, intraperitoneal, and subcutaneous injections and by stomach tube. It was found that no radioactivity could be detected in the respiratory carbon dioxide, indicating that the 9- and 10-carbons of the carcinogen do not participate in many normal pathways of metabolism; the same holds true for 3,4-benepyrene-5-C l 4 (Heidelberger and Weiss, 1951). When labeled dibenzanthracene was administered intravenously as an aqueous colloid, the radioactivity quickly appeared in the liver, from which it passed through the intestinal tract and into the feces. Table I TABLE I Initial Steps in Distribution 0.5 mg. of aqueous DBA colloid injected intravenously % ' of dose
Liver Spleen Plasma Red cells Intestinal contents and feces Lungs Total recovery
4 hr.
1%hr.
24 hr
89. 1.9 0.24 1 .oo 1.9
82. 2.3 0.10 '0.71 7.6 0 97.0
3.5 0
0.43
94.7
0 0 94.
0.37 98.8
shows this sequence of events as well as the amount of CI4 in the lungs. Although there is very little radioactivity in the lungs, pulmonary tumors have been induced in mice by carcinogens administered by this route. The mechanism of this phenomenon remains unexplained. When the bile duct was cannulated, the radioactivity was excreted into the bile (Table 11). Similar results were obtained with benzpyrene, although there was more absorption into body fat than was found with dibeneanthracene. Table I11 gives the data obtained with labeled benepyrene following intravenous administration and illustrates the quantitative recovery of the administered dose. The radioactivity was measured in several tumors induced by subcutaneous injection of labeled dibeneanthracene, and the results are presented in Table IV. It is significant that even after six months there
282
CHARLES HEIDELBERGER
TABLE I1 Bile Data from Intravenously Injected Colloid Per cent total activity given, 24 hours Normal Mouse Feces Stomach and contents Intestinea Intestinal contents Bile Total
5.62 4.15
0 48.30
None 58.1
Bile Fistula Mouse 0 0 0 0 53.2 63.2
TABLE I11 Distribution of Radioactivity Following Intravenous Injection of an Aqueous Colloid of 0.45 mg. Benrpyrene-5-C14 90 Minutes
Dry Wt. g.
Dose Respiratory C 0 2 Feces Urine Gastrointestinal tract contents Carcass fat tissue Abdominal fat tissue Liver Kidneys Lungs Heart Stomach Salivary glands Plasma Red cells Carcass bile and gall bladder Recovery
+
+
+
+
Total ct./min.
24 Hours
%of Dose
Dry Wt. Total g. ct./min. 1,210,000
1,390,000 <400 <0.03
None 3,300 0.834 1.746 1.778 0.371 0.083 0.043 0.029 0.142 0.029 0.085 0.178 7.038
1. O l 1.36 0.31 0.072 0.034 0.14 0.022 0.22 0.022
1,000,000 72.0 1,344,000 97.0
<0.008
0.575
794,000 210,000
0.532 0.982
16,600 14,200
1.37 1.17
0.971 0.270 0.078 0.039 0.031 0.066 0.035 0,072 0.179
16,100 20,900 4,100 890 350 3,700 540 1,700 590
1.33 1.72 0.34 0.071 0.029 0.31 0.045 0.14 0.049
5.614
92,100 1,176,000
0.24
163,000 11.7 133,000 9.6 14,000 18,900 4,300 1,000 480 2,000 300 3,000 310
% of Dose
65.5 17.3
7.60 97.2
RADIOISOTOPES APPLIED TO TUMOR STUDIES
283
was an appreciable quantity of radioactivity appearing in the tumor, indicating that the carcinogen is present in appreciable quantity throughout the period of induction of the tumor. The tumors produced by the labeled carcinogen had the same incidence and latent period as those produced by the nonradioactive control, and there were no detectable histological differences among them. Thus it appeared that there was no biological effect exerted by the radioactivity of the carcinogen, in agreement with the calculations which indicated a very slight effective TABLE IV Radioactivity in Tumors Produced by Dibenaanthracene Time, Months
Solvent
Tumor
Counts per Minute Per Cent
Spindle-cell sarcoma 6 Mouse fat 10 Mouse fat Spindle-cell sarcoma 43 Tricaprylin Hyperkeratosis of the skin with early squamous-cell carcinoma 6 Tricaprylin Hyperkeratosis of skin with early subcutaneous spindle-cell sarcoma 6 Tricaprylin Spindle-cell sarcoma 8 Tricaprylin Spindle-cell sarcoma with marked hyalinization
I250 980 1460
3.7 2.9 5.75
745
1.2
5700 4000
9.25 7.8
radiation dosage of the tissue surrounding the initial injection site. Dauben and Mabee (1951)have found similar amounts of radioactivity in tumors induced by 20-methylcholanthrene-11-C14. 3. Chemical Fractionation of Metabolites
Thus far only the distribution of radioactivity in tissues and excreta had been considered, No information was available as to the chemical nature of the radioactive metabolites. A program was then initiated to characterize and identify the metabolites. As a first step in this program an extensive fractionation procedure was adopted by Heidelberger et al. (1948)for a study of the metabolism of labeled dibenzanthracene. The same procedure was used by Dauben and Mabee (1951)with methylcholanthrene. This method gave separations of neutral, acidic, and phenolic radioactive metabolites in various stages of conjugation. I n each case the neutral fractions were analyzed quantitatively by carrier technic for unchanged hydrocarbon. I n liver and excreta, dibenzanthracene was very extensively degraded, particularly to acidic products, and only 2 to 8% of the radioactivity was present as the unchanged carcinogen. On the other hand, in subcutaneous tissue and tumors the radioactivity was preponderantly due to dibenzanthracene itself ,'although
284
CHARLES HEIDELBERQER
small amounts of other types of metabolites were present. The same situation was observed with methylcholanthrene (Dauben and Mabee, 1951). These fractionations have furnished information on the chemistry of various metabolic products and is invaluable in their identification. Shay et al. (1950) detected radioactivity in the stomach of young rats, nursed by mothers given gastric instillations of labeled methylcholanthrene. The absorption spectrum of methylcholanthrene was associated with the radioactivity, showing that in all probability, methylcholanthrehe was excreted into the mother’s milk, to cause the tumors that have been observed in the sucklings.
4. Rates of Elimination Because a great many studies on the evaluation of the potencies of carcinogenic hydrocarbons have involved subcutaneous injection, it appeared important from the start of this work to determine the rate of elimination of the compounds from the site of subcutaneous injection. The data available a t the present time are shown in Fig. 1, in which the logarithm of the per cent of the radioactivity remaining a t the site is plotted against time. The curve for dibenzanthracene-9, 10-C1*is from Heidelberger and Jones (1948)) that for methylcholanthrene-11-C14 is from Dauben and Mabee (1951))and the benzpyrene-5-C1*curve is from Heidelberger and Weiss (1951). In all cases the same dose and solvent (tricaprylin) were used. It is clear that the three compounds are eliminated at different rates and that the radioactivity is excreted through the feces. At the dosage level of this experiment, methylcholanthrene is the most potent carcinogen, benzpyrene is intermediate, and dibenzanthracene is the weakest. There is no direct correlation between the rate of elimination, which is probably a measure of the rate of metabolism and the carcinogenic potency of these three compounds. However, it has been shown (Bryan and Shimkin, 1943) under similar conditions that at very low doses, dibenzanthracene is the most potent, methylcholanthrene, intermediate, and benzpyrene the least potent compound of this series. This is an inverse correlation with the rate of elimination and strongly suggests that a t minimal dosage, dibenzanthracene is the only carcinogen of the three that is retained in sufficient quantity to be effective in tumor production. In the earlier work (Bryan and Shimkin, 1943) the interpretation was made that dibenzanthracene was the most potent of the carcinogens. However, that interpretation would seem untenable in view of the present data. Since benzpyrene and methylcholanthrene at somewhat higher doses produce tumors with considerable more rapidity than dibenzanthracene, in spite of the fact that they are eliminated faster, it would seem that the former are actually more
285
RADIOISOTOPES APPLIED TO TUMOR STUDIES
powerful carcinogens. Thus, for proper evaluation of carcinogenic potency, it is necessary to know the rates of elimination of the carcinogens. This information is now a t hand for the above-mentioned compounds and can best be obtained for other compounds by means of radioactive technics. It has long been known qualitatively that carcinogens applied directly to the skin disappear rapidly from the area. However, it has also been recognized that the mice lick the site of application and rub it vigorously against the cage, and carcinogenic hydrocarbons ingested orally are
-
0.4-
,,:
I
0.2-i
0.15-'
*-• Benzpyrene.C14 t 1/2 = 1,3/4 weeks *.----o Benzpyrene accumulated activity in feces c-Dibenzanthracene-C14t 1/2 = 12 weeks Methylcholanthrene-C14t 1/2 = 3,1/2 weeks
-.-
1
2
3
4
5
6
7
8
9
-
10
11
12
Weeks
FIG.1. Rate of disappearance of radioactivity following subcutaneous injection of carcinogenic hydrocarbonsin tricaprylin.
rapidly eliminated in the feces (Heidelberger and Jones, 1948). It appeared necessary to study this elimination uninfluenced by these mechanical factors, and Heidelberger and Wiest (1951s) developed a cage which restricted the movement of the mouse so that ingestion and rubbing of the site were prevented. They were able to show that radioactivity was eliminated in the feces, thus proving that the carcinogen or a metabolite was actually absorbed through the skin. It was also demonstrated that in mice under chronic caloric restriction, dibenzanthracene was eliminated considerably more slowly than in mice fed ad libitum. The significance of this finding in its relationship to carcinogenesis is not yet clear. The rate of elimination of labeled dibenzanthracene and
286
CHARLES HEIDELBERGER
benzpyrene from the site of a single application to the skin has been measured by Heidelberger and Weiss (1951). 6. IdsntiJication of Metabolites
The use of the radioactive carrier teehnic has been invaluable for the identification of metabolites of carcinogenichydrocarbons, It is possible
@.
Dibenzanthracene
J
/
HO
/
No
\
\
\
/
4',8'-Dihydroxydibenzanthracene
Dibenzanthracene-3,4-quinone (metabolite)
4'S'-Dihydroxydibenzanthracenk9,lOquinone '
Dibenzanthracene-3,4,7,8-quinone (not a metabolite)
HOOC 5-H~d~0~s-l.2-naphthalic acid FIG.2. Metabolism of dibenzenthracene.
to conduct a suitable series of liquid-liquid distributions of the radioactivity of metabolic products and derive considerable information about their chemical nature. One can then guess the identity of the metabolite
RADIOISOTOPES APPLIED TO TUMOR STUDIES
287
and synthesize it. This carrier compound can then be added to a solution of the labeled metabolite, and if radioactivity is maintained after rigorous purification, identification of the metabolite is certain. In order to establish the dihydroxy compound illustrated in Fig. 2 as a metabolite, the excreta of large numbers of animals given enormous doses of the carcinogen had to be processed and the compound isolated, purified, characterized, and finally identified by synthesis. By means of carrier technic, once the compound is available, it is possible to carry out an accurate quantitative analysis as well as qualitative identification of the metabolite in a single animal given a minimal dose of the carcinogen. In this way two new metabolites have thus far been identified and shown to occur in skin, liver, and feces. One metabolite is 5-hydroxy-1,2naphthalic acid (Heidelberger and Wiest, 1951b)) and the one of the many criteria of purity used in its identification by carrier technic was the solubility distribution coefficient, illustrated in Table V. The TABLE V Solubility Distribution Coefficient, K =
mg. anhydride/ml. of ether C 2= CZ mg. anhydride/ml. of aqueous phase 3.2 mg./0.9 ml. = 4.2 = 1.2 mg./1.4 ml.
Radioactivity Distribution Coefficient, H counts/min. /ml. of ether H = -c. ? = Cz counts/min./ml. of aqueous phase 112 ct./min./0.9 ml. = 4.5 €I= 39 ct./min./l.4 mi.
probable sequence of reactions leading to the production of this compound is shown in Fig. 2. It has been proved that the hydroxylation takes place before the central ring is cleaved, and although 4’,8’-dihydroxydibenzanthracene-9,lO-quinonehas not yet been shown to be a metabolite,* it is more than likely that it is produced as an intermediate at some time during the course of metabolism. The hydroxy naphthalic acid is neither carcinogenic nor does it inhibit the growth of tumors, and hence is probably a detoxication product. The other new metabolite that has been identified is dibenzanthracene-3,4-quinone (Wolf and Heidelberger, 1951)) and it is interesting to note that the symmetrical compound, dibenzanthracene-3,4,7,8-quinone has not been shown t o be a metabolite. This is also the first case in *Note Added in Proof. This compound has recently been demonstrated as a metabolite, as has dibeneanthracene-9,10-quinone. (Heidelberger and Hadler, unpublished.)
288
CHARLES HEIDELBERGER
which the metabolite of a carcinogenic hydrocarbon has been found that is substituted on the phenanthrene double bond, considered to be necessary for carcinogenic activity. However, interpretation of this finding is obscured by the fact that there is another unsubstituted phenanthrene double bond in the molecule, and also because this metabolite is only found in very small amounts. Dibenzanthracene is not carcinogenic in the rabbit, and it is particularly interesting that a dihydroxydibenzanthracene, different from the 4',8'-dihydroxydibenzanthracene produced by susceptible rats and mice, has been isolated as a metabolite in this species by Levi and Boyland (1937), and Dobriner et al. (1939). This compound has not yet been identified, although the matter is still under investigation. It is t o be hoped that when this compound has been fully characterized, the exact knowledge of the difference in metabolism of a carcinogen in susceptible and resistant species may provide important clues as to the mode of action of the compound. Thus for this reason and many others it appears to be important to continue to identify metabolites and test them for carcinogenic activity, since there is no compelling evidence as yet to exclude the possibility that some metabolite is the actual carcinogen. Since it has been shown that cystine in the diet counteracted 8ome growth inhibitory properties of dietary carcinogen, it had been postulated that the cystine reacted with the hydrocarbon and the resulting mercapturic acid was excreted. It has now been conclusively shown by Gutmann and Wood (1950a,b) that benzpyrene is not excreted as a mercapturic acid. This was carried out by the addition of nonlabeled benzpyrene to animals on diets of cystine-Sab or methionine-SS6. No radioactivity in the urine could be detected in the form of mercapturic acid. A somewhat similar experiment (Heidelberger and J. White, unpublished) with labeled dibenzanthracene also indicated that no mercapturic acid was excreted. 6 . Interaction with Tissue Components
An extremely important discovery was made by E. C. Miller (1951), who demonstrated that benzpyrene applied to the skin of mice was bound to protein. This was confirmed by Heidelberger and Weiss (1951) with labeled benzpyrene and was also shown to occur with dibenzanthracene. It would seem almost axiomatic that a chemical carcinogen should produce its biological effect as a result of a chemical interaction with some important cellular component, and the demonstration that such an interaction actually does take place provides the impetus for a new approach to the problem. Technics have now been developed (Wiest
RADIOISOTOPES APPLIED TO TUMOR STUDIES
289
and Heidelberger, in press), which make possible the study of such an interaction with various cell fractions and nucleic acids derived from skin and from submaxillary glands of mice. There appear to be distinct advantages in working with the latter tissue. It is sensitive to carcinogenesis, is relatively homogeneous histologically, and can be fractionated into well-defined nuclear, large granule, small granule, and supernatant fractions. By means of these technics investigations are now under way (Wiest and Heidelberger, unpublished), to ascertain the exact components of the cell with which the hydrocarbon reacts. I t may be possible by experiments of this sort to determine whether the hydrocarbons are reacting with desoxyribose nucleic acid and causing mutations, as has recently been proposed by Boyland (1951), or whether their action is primarily on the cytoplasm possibly by causing the deletion of some essential enzyme. I n any event, it is hoped that studies of this sort will make possible a clearer understanding of the mode of action of carcinogenic hydrocarbons. It had been hoped that some progress in the study of the metabolism of carcinogenic hydrocarbons would result from in vitro studies. However, after considerable effort was expended on the study of skin slice and of liver slice and homogenate systems, it was concluded (Wiest and Heidelberger, Cancer Res. 12,308 (1952)),that it was not possible to obtain any significant metabolism, and this line of investigation was abandoned. Another aspect of hydrocarbon carcinogenesis of considerable importance is to study the effect of applications of the compounds on the metabolism of the skin. This type of study has been pioneered by Carruthers and others of the St. Louis group. Now that technics are available for the determination of nucleic acids in skin, a program has been undertaken to study the changes in nucleic acid content of the skin under the influence of various carcinogenic hydrocarbons (Heidelberger, Rusch, Boutwell, and Gilbert, unpublished). If this approach appears promising, it will doubtless be extended to include nucleic acid turnover studies with radioactive precursors. In summary, the use of radioactive isotopes is contributing to our knowledge of hydrocarbon carcinogenesis by: (1) providing quantitative information on the distribution, excretion, and rates of elimination of carcinogens; (2) identification of metabolites and metabolic sequences of carcinogens; (3) determination of the site or sites of interaction of carcinogens with the cell; (4) providing methods for studying the effect of the carcinogens on the metabolism of sensitive and resistant tissue. It is to be hoped that this rather comprehensive approach t o the problem may provide some clues not only to the mechanism of carcinogenesis but also to methods of preventing or controlling the neoplastic transformation.
290
CHARLES HEIDELBERQER
111. OTHER CARCINOGENIC COMPOUNDS 1. Carcinogenic Azo Dyes Since the subject of the carcinogenic azo dyes is reviewed in another chapter of this volume (Miller and Miller) only very brief mention will be made of the applications of radioisotopes in this field. Boissonnas et al. (1949) studied the metabolism of 4-dimethylaminoazobenzene-Nmethyl-C14 (I) in a single rat. They found an appreciable oxidation of ~ - N = N - ~ - N ( c H ~ ) ~ I
the methyl carbon to respiratory carbon dioxide. Moreover, they were unable to demonstrate any radioactivity in the methyl groups of choline or creatinine and concluded that no transmethylation had occurred. On the other hand, Miller et al. (1951)found that radioactivity was present in the serine methyl groups of choline and creatinine following administration of 3'-methyl-4-dimethylaminoa~obenzene-N-methyl-C~~ (11). CHs I
I1
They were also able to demonstrate considerable radioactivity in the P-carbon of serine, and since others have shown that formate and formaldehyde are metabolically transformed into the P-carbon of serine, it is likely that the methyl group of the azo dye is oxidized via those intermediates to respiratory carbon dioxide. It is also likely that the radioactivity in the choline has arisen not from direct transmethylation, but from biological synthesis of methyl groups from formate or its equivalent. The same compound, labeled in the ring-methyl group, 3'-methylC14-4-dimethylaminoazobenzene (111) has been prepared by Salzberg et al. (1950)and its metabolism reported on (Salzberg et al., 1951). &Ha I
I11
They were able to detect protein-bound radioactivity in the liver, and thus confirmed Millers' observations, and also found considerable C l4 that could not be accounted for as azo dye.
RADIOISOTOPES APPLIED TO TUMOR STUDIES
291
It is apparent that the use of labeled azo dyes will become more common and that this tool will materially advance the progress in certain aspects of the investigation of liver carcinogenesis by this class of compounds. 2. Acetylaminojluorene and Derivatives
The carcinogen 2-acetylaminofluorene has the interesting property of inducing a variety of tumors a t different sites in experimental animals. 2-Acetylaminofluorene-9-C14 IV and ~0-c'~ (V) have been prepared by
Ray and Geiser (1950) and the former has also been synthesized by
V Heidelberger and Rieke (1951). The distribution and excretion of radioactivity following the administration of the carcinogen labeled in both positions has been reported by Morns et al. (1950) and by Weisburger et al. (1951). The compound was administered by stomach tube to rats, the excreta and respiratory carbon dioxide were collected, and the animals were sacrificed after various time intervals. The organs were dried and combusted to COe, and the radioactivity was determined. The results of the distribution of radioactivity six hours after administration are compared for the ringlabeled (AAF-9-C14) and side-chain labeled (AAF-w-C 14) carcinogen and are shown in Fig. 3. One striking difference is that the labeled carbon atom of AAF-9-C14 was not converted into respiratory COz, whereas there was appreciable metabolic oxidation of the carbon in the acetyl group. This difference was observed a t all the time intervals studied and, together with the more generalized distribution of the radiocarbon derived from the AAF-w-C14, suggests that the amino group was deacetylated in the animal. The resulting labeled acetate would then be rapidly oxidized to carbon dioxide. It has been found that the acetylated compound is more carcinogenic than the free amine, and that deacetylation does occur to a significant degree. Yet these results-are
CHARLES HEIDELBERGER
292
in no way contradictory, since there is ample acetylated compound left to be effective, if indeed it is the true carcinogen. It will also be seen from Fig. 3 that radioactivity from the 9-labeled compound remained in the stomach for a significantly longer time than that from the w-labeled compound. This observation suggests to the reviewer that deacetylation must take place proponderantly in the stomach, aided perhaps by the gastric acidity, since aromatic acetylamino groups are readily hydrolyzed by acid. Otherwise, the radioPERCENT Of TOTAL COUNTS RECOVERED
STOMACH SMALL INTESTINE CAECUM
URINE CARCASS KIDNEY
LIVER
co2
COMPARISON OF OlSTRlSUTlON
=
AFTER ADMINISTRATION OF: 2-ACETYLAM lNOf LUORENE -0 -Cod
Z-*CETYUMINO~LUORENE-W-C~~
OTHERS
FIQ.3. The distribution of radioactivity six hours after administration of AAF-Qand -w-C".
activity is distributed in small quantities widely throughout the body and does not appear to be concentrated to a significant extent in any organ, susceptible or not. Thus it is clear that the mere presence of radioactivity in a tissue does not suffice to give information as t o the mode of action of the carcinogen. It will be necessary to await further information on the chemical form of the radioactivity before many conclusions as to mechanism can be reached. An interesting lead in this direction is the observation that there is considerable C14 in the feces following administration of the 9-labeled compound, whereas no detectable diazotizable amine group is present. This strongly suggests that gome metabolite no longer containing an aromatic amine group is formed,
RADIOISOTOPES APPLIED TO TUMOR STUDIES
293
Ray and Argus (1951) have studied the distribution of Sas-labeled 2-(p-toluenesulfonyl) aminofluorene (VI), which is noncarcinogenic. This compound was fed, and after 66 hours over 90% of the radioactivity was found in the feces as the original compound.
VI
Since this compound is not carcinogenic, and the p-toluenesulfonyl group is not hydrolyzed in the rat, support is given to the hypothesis that 2-aminofluorene is the actual carcinogen. However, the rapid and extensive excretion makes it possible that the compound was not absorbed to any significant extent. Further evidence is required before this point can be considered settled, and it is hoped that continued research on the metabolism of these interesting compounds will be forthcoming. 3. Carcinogenic Benzacridines
The French workers, Daudel et al. (1951) have prepared a labeled benzacridine, 3,10-dimethyl-5,6-benzacridine-10-C14 (VII) .
VII
They will initiate metabolic studies in this series of compounds, which they discovered to be carcinogenic. If enough profitable research is carried out on the metabolism of several different series of carcinogenic compounds, it is possible that some mechanism of tumor production, common to all, may emerge to clarify our understanding of this important, but as yet obscure problem. IV. OXIDATIVEMETABOLISM OF TUMORS 1. Dejiciency in Tumors?
Among the most important experiments that pioneered the field of cancer biochemistry were those of Warburg during the 1920’s. He concluded (1930) from the results of his extensive studies that the high
294
CHARLES HEIDELBERGER
aerobic glycolysis he observed in tumors was caused by a respiratory defect and that interference with respiration is the cause of tumors. However, since the time of Warburg’s earlier experiments, a high anaerobic glycolysis has also been observed in several nprmal tissues, which diminishes somewhat the force of the original argument. Nevertheless, experiments undertaken with the homogenate technic showed a diminished level in tumors of cytochromes and certain enzymes involved in Krebs cycle oxidations compared with the more metabolically active normal tissues. Under certain circumstances, the failure to observe Krebs cycle oxidation in homogenates suggested the possibility that one or more key steps in the Krebs cycle might be very low or absent in tumors. A difference as fundamental as this between tumors and normal tissues would be of great importance, and this intriguing possibility has stimulated further investigations along these lines. One observation that was unexplained was that tumor slices had an appreciable oxygen uptake, which, however, could not be stimulated significantly by the addition of Krebs cycle substrates. Thus it was not clear whether the oxidation was taking place by other pathwaw altogether, or whether the oxygen uptake was caused by such a high endogenous level of Krebs cycle intermediates that further addition of these substrates failed to cause further stimulation. An obvious way to test these alternatives was t o add labeled Krebs cycle intermediates to tumor slices and measure the rate of their conversion to C1402. Thus isotopic tracer studies were initiated in this field. 2. Krebs Cycle in Tumors
Pardee, Heidelberger, and Potter (1950) measured the oxidation of acetate-l-C14 to CI4O2in slices and homogenates of a number of normal tissues. There was a significant conversion of acetate to carbon dioxide in slices of the normal tissues studied, and considerable evidence was obtained to indicate that the oxidation took place via the Krebs cycle. However, in slices of transplantable rat tumors less than one twentyfifth as much C1402was produced than in kidney, an amount well below the magnitude that would be significant in oxygen uptake experiments. Some of these experiments are summarized in Table VI. It was concluded that either the Krebs condensation reaction takes place to only a very limited extent in tumors, or that tumors are less able t o activate acetate than several normal tissues. While this work was in progress, an abstract by Olson and Stare (1949) announced that pyruvate-2-C14 was oxidized to C1402to about an equal extent by slices of normal liver and primary hepatoma. This finding was confirmed in transplantable tumors (Potter, Watson, and Heidelberger, unpublished), and the results
RADIOISOTOPES APPLIED TO TUMOR STUDIES
295
were consistent with the idea that pyruvate was oxidized via the Krebs cycle, although once again it was found that acetate was only slightly oxidized under these conditions. It seems likely that the conditions for maximum conversion of acetate-l-C14 to C1402 are not the same as for ~ ~C1402in tumor slices, and further the conversion of p y r ~ v a t e - 2 - Cto studies on this point are needed. TABLE VI COZProduction from Acetate by Various Rat Tissue Slices The incubation time was 150 minutes. The specific activity of the acetate WM 1050 ct./min. per pl. The figure in the last column was obtained by dividing the counts found in the center well by the specific activity of the acetate.
Tissue Kidney Kidney Liver Liver Lung Lung Flexner-Joblingcarcinoma Flexner-Jobling carcinoma Flexner-Joblingcarcinoma Walker carcinosarcoma No. 256 Walker carcinosarcoma No. 256
Total Wet Initial Counts ConverWeight Rate of 0 2 in per Min. sion of per Acetate Oxygen 150 in Center Acetate Flask* Added Uptake Min. Well to COI 47 35 58 50 32 12 30 44 28 22 26
10 4 10 10 4 4 4 10 4 10 4
84 51 32 33 17 7 17 20 13 17 20
470 590 370 380 195 69 215 263 125 196 254
10,100 3,920 1,680 2,570 680 285 184 103 140 152 174
96 92 16 25 16 6.7 4.1 0.98 3.3 1.5 4.1
* In the experiments the weight given WBB the final wet weight of the blotted slices after treatment in the flask with HC104. Experiments showed that the initial dry weight waa 0.48 of this value for both kidney and the Walker carcinosarcoma No. 266. A more direct demonstration that oxidation occurred by means of the Krebs cycle was provided by Weinhouse et al. (1950), who were able to isolate isotopic citric acid following incubation of labeled glucose, acetate, and palmitate with slices of various mouse tumors. Their data are presented in Table VII, and taken together with their demonstration of the presence in tumors of the enzyme that causes the condensation of acetate and oxalacetate to citrate, the evidence that the Krebs cycle does occur in tumor slices appears conclusive. Further lines of evidence based on enzymatic assays have been discussed by Weinhouse (1951a) but cannot be dealt with here. Weinhouse (1951a) has also shown that CI4OZis produced from labeled glucose, palmitate, lactate, and acetate to about the same extent in slices of tumor and a number of normal tissues. There is at present no
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CHARLES HEIDELBERGER
explanation for the discrepancy between his acetate data and those of Pardee et aE. (1951). Weinhouse has expressed his results in units expressed in terms of substrate oxidized to COz/g. dry tissue/hour, and has called them “oxidative capacity.” This is a somewhat ambiguous term, since it is based on experiments with slices where factors of permeability and phosphate turnover make it impossible to measure the true enzymatic capacity of the tissue. TABLE VII Radioactive Citrate from Tumor Oxidations
Tissue
Substrate
Normal, mouse Heart Liver Kidney
Glucose Glucose Glucose
Neoplastic, mouse Hepatoma Mammary tumor Rhabdomyosarcoma Ehrlich ascites Mammary tumor Mammary tumor Hepatoma Rhabdomyosarcoma
Glucose Glucose Glucose Glucose Acetate Palmitate Palmitate Palmitate
Activity (ct./min.)
Quinidine Citrate Activity (ct./min.) ..
6.5 X lo6 5.5 x 106 5.5 X lo6
150 126 2,480
1.37 X lo6 1.37 X 106 5.5 x 106 5.5 x 106 1.4 X 106 66,200 66 ,200 66,200
1,225 1,750 1,000 910 293 74 138 83
Olson (1951) has presented considerable data on the oxidation of various labeled substrates in slices of normal rat liver and hepatoma. I n addition to measurements of C1402, he has also determined oxygen uptake and substrate disappearance and has expressed his results as Q values (micromoles/mg. dry weight/hour). He carried out his experiments on C14-labeled pyruvate, lactate, glucose, acetate, and succinate. In most cases the & c t 4 0 1 values for liver and hepatoma were of the same order of magnitude. The data for succinate are presented in Fig. 4. These results show, that for this particular Krebs cycle intermediate, the situation in the liver differs markedly from that of the tumor, as had been concluded earlier by Schneider and Potter (1943) on the basis of studies with homogenates. Although the amount of C1402in the two cases is not very different, owing to the fact that no COz is formed when succinate is oxidized to fumarate, there is an enormous oxygen uptake and succinate disappearance in the liver, which evidently did not reach
RADIOISOTOPES APPLIED TO TUMOR STUDIES
297
its maximum in this experiment. These data may be compared with the work of Rosenthal (1937) who was able to obtain QY20"inste greater than 100 in liver slices under optimal conditions. This fact shows that the liver has a much higher capacity for succinate oxidation than does the tumor, and it is significant, as will be discussed below, that succinate oxidation can proceed independently of phosphate turnover. Olson concludes that the route of terminal oxidation of substrates in tumors is the same as that for normal tissues. 4kot
501251
HEPATOMA
SUCC I NATE mWL
SUCCINATE m M R
FIG.4. The metabolism of carboxyl-labeledsuc~inate-C1~ in slices of hepatoma and rat liver. Metabolic quotient is plotted against substrate concentration. Substrate disposition and gas exchange are plotted on different scales on the ordinate so arranged that 1-pl. change in 02/C1402 exchange is equivalent to 2-111. change in substrate utilization. Each point represents at least four determinations.
Zamecnik et al. (1951) have also studied the metabolism of labeled glucose in slices of liver and hepatoma, and in agreement with Olson, find that there is a greater production of radioactive carbon dioxide from glucose by hepatoma, than by liver slices. It must be emphasized that rat liver slices have an especially low activity for this conversion (cf. Olson). There was a less marked difference between tumor and liver when labeled fructose was the substrate. Zamecnik et al. found a greater incorporation of 0 4 into hepatoma protein than into liver protein and obtained labeled glutamic and aspartic acids, alanine, glycine, serine, and proline by hydrolysis. Their information also suggests that there is no significant difference in the connections between carbohydrate and protein metabolism of hepatoma and normal liver. Zamecnik also con-
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CHARLES HEIDELBEROER
cluded that there are no qualitative differences in oxidative pathways in hepatomas and liver and that synthetic processes such as protein synthesis occur more rapidly in tumors. The latter topic will be discussed in some detail in Sec. V. In the course of an intensive study of the metabolism of cell suspensions of the Gardner lymphosarcoma, Kit and Greenberg (1951a) investigated the oxidation of labeled acetate and several amino acids. They found that acetate was oxidized to C140a to about one-third the extent in tumor than in spleen and were able to show that citrate production in the presence of fluoroacetate was no greater than in its absence although the CI4O2 output was diminished. This is further evidence that the Krebs cycle proceeds in tumor tissue, and also indicated that it is of a low order of activity, since it is less than spleen, one of the normal tissues of relatively low oxidizing capacity. 3. Present Status What then is the present status of oxidative metabolism in tumors? It has now been definitely established, primarily by means of isotopic experiments, that the oxidation of substrates in tumors does take place by means of the Krebs cycle. There appears to be no qualitative difference in the overall oxidative metabolism of tumors and normal tissues. Furthermore, there seems to be the opportunity for the tumor to carry out oxidations that are ample to satisfy the requirements of energy for the synthetic processes that are constantly demanded by a rapidly growing tissue. On the other hand, there is some evidence to show that there is a t least a quantitative difference in the oxidative capacity of some tumors and the more metabolically active of the normal tissues. Most of the work has been concerned with a direct comparison of normal liver and primary hepatoma tissue. It has been known for some time that the enzymes of the Krebs cycle are located within the mitochondrial fraction of the cell, and it has now been established:,(Weinhouse, 1952; and Kielley, 1952) that the mitochondria of tumors are capable of carrying out oxidation of Krebs cycle substrates. However, since it has been shown that the content of mitochondria in hepatomas is considerably lower than that of liver, the amount of oxidation in these tumors on a cellular basis must necessarily be lower than in normal liver. Since these differences in mitochondrial content or the amount of other enzymes, such as cytochrome oxidase, are not paralleled by the data obtained with slices utilizing pyruvate or glucose, the slice experiments do not measure the capacity of the tissue to carry out these reactions.
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299
Weinhouse (1951) has redetermined the quantity of lactic, isocitric, and malic dehydrogenases in transplantable tumors and has shown that the amounts of these enzymes are of the same order as are found in liver. He has also reported that the poor oxygen uptake of tumor homogenates can be stimulated by the addition of high concentrations of diphosphopyridine nucleotide (DPN). With the proper addition of DPN, the oxygen uptake of mouse hepatoma and liver mitochondria on a weight basis were found to be comparable (Weinhouse, 1952). He concluded that the high DPN requirement makes it appear that tumor mitochondria are considerably more fragile than those of liver, and that “it seems probable that differences in enzymatic activity are due t o various degrees of loss or inactivation during preparation rather than t o different concentrations in the intact cell” (Wenner, Spirtes, and Weinhouse, 1952). Williams-Ashman and Lehninger (1951) have shown that oxidative phosphorylation occurs in tumor mitochondria with succinate as the substrate, another close similarity to liver. Potter (unpublished) has also shown that tumor mitochondria are more fragile or permeable than their counterparts in liver, and as a consequence oxidative phosphorylation is more difficult to maintain. It is doubtless for this reason that earlier failures of tumor homogenates to carry out oxidations in the absence of fluoride or large amounts of DPN can be explained. Although all the data so far mentioned indicate that there is no quantitative difference between tumors and normal tissues, there are other experiments that remain to be explained. Experiments involving whole animals poisoned with fluoroacetate show that citrate is not accumulated in tumors as it is in most normal tissues, and suggest that some additional factors operate in the intact animal to regulate either the Krebs cycle oxidations or a closely related process. As Potter (1951) has stated, “The comparison of results obtained by means of homogenates, slice technics involving isotopic tracers, and whole animal studies lead inescapably to the conclusion that these studies measure different rate-limiting phenomena, and that any explanation of cancer metabolism based on one technic must be examined by the supplementary data provided by other technics.” It is clear from Fig. 4 that the capacity for succinate oxidation in liver is much greater than that of the hepatoma, and other evidence indicates that the capacity of tumor to undergo various reactions is lower than liver. These considerations have led to the view (Potter, 1951) that the rate-limiting factor in the tumor is the quantity of some component of the oxidative enzyme system, and in normal tissues, such as liver and kidney, the rate-limiting factor is the amount of substrate. This view is summarized in Fig. 5 (Potter, 1951), where in the kidney, for example, the
300
CHARLES HEIDELBEROER
rate-limiting factor is either the substrate, phosphate acceptor, or inorganic phosphate. In the tumor the enzymes are limiting, and the substrates are in excess. This interpretation of Potter’s, if correct, would mean that building blocks for growth might be available at higher concentrations in tumor than in nongrowing tissues. It appears that more work will have to be done before there is general agreement as to whether there is a distinctive pattern of oxidative metabolism in tumors. At present the bulk of evidence based on fluoroacetate poisoned whole animals, defects in electron transport enzymes and oxidative phosphorylation, and the lowered content of mitochondria favors the existence of such a pattern. It is also likely that considerable variation Kidney (Enzyme excess)
Homog: (195) Slice:
(21)
Tumor (Substrate excess)
Aperture = Enzyme amount Outflow =Enzyme activity
(18) (7)
FIG.5. Comparison of data from slices and homogenates. The figures 195 and 18 refer to the succinoxidase Qo,values for homogenates of rat kidney and FlexnerJobling tumor, respectively, while the figures 21 and 7 refer to the Qosof the two tissues using the slice technic with pyruvate as substrate.
will be observed among different types of tumors, which will perhaps depend on the rate of growth and degree of malignancy of the cancers. In this regard, generalizations based on experiments with “tumors” may well be meaningless unless the particular neoplasm is specified and unless a whole spectrum of tumors is studied. Another aspect of intermediary metabolism of tumors was studied by Stoesz, Heidelberger, and LePage (1950). They were able to show that in glycolyzing tumor homogenates under anaerobic conditions considerable amounts of pyruvate could not be accounted for as pyruvate or lactate. By the use of pyruvate-l-C14 and pyruvate-2-C14 it was found that there was no production of carbon dioxide, acetate, formate or alanine, no fixation of carbon dioxide, and that propanediol and propanediol phosphate were produced. The significance of these findings to the overall metabolism of the tumor is not yet clear.
RADIOISOTOPES APPLIED TO TUMOR STUDIES
30 1
V. INCORPORATION OF AMINOACIDSINTO TUMOR PROTEINS 1. Introduction One area of biochemical study that was almost untouched before the use of isotopes was the problem of protein synthesis. The technics whereby such investigations could be carried out were almost completely lacking until Schoenheimer and Rittenberg turned their attention towards protein synthesis and turnover. They showed that N16-labeled amino acids, administered to animals, were incorporated into tissue proteins, and later it was shown that the concentration of isotopic nitrogen decreased at different rates in different tissues (Shemin and Rittenberg, 1944). In that study transplanted tumors were compared with several normal tissues, and the same comparison has been made since that time with a variety of amino acids in whole animals and in tissue slices. The use of C14-labeled amino acids has become commonplace for studying these processes and a recent review by Zamecnik (1950) discussed their use in cancer research. Since there is seldom an actual net increase in protein in many normal tissues, the rapidly growing tumor represents an ideal case to study true protein synthesis. It is now possible to measure the incorporation of labeled amino acids into more or less well-defined proteins, and while it can be inferred that the amino acids are held in peptide linkages, it is impossible to say whether new protein has actually been synthesized or whether amino acids are exchanged without true synthesis of protein. In the case of tissue slices and homogenates the situation is further complicated by the likelihood that protein is actually breaking down under the experimental conditions. Thus one can study the incorporation of amino acids into proteins, but any statements in the literature purporting to show “protein synthesis’’ by this type of experimentation should be very carefully scrutinized for unequivocal evidence that de novo synthesis has occurred. Although we are ignorant of the exact manner in which the incorporation into the complex protein molecule takes place, nevertheless a considerable amount of valuable information has already been obtained. 2. In vivo Studies
Reid and Jones (1948) studied the radioactivity in organs of mice bearing melanosarcomas following the administration of DL-tyrosine8-Cl4. Although they found no appreciable concentration of C14 in the tumor, it is difficult to draw conclusions from organ radioactivity alone. Winnick et al. (1948) measured the incorporation of labeled tyrosine into the tissue proteins of normal and tumor-bearing rats, and the highest
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CHARLES HEIDELBERGER
specific activities were found in the intestinal mucosa, kidney, and tumor. They also demonstrated that almost all the radioactivity in the proteins was present as tyrosine itself. A closely similar study was carried out by Greenberg and Winnick (1948) with glycine-l-C1*, in which, however, no tumor-bearing animals were used. into Griffin et al. (1950) compared the incorporation of gl~cine-2-C'~ the proteins of noncancerous portions of rat livers bearing primary hepatoma nodules and the tumor proteins. They found a maximum specific activity in the noncancerous liver two hours after administration, whereas the tumor reached a maximum at twelve hours, a t which time the specific activities of the two tissues were the same. The distribution of radioactivity in tissue proteins of mice bearing various neoplasms following administration of DL-methionine-Sa6 has been determined by Kremen et al. (1949) and by Bloch et al. (1951). They showed that the peak specific activity of several normal tissues and of tumors occurred at about six hours. Furthermore, when a number of normal tissues and tumors were compared twenty-four hours after administration of the methionine, the liver was invariably found to have the highest specific activity. The specific activities of kidney, tumor, and gastrointestinal tract were intermediate, and a number of other tissues, such as muscle were definitely lower. A similar type of study was carried out by Norberg and Greenberg (1951), and comparable results were obtained. In addition, they noted a systemic effect of the tumor that caused a marked increase in the specific activity of spleen (which enlarges in tumor-bearing animals) and plasma proteins over those of the normal animals. Tyner et al. (1952) measured the specific activity of rat kidney, liver, and transplanted Flexner-Jobling carcinoma proteins at varying time intervals after the administration of glycine-2-CI4. The results are shown in Fig. 6 and show that the incorporation into liver protein was more rapid and extensive than into the proteins of tumor and kidney. In contrast to the results of Griffin el al. (1950) the highest specific activity was found at twelve hours in all the tissues studied. In liver the decrease of specific activity, a reflection of protein catabolism in nongrowing tissues, was considerably more rapid than in tumor. Since the tumor increased in weight throughout the experiment, it could not be determined whether protein catabolism, in addition t o protein synthesis, was occurring in the tumor. In order to test this important point, a series of experiments were carried out by LePage et al. (1952). They measured the weight changes of tumor, liver, and kidney in fed and starved rate bearing FlexnerJobling carcinoma transplants. They also measured the specific activity
303
RADIOISOTOPES APPLIED TO TUMOR STUDIES
and total radioactivity in the proteins of these three tissues. Whereas the total CI4 in liver and kidney decreased with time, the total isotope increased greatly in tumor, particularly in the starving animals, as shown in Fig. 7. Thus it has been clearly shown that the tumor grows by deriving amino acids for protein synthesis from the breakdown of the proteins of normal tissues and utilizing them for its own growth. This
12
+-
4
6
-24 2
12
HOURS
r
4
12
DAIS
d
TIME
FIQ.6. Specific activity of tissue protein (logarithmic ordinate) versus time. x 10-3
X
Liver
I
130
90
-.--50
0
2
4
Fed control rats Fasted control rats
6
10-2
X
Kidney
10-3
-
0
o-
2
4 Days
Tumor
I
6
t
0
2
Fed tumovbearing rats rats
I--Fasted tumor.bearing
FIQ.7. Total radioactivity per organ versus time. 0 0 fed control rats @--a fasted control rats 0 0 fed tumor-bearing rats .--------a fasted tumor-bearing rats The zero time is twelve hours after administration of the labeled glycine.
4
6
304
CHARLEB HEIDELBERGER
effect is particularly marked in the starved animals, where the parasitic nature of the tumor, which gains weight even under these conditions, is most in evidence. These experiments show further that there is little if any catabolic activity in rapidly growing tumor, and thus it is unable to mobilize its own protein for the production of energy. These conclusions should be very significant in the future development of our knowledge about the mechanisms of cancerous growth. Barton and Rusch (1951) found that the incorporation of labeled glycine into the proteins of mouse liver, kidney, intestine, and muscle was diminished in animals treated with adrenal cortical extract or cortisone. However, these hormones had no effect on the incorporation of isotope into the protein of a transplantable mammary adenocarcinoma. This type of research appears to be a promising approach to the understanding of the mode of actions of hormones in cancer. Kogl (1949) has reported additional evidence, based on tracer experiments, to support his contention that tumors contain D-amino acids. He administered CI4-labeled D-glutamic acid to normal and tumorbearing rats, isolated the excretory product, D-pyrollidone carboxylic acid, and measured its radioactivity. In the compound excreted by normal rats there was essentially no dilution of the isotope, whereas there was some dilution (94.8% of the original) in the compound excreted by the tumor-bearing animals. This is purported to be a crucial experiment in the proof that D-glutamic acid is present in tumors. However, in the absence of further information as to the actual radioactivity measurements and criteria of optical purity of the D-glutamic acid used in the experiment, a final decision in this matter will have to be held in abeyance. Miller (1950) has presented a critical review of this controversy. 3. In vitro Studies
One of the most promising approaches to an understanding of the processes leading to the synthesis of proteins has been the investigation of the incorporation of labeled amino acids into proteins in tissue slices and homogenates. A considerable body of work has been carried out along these lines, particularly in liver systems, and Borsook (1950) has presented a critical review of the subject. The earlier work produced a discouraging series of complications. It was extremely difficult to establish that true incorporation into protein had occurred, and there were numerous investigators that were plagued by mechanical adsorption or coprecipitation of radioactivity; even now there are some who believe that no one has yet obtained true incorporation. The early results obtained with homogenates were often ambiguous and inconsistent; these troubles were undoubtedly caused by improper prepara-
RADIOISOTOPES APPLIED T O TUMOR STUDIEB
305
tion of the homogenates. However, there are some conclusions that have been reached by studies of this sort that appear to be well established: (1) The incorporation is an enzymatic process. (2) The synthesis of peptide bonds occurs, but little or nothing is known of the pattern of incorporation or the structure into which the amino acid is built. (3) Oxidative processes are necessary to furnish the energy for the reactions. (4) The enzymatic activity is associated with the particulate matter of the cell. Zamecnik et al. (1948) presented the first report on the incorporation of ~i1anine-l-C’~ and glycine-l-C14 into the proteins of rat liver and primary hepatoma slices. They found considerably more incorporation of radioactivity into the protein of the hepatoma nodules than into normal liver. The noncancerous portion of the livers bearing tumors was intermediate in its ability to incorporate the amino acids. Their data are given in Table VIII, in which the word “amounts” should be TABLE VIII Comparison of Rates of Incorporation into Liver Proteins of C14 Activity Derived from Alanine and Glycine Type of Liver Tissue Type of Labeled Amino Acia Counts per Minute Control Control Control-hepatoma* Control-hepatoma * Hepatoma Hepatoma Control Control Control-hepatoma Control-hepatoma Hepatoma Hepatoma
DL-Alanine m-Alanine DL-AlaniDe DL-Alanine DGAlanine DG Alan& Glycine G1ycine Glycine G1ycine Glycine Glycine
36 57 94 145 329 275 180 138 270 338 505 563
Note: The same two rats furnished hepatic tissues for both types of experiment. 10.000 ct./min., contained in 0.44 mg. of DL-alanine, were added t o the appropriate vessels, and 10,000ct./min., contained in 0.37 mg. of glycine, were similarly added. While the same number of moles of DL-alanine and glycine waa used, the L-alanine represented only half the concentration of the glycine. A strict comparison of the rates of uptake would require the use of L-alanine, free from the presence of the D form. One cannot multiply the alanine values given by 2 because of the possibility of conversion of labeled n-alanine to L-alanine via deamination and transaminstion. Control part of hepatoma-containingliver.
*
substituted for “rates.” They further found the incorporation of alanine into fetal liver was of the same order as into hepatoma and that the radioactivity incorporated into hepatoma slices was due almost entirely to alanine.
306
CHARLES HEIDELBERGER
The uptake of labeled glycine and alanine into protein of tumor homogenates has been reported by Winnick (1950). He compared the amount of incorporation of radioactivity into unfortified homogenates of mouse fetal and adult liver and transplanted mammary carcinoma. He observed an incorporation in tumor five to six times as great as in liver, and in the case of alanine very little radioactivity was lost when the protein was dissolved in alkali and reprecipitated. No radioactivity was released by treatment with ninhydrin until after hydrolysis, which is evidence that the amino acids are incorporated into peptide bonds. However, the tumor homogenates were not cell free and prolonged homogenization resulted in almost complete inactivation (T. Winnick, personal communication), Therefore, the incorporation that was observed in these experiments was very probably due to intact tumor cells, and it may be stated that a t the time of this writing, incorporation of labeled amino acids into tumor proteins has not yet been reported in cell-free system s. Farber, Kit, and Greenberg (1951) have made a very thorough study of the incorporation of labeled glycine into the proteins of cell suspension of the Gardner lymphosarcoma. A summary of some of their results is given in Table IX, which shows the effect of various treatments on the TABLE IX Effect of Various Treatments on Glycine Uptake of Tumor* Treatment
Counts per Minutet
Control4 TCA added at start of incubation Control Homogenized 3 min. Control Frozen at acetone-dry ice temp., thawed after 5 min. 95 % Or5 % COZ,0.025 M glucose (control) 95 % Nz-5 COz,0.025 M glucose 95 % Oz-5 % COz, 0.025 M pyruvate 95 % Nz-5 % COZ,0.025 kfpyruvate 95 % N2-5 % COz, 0.025 M lactate
205 0.5 69 19 91
* 0.002 d4 glyoine-2-C:l' (3.46 po./mg.) t Ccunts/min./mg.
1 35 0
7
15 1 1
were in eaoh flask;temp. 8 8 O incubated 100 min.
t 0.6 m M glycine-2-Cl' used, incubated 60 min. Radioaotivity in
pg.
Cl4/g. protein in this series of experiments.
glycine uptake. Marked inhibition was obtained by homogenization, anaerobiosis, freezing, and thawing and by oxidative inhibitors. This evidence makes it clear that an actual enzymatic reaction occurred and
RADIOISOTOPES APPLIED TO TUMOR STVDIES
307
that the results could not be explained solely by a mechanical adsorption of the labeled amino acid. Kit and Greenberg (1951a) showed the oxidation of labeled glycine, leucine, and alanine, to C1402, which is further evidence for the Krebs cycle in tumors. The same authors (Kit and Greenberg, 1951b) compared the uptake of labeled amino acids in this tumor and in normal spleen cell suspensions. The incorporation was about the same in the two tissues. Zamecnik et al. (1951) have carried out additional experiments which support their earlier observations of a greater amount of incorporation of amino acids into protein in hepatoma slices than in liver slices. They found a considerably greater uptake of radioactivity from glucose-CI4 into protein of hepatoma slices than of liver and also found that the isotope was present largely as aspartic and glutamic acids, indicating that the Krebs cycle is the most likely path for the conversion of carbohydrate into protein. It will be recalled from the earlier discussion that in contrast to the in vitro experiments, amino acids are incorporated somewhat more rapidly into liver protein than into tumor protein in the whole animal. In order to account for this difference it was postulated that (Griffin, 1950; Zamecnik et al., 1951) although the tumor has a greater capacity to incorporate amino acids into protein than liver, the results in the intact animal are caused by a relatively poor blood supply. I n order to test this idea, an ingenious experiment was carried out (Zamecnik et al., 1951). One minute after the administration of labeled alanine to a rat, the liver was removed and a hepatoma nodule was frozen and sectioned. The radioactivity of successive slices of this nodule is shown in Fig. 8 and the highest activities were found a t the periphery. This demonstration of circulatory inadequacy was confirmed by radioautography, and the conclusion may be drawn that one factor controlling the rate of protein synthesis in the whole animal is the circulation in the individual tissues. Siekevitz and Zamecnik (1951) have shown that the enzymes responsible for the incorporation of labeled alanine into protein are localized within the microsomal fraction of the cell, but require energy supplied by the oxidative enzymes of the mitochondria. They were unable to attain sufficient oxidation in hepatoma homogenates to permit the incorporation of alanine into protein. However, when liver mitochondria were added to hepatoma microsomes, incorporation was observed. It is of interest that Borsook et al. (1950) and Keller (1951) have shown in vivo that the radioactivity following the administration of several labeled amino acids was found in the microsome fraction of liver. No such experiment has as yet been reported in tumors. It may be seen that some progress has been made in our understand-
308
CHARLES HEIDELBERQER
CONSECUTIVE SLICES FROM HEPATOMA NOOULE IN VNO ALANINE
FIG.8. Consecutive slices from hepatoma nodule. Chart represents counts found in consecutive slices from the large hepatoma nodule (A). Rat was given 0.45 cc. alanine-cld containing 50,000,000 c.p.m. in 50 mg. by jugular vein. One minute later the large hepatoma nodule and a normal lobe were removed and frozen on Cot snow. A cylinder of frozen tissue going from one edge of the nodule to the other was cut out of the hepatoma. A No. 8 cork borer was used. This gave a diameter of about 1 cm. The “whole slice” counts were obtained by placing a frozen 25-micron slice on a copper planchet. The slice was dried a t room temperature and counted. The pooled 6 0 4 c e fractions were homogenized in 3 cc. of HsO, and the tubes were immersed in boiling water for seven minutes to precipitate the protein. The supernatant obtained after centrifugation was plated on planchets. This represented the nonprotein fraction. The protein was washed in the usual way and plated on disks for counting.
RADIOISOTOPES APPLIED TO TUMOR STUDIES
309
ing of the processes involved in the uptake of amino acids into proteins. However, much remains to be done. As Zamecnik (1950) has stated, “The key problem still remains unsettled as to whether the amino acids become incorporated solely by a process of ‘de novo’ synthesis of the entire peptide chain, or whether the peptide chain can break at two peptide bond linkages and permit single amino acids to be exchanged with no net change in protein mass or structure occurring. Upon the solution to this question rests the interpretation of all the results obtained with labeled amino acids in tissue slice and homogenate experiments and of a share of the results in whole animals.”
VI. NUCLEIC ACIDS 1 . Introduction Few substances are of greater importance in the biochemistry of cancer than the nucleic acids. There is a definite association of the nucleic acids with the growth processes, yet as Davidson and Leslie have stated (1950), “although the information available on this subject has increased abundantly, we are still unable to explain the precise biological function of the nucleic acids. While it is clear that they play an important and probably fundamental part in the processes of cellular growth and division and in the synthesis of proteins, their exact mode of action has not yet been satisfactorily explained.” The whole field of nucleic acid chemistry and metabolism is complicated a t the present time. The molecules involved are complex, and although their component parts have been well characterized, their structures are not fully known. The nucleic acids occur within the cell as nucleoproteins and can be separated from the protein moiety by mild conditions. These nucleic acids are polynucleotides, each nucleotide consisting of a purine or pyrimidine base, a pentose, and one phosphoric acid group. One type of nucleic acid contains only 2-desoxyribose as its carbohydrate constituent and is known as desoxyribosenucleic acid ( D N A ) . This type of nucleic acid has a molecular weight of around 7 million, and is found only within the nucleus of the cell; it is an important constituent of the chromosomes. The other type of nucleic acid contains only &ribose as the carbohydrate, and is known as ribosenucleic acid ( R N A or PNA). This type of nucleic acid has a molecular weight of the order of 10,000 and is found in the cytoplasmic fractions of the cell, as well as in the nucleus. In DNA the purines, adenine and guanine, and the pyrimidines, cytosine, thymine, and 5-methylcytosine are found; in RNA the same purines occur and the pyrimidines, cytosine and uracil. It used to be thought that the nucleic acids were tetranucleotide polymers, containing one nucleotide of each principal purine and pyrimidine base.
310
CHARLES HEIDELBERGER
However, largely as a result of the extensive analytical data compiled by Chargaff and his associates (1951), that view is no longer held. The study of the biosynthesis and metabolism of nucleic acids has been made possible almost entirely by the use of isotopes, and in the last few years a large body of literature pertaining to this subject has been accumulated. It is clearly impossible to cover adequately the tremendous amount of fundamental work in this area, and the present discussion will therefore be strictly limited to those cases in which the nucleic acid metabolism of tumors has been investigated. There has thus far been a complete separation of studies of the biosynthesis of nucleic acids studied with radioactive phosphorus on one hand, and with the purine and pyrimidine bases and their precursors on the other. This separation will be kept in the ensuing discussion. No work on the metabolism of the carbohydrate components of tumor nucleic acids has as yet come to the attention of this reviewer. An excellent compilation of recent information on the chemistry and metabolism of nucleic acids may be found in a “Symposium of Biochemistry of Nucleic Acids” (Symposium, 1951). Davidson and Leslie (1950) have reviewed the information on the correlation of nucleic acids and growth, Hevesy (1948a,b) has discussed the Paz work. Recent research in various aspects of the field are summarized by Holmes, Howard and Pelc, Davidson, Jeener, Brown, Wilson, and Hammarsten, and informal discussions are transcribed in “Isotopes in Biochemistry” (Ciba, 1951). One word of caution before the actual material is presented: In few cases has there been such a state of flux in the technical methods of isolation, analysis, and characterization of substances as there has been in isotopic experiments on nucleic acid biosynthesis and metabolism. As refinements in these technics have been developed, new pitfalls were encountered in the guise of new compounds and highly radioactive impurities. Because of this situation it is quite possible that the data derived from the early experiments in this field may be of little or no value. It is imperative that only by the most rigorous examination and analysis of experimental procedures and by work with compounds as well defined as possible can truly reliable information be obtained. The possibility should constantly be borne in mind throughout the following discussion that many of the facts and quantitative results presented may have to be revised when they shall have been more painstakingly reinvestigated. 2. Radioactive Phosphorus Studies
Tuttle et al. (19414 studied the uptake of Pa2into the acid-soluble, phospholipid, and “nucleoprotein” fractions of liver, spleen, and lymph
311
RADIOISOTOPES APPLIED TO TUMOR STUDIES
nodes of normal and leukemic mice and of lymphoma. It wm found that there was considerable incorporation of isotope into these fractions and that the “nucleoprotein” radioactivity was higher in all cases in the leukemic than in the normal animals at all the times that were studied. Similar results were obtained with rats by Kohman and Rusch (1941) who found that livers bearing hepatomas incorporated more P32into the residual “nucleoprotein ” fraction that did normal livers. Tuttle et al. (1941b) showed that a smaller percentage of the dose was incorporated into the same three fractions they studied before when 80 microcuries were given than with 8 microcuries, showing that there was a significant radiation effect. Marshak (1941) has compared the uptake of P32into the whole tissue and nuclei of liver and lymphoma of mice after a variety of times following administration. The results are given in Table X and show that the TABLE X Lymphoma Activity as per cent injected dose Nuclei
Tissue
Nuclei/Tissue
Time after Injection
Liver
Tumor
Liver
Tumor
Liver
Tumor
1 hr. 3 hr. 5 hr. 12 hr. 1 day 2 days 3 days 4 days 5 days
2.995 1.76 1.66 1.25 1.23 0.945 0.97 1.03 0.71
1.255 2.57 4.85 6.41 6.395 7.17 7.02 6.09 5.47
9.57 6.24 5.825 3.45 3.035 1.96 2.12 2.34 1.64
3.72 3.90 4.155 3.70 4.11 4.06 3.68 2.80 2.27
0.311 0.286 0.284 0.371 0.412 0.398 0.461 0.449 0.438
0.337 0.689 1.13 1.37 1.59 1.78 1.91 2.19 2.35
Note: The value a t each time interval is the mean of 2 to 4 determinations with 10 mice. activity of the nuclei is given a6 per cent/om.*, and that of the tissue aa per cent/g.
The
whole lymphoma, particularly the nuclei, take up a considerably larger amount of P32than liver. It was shown that regenerating liver also incorporated a larger amount of radioactivity than did normal liver, indicating that this is a property of growing tissue and not a specific characteristic of tumors. On the basis of the radioactivity measurements and total phosphate analysis he calculated that the nuclear phosphate was replaced anabolically completely every twenty-seven hours. He further showed that the residual “nucleoprotein” fraction contained about 70 % of the nuclear radioactivity.
CHARLES HEIDELBERQER
312
In the pioneering experiments mentioned thus far the radioactivity was determined on ill-defined “ nucleoprotein )’ fractions of unknown composition, and no attempt was made to isolate or separate the two types of nucleic acids. Therefore, a considerable advance was made by Brues, Tracy, and Cohn (1944), who carried out studies on the incorporation of radioactive phosphorus into the DNA and RNA of normal and regenerating liver and of hepatoma. Their work was carried out at the same time as that of Hevesy and his colleagues, which will be described below. Their results (Brues et al., 1944) are summarized in Table XI, in TABLE XI Specific Activities of Nucleic Acid Phosphorus Expressed as Per Cent of Inorganic Phosphate Specific Activities (Brues et al., 1944) Organ Resting liver Regenerating liver Hepatoma
Days after Injection
Ribosenucleic Acid
Desoxyribosenucleic Acid
3 8 3 13 3
54.9 123 230 314 171
10.6 20.8 180 04.0
which the term “thymonucleic acid” is synonymous with DNA. These data show a considerably higher incorporation of radioactivity into the DNA of the rapidly regenerating liver and the more slowly growing hepatoma than into the DNA of the nongrowing liver. There was a greater incorporation into the RNA than into the DNA at the times studied. This apparent stability of the DNA, is in contrast to most of the other metabolic systems that have been studied, and this has often been correlated with the philosophically attractive hypothesis of the biochemical stability of the chromosomes and genes. There are, however, certain situations, as will be seen below, in which this correlation does not appear to hold. A tremendous body of data on various aspects of nucleic acid metabohas been amassed by Hevesy and his group. It lism, studied with Pa%, would be impossible to do justice to this work in the allotted space, but fortunately the subject has been comprehensively reviewed (Hevesy, 1948a,b). The preliminary experiments of Hahn and Hevesy (1940) indicated a very small incorporation of radioactivity into the DNA of rabbit liver, and since the possibility of contamination by small amounts of highly radioactive impurities was not ruled out, the experiments were repeated and amplified (Hevesy and Otteson, 1943) under more carefully controlled conditions. They measured the specific activity of the free
RADIOISOTOPES APPLIED TO TUMOR STUDIES
313
inorganic phosphate of a number of tissues at various times after administration of Pazand computed an “average value” for each tissue. This “average value” was then compared to the specific activity of the DNA of each tissue at a single time and the percentage ratio, corrected for extracellular phosphate, was taken as the percent “renewal ” and the “rate of renewal.” The result of the experiments is shown in Table XII. TABLE XI1 Specific Activity * of the Nucleic Acid Phosphorus Extracted from Different Organs of 8 Rats 4 Days after the Administration of Labeled Phosphate x 1000
I
I1
Percentage Ratio of the Specific Activity of Nucleic Acid P and Free P (Percentage Renewal)t
12.7 5.93 1.05 1.01 0.83 0.60 0.09
15.4 6.24 1.42 1.66 0.97 0.67 0.22
59 23 8.8 4.2 10 2.1 2.3
Specific Activity X 1000 Organ Small intestinal mucosa Spleen Muscle Liver Testes Kidney Brain
* Percentage of the Par administered present in 1 mg. P.
t When calculating the above ratio we must take into account that the nuoleic acid has been extracted from the organs of 8 rats.
These values are then used to calculate the quantity of nucleic acid synthesized during a day. Since these calculations form the basis for the subsequent experiments by this group, it is desirable to examine the calculations and assumptions in somewhat more detail. In the paper by Hevesy and Otteson (1943) the specific activity of the free phosphate of various tissues rapidly reached a maximum and then declined, as do all such curves. The “average value” is taken as the basis for the calculations, without any statement as to how this value is computed. The justification for the use of this quantity is not clear. As Zilversmit et al. (1943) have pointed out, the relation of a known precursor to its product can only be derived in terms of the differential equations of the rate laws, in which rates can only be determined by measurements a t a series of times. In the present treatment, the “average value” of the specific activity of the free phosphate over the duration of the experiment is taken, and the specific activity of the product (DNA) is measured at only a single time, four days after administration of the Paz. Therefore, while the ratio of these specific activities is an experimentally determinable quantity, there is little or no justification in assuming that this quantity measures the “rate of renewal,”
314
CHARLES HEIDELBERQER
since no rate is in fact being measured. Furthermore, it was assumed that phosphate is the immediate precursor of the nucleic acid, yet a more recent kinetic analysis of the situation (Barnum and Huseby, 1950) makes this appear to be an oversimplification. Another important factor which affects the situation is the permeability of the various tissues, which may differ with respect to inorganic phosphate and a more proximal nucleic acid precursor. This permeability will be the resultant of a number of physiological factors determining the amount of glycolysis and respiration in the tissue, which in turn govern the availability of the energy required for the synthetic process. Because of these and doubtless other complications, it is impossible a t the present time, even with careful kinetic analysis, to calculate the true turnover rates or rates of renewal of nucleic acids or their precursors. This is in no way meant as disparagement of the prodigious amount of experimentation of the Hevesy group. Their data are of the utmost importance in the study of nucleic acid metabolism and are undoubtedly correct. Nevertheless, it must be pointed out that their calculations of absolute rates of renewal are based on gross oversimplifications. A more complete discussion of similar points has been given by Chaikoff (1942). Euler and Hevesy (1944) studied the incorporation of Psz into the DNA of the Jensen sarcoma. They found a greater incorporation into the DNA of the tumor than of the liver. This uptake was greatly diminished in animals given moderately high whole-body x-radiation. They also found a greater incorporation in the fresh than in necrotic parts of the tumors. The incorporation of radioactive phosphorus into the DNA of rapidly growing and spontaneously regressing Jensen sarcomas was compared by Ahlstrom et al. (1947). No significant difference in the ratio of the specific activities of the DNA to the inorganic phosphorus in the two cases was found. This result implies that although the tumor is regressing, the enzymes involved in the synthesis of DNA are not disturbed. Unfortunately, experimental details are lacking; it is not stated whether or not the necrotic portions of the tumor were used, and the time interval at which the measurements are made is not stated. Thus it is difficult to evaluate the significance of these interesting findings. It was also shown that greater incorporation occurred in transplanted benzpyrene-induced tumors than in liver, and furthermore, that colchicine inhibited the incorporation of labeled phosphorus into the DNA of Jensen sarcomas. Ahlstrom et al. (1945) studied the effect of x-radiation on rats bearing two Jensen sarcomas. One tumor was shielded from the radiation, and the other was not. The incorporation a t two hours of P32 into DNA of both tumors was compared, and it was found that although there was a
RADIOISOTOPES APPLIED TO TUMOR STUDIES
315
marked inhibition of uptake in the unshielded tumor as compared to the shielded one, nevertheless the shielded tumor incorporated less radioactivity than was found in tumors of nonirradiated animals. This demonstrated that there is an indirect effect of x-radiation on DNA, which was also paralleled by regression of the tumors. In an attempt to characterize further the nature of this indirect effect, Ahlstrom et al. (1946a) transfused blood from a growing rabbit that was heavily irradiated into an untreated sister rabbit, which was then treated with a tracer dose of Psz. There appeared to be a significant diminution of incorporation of radioactivity into the DNA of kidney in the recipient animals over the controls, but no change in the DNA of liver and intestinal mucosa. The fate of labeled DNA obtained biosynthetically was studied after administration to rats by Ahlstrom et al. (1946b). They found that the majority of the radioactivity was found in the acid-soluble fraction of liver. No attempt was made to isolate DNA from the recipient animals. It was also shown that liver slices were capable of splitting inorganic phosphorus from the labeled DNA. Holmes (1949, and in Ciba, 1951) studied the effects of x-radiation on shielded and unshielded Jensen sarcomas in the same animal. She confirmed the observations of Ahlstrom et al. (1945) that the incorporation of PS2into DNA was inhibited in the unshielded tumor to a marked extent, and in the shielded tumor to a lesser extent. I n addition she studied the effect of radiation on RNA synthesis, which was unaffected. Interpretation of these experiments is complicated by the sensitivity of the DNA incorporation to trauma, t o the impairment of circulation by x-rays under certain conditions, and to variable mitotic effects. In one series of experiments, PS2and S36were administered simultaneously to irradiated tumor-bearing rats. The Paz incorporation into DNA and RNA was measured, as was the SaSuptake into heat-coagulated proteins, “ riboproteins,” and nuclear histones. The results are given in Table XI11 and show that although incorporation of the Pa2into the DNA was diminished, the uptake of SS6into the histones, a principal component of the nucleoprotein protein, was unaffected. It was concluded that “if the histone is actually attached to the DNA, the lack of correspondence in x-ray effect on the two substances is remarkable.” The indirect effect of irradiation has also been demonstrated by Kelley and Jones (1950a), who measured the specific activity of mammary carcinoma DNA of animals whose livers were selectively irradiated with a colloid of radioactive yttrium. There was a definite diminution of specific activity in the tumor under these circumstances as shown statistically in large numbers of animals. A systemic effect of tumors on the
316
CHARLES HEIDELBERQER
TABLE XI11 Pa* a8 Inorganic Phosphate and Sss Methionine Injected Simultaneously
Nucleic acids
Whole tissue Heat
Acid Acid coag. soluble insol. protein
Riboprotein
Histones
Experiment
Ribo- Desoxynucleic ribonucleic
-
0 ) 332 +) 426 0 ) 321 433
+) 278 467 0 ) 198 380
+) 348 260
0) 1333 1332 +) 453 1094
0) 208 490 +) 260 328 0) +)
509 706
Injected, irradiated +) 441 +) 418 +) 39* Killed 1 4 hr. later 0) 9.1 * Injected, irradi0) 368 0) 576 ated +) 403 +) 358 +) 3.9, Killed 13 hr. later 0) 10.4* Injected, irradi0) 528 0) 280 ated +) 273 +I 158 +) 15* Killed 14 hr. later 0) 1332 0) 370* 0) 53.8* Injected, irradiated +) 1094 +) 238* +) 13.5* Killed 13 hr. later 0) 263 0) 316 0) 4.1 * Injected, irradiated +I 299 +) 335 +) 6.7* Killed 14 hr. later 0) 156 0) 156 0) 6 . 5 Irradiated May 27/50 +) 221 +) 115 +) 15.6 Injected May 29/50 Killed 14 hr. later 0) 319
0) 331
0) 44'
0) 140
0) 68
f)139
+) 26
0) 214
0) 40
+) 211 +)
0) 427
0) 117
+) 465 +)
0) 221
62
0) 86
+) 282 +)
0) 367
34
32
0) 9 8 . 2
+) 364 +) 46.3
0) 90 +)
0) 16.4
57 +)
9.0
0) signiiies oontrol turnom. +) signifieu irradiated tumors.
Pal oontent of nuoleio aoida expraased aa oounts per 0.1 mg. P.
SsS content of proteins expreuned aa oounts per 1.0 mg. BaSOr preoipitate. Fraotions marked are derived from small amounts of protein and contain a large proportion of
*
inert sulphate added aa owner. SU oontent of whole tissue serves aa oontrol.
specific activity of the liver DNA has been demonstrated by Kelley, Jones, and their colleagues (1960b, 1951). They found a marked increase in the specific.activity of the liver DNA in rats and mice bearing a variety of neoplasms. A similar effect was also observed in spleen, but essentially no change waa found in small intestine and kidney. The increase
3 17
RADIOISOTOPES APPLIED TO TUMOR STUDIES
$2%%%%,10Q
A
P 3 2 NUCLEAR PROTLlw ud NUCLEIC A C W P S wno~r ~ TISSUEmmn X loo lad NUCLEIC ACllw
C
r4
D
FIQ.9. Per cent uptake of Pa* in nuclear fractions with respect to whole-tissue homogenate or fractions. Solid, shaded, and unshaded areas represent normal, precancerous, and tumor tissues, respectively.
activity of acid-soluble, lipid, DNA and PNA phosphorus in the nuclei and cytoplasm or normal, and “precancerous” livers and of the liver tumors induced by 3’-methyl-4-dimethylaminoazobenzene. The results are shown in Fig. 9, and a great increase over the normal value for incorporation of Pszinto the DNA in the precancerous livers and tumors was observed. This type of approach appears to be of great promise in the elucidation of the mechanism whereby carcinogens exert their action.
318
CHARLES HEIDELBERQER
The in vitro uptake of Pa2 into total nucleic acid and phosphoprotein was studied in slices of liver, kidney, and tumor by Mann and Gruschow (1949). They found a marked inhibition of uptake in liver and kidney under anaerobic conditions with and without the addition of glucose. On the other hand, the uptake in tumor was only slightly inhibited by anaerobiosis in the presence of glucose. These results suggest that the high level of glycolysis in tumors may be sufficient to supply the energy for the incorporation, whereas aerobic oxidations are necessary in normal tissues. Considerable progress is now being made toward a better understanding of the mechanisms of nucleic acid synthesis and metabolism by the work now being done with cell fractionations and P32uptake largely through the efforts of Davidson, Marshak, Barnum and Huseby, and Jeener. These experiments have largely been carried out in liver, and cannot be discussed here. It appears to be of great importance to extend these studies to tumor tissue as well, and a beginning in this direction is now being made by this author in collaboration with Tyner and LePage. 3. Nucleic Acid Purines
The nature of the biochemical building blocks of the purines was elucidated by the cIassic investigations of Buchanan and his co-workers (1948) on the biosynthesis of uric acid in birds. They found that formate is incorporated into the ureide carbons, 2 and 8; carbon dioxide is fixed into carbon 6; and glycine is the precursor of carbons 4 and 5 and nitrogen 7. The same compounds have been shown to be precursors of the purines adenine and guanine in mammalian nucleic acids (Heinrich and Wilson, 1950).
Uric acid
Adenine
Guanine
Brown and his group have investigated the incorporation of purines and pyrimidines into nucleic acids. They have shown that adenine is incorporated into nucleic acid adenine and guanine and that guanine is not an important precursor of nucleic acid guanine. In order to gain information as to the intermediate between adenine and guanine, 2-oxyadenine and 2,6-diaminopurine were investigated, and only the latter compound was incorporated into nucleic acid guanine. However,
RADIOISOTOPES APPLIED TO TUMOR STUDIES
319
adenine, which is incorporated efficiently into the nucleic acids, occurs in the diet to only a very slight extent. When a mixture of labeled mononucleotides was fed to rats the incorporation into nucleic acid took place to a much lesser extent than was found with the free purines. Similar results were found with orally administered yeast nucleic acids. However, intraperitoneally injected guanylic acid was incorporated into polynucleotide guanine, whereas guanine and guanosine were not. Results similar to those of Brown have been obtained by Hammarsten and his colleagues. I n view of the aforementioned findings it seems possible that the small-molecule precursors of nucleic acid purines play a larger role in the everyday economy of the animal than do the purines themselves. Or, as Graff et al. (1951) have stated, “It is abundantly clear that the ultimate precursors of the nucleic acids in the Metazoa are to be found among the amino acids. Neither nucleic acids, nor purines and pyrimidines are essential nutrients in these species.” All the findings mentioned above have been carried out either in mixed viscera or liver, and more recently the incorporation of adenine and 2,B-diaminopurine into DNA and RNA has been demonstrated. These studies have been reviewed by Brown (Symposium, 1951; Ciba, 1951). The only published work of Brown et al. (1949) involving tumors demonstrated that labeled guanine was incorporated t o a very small extent into the nucleic acid guanine of viscera and mammary adenocarconoma of mice, whereas no such incorporation was observed in rats. Graff et al. (1951) have found no detectable incorporation of guanine-8C14into polynucleotides of liver, kidney, small intestine, one spontaneous tumor, and two transplanted tumors in mice. They also found that adenine was incorporated into tumor nucleic acids to about the same extent as in the above-mentioned normal tissues. The incorporation of glycine-2-C14 into the purines of DNA and RNA in liver, thymus, and Flexner-Jobling carcinoma in rats has been studied by LePage and Heidelberger (1951). They found more radioactivity in the purines of regenerating liver and tumor than in those of thymus and nongrowing liver. Furthermore, they found an incorporation of C14 into the purines of DNA of nongrowing liver of the same order of magnitude as into the RNA. This is in sharp contrast to the results obtained with Pa2 (Brues et al., 1944) and with adenine (Furst et al., 1950), where a considerably smaller uptake into DNA than into RNA was found. This same result of a high incorporation into DNA of resting liver was found independently with glycine and serine (Elwyn and Sprinson, 1950) and formate (Totter et al., 1951). This finding was
320
CHARLES HEIDELBERGER
confirmed and extended by Furst and Brown (1951), who showed that when g1ycine-Nlb and adenine-8-C l 4 were administered simultaneously to rats, the glycine was incorporated extensively into the DNA of liver, whereas the adenine was incorporated to a considerably lesser extent. They suggested that either two mechanisms of synthesis of DNA must exist or that there are two types of DNA. In order to exclude the possibility that formate, produced from glycine, was entering the nucleic acid purines solely by exchange of the ureide carbons, Heidelberger and LePage (1951) degraded the purines obtained from animals given labeled glycine. It was found that more than 50% of the radioactivity of the guanine and adenine of liver and tumor DNA and RNA was present in carbond of the purines, showing that the glycine was incorporated as such. They stated “Thus, it may be concluded that at least two pathways of DNA synthesis exist. One involves the incorporation of preformed adenine together with desoxyribose and phosphorus into the nucleic acid. (This pathway only operates when there is a net synthesis of DNA.) The other, and perhaps preponderant route, involves the incorporation of small-molecule precursors of purines into a skeleton to which the desoxyribose and phosphorus have already been afKxed. This incorporation must take place in such a way that the total quantity of DNA in resting tissue remains essentially static.” The incorporation of radioactivity into the purines of DNA and RNA of liver and tumor at several time intervals after the administration of glycine-2-CI4 was investigated by Tyner et al. (1952). The results are shown in Fig. 10 and should be compared to the protein data from the same experiments, Fig. 6. Since the immediate precursor of nucleic acid purines is not known, it was not possible to calculate true turnover rates in these experiments. Although the isotope was incorporated more rapidly into the proteins of ‘liver than into those of tumor, the inverse is true for the nucleic acid purines. A distinct lag was found from the time free glycine-2-C14 was known to be in the tissue to the appearance of radioactivity in the purines, and the rates of incorporation and decay of the adenine and guanine of both DNA and RNA are strikingly parallel. These results may suggest that protein synthesis is not necessarily synchronized with RNA synthesis. The effect of x-radiation on the incorporation of the radioactivity from acetate-l-CI* into the purines of DNA was studied by Hevesy (1949). He found a marked diminution of radioactivity in the irradiated animals, an effect similar to that he had earlier demonstrated with Paz. However, since the time of Hevesy’s experiments it has been found that acetate is not a precursor of purines, and it seems likely that Hevesy observed the fixation of carbon dioxide derived from the oxidation of acetate. In order to check this point Skipper and Mitchell (1951)
32 1
RADIOISOTOPES APPLIED TO TUMOR STUDIES
-
LIVER 6000+
7;
3000
$:,,
2000; j
:
IOOOi
A,,
"4.
........
A
......%..
......
600;
r C 3 3002
:
-
PNA GUANINE PNA ADENINE ---o DNA GUANINE b-4 DNA ADENINE &------a F R E E GLyClNE &................A
h.%..
L
-
.....
.-A . .......
............
-
-r?.-Y 0
36
1
+-L:
y-. ...... ...... -
"'P...
c
-
.....
--u.\
II
I
2
4
..............b I ..... -
-.:.5
5- - 2
"'b
--\ -
12
7
1
TUMOR
L, r { I
A-
-- -- ---.......... - --- -- - - -----____ ---3R\. I 0
0.. .........
I--
PNA
Q U I N I N E 0-0
PNA
ADENINE
++
DNA
QUANINE
&-4
DNA
ADENINE D - - - - - - O
FREE
c-
- _____-..,-
-
-\0 -
A
GLYCINE
HOURS TIME
-DAYS
Fro. 10. Logarithm of specific activity of free glycine and of DNA and PNA purines in liver and tumor versus time.
322
CHARLES HEIDELBERQER
TABLE XIV The Effects of Certain Known Anticancer Agents on Incorporation of C14 (from Formate) into Viscera, Nucleic Acids, and Nucleic Acid Purines Specific Activity *
CornExp. No.
Treatment
Viscera bined homog- nucleic Dosage (mg./kg.) enate acids 57.6 71.8 42.4
138.4 176.8 187.2
(Average) 4.6
6.6
57.3 57.2
167.5
2.7
6.8 7.4
39.8 45.4
135.2 148.2
5 .O 5.0
8.4
19.8
132.8
6.3
5.6
23.3
74.8
7.6
5.0
38.3
161. O
3.1
4.2 6.6
22.5 52.4
79.8 172.4
5.3 3.8
5.6 5.8
39.3 53.2
163.5 98.6
3.4 5.8
7.0
18.5
65.6
10.7
3.7 4.4 6.3 5.3
27.2 36.7 49.0 31.9
99.2 88.0 133.5 98.0
5.0 5.0 4.7 5.4
1 Controls 2 Controls 3 Controls 4 Controls 5 Controls
4.4 4.4 5.2 4.4
4.0 0.75(6X) 4.0(1X) 8 Urethan 1,800.0 450.0(6X) 9 Urethan 1,800.0(1X) 10 Urethan nitrogen 0.5(6X) 225 mustard 900 2 . 0 0 X ) 11 Benzene 250.0(6 X ) 1,150.0(1 X ) 12 KAsOa 4.5(6 X ) 9.0(1X) 2.4 13 Colchicine 0.63(6 X ) 14 Colchicine 2.4(1X) 100.0(7X) 15 2,&Diaminopurine 16 2,6-Diaminopurine 100.0(6X) 248.0(1 X ) 17 8-Azaguanine 31.3(6X) 250.0 (1X ) 18 Cortisone 44.0 19 Cortisone 44.0 44.0(3 X ) 20 Cortisone 6 Nitrogen mustard 7 Nitrogen mustard
+
Ratio of CornSpecific bined Activities nucleic of Viscera acid t o Purines ( X loz) purines
+ +
* Specific activities in rc./mole of carbon.
Ratio of the apecifia activitiee of the viscera homogenate to the combined nualeic acid purinea isolated therefrom. Number of injectiona indicated in parenthesea under dosage.
RADIOISOTOPES APPLIED TO TUMOR STUDIES
323
measured the incorporation of labeled bicarbonate and formate into the DNA purines. Their findings check those of Hevesy quantitatively for both precursors and suggest that the anticancer activity of x-rays is due at least partly to an inhibition of nucleic acid synthesis. Skipper and his group have carried out an extensive series of investigations on the effect of a number of inhibitors of various types on the incorporation of labeled formate into nucleic acid purines. They found (Skipper et al., 1950) that the folic acid antagonists, aminopterin and A-methopterin, produced an inhibition of the incorporation of formate and carbon dioxide into the nucleic acid purines, but did not inhibit the fixation of COz into the tissues. These data support the contention that folic acid acts as a coenzyme for formate transfer. Bennett et al. (1950) synthesized 8-azaguanine-2-C14 (guanazolo), a compound that inhibits the growth of certain tumors in mice, and studied its distribution. No selective uptake by tumors was observed. They showed (Mitchell et al., 1950) that radioactivity was incorporated into the nucleic acids of tumors and viscera, and some labeled 8-azaguanine was actually isolated from the purine fraction after hydrolysis, showing that some of the compound was incorporated intact into the nucleic acids. By measuring the incorporation of labeled formate into nucleic acid purines, Skipper et al. (1951b) found that inhibition was produced by 2,6-diaminopurine, 8-azaguanine, cortisone, potassium arsenite, urethan, and nitrogen mustard. Benzene and the mitotic poison colchicine produced no inhibition. Leukemic mice fixed more formate into visceral nucleic acids than did normal animals. The data are shown in Table XIV.
4. Nucleic Acid Pyrimidines The principal pyrimidine bases that are constituents of nucleic acids are cytosine, found in both DNA and RNA, uracil, found only in RNA, and thymine, found only in DNA. Brown and his colleagues have shown that whereas free pyrimidines are not utilized for nucleic acid synthesis, the pyrimidines from nucleic acids and nucleotides are utilized. This is in contrast to the purines, as already stated. Hammarsten’s group has shown that cytidine in an effective precursor of both cytosine and uracil of nucleic acids, whereas uridine is ineffective as a precursor. They also found that orotic acid was an efficient precursor of pyrimidines. Elwyn and Sprinson (1950) have shown that formate is a precursor of the methyl group of thymine, and Wright et al. (1951) have proved that ureidosuccinic acid is a pyrimidine precursor in bacteria. Carbon dioxide is the precursor of the ureide carbon of pyrimidines (Wilson, Ciba, 1951). All these factshave been gained through the use of isotopes, but tumor material was not included.
324
CHARLEB HEIDELBERGER
Cytosine
Uracil
o=cI /NHa
I
N 6OOH
AHn
\/
LOOH Ureidosuccinic acid
Orotic acid
TABLE XV Specific Activities of Pyrimidine Nucleotidea of Nucleic Acid from Tissue Slices Incubated with Radioactive Orotic Acid Thymidylic Acid
Tissue Rat liver (1) (2) Cat liver Rat Walker carcinoma 256 Rat Walker carcinoma with Amethopterin Gastric carcinoma Gastric carcinoma with A-methopterin Fibrosarcoma Carcinoma of large intestine Teratoma of testis Spleen from patient bearing gastric carcinoma Regenerating liver
Uridylic Acid
RNA Cytidylic Acid
Ct.1 ct./ ct./ min.1 min. / min./ mg. mg. mg. Amt. free Amt. free Amt. free (rg.1 base (rg.) base (rg.1 base 120
21
103 224
45 37
186
56
66 163 120 206* 128 136* 63
124 32 48 24 81 149 246
Rat liver (tumor-bearing animal) *Eluted from Dowex-1 oolumn with 0.1 N HCI. Eluted from Dowex-1 oolumn with 0.01 N HCI.
1,835 1,460 1,088 622
358 410 795 920
3,006 85 2,280 207 1,946 224 1,410 99
750 296 268 218 370 169 444
905 1,150 107 1,130 536 122 1,033 480 123 2,300 252 348 1,070 650 94 1,346 326 187 309 950 43
2,680
630 4,770 216
1,430
276 2,890
69
RADIOISOTOPES APPLIED TO TUMOR BTUDIEB
325
Weed and Wilson (1951) obtained incorporation of orotic a~id-2-C’~ into the nucleic acid pyrimidine nucleotides on incubation with liver and tumor slices. No radioactivity was found in the purines. The incorporation was greater into uridylic than into cytidylic acid, and greater in Walker 256 carcinoma than in liver. Weed (1951) extended his studies to a number of tumors, Table XV, and found extensive incorporation in all cases. A particularly interesting feature of this work is the comparison of the slices of tumors of various species, including human, as to their ability to incorporate orotic acid. Hurlbert and Potter (1952) have investigated in vivo the metabolism of orotic a~id-6-C’~.They found that the compound is a highly specific
”’i 10
I 72
1
4
I
8
I
I
12 20 Hours
I
91
FIG.11. Specific activity of RNA pyrimidine nucleotides in liver cell fractions versus time. The “nuclear,” “mitochondrial,” and “supernatant” cell fractions are designated by initials. UR is uridylic acid, CY is cytidylic acid. pyrimidine precursor and is incorporated efficiently. A relatively small amount of incorporation was found in tumor, compared to liver, and this was attributed to the great efficiency of the liver in absorbing and utilizing the compound. In the liver, only a very small amount of radioactivity was observed in the DNA, and in this respect orotic acid resembles phorphorus and adenine, rather than glycine and formate. The livers were fracttionated into nuclear, mitochondrial, and supernatant fractions, and the specific activity of the pyrimidine nucleotides of RNA of each fraction was determined. The results shown in Fig. 11 demonstrate a striking inhomogeneity of the RNA in different parts of the cell. Such observations have previously been made with Ps2(cf. Barnum and
326
CHARLES HEIDELBERGER
Huseby, 1950) and are compatible with the concept that nuclear RNA is the precursor of cytoplasmic RNA. It appears self-evident from a consideration of the work that has been done on nucleic acids that these compounds are of great importance in the growth process. Before the true function of these substances can be established, much work remains to be done. It would appear that the most promising lines of research to follow in the near future might be the careful comparison of the in vivo changes in specific radioactivity with time of the products derived from the more important precursors. Equally important is the in vitro study of the enzymatic systems responsible for the synthesis of nucleic acids and their components. Knowledge of the sequence of events and mechanisms of these various processes within the cell, and correlations of various tumors with growing and nongrowing normal tissues should provide many important clues which should eventually lead t o an understanding of the mechanisms of the control of growth.
VII. MISCELLANEOUS COMPOUNDS A number of papers in the literature deal with the distribution of labeled iodine and phosphorus in tumor-bearing animals, but their relationships to the main topics of this review are so tenuous that they will not be discussed here. There are, however, several compounds, which by themselves merit consideration because of their general importance or direct connection with tumor metabolism. They will be discussed in this section. 1. Iodinated Polysaccharide from Serratia marcesnes
The discovery by Shear and Turner (1943)that a substance isolated from Serratia marcesnes produced necrosis and destruction of moiise sarcomas launched a series of investigations into its chemical and pharmacological properties. It was found that the compound was a polysaccharide, and when given to humans produced an acute febrile response and was highly toxic. I n an attempt to diminish the toxicity of the substance, it was iodinated, and this derivative lost no tumor necrotizing potency in mice (Seligman et al., 1948). The substance was iodinated with I1*l,and the distribution of radioactivity in various mouse tissues was studied. No concentration was found in tumor. The rate of disappearance of radioactivity from the blood of human patients was also determined. However, the toxicity of this derivative was high, and no effect could be observed on human tumors. I n another attempt t o improve the properties of the substance, Seligman and Sack (1949)
RADIOISOTOPES APPLIED TO TUMOR STUDIES
327
treated the polysaccharide with radioactive piodobenzene diazonium chloride. The resulting azo compound was also highly toxic and apparently ineffective on human tumors. The rate of disappearance of radioactivity from the blood of patients was again measured, but because of the disappointing clinical results, further work along these lines was discontinued. 2. A Radioactive Oxazine Dye The distribution of radioactivity in animals receiving a dose of iodinelabeled Nile blue 2B was investigated by Sloviter (1949).
N
It had previously been found that compounds of this type stained mouse tumors and produced a retardation of their growth. The specific activity of the tumors in mice was in general comparable to that of the liver, kidney, and spleen, so that no selective uptake was observed. However, a significantly longer survival was found in mice treated with the nonradioactive dye as shown in Table XVI. Thus the radiation of the iodine had an effect additive to that of the retardation of tumor growth of the dye itself. This indication of an irradiation effect by a compound that is localized in the tumor is a promising one, although this particular substance is not selectively localized in the tumor. If it were possible TABLE XVI Oral Administration of Radioactive Dye to Mice with Mammary Carcinomata Radioactive Dye Group
Control Dye Group
Days dye fed Days survived
Days dye fed Days survived
17 17 18 17 18 14
58 58 62 83 90
92 Av. 74
13 15 16 18 18 14
27 28 29 37 44 50 Av. 36
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CHARLES HEIDELBEROER
to find a compound that concentrated exclusively in tumors, then a radioactive element in the molecule might selectively destroy the neoplasm. Tritium, which has a very short path of ionization, would be an ideal isotope for such a purpose. However, in order for a compound to locali~e exclusively in tumors, some qualitative difference between tumors and all normal tissues would have to exist. It is an unfortunate fact that no such difference has as yet been discovered. 3. Stilbamidine
The distribution and excretion of stilbamidine-C14in mice was studied NH
NH
by Reid and Weaver (1951). This compound was of interest because of some effect that was observed in the clinical treatment of multiple myeloma, and the suggestion had been made that it is selectively taken up by the plasma. The radioactivity was excreted rapidly in both urine and feces following administration to mice, and the C14 in a number of tissues was determined at various time intervals. There were no indications that the compound was concentrated in the blood or the hematopoietic system. Weaver et al. (1951) investigated the amount of radioactivity in the livers of normal and tumor-bearing mice ninety-six hours after administration of the labeled stilbamidine. They found that there was a considerably greater isotopic content in the livers of tumor-bearing mice than in normal mice. The nature of the chemical combination of the radioactive substance with the tissue is under investigation, and it was tentatively suggested that the complex is between stilbamidine and nucleic acids. 4. Triphenylbromoethylene The selective absorption of radioactivity in the ovaries of mice injected with the synthetic estrogen, triphenylbromoethylene-BrE2was reported by Daudel et al. (1946), who also found that the tissues of male mice had less radioactivity than the blood. A very high concentration of radioactive bromine was found in ovaries, thyroid, adrenals, and pituitary eighteen hours after the administration of the compound to mice (Daudel et al., 1950). It had previously been shown that this substance is carcinogenic and that the tumor production was related to its estrogenic activity. The importance of the high concentration in the ovaries and other endocrine organs of this hormone is obvious. In an attempt to check these observations and gain more information about
RADIOISOTOPES APPLIED TO TUMOR STUDIES
329
male mice, Twombly et al. (1951) prepared the compound and studied the distribution of radioactivity in various mouse tissues. They failed to confirm the observation of the selective adsorption of radioactivity in the ovaries, adrenals, and pituitary, and only found a high specific activity in the kidney, which they attributed to the active excretion of the compound, There is no explanation for the different results obtained in the two investigations, and further work will have to be done before the discrepancy is resolved. 6. Diethylstilbestrol
A preliminary study of the metabolism of another synthetic estrogen, diethylstilbestrol, has been reported by Twombly and Schoenewaldt (1951). They labeled the compound with C14 and studied the distribution of radioactivity in the tissues and excreta of mice. ~H*CH*
As in the previous examples, no selective absorption into any of the target organs, uterus, pituitary, or mammary gland was found. The radioactive material was absorbed rapidly and excreted through the bile into the feces. There was no significant appearance of radioactivity in the respiratory carbon dioxide. The excretion of radioactivity in urine of human cancer patients was also reported (Schoenewaldt and Twombly, 1951). 6. Nitrogen Mustards
The nitrogen mustards represent a series of compounds of interesting biological activity. They have a pronounced antileukemic action and have recently been shown to be carcinogenic as well. It has been postulated on theoretical chemical considerations that the nitrogen mustards exert their action by cyclization to the ethyleneimmonium cation, which is a powerful alkylating agent. In order to test this
330
CHARLES HEIDELBERGER
hypothesis in the animal, a monofunctional mustard of relatively low toxicity was labeled with radioactive iodine by Seligman et al. (1949). The compound chosen was diethyl-fl-iodoethylamine-I'31(I),which on cyclization releases the halogen as inorganic iodide and forms the cyclic salt (11). CzHa
\
/NCHzCHzl* CzHs (1)
caHs\N'r /+\
CzHa *I-
Hz
(11)
The excretion and distribution of radioactivity in various tissues of tumor-bearing mice following administration of the mustard was compared with that observed when sodium iodide was given. The extensive data that were collected show that the radioactivity patterns in most organs were almost identical for the two substances. This indicates that cyclization does take place to a marked extent in the animal. However, a small amount of was retained in most tissues and blood following dosage of the mustard, which was not found when inorganic iodide was given. The highest concentration of this residual material was found in blood, lung, and lymph nodes, the loci of clinical activity of the mustards. TABLE XVII Ratios of the Specific Activities of Organs and Tissues from Leukemic Mice to Control Mice Injected with Methyl-Labeled Nitrogen Mustard Ratio Tissue Whole blood Serum Cells Thymus Lymph nodes Spleen Adrenals Liver Kidneys Testes Brain Jejunum Bone Bone marrow Muscle
At 6 hr.
At 24 hr.
1.4 1.6 1.4 2.8 5.2 1.1 5.4 1.4 0.5 3.3 2.2 0.5 1.4 1.6 1.9
1.4 1.5 1.2 2.0 1.2 1.3 4.5 0.9 0.9 0.9 3.5 1.3 1 .o
0.7 0.5
RADIOISOTOPES APPLIED TO TUMOR STUDIES
331
The distribution of radioactivity following the administration of a physiologically active nitrogen mustard, methylbis(2-chloroethy1amine)methyl-C14 (111) was studied by Skipper et al. (1951~). They gave the
*
CHI-N
/CHzCH2C1
\
CHzCHiCl
(111)
labeled compound to normal and leukemic mice. It was found that 10 to 18%of the dose was converted into respiratory carbon dioxide, and about the same amount of radioactivity appeared in the feces. The radioactivity was distributed throughout the body, but there was a considerably higher specific activity of adrenals, lymph nodes, testes, thymus, and brain in the leukemic animals compared with the normals, as shown in Table XVII. This is a striking observation, but further studies must be carried out before its significance can be assessed. 7. Urethan The study of the metabolism of urethan has been carried out by Skipper and his associates. This compound, like the nitrogen mustards, produces some effect in leukemias and is also carcinogenic. I n the first paper (Bryan et al., 1949) a very thorough study of the distribution of radioactivity in excreta and tissues of mice following dosage of carbonyllabeled urethan (I) was reported.
H~NEOOC~H~ (1)
It was found that in the course of twenty-four hours essentially a complete conversion into respiratory carbon dioxide occurred in normal mice, and only minute amounts of radioactivity could be detected in all tissues. There was no evidence for any selective localization of radioactivity. The significant observation was made that in mice bearing various neoplasms there was a much greater retention of isotope in the tissues than was found in the normal animals. Since the radioactivity remaining in these mice was found in the tumor, and other tissues as well, it appears that this retention is due to a systemic effect of the tumor on the host. However, there was no selective retention by malignant cells. A good correlation was observed (Mitchell et al., 1949) between the amount of radioactivity in the tissues and the quantity of urethan determined by chemical analysis. It seemed likely that the urethan was hydrolyzed in the body to
332
CHARLES HEIDELBERGER
carbon dioxide, ethanol, and ammonia, and in order to test this hypothesis, Skipper et al. (1951a) compared the fate in mice of carbonyllabeled urethan (I),methylene-labeled urethan (11),methylene-labeled
HOURS FIG.12. Rate of expiration of Cl4 following injection of certain C'clabeled compounds. The broken line for NaHC1400sis the calculated rate of expiration following continuous injection.
ethanol (111), labeled bicarbonate and urea (IV). The data obtained from the respiratory carbon dioxide are shown in Fig. 12. H~NCOO~H~CH~ H O ~ H ~ C H ~ I1 I11
H~NC~ONH~ IV
These data and the retention in the body support the view that the urethan is hydrolyzed in the body. The authors also present calculations of the amounts of radioactivity that would be excreted if the compound were administered continuously. Experiments bearing more closely on the mechanism of action of urethan were carried out by Cornman et al. (1951). They studied the uptake of labeled urethan in sea urchin eggs and sperm, and in general found accumulation of the compound in concentrations at least equal to the surroundings. There was a greater concentration in fertile than in nonfertile eggs, and sperm concentrated more than either. These results suggest some interaction of urethan and nucleoprotein, but direct evidence for such combinations has not as yet been reported.
RADIOISOTOPES APPLIED TO TUMOR STUDIES
333
8. Colchicine Radioactive colchicine has been obtained from plants grown in an atmosphere of C1*02,and its distribution in mice has been studied by Back et al. (1951). This compound is of interest because of its effectiveness as a mitotic poison. The tissues were processed in a specific way for the isolation of colchicine, and in this study it is likely that the radioactivity measured was actually present as colchicine, although the direct proof by a carrier experiment was not furnished. It was found that four hours after the injection, no CI4 could be detected in blood, brain, muscle, and heart. The greatest amount of labeled colchicine was found in kidney, spleen, and intestine. The amounts found in liver and sarcoma 180 were equivalent. An interesting observation is that whereas the spleen of normal mice contained about 40% of the dose, no radioactivity could be detected in the spleens of tumor-bearing animals. It should be emphasized that in all the studies reported in this section, with the exception of the work on colchicine, only radioactivity following the administration of labeled compounds is measured. No information is available as to the chemical nature of the radioactive materials, and hence no definite conclusions can be reached as to the mechanisms whereby these compounds exert their biological effects. I n this respect all these researches must be considered as preliminary, and it will be of considerable interest t o watch for further work on these compounds that may produce direct evidence as to their modes of action.
VIII. CONCLUSION Although this chapter emphasizes the basic fallacy of attempting to review the applications of an important and general technic, used in many fields, it does permit the coverage of several areas of oncology to which isotopes have made substantial contributions. This coverage, because of space limitations, has of necessity been superficial, yet has allowed the author to express his opinion as to the present status and important trends of research in several subjects of general metabolic importance. Potter and Heidelberger (1950) wrote, “Since the isotopic tracer technic has only recently become available as a tool that can be used in almost any laboratory concerned with problems in metabolism the application of the technic to special metabolic situations is just beginning. It seems likely that in the not too distant future there will be available a description of the metabolism of nearly every individual metabolite in terms of the alternate pathways that it pursues in different organisms, in different tissues, and under a wide variety of special situations. With this knowledge may come the ability to influence metabolism along
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CHARLES HEIDELBERGER
specific lines by means of substitution in the case of deficiencies and by the use of antimetabolites and ot,her types of enzyme inhibitors that will modify enzyme action.” It is this reviewer’s firm belief that intensive and productive research along the lines here described will eventually supply the answer to many of the enigmas that are collectively known as cancer. ACKNOWLEDGMENT The author wishes to express his profound gratitude to his many colleagues a t the McArdle Memorial Laboratory for their unstinting cooperation. council, and criticisms during the preparation of the manuscript. REFERENCES Ahlstrom, L., Euler, H., and Hevesy, G. 1945. Arkiv. Kemi Mineral. Geol. 19A, No. 13. Ahlstrom, L., Euler, H., Hevesy, G., and Zerahn, K. 1946a. Arkiv. Kemi Mineral. Geol. 22A, No. 7. Ahlstrom, L., Euler, H., Hevesy, G., and Zerahn, K. 1946b. Arkiv. Kemi Mineral. Geol. 28A, No. 10. Ahlstrom, L., Euler, H., and Hevesy, G. 1947. Arkiv. Kemi Mineral. Geol. 24A,
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Keller, E. B. 1951. Federation Proc. 10,206. Kelley, L. S.,and Jones, H. B. 1950a. Proc. SOC.Exptl. Biol. Med. 74, 493-97. Kelley, L. S., and Jones, H. B. 1950b. Science 111, 333. Kelley, L. S., Payne, A. H., White, M. R., and Jones, H. B. 1951. Cancer Research 11, 694-96. Kielley, M. 1952. Cancer Research 12, 124-8. Kit, S.,and Greenberg, D. M. 195la. Cancer Research 11, 495-99. Kit, S., and Greenberg, D. M. 1951b. Cancer Research 11,500-04. KSgl, F. 1949. Experientia 5, 173-220. Kohman, T. P., and Rusch, H. P. 1941. Proc. SOC.Exptl. Biol. Med. 46, 403-04. Kremen, A. J., Hunter, S. W., Moore, G. E., and Hhhcock, C. R. 1949. Cancer Research 9, 174-76. LePage, G. A., and Heidelberger, C. 1951. J . Biol. Chem. 188, 593-002. LePage, G. A., Potter, V. R., Busch, H., Heidelberger, C., and Hurlbert, R. B. 1952. Cancer Research 12, 153-7. Levi, A. A., and Boyland, E. 1937. Chemistry & Industry 16, 440. Mann, W., and Gruschow, J. 1949. Proc. SOC.Exptl. Biol. Med. 71, 658-60. Marshak, A. 1941. J. Gen. Physiol. 20, 275-91. Miller, E.C. 1951. Cancer Research 11, 100-08. Miller, E. C., Plescia, A. M., Miller, J. A., and Heidelberger, C. 1951. Cancer Research 11, 268-69. Miller, J. A. 1950. Cancer Research 10,65-72. Mitchell, J. H., Jr., Hutchison, 0. S., Skipper, H. E., and Bryan, C. E. 1949. J . B i d . Chem. 180, 675-80. Mitchell, J. H., Jr., Skipper, H. E., and Bennett, L. I,., Jr. 1950. Cancer Research 10, 047-9. Morris, H. P., Weisburger, J. H., and Weisburger, E. K. 1950. Cancer Research 10,620-24. Norberg, E., and Greenberg, D. M. 1951. Cancer 4, 383-86. Olson, R. E. 1951. Cancer Research 11, 571-84. Olson, R. E.,and Stare, F. J. 1949. Abstracts, 116th Meeting, Am. Chem. SOC.61C. Pardee, A. B., Heidelberger, C., and Potter, V. R. 1950. J . Biol. Chem. 186,625-35. Potter, V. R. 1951. Cancer Research 11, 565-70. Potter, V. R., and Heidelberger, C. 1950. Physiol. Revs. SO, 487-512. Pressman, D. 1951. Federation Proc. 10, 568-9. Pullman, A., and Pullman, B. 1946. Rev. sci. 84, 145-58. Quimby, E. H. 1951. Advances in Biol. Med. Physics 2, 243-68. Ray, F. E., and Argus, M. F. 1951. Cancer Research 11, 274. Ray, F. E., and Geiser, R. C. 1950. Cancer Research 10, 616-9. Reid, J. C., and Jones, H. B. 1948. J . Biot. Chem. 174, 427-37. Reid, J. C., and Weaver, J. C. 1951. Cancer Research 11, 188-94. Rittenberg, D., and Foster, G. L. 1940. J . Biol. Chem. 133, 737-45. Rosenthal, 0. 1937. Biochem. J . 31, 1710-19. Rusch, H. P., andLePage, G. A. 1948. Ann. Rev. Biochem. 17, 471-94. Salzberg, D. A., Nye, W., and Griffin, A. C. 1950. Arch. Biochem. 27, 243-4. Salzberg, D.A.,Hane, S., and Griffin, A. C. 1951. Cancer Research 11, 276. Schneider, W.C.,and Potter, V. R. 1943. Cancer Research 3, 353-57. Schoenewaldt, E. F., and Twombly, G. H. 1951. Cancer Research 11, 277. Schoenheimer, R. 1946. The Dynamic State of Body Constituents. Harvard University Press, Cambridge, Mass.
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Skipper, H. E., and Mitchell, J. H., Jr. 1951. Cancer 4, 363-66. Skipper, H. E., Bennett, L. L., Jr., Bryan, C. E., White, L., Jr., Newton, M. A., and Simpson, L. 1951a. Cancer Research 11, 46-51. Skipper, H. E., Mitchell, J. H., Jr., Bennett, L. L., Jr., Newton, M. A., Simpson, L., and Eidson, M. 1951b. Cancer Research 11, 14549. Skipper, H. E., Bennett, L. I,., Jr., and Langham, W. H. 1951c. Cancer 4, 1025-27. Sloviter, H. A. 1949. Cancer Research 9, 677-80. Stoesc, P. A., Heidelberger, C., and LePage, G. A. 1950. Cancer Research 10, 243. Symposium on Biochemistry of Nucleic Acids. 1951. J . Cellular Comp. Physiol. 88, Supp. 1. Totter, J. R., Volkin, E., and Carter, C. E. 1951. J . Am. Chem. SOC.73, 1521-3. Tuttle, L. W., Erf, L. A., and Lawrence, J. H. 1941a. J . Clin. Invest. 20, 57-61. Tuttle, L. W., Erf, L. A., andLawrence, J. H. 1941b. J . Clin. Inuest. 20, 577-81. Twombly, G. H., and Schoenewaldt, E. F. 1951. Cancer 4, 296-302. Twombly, G. H., Schoenewaldt, .E.- F., and Meisel, D. 1951. Cancer Research 11, 287.
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The Carcinogenic Aminoazo Dyes* JAMES A. MILLER
AND
ELIZABETH C. MILLER
The McArdle Memorial Laboratory for Cancer Research, The Medical School, University of Wieconsin, Madison, Wisconsin CONTENTS
Page I. General Introduction. . . . . . . . . . . . . . . ..................... 340 11. Early Observations.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 111. 4-Dimethylaminoazobenzene and Its Derivatives. . . . . . . . . . . . . . . . . . . . . . . . 342 1. General Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342 2. Histology ....................... ........................ 344 3. Dietary Effects.. . . . . . . . . . . . . . . . . .................... 346 A. Vitamins ....................... ..................... 346 B. Cystine, Methionine, and Choli ..................... 348 C. Protein ...................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 ............ .................... 350 F. Relrttionship of Diet to Liver C n s . . . . . . . . . . . . . . . . . . 351 4. Structure and Carcinogenicity.. ... .................... 351 A. Assay Procedures.. . . . . . . . . . . . B. Linkage Requirements.. . . . . . . .................... 352 C. Amino Substituents.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Ring Substituents.. . . . . . . . . . . ..................... 356 5. Metabolism by the R a t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Overall Metabolism.. . . . . . . . . . ..................... 359 B. Reduction of the Azo Linkage., . . . . . . . . . . . . . . . . . . . . . . . . C. Hydroxylation of the “Aniline” Ring.. ..................... 363 D. N-Demethylation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. The Formation of Protein-Bound Dyes.. . . . . . 6. Alterations in Chemical Composition Following Ingestion of the Dyes. . . 371 IV. Studies on the Hepato-Carcinogenicity of Other Azo Dyes. . . . . . . . . . . . . . . 379 oluene, AAT) and Re1. 2’,3-Dimethyl-4-aminoazobenzene@-Amino lated Compounds.. . . . . . . . . . . . . . . . . . . . . . . ..................... 379 2. Azonaphthalene Series. . . . . . . . . . . . . . . . . . . ..................... 380 3. Trypan Blue.. . . . . . . ................................. 381 4. Commercial Food Dyes.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 V. On the Mechanism of Azo Dye Carcinogenesis.. . . . 1. General Considerations. . . . . . . . . . . . . . . . . . . . . . .
* The work of the authors in this field has been supported by grants from the National Cancer Institute, Public Health Service, the American Cancer Society, the Jane Coffin Childs Memorial Fund for Medical Research, and the Alexander and Margaret Stewart Trust Fund. 339
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Page 384 A. The Benzidine &arrangement Hypothesis. ....................... 384 B. The Split Product Hypothesis.. ................................. 386 C. The Methyl Deficiency Hypothesis.. ............................ 387 D. The Protein (or Enzyme) Deletion Hypothesis.. . . . . . . . . . . . . . . . . . . 388 VI. References.. ...................................................... 390 2. Specific Hypotheses.. .............................................
I. GENERAL INTRODUCTION One of the major achievements of cancer research in the last twenty years has been the discovery of a great variety of chemical carcinogens. These agents have become convenient tools for the production of tumors and for the study of carcinogenesis. However, it must be admitted that as yet little is known about the fundamental nature of the carcinogenic process, and no tests are available which enable one t o identify with any precision successive stages in the genesis of tumors. The only certain indicator that the carcinogenic process has occurred or has been in progress in a given tissue is the eventual emergence of a gross tumor. Consequently the task of defining the carcinogenic process is still largely restricted to the correlation of events in the target tissue with the ultimate appearance of gross tumors. Since it seems to be generally true that the presence of a carcinogen in the target tissue is not required throughout the whole process, the study of the interaction of carcinogen and tissue will probably be useful chiefly in defining the initial phases of the process. However, even an understanding of these early phases seems far away. The great number and, more especially, the wide variety of the chemical carcinogens that are now known, even for a single organ such as the rodent liver, might persuade one to the view that many different carcinogenic processes exist. Of course, chemically dissimilar carcinogens may and probably do attack tissues initially in different ways. But, for the time being at least, it seems to be useful to employ the hypothesis that all these agents subsequently produce essentially the same biochemical changes leading to tumor formation. The general uniformity of the properties of tumors (Greenstein, 1947) is in agreement with this hypothesis. On this basis an intensive study of those carcinogens which easily initiate tumors in sites particularly amenable to biochemical analysis should advance our knowledge of the general problem. The induction of tumors in the livers of certain rodents by various aminoazo dyes seems to us to be particularly suitable for this purpose. The specificity of these dyes for the liver and the relative ease with which both the dyes and the liver may be subjected to analysis are evident advantages. Furthermore, the great and rapidly increasing store of biochemical information
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on the liver will surely aid the study of carcinogenesis in this organ. Accordingly, the following review is devoted t o these hepatic carcinogens. Some of the general aspects of this problem have been discussed by Kinosita (1937),Rusch et al. (1945), and Greenstein (1947), while the dietary problems have been reviewed by J. A. Miller (1947), Kensler (1947),Baumann (1948),and Sugiura (1951). 11. EARLYOBSERVATIONS The studies on the carcinogenic aminoazo dyes date back t o an observation by Fischer (later known as Fischer-Wasels) in 1906. He found that the injection of scarlet red (I) into the ears of rabbits caused the formation of atypical epithelial growths which were difficult to distinguish histologically from cancers. However, unlike true cancers these growths always receded when the chemical stimulus was removed. Following Fischer’s report numerous other investigators confirmed this observation (for references, see Woglom, 1913,and Shear, 1937),and as a consequence scarlet red was wed clinically to stimulate the healing of epithelial tissues. In 1924 Schmidt, who was feeding scarlet red to mice as an in vivo fat stain, noticed that the dye caused an extensive proliferation of the epithelial cells of the liver. Large liver masses, which Schmidt considered to be both adenomatous and sarcomatous in nature, were found following prolonged administration of the dye. However, the first conclusive demonstration of the carcinogenicity of an azo dye was furnished by Yoshida (1933) and Sasaki and Yoshida (1935). These investigators employed 2’,3-dimethyl-4-aminoazobenzene (0-aminoazotoluene, AAT) (11) which is a part of the scarlet red molecule and which had been shown earlier (see Shear, 1937) to be as effective as scarlet red in inducing epithelial proliferation in experimental animals and in patients. The structural relationships between these compounds are shown in Fig. 1. Hyperplastic proliferation of the liver cells was noted after rats had been fed a rice diet containing 1 mg. of the dye per gram of food for two months, and adenomatous changes were seen after five to eight months of dyefeeding. Hepatomas or cholangiomas were found in all the rats which survived for more than 255 days. Early attempts by German workers (Fischer-Wasels, 1936; Heep, 1936) to confirm this observation were relatively unsuccessful, probably because of the protective nature of the diets used, but subsequent studies have repeatedly demonstrated the carcinogenicity of AAT for the livers of both rats and mice (Hartwell, 1941). In the next few years many related compounds were also tested for carcinogenic activity (see Kinosita, 1937), and in 1936 Kinosita reported that 4-dimethylaminoazobenzene (DAB) (111)was a much more active hepatic carcinogen for rats than AAT. This dye and certain of its
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derivatives have been employed in most of the recent studies on aminoazo dye carcinogenesis.
scarlet red
2’,3-dimefhyl4-ominoorobenzene (o-ominornotoluene ,AAT )
4-dlmelhylarnimzobenzene (DAB)
4monomethylominoozobenzene (MAB)
P
QN=N@H~
4-aminoozobenzene ( A 8 1
FIG.1. The structures and commonly used abbreviations for several of the cercinogenic aminoazo dyes and their derivatives.
111.
4-DIMETHYLAMINOAZOBENZENE AND
ITS DERIVATIVES
1. General Aspects For efficient tumor induction DAB has generally been fed to rats in the diet a t a level of about 0.06%, and under appropriate conditions a high incidence of liver tumors can be obtained in 120 to 180 days. In the authors’ laboratory rats are routinely fed for 4 t o 434 months a diet consisting of crude casein, 120 g.; Vitab (a rice bran concentrate), 20 g.; salt mixture, 40 g. ; glucose monohydrate, 770 g. ; corn oil, 50 g. ;halibut liver oil, 310 mg.; DAB, 600 mg.; and riboflavin to a total content of 1.5 or 2.0 mg. per kilogram of diet. Generally laparotomies are performed a t the end of the dye-feeding period so that the progress of the carcinogenic process can be assessed, and the rats are then fed the same diet without the dye for an additional two months. The latter period allows undetected tumors to grow to a recognizable size while the gross cirrhosis recedes. If the total riboflavin content of the diet (including that in the crude casein and Vitab) exceeds 2.0 mg. per kilogram (see below) or if other strains of rats are used (Sugiura and Rhoads, 1941), the required period of dye-feeding may be increased. On the other hand, if the more active
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derivative 3’-methyl-4-dimethylaminoazobenzene (3’-M:e-DAB) is fed at a level of 0.05 t o 0.06%, a dye-feeding period of .three months is adequate and the level of dietary riboflavin is less critical (Giese et al., 1946). In general the tumor incidences are about the same whether male or female rats are used. However, Rumsfeld et al. (1951) found that with 3’-Me-DAB and 4’-fluoro-4-dimethylaminoazobenzene(4’-F-DAB) male rats are more susceptible than female rats when a low level of dye is fed continuously or a high level is fed intermittently. Both lower and higher levels of DAB can be used although the latent period may become excessive in the first case and the mortality may be high in the latter case. For example, reducing the level of DAB from 0.06 to 0.03% of the diet reduced the tumor incidence from 100 to 16% in one experiment and from 87 to 0% in another (MacDon’ald et al., 1952a). Maruya (see Hartwell, 1941) finally obtained hepatomas in two rats fed 0.006% DAB for at least 371 days while no tumors were found in rats fed 0.002% of the dye for 400 days. Dmckrey and Kupfmuller (1948) failed to obtain tumors in rats given 0.1 or 0.3 mg. of DAB daily for 877 days. When these authors administered daily doses of 3 to 30 mg. of DAB per day, they found that the observed minimum latent period ranged from 41 days for the highest dose to 363 days for the lowest dose. Despite this wide range of daily dose levels the total amount of dye administered up to the time when approximately 20% of the rats had tumors varied only from 950 to 1050 mg., and the authors concluded that the total dose of DAB determined the final tumor incidence while the daily dose determined the latent period. However, when only 1 mg. of dye was administered daily, the total amount required for tumor formation decreased to 650 mg. Liver tumors can also be induced in rats by repeated subcutaneous injections of an oil solution of the dye, but the rate of tumor formation is much slower (Kinosita, 1937; Sugiura, 1951). Attempts to produce tumors by intravenous injection of emulsions of the dye or by implantation of cholesterol pellets containing DAB into the liver have been unsuccessful (Kinosita, 1937, 1940a). In general tumors have been found only in the livers of rats, although histological changes also occur in other organs, especially the spleen (Kinosita, 1937; Orr, 1940; Edwards and White, 1941). Recently, however, Hoch-Ligeti (1949) found pancreatic tumors in three rats fed DAB for 12 to 15 months in diets which protected against the formation of liver tumors, and Lowenhaupt (1949) reported that lymphoblastic lymphosarcomas developed in the spleens of 5 out of 28 rats with intrasplenic implants of the dye which survived for over 400 days. Although the liver of the rat is relatively susceptible to the carcinogenic action of DAB, the livers of the other species which have been tested
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are much more resistant. Thus, mice develop liver tumors following either oral or subcutaneous administration, but the latent period is at least twice as long as with rats and the incidences are low (Kinosita, 1937; Law, 1941; Andervont and Edwards, 1943a; Andervont el aZ., 1944; Kirby, 1945b; J. A. and E. C. Miller, 1952a). The survival of mice fed DAB is frequently poor. The livers of rabbits fed the dyes show hypertrophy, hyperplasia, and adenomatous proliferation, but attempts to induce tumors in this species have been unsuccessful (Kinosita, 1937). DAB has also failed to induce tumors in squirrels (Kinosita, 1940a), chickens (Kinosita, 1937), guinea pigs (Orr, 1940), hamsters (E. C. Miller et al., 1949b), chipmunks (J. A. and E. C. Miller, 1952a), and cotton rats (E. C. and J. A. Miller, 1947). In each of these cases oral administration of the dye was continued for long periods of time. 8. Histology The feeding of the azo dyes to rats results in early and progressive changes in the histology of the liver. Kinosita (1937) regarded the hepatic changes as being primarily of a hyperplastic nature and reported that an extensive proliferation of the cells of the periportal region occurred after only a few days of feeding of DAB. He concluded that the continued proliferation of these cells and the resulting pressure on the parenchymal cells were responsible for the degenerative changes and the loss of regular structure of the liver. Kinosita could not decide whether the proliferant cells were derived from parenchymal or bile duct cells; some appeared to give rise to aberrant ducts and eventually to cholangiomas while others proliferated as strands and gave rise to hepatomas. In contrast Orr (1940) concluded that the primary action of DAB on the liver was to damage the parenchymal cells and that the proliferation was a reparative process. Support for this view was drawn from experiments by Orr and Price (1948) in which hepatic degeneration was evident in as little as twenty-four hours after massive doses (50-250 mg.) of DAB were administered. It is obvious, however, that the effects of repeated moderate doses of the dye might be quite different from those of single massive doses. Opie (194413, 1946, 1947a,b) also considered the primary change to be one of degeneration; the degeneration was characterized by a reduction in the amount of cytoplasmic nucleic acid and was followed by a focal regeneration of some of the parenchymal cells around the portal spaces. These new cells were characterized by increased levels of pentosenucleic acid and appeared to be the source of the hepatomas, while the cholangiomas appeared to arise from newly formed hyperplastic bile ducts. Differences in the experimental conditions may have been responsible for some of the divergent observations cited
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above, since Kinosita obtained tumors much more rapidly than the other investigators. Price et al. (1952) made a detailed study of the histological changes which occur in the livers of rats fed the more active carcinogens 3I-MeDAB and 4’-F-DAB. They saw hyaline droplets or inclusions in the cytoplasm of the parenchymal cells after only seven days of dye-feeding, and the number and size of these bodies increased up t o about the twentyfifth day. The inclusions were found principally in the peripheral parts of the lobule when 3‘-Me-DAB was fed and in the central part when 4’-F-DAB was given. On reexamination of sections from earlier studies similar inclusions were also seen in the peripheral cells of the liver lobules from rats fed DAB. Considerable proliferation of the bile duct cells was seen in the livers of rats killed after four t o six weeks of feeding of either 3’-Me-DAB or 4’-F-DAB, while fewer bile duct cells and more parenchymal cells were seen in those killed between the sixth and eighth weeks. In view of the low mitotic index in these areas and because of certain histological characteristics, the most probable explanation for the apparent change in cell type appeared to be a conversion of the hyperplastic bile duct cells into cells which resembled parenchymal cells. Kinosita (1937) suggested a similar transition of cell-type in the livers of rats fed DAB. Scattered areas of hyperplastic bile ducts persisted, however, whether or not the feeding of the dye was continued. These areas were classified as hyperplasia or cholangiofibrosis (Opie, 194410) and appeared to be the areas from which the tumors arose. Brock et al. (1940) and Langer (1942) considered the increased nuclear size and variability to be one of the striking histological changes in the livers of rats fed DAB. Similar alterations were seen in the livers of rats fed 3’-Me-DAB by Richardson and Borsos-Nachtnebel (1951). The origin and classification of the tumors induced by these dyes have been the subject of some debate. Kinosita (1937) concluded that all the DAB-induced tumors which he studied had areas of hepatoma and adenocarcinoma derived from the hepatic cells and that about half of the tumors also contained areas of cholangioma. Edwards and White (1941) classified the tumors induced by DAB as hepatomas and adenocarcinomas, and both they and Dalton and Edwards (1942) felt that both types of tumors were derived from the hepatic parenchymal cells. Orr (1940) and Opie (194413, 1946), on the other hand, believed that carcinomas developed from both parenchymal and bile duct cells, and in Opie’s experiments (1946) feeding a low protein diet appeared to favor the development of cholangiomas. Most of the tumors induced by 3I-MeDAB have likewise been classified as cholangiomas, hepatomas, or mixed types (Cortell, 1947; Richardson and Borsos-Nachtnebel, 1951; Price et al.,
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JAMEB A. MILLER AND ELIZABETH C. MILLER
1952). However, Price et al. (1952) concluded that the classification seemed arbitrary, since in the mixed tumors there was a gradual transition from one type of structure to the other and since all types, whether classified as hepatoma or cholangioma, appeared to have a common origin in areas of cholangiofibrosis. Metastases, especially to the lungs and mesentery, are relatively common among animals fed any of these dyes if the primary tumors are allowed to grow to a large size (Kinosita, 1937; Brock et al., 1940; Orr, 1940; Edwards and White, 1941; Opie, 194413; Cortell, 1947; Richardson and Borsos-Nachtnebel, 1951). Kline and Rusch (1944) also found small metastases in the areas around healing laparotomy wounds in rats bearing primary liver cancers, but these tumors usually regressed in the later stages of the healing process. Of considerable interest is the recent demonstration by Breedis and Young (1949) that liver tumors induced by DAB, as well as several other types of primary and metastatic tumors in the liver, received no blood from the portal system and were supplied only by the hepatic artery. 3. Dietary Efects
In most of the early work rats were fed unpolished rice to which an oil solution of DAB was added so that the final concentration of dye in the diet was 0.06%, and this diet was supplemented by a piece of fresh carrot per rat per day. However, it was soon found that the rate of tumor formation was markedly altered by the addition of various natural materials to this diet. These studies led to the use of purified diets in which the contents of protein, vitamins, fat, and other factors could be varied independently. Much of the early literature on this subject has been reviewed before (Kinosita, 1937, 1940a; Rusch et al., 1945; J. A. Miller, 1947; Baumann, 1948; Sugiura, 1951) and will be considered in less detail here. A. Vitamins. Riboflavin is one of the most potent inhibitors of the induction of tumors by DAB. This effect was first reported by Kensler et al. (1941), who obtained marked inhibition when daily supplements of 200 pg. of riboflavin and 2 g. of casein per rat were added to the rice-carrot diet. Little or no retardation was noted when either riboflavin, even in amounts up to 5 mg. per rat per day, or casein alone was administered. Miner et al. (1943) used purified diets containing 12 t o 18% of casein and found that 10 mg. of riboflavin per kilogram of diet (or approximately 100 pg. per rat per day) prevented the formation of liver tumors by six months, although 60 to 100% of the rats fed the same diets with only 2 mg. of riboflavin per kilogram developed tumors by this
THE CARCINOGENIC AMINOAZO DYES
347
time. However, high levels of riboflavin only slowed down the rate of tumor formation, and high incidences have been observed when rats were fed the dye in such diets for long periods of time (Harris et al., 1947a). The strong protective effect of riboflavin has been repeatedly confirmed with DAB as the carcinogen (Antopol and Unna, 1942; Griffin and Baumann, 1948b; Harris et al., 1947a; J. A. Miller, 1947), and similar results have been obtained with both 4-monomethylaminoazobenzene (MAB) (E. C. Miller and Baumann, 1946) and 4l-fluor0-4-dimethylaminoazobenzene (J. A. Miller et al., 1949). However, much less protection was obtained with either 2~-methyl-4-dimethylaminoazobenzene (2’-Me-DAB) or 3I-Me-DAB (Giese et al., 1946; Griffin et al., 1949). The protection offered by high levels of riboflavin is apparently explained by the participation of flavin-adenine-dinucleotide in the reductive cleavage of the azo linkage of the dyes (Mueller and Miller, 1950). This reaction converts the dye into two noncarcinogenic amines and is more rapid in liver slices from rats supplemented with high levels of this vitamin than in rats receiving minimal amounts (Kensler, 1948, 1949). This cleavage is also probably the reason for the lower levels of protein-bound dyes in the livers of the riboflavin-supplemented rats (E. C. and J. A. Miller, 1947). With DAB as the carcinogen Miner et al. (1943) found approximately the same tumor incidence whether the diet contained high or low levels of thiamine, pantothenic acid, or nicotinic acid. In these studies fewer tumors were obtained when little or no pyridoxine was added to the diet, but :later studies showed that relatively deficient animals were needed in order to obtain a marked inhibition of tumor formation (E. C. Miller.et al., 1945). Recently Day et al. (1950) reported that rats fed from weaning a soy protein diet containing DAB developed tumors more rapidly if vitamin Biz was also included in the diet. The number of animals used in this experiment was*small, but similar results with more adequate numbers of rats have since been obtained in the authors’ laboratory (E. C. Miller et al., 195213). Whether or not this effect is related to the role of vitamin Bu in methylation reactions as suggested by Day et al. remains to be investigated. Using highly protective diets containing 10 or 20 mg. of riboflavin per kilogram du Vigneaud and his associates (1942) observed a stimulation of tumor induction by DAB when the rats received daily supplements of 2 t o 4 pg. of biotin. Thus in two series none of five and none of fourteen control rats developed tumors by 150 to 210 days while three of five and eleven of sixteen rats fed the same diets plus crystalline biotin had tumors at this time. Harris et al. (1947b) confirmed the stimulating effect of
348
JAMES A. MILLER AND ELIZABETH C. MILLER
biotin in a protective diet, but found no acceleration of tumor development when biotin was added to a diet which permitted 70% of the rats to develop tumors by 200 days. J. A. and E. C. Miller (1952a) also found no stimulation in tumor development when biotin was added to a nonprotective diet containing 12% of casein and 2 mg. of riboflavin per kilogram. Furthermore, the substitution of egg albumin for casein strongly retarded the induction of tumors by DAB, but the inhibition was not altered by heating the albumin to destroy the avidin or by supplementing the diet with biotin (Harris, 1947b; Kline et al., 1945). B. Cystine, Methionine, and Choline. Numerous studies have been reported in which the levels of these dietary constituents have been varied. In general supplementation of diets containing DAB with cystine has resulted in a decreased tumor incidence (Gyorgy et al., 1941; Harris et al., 1947a; E. C. Miller et al., 1948). However, when the basal diet contained only 6% of casein, cystine supplementation gave an increased tumor incidence when ad libitum feeding was employed (White and Edwards, 1942a,b) or no change in incidence when paired-feeding was used (White and White, 1946). Mori (1941b) found no effect of cystine on tumor production in a polished rice-carrot diet. A less pronounced decrease in tumor incidence has been obtained by supplementing diets containing DAB (GrifIin and Baumann, 1948b; E. C. Miller et al., 1948; Griffin et al., 1949; Day et al., 1950) or its 2’-methyl or 3’-methyl derivatives (Griffin and Baumann, 1948b) with methionine. Supplementation of diets containing DAB or MAB with choline reduced the amount of gross cirrhosis but had little or no effect on the tumor incidence (White and Edwards, 194213; Miner et al., 1943; E. C. Miller and Baumann, 1946). The extent of gross cirrhosis was increased when either nicotinamide or guanidoacetic acid was fed with MAB without affecting the rate of tumor induction (E. C. Miller and Baumann, 1946). C . Protein. Variable results have been obtained in experiments designed t o test the effect of alterations in the level of dietary protein (purified casein) on the rate of tumor induction. Thus, with DAB Miner et al. (1943), E. C. Miller el al. (1945), Opie (1944a), Harris et al. (1947a), and Harris (1949) obtained essentially the same tumor incidences with dietary casein levels ranging from 10 to 48%. However, Opie (1944a) concluded that more of the tumors were of a cholangiomatous type when a low protein diet rather than a high protein diet was fed. On the other hand Gyorgy el al. (1941) reported a definite but not regular protection against liver damage due to DAB with increased levels of dietary casein, and GrifIin et al. (1949) concluded that rats usually developed fewer tumors with DAB or its 2’- or 3’-methyl derivatives if the casein content was 2401, rather than 12%. The differences were generally small, how-
T H E CARCINOGENIC AMINOAZO DYES
349
ever, and in one experiment with DAB the rats fed 36% of casein developed more tumors than those fed either 12 or 24%. D. Fat. The quantity and type of fat added to diets containing DAB also have a marked effect on the rate of tumor induction. Thus diets containing hydrogenated coconut oil, its fatty acids, or lauric acid permitted only a small number of rats to develop tumors by six months, while diets containing corn oil or its fatty acids elicited high tumor incidences (J. A. Miller et al., 1944a,b; Kline et al., 1946a). When trilaurin, raw coconut oil, or olive oil was used the tumor incidence was generally a little lower than if the diet contained corn oil; tumor induction in fat-free diets was variable with very low to moderately high tumor incidences resulting (J. A. Miller et al., 194413; Kline et al., 1946a). Furthermore, if the diets contained 10 t o 20% of corn oil, tumors developed faster than with only 5% of corn oil in the diet (Kline et al., 1946b; Harris, 1949); similar results were obtained by Opie (1944a) when he varied the level of hydrogenated vegetable oil in his diets. On the other hand, rats fed diets containing 20% of olive oil developed fewer tumors than those receiving diets containing 5 or 10% of this oil (Harris, 1949). Substitution of hydrogenated coconut oil for corn oil also resulted in lower tumor incidences when MAB or 2’-Me-DAB was fed, but little or no difference was observed between the two fats when 3’-Me-DAB was the carcinogen (E. C. Miller and Baumann, 1946; Giese et al., 1946). J. A. Miller et al. (1946) also found that the addition of 0.25% of either of two synthetic detergents strongly inhibited tumor formation due to DAB. The differences in the linoleic acid and antioxidant contents of the fats did not appear to be involved in these effects, since supplementation with either ethyl linolate or a-tocopherol did not alter the tumor incidence (J. A. Miller et al., 1944a,b). Nor were the pro- and anticarcinogenic effects due to destruction of the dye in the diets, since analyses showed DAB to be stable for many weeks in purified diets containing various fats (J. A. Miller et al., 1944a). However, DAB was found to be much less stable in the brown rice diet (Kensler, 1947). Within twenty minutes after mixing DAB into this diet appreciable amounts of MAB could be detected; however, by seven days the total level of methylated dyes in the diet had decreased by only 7%. Since these dyes are equal in carcinogenic activity, the overall activity of the dyes in a brown rice diet does not decrease greatly upon storage for reasonable times. DAB has also been found to undergo destruction in a diet containing linoleic acid (Gyorgy et al., 1942). A similar oxidation of the N-methyl groups and of the azo linkage has been studied i n vitro using dyes dissolved in films of autoxidizing linoleic acid (Rusch and Miller, 1948). This confusing series of results with diets containing various fats,
350
JAMES A. MILLER AND ELIZABETH C. MILLER
different levels of a given fat, or a detergent now appears to be largely resolved in terms of the amounts of hepatic riboflavin which rats can maintain when these dietsarefed (J. A. Miller, 1947; Griffin and Baumann, 1948b; E. C. Miller et al., 1948). Thus, rats fed dye-free diets containing hydrogenated coconut oil, detergents, or high levels of riboflavin maintained higher levels and those fed a diet containing 20% of corn oil maintained a lower level of riboflavin in the liver than rats fed a diet containing 5 % of corn oil. When DAB was added to any of these diets, the level of hepatic riboflavin decreased, but the levels of riboflavin in the liver were still highest when the protective diets were fed and lowest when the procarcinogenic diet high in corn oil was fed. Except for the high riboflavin diet all these diets contained the same level, 2 mg. per kilogram, of riboflavin. While the levels of biotin and vitamin B6in the livers were also decreased by the ingestion of the dye, there was no correlation between the levels of these vitamins which were maintained on certain diets and the probable tumor incidences (E. C. Miller et al., 1948). Furthermore, the lack of sensitivity of 3'-Me-DAB to dietary control was correlated with an inability of rats fed this dye to maintain high levels of hepatic riboflavin even when diets containing hydrogenated coconut oil or 10 mg. of riboflavin per kilogram were fed (Griffin and Baumann, 194813). Recently Clayton and Baumann (1949) have studied the effect of diet on tumor induction with 3'-Me-DAB in another way. They found that the tumor incidence in rats fed this dye for two four-week periods separated by a two- to twelve-week period in which the rats were not fed the dye was inversely related to the length of the intermediate period and depended to some extent on the diet fed during this time. Thus, rats fed a diet containing 20% of corn oil during the period in which they did not receive the dye developed more tumors than those fed the 5 % corn oil diet, even though all the rats were fed the 5% ' corn oil diet during the two terms of dye-feeding. In a similar type of experiment Sugiura (1951) fed DAB in the brown rice diet to rats for various times and then transferred the animals to dye-free diets of varying composition. The tumor incidence in the animals fed the dye for sixty days was a function of the nature of the dye-free diet fed afterwards and was highest when the brown rice diet was used and lowest if 15% of dried milk or yeast was added to it. If the dye was fed for more than eighty-five days, the tumor incidence was 100% regardless of the subsequent dye-free diet. E. MisceZlaneous. The basal metabolic rate of the animal apparently haslittleeffect on the rate of tumor induction by 3'-Me-DAB (W. L. Miller and Baumann, 1951). The incidence of tumors in rats made hyperthyroid with iodinated casein or hypothyroid with 2-thiouracil was higher than
THE CARCINOGENIC AMINOAZO DYES
351
that of the controls. The tumor incidence in rats made hypothyroid with 6-n-propyl-3-thiouracil was essentially the same as in untreated animals. Richardson et al. (1952) reported that the administration of 20-methylcholanthrene t o rats receiving 3’-Me-DAB markedly inhibited liver tumor formation. This striking observation has been repeated and extended by the authors (E. C. Miller et al., 1952a). While 75% of the rats fed 0.054% of 3‘-Me-DAB developed liver tumors by three months of dye-feeding and all the rats had tumors after two months on the basal diet, none of the rats fed 0.0033% of 20-methylcholanthrene in addition to the dye had tumors by the end of the experiment. Similar results have been obtained when 1,2,5,6-dibenzanthracene,3,4-benzpyrene, or l12-benzanthracene were added to the diet at equivalent levels; pyrene had no effect under these conditions. Although the mechanism by which the hydrocarbons act has not been worked out, the metabolism of the dye must be accelerated when they are fed. Thus, the levels of free dyes in the livers and blood and of protein-bound dye in the livers of rats fed the dye with one of the protective hydrocarbons were about half of the levels obtained in the absence of the hydrocarbons and similar to those obtained when only half as much dye was fed. Simultaneous feeding of low levels of 3’-Me-DAB and 2-acetylaminofluorene has resulted in a higher incidence of liver tumors than the sum of the incidences obtained when the two hepatic carcinogens were fed separately (MacDonald et al., 1952a). The mechanism of this synergistic response is obscure at present, but the data are consonant with the hypothesis that the carcinogenic processes induced by both compounds have one or more common features. F. Relationship of Diet to Liver Cancer in Humans. This problem is discussed fully in the excellent and authoritative book on primary carcinoma of the liver by Berman (1951). Suffice it to say here that it now seems probable that the liver damage and high predisposition to liver cancer found in humans in Africa and the Orient are due t o food deficiencies. The similarities between the condition of the livers in experimental animals fed deficient diets, with and without carcinogens, and those of poorly fed human beings are too striking to be ignored.
4. Structure and Carcinogenicity A. Assay Procedures. To determine how any compound acts as a carcinogen it is important to determine what compound or set of compounds acts as the primary carcinogen (i,e., the compound which directly initiates the carcinogenic process) when the parent compound is administered. One approach to this problem is through an analysis of the
352
JAMES A. MILLER AND ELIZABETH C. MILLER
structural features required for carcinogenic activity. Some experiments of this type were carried out by the Japanese from about 1935 to 1942 (Kinosita, 1937, 1940a,b; Nagao, 1941a,b; Hartwell, 1941), but they were generally inadequate from a quantitative standpoint. In most cases the use of the rice-carrot diet permitted only a poor survival of the animals, and the compounds were administered a t various dosage levels in different experiments and at different times in the same experiment. We found the comparisons to be more satisfactory when the compounds were incorporated at equimolar levels (usually equivalent t o 0.06% DAB) in a 12% casein diet which permitted both a good survival and rapid tumor induction with the more active compounds. Each compound was fed to a group of twelve to fifteen rats for eight months or until tumors developed, and each series of compounds was controlled by a group of rats fed DAB for three t o four months. At the end of the dyefeeding periods the rats were maintained on the basal diet for an additional two months to allow the growth of latent tumors to a recognizable size. The liver of each rat was examined by laparotomy at the end of the dye-feeding period and at autopsy, and the following formula was used to calculate a rough index of the relative activities of the various compounds. Relative activitv - 6 X Mos. DAB feeding X % Tumors with test compound Mos. feeding test compound X % Tumors with DAB For these calculations DAB was arbitrarily assigned an activity of 6, and the tumor incidences at the end of the dye-feeding periods were used for calculation unless no tumors were noted at this time; in the latter case the tumor incidences at the end of the experiment were substituted. In the experiments by Sugiura (1948) and Sugiura et aZ. (1945) the use of the rice-carrot diet was retained, but under their conditions the survivals were good. These investigators fed the dyes continuously until the rats died or were killed with tumors or until the experiment was termina.ted after about one year. A rather large number of compounds structurally related to DAB have now been tested for carcinogenic activity. From these results it appears that in order to have an activity of greater than 1 the compound should have (1) either an azo or ethylene group joining the two aromatic rings, (2) at least one N-methyl group together with the proper second amino substituent, and (3) either no ring substituent or only certain substituents, preferably in the 3' position. B. Linkage Requirements. The requirement,' for the az; linkage is shown by the data in Table I. Thus, neither of the analogous Schiff
353
THE CARCINOUENIC AMINOAZO DYEB
TABLE I The Relative Carcinogenic Activities of Compounds Containing the Rings Present in 4-Dimethylaminoazobenzene
X
Relative Activity*
-N=N-
6 (reference compound) 0 0 0
-N=CH-CH=N-CLNH-
tl
+
-NH2 H2N-CH=CH-
0 (6, see text) ~ ~ _ _ _ _
* 8ee J. A. Miller and Baumann (1846b),J. A. Miller and E. C. Miller (1948), and Haddow et ol. ~
(1848).
bases with the -CH=Nlinkage nor N’-benzoyl-N,N-dimethyl-pphenylene diamine with the -C-NHlinkage were active (J. A. Miller
a I1
and Baumann, 1945b; J. A. Miller and E. C. Miller, 1948). The amines obtained by complete reduction of the azo linkage also appear to be inactive. Neither liver tumors nor liver damage were found when the pairs N,N-dimethyl-p-phenylene diamine dihydrochloride plus aniline hydrochloride (from DAB), N-monomethyl-p-phenylene diamine dihydrochloride plus aniline hydrochloride (from MAB) or N,N-dimethylp-phenylene diamine dihydrochloride plus m-toluidine hydrochloride (from 3’-Me-DAB) were fed for nine months at three times the molar level used for the azo dyes (J. A. and E. C. Miller, 1948). Kinosita (1940a), Sugiura et al. (1945), and F. R. White et al. (1948) also failed to induce tumors in rats by feeding N,N-dimethyl-p-phenylene diamine. Druckrey (1950a,b) administered 22 mg. of aniline hydrochloride daily to rats for 750 days in the drinking water but found neither liver damage nor tumors. The only evidence for the carcinogenicity of aniline was reported by F. R. White et al. (1948). Out of forty-three rats fed 0.033% of aniline hydrochloride in a diet containing 6% casein and 0.5% cystine for 420 to 1032 days four developed hepatomas. However, no tumors were observed in twenty-one rats fed the same level of aniline hydrochloride plus 0.056 % of the hydrochloride of N, N-dimethyl-p-phenylene diamine. The authors concluded that aniline was not directly involved in carcinogenesis by DAB.
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JAMES A. MILLER AND ELIZABETH C. MILLER
The azo linkage can be replaced by an ethylene bridge, but the resulting carcinogen, 4-dimethylaminostilbene, has quite different properties. It appears to have about the same activity as DAB but it is difficult t o make a comparison from the data available. Although 4-dimethylaminostilbene is so toxic that Haddow et al. (1948) could feed only 0.5 mg. per rat per day for the first two months and 0.25 mg. per rat per day for the next four months, four of twelve rats developed cholangiomas after 330 t o 424 days. DAB is usually fed at a level of 5 to 6 mg. per rat per day, and a 30% incidence of tumors is obtained at 100 to 120 days; if the rats are then maintained on the basal diet the tumor incidence approaches 100%. Maruya (see Hartwell, 1941) obtained two liver tumors when he fed DAB at a level of 0.006% of the diet (or approximately 0.6 mg. per rat per day) for 371 days and no tumors when he fed the dye at a level of 0.002% for 400 days. Unlike the azo dyes 4-dimethylaminostilbene also induces tumors in other tissues, especially carcinomas of the acoustic duct and the mammary ducts, and in this respect is similar t o 2-acetylaminofluorene (see for example Wilson et al., 1941; E. C. Miller et al., 1949a). C . Amino Substituents. From Table I1 it is evident that no compound without at least one N-methyl group has carcinogenic activity, and the compounds in which the other nitrogen substituent was H, methyl, or ethyl all had the same high activity. The compounds in which the other nitrogen aubstituent was benzyl or j3-hydroxyethyl were noncarcinogenic while the compound with a N-formylmethyl group was only weakly carcinogenic. Thus, at least one N-methyl group is a necessary but not sufficient condition. These data are more readily interpreted in view of the metabolism of the dyes in vivo. Thus, whether DAB or MAB is fed both of these dyes and AB are found in the liver (J. A. Miller TABLE I1 The Relative Carcinogenic Activities of Certain N-Substituted Derivatives of 4-Aminoazobenzene Relative Activity
n - N = N - m -
Reference*
6 (reference compound) a-f
6
b, d
355
THE CARCINOGENIC AMINOAZO DYES
TABLE I1 (Continued)
D - N
=
N
-
o-
Relative Activity 0
Reference*
b
N
\
CHzCHaOH
N /CHa
0
1-2
N
‘CHO
N /CHzCHa
\ N
\
CHzCHs
CHaCHzOH
N /(CHz)z-4CHs
\
(CHds-rCHa
N/CHa
\H N /CH2CHa
\H
0
N /CHo
\I3 N /H
H ‘
*
\Very weak
g1
i
(a) E. C. Miller and Baumann (1946); (b) J. A. Miller and E. C. Miller (1948); (a) J. A. Miller e f o l .(1949); (d) Bugiura (1848); (e) Sugiura et al. (1945); (f) F. R. Whiteetal. (1948); (g) Kirby (18470); (h) J. A. Miller and Baumann (194613); (i) Kirby and Peacock (1847).
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JAMES A. MILLER AND ELIZABETH C. MILLER
et al., 1945). When 4-ethylmethylaminoazobenzene was fed, one or more secondary aminoazo dyes and AB were likewise present in the liver, but when 4-/3-hydroxyethylmethyl- or 4-benzylmethylaminoazobenzene was fed, very little dye other than the one ingested could be detected in this organ (J. A. Miller and E. C. Miller, 1948). These observations suggest that MAB may be a key intermediate in the carcinogenic process induced by these dyes either directly or through conversion to DAB or some other metabolite. The inactivity of AB with feeding periods of eight to eleven months (Kinosita, 1937; J. A. Miller and Baumann, 1945b; E. C. Miller and Baumann, 1946; F. R. White et al., 1948) is explainable on the same basis, since attempts to demonstrate methylated dyes in the livers of rats fed AB with or without high levels of dietary choline have been negative1 (J. A. Miller et al., 1945; E. C. Miller and Baurnann, 1946). In one experiment hepatomas were eventually induced in seven of sixteen rats fed high levels (three to six times the molar level used with DAB) of AB for thirteen to twenty-four months (Kirby, 1947b; Kirby and Peacock, 1947). However, in another experiment and with a different diet these workers obtained no tumors with this compound. Kirby (1945a) also failed to induce liver tumors in mice given subcutaneous injections of the dye. D. Ring Substituents. Large alterations in carcinogenic activity can also be made through the introduction of ring substituents into DAB, and the activities of the resulting compounds are dependent on both the nature of the group and its location (Table 111). Early studies showed that the 4’-sulfonic acid (methyl orange) (Kinosita, 1937), the 2’-carboxy (methyl red) (Kinosita, 1937), the 4’-arsenic acid (Kinosita, 1940a), and the 4’-amino (Nagao, 1941b) derivatives of DAB were inactive. Nagao (1941a) found that 2-Me-DAB was a very weak carcinogen, while 4’-Me-DAB, although weak, finally produced a high incidence of liver tumors when it was fed at high levels for long periods. When all the ring-methyl derivatives of DAB were tested under the same conditions, the 3’-methyl derivative was found to be about twice as active as DAB while the 2’-methyl and 4’-methyl derivatives were onethird to one-half and less than one-sixth, respectively, as carcinogenic as the unsubstituted dye; 2-Me-DAB and 3-Me-DAB were both inactive (J. A. Miller and Baumann, 194513; Gieae et al., 1945; J. A. Miller and 1 More recently with improved methods it has been possible to demonstrate that AB, 3‘-Me-AB and 4’-F-AB are methylated, chiefly to the N-monomethyl derivatives, to a small extent in Viuo (J. A. Miller and E. C. Miller, 1962b). The latter two compounds were found to have low but easily demonstrable carcinogenic activities in proportion to the extent to which they were methylated and formed protein-bound dye. AB was methylated to the smallest extent and again was not carcinogenic under our conditions.
357
TEE CARCINOGENIC AMINOAZO DYES
TABLE I11 The Fblative Carcinogenic Activities of Certain Ring-Substituted Derivatives of 4Dimethylaminoazobenzene
3’
2’
2
3
-
’
4 ’ a - h ’ = N a ’ - N ( C X & ) I
5
6
\GI ~~~~
~
g
\5
Relative Activities* (unsubstituteddye = 6)
Position
0 0 0 0
0 0 0 0
c1-
F-
CFs-
_ _ _ _ ~
~
~~
4’ 3’ 2’ 2 3 2’,4‘ 2’,5’ 3’,5‘ 2‘,4’,0’
N o r
CHI-
HO-
0
5 (9t) 3
1-2 5-6 2
0
0
10-12 10-12 7
> 10
0 0 0
> 10 -10
* The experimental data are recorded in J. A. Miller and Baumann (1946b),J. A. Miller and E.C. Miller (1948), J. A. Miller et 02. (1949), and J. A. Miller st 02. (1961b). t Figure corrected for poor absorption of dye from gastrointestinal tract. E. C. Miller, 1948). In conformity with the equal carcinogenic activities of DAB and MAB the 3-, 2‘-, 3’-, and 4I-methyl derivatives of MAB each had essentially the same activity as the corresponding dimethyl compounds (J. A. and E. C. Miller, 1948; Sugiura, 1948). Further studies showed that the nitro- and chloro-substituted derivatives of DAB also formed activity series of the order 3’ > 2’ > 4‘ (J. A. Miller and E. C. Miller, 1948). When methyl groups were substituted in the 3’ and 5’, 2’ and 5’, 2’ and 4’ (J. A. Miller and E. C. Miller, 1948), 2 and 4’ or 3 and 4’ (Nagao, 1941a) positions or when chloro groups were introduced into the 2’ and 5’ or 2’, 4’, and 6’ positions of DAB (J. A. Miller et al., 1949), the resulting compounds were inactive. The 3’-bromo (Kuhn and Quadbeck, 1949) and 2’,4’,6‘-tribromo (J. A. Miller et al., 1949) derivatives of DAB were also found to be noncarcinogenic; however, the tribromo derivative was only poorly absorbed from the gastrointestinal tract. Similarly, the 3‘-ethosy derivative of DAB had an activity less than one-sixth that of the parent dye (J. A. Miller and E. C. Miller, 1948). Introduction of a fluorine atom in the 2, 2‘, 3‘, or 4‘ positions of DAB resulted in compounds which were at least as active as the unsubstituted dye, and the activity series was very different from that obtained by the
358
JAMES A. MILLER AND ELIZABETH C. MILLER
introduction of a methyl, chloro or nitro group. Thus, the 2-, 3‘-, and 4’-fluoro dyes were about equally active and were approximately twice as carcinogenic as DAB, while the potency of the 2’-fluoro derivative was similar to that of the original compound (J. A. Miller et al., 1949, 1951b). 2’,4’-Difluoro- and 2’,4’,6’-trifluoro-DAB were also twice as active as the unsubstituted dye, and they are the only known carcinogenic polysubstituted derivatives of DAB (J. A. Miller et al., 1951b).2 The high carcinogenic activities of these compounds are particularly significant since they constitute evidence against the benzidine rearrangement theory of DAB carcinogenesis as suggested by Elson and Warren (1944) and Elson and Hoch-Ligeti (1946) (see Sec. V). The introduction of a trifluoromethyl group in the 2’, 3’, or 4’ positions (J. A. Miller et al., 1949) or of a hydroxy group in the 2, 2‘, 3’, or 4’ positions (J. A. Miller and E. C. Miller, 1948; J. A. Miller et al., 1949; Sugiura, 1948) has resulted in inactive compounds. The hydroxy compounds are of special interest since each could theoretically be formed in vivo from DAB. The formation of 4’-hydroxy-DAB following incubation of the dye with liver homogenates (Mueller and Miller, 1948) or liver slices (Kensler and Chu, 1950) has been demonstrated. The 4’-hydroxy derivatives of MAB and AB, both of which appear to be excreted in the urine of rats fed DAB, were also inactive (J. A. Miller and E. C. Miller, 1947, 1948). Several known and possible metabolites of DAB have been tested for carcinogenic activity, and, with the exception of MAB, all have failed to induce tumors with feeding periods of eight to twelve months. In addition to those compounds already discussed these include 4-hydroxyazobenzene, p-phenylene diamine, o-aminophenol, p-aminophenol, hydroquinone (J. A. Miller and E. C. Miller, 1948), and diacetyl-p-phenylene diamine (Sugiura et al., 1945). No tumors were obtained after feeding 2,4’-diamino-5-dimethylaminobiphenyl,the derivative obtained by the benzidine rearrangement of 4-dimethylaminohydrazobenzene in strong acid, for ten months at twice the molar level used for the azo dye (J. A. Miller and E. C. Miller, 1948; J. A. Miller et al., 1949). 3-Dimethylaminocarbazole was also found to be noncarcinogenic (J. A. Miller and E. C. Miller, 1948). This compound was tested since it has been postulated that 2,2’-aaonaphthalene, a hepatic carcinogen for mice, is converted in vivo t o 3,4,5,6-dibenzcarbazole,which is also carcinogenic for mouse liver (Boyland and Brues, 1937; Strong et al., 1938) and which has been a Since this manuscript was submitted 3’,5‘-difluoro-DAB and 2’,5’-difluoro-DAB have also been found to be stronger carcinogens than DAB (J. A. Miller and E. C. Miller, 1952a). Hence it now seems certain that none of the positions on the prime ring of DAB can be directly involved in the carcinogenic process.
THE CARCINOGENIC AMINOAZO D Y E S
359
indicted as the primary carcinogen in this case (Cook et al., 1940). 3-Dimethylaminocarbazole could bear a similar relationship to DAB. Finally, neither cirrhosis nor tumors were obtained when a mixture of the following known and possible metabolites was fed for eleven months: 0.04% of AB and 0.01% (calculated as the free base) each of 4’-hydroxyDAB, 4’-hydroxy-MAB1 4’-hydroxy-AB1 N,N-dimethyl-p-phenylene diamine dihydrochloride, N-monomethyl-p-phenylene diamine dihydrochloride, p-phenylene diamine, aniline hydrochloride, and p-aminophenol (J. A. Miller and E. C. Miller, 1948). 6. Metabolism b y the Rat
If detailed studies on the metabolism of chemical carcinogens are pursued with enough vigor, it is inevitable that at least the initial reactions in the carcinogenic process will be brought to light. Metabolic studies on such agents must be directed not only toward determining the action of the tissue on the carcinogen but also toward discovering the direct biochemical attack of the agent and its metabolites on the target tissue. As pointed out in the Introduction the importance of metabolic pathways in the reactions leading to carcinogenesis can only be assessed a t present by correlating their occurrence and intensity with the time and frequency of appearance of gross tumors in the tissue. As these correlative guideposts become established, the investigator should find it. easier to fill the gaps in our biochemical knowledge of the sequence of events which make up the carcinogenic process. A. Overall Metabolism. The metabolism of DAB in the rat is known in greater detail than that of any other carcinogen. This is largely due to the relative simplicity of the analytical problems involved, the relatively high carcinogenic dose required, and the ease with which large amounts of the susceptible tissue, the liver, can be obtained, The first conclusive study on the metabolism of this dye was that of Stevenson et al. (1942) who found that following the administration of the dye to rats isolable amounts of the N-acetyl derivatives of p-aminophenol and p-phenylene diamine were excreted in the urine. Approximately 50 to 60% of the dye can be accounted for in the urine in the form of acid-hydrolyzable conjugates of these two amines (J. A. Miller and E. C. Miller, 1947). The excretion of large quantities of these metabolites proves that extensive reduction, hydroxylation, and demethylation of the component structures of the dye occur in the rat. It is probable that all possible sequences of these three reactions occur t o some extent, but in general the demethylation reaction apparently precedes the other two (J. A. Miller et al., 1945). That most of the dye is subject t o reductive cleavage of the aao linkage at some stage in its metabolism is evident from the large
360
JAMES A. MILLER AND ELIZABETH C. MILLER
amounts of monophenyl amines excreted in the urine and the low levels of azo derivatives which can be detected in the tissues and excreted during the steady state. At least a part of the dye undergoes a fourth reaction in which it is chemically bound to the liver protein. It is this reaction which, though its exact nature is not known, appears t o be of importance in the carcinogenic reaction induced by the dye. Rats are usually fed approximately 5000 to BOO0 pg. of DAB per day to produce tumors but only 2 to 5 pg. of this dye, 1 to 5 pg. of MAB, and 6 to 10 pg. of AB are found free in the liver (J. A. Miller et al., 1945; Silverstone, 1948). About 25 t o 50 pg. of dye are found in combination with the liver protein. None of the other tissues contains the methylated or the protein-bound dyes. The rat also contains 200 to 300 pg. of AB, principally in the red blood cells, and approximately 25 pg. of this dye and the two methylated dyes are excreted in the urine and feces daily (J. A. Miller and Baumann, 1945a; J. A. Miller et al., 1945; E. C. Miller and J. A. Miller, 1947). In a study of the distribution of 3’-Me-CI4-DAB in the rat Salzberg et al. (1951) found that of the blood fractions tested, the formed elements had the greatest radioactivity; presumably this was due at least in part to the 3’-Me-AB known to be present. However, while they could not detect dye in the feces by colorimetric mcana, approximately 20% of the isotope was excreted by this route. Since it is known that AB is excreted in the bile by rats fed DAB (J. A. Miller et al., 1945), it seems likely that in the case of 3’-Me-C14-DAB bacterial destruction of the 3’-Me-AB in the tract leads to the excretion of nondye fragments containing CI4in the feces. The known and several possible metabolic pathways of DAB and its metabolites in the rat are shown in Fig. 2. B. Reduction of the Azo Linkage. The reductive cleavage of the azo linkage at some stage in the metabolism of DAB was first conclusively demonstrated by Stevenson et al. (1942), who isolated N-acetyl-paminophenol and N,N’-diacetyl-p-phenylene diamine from the urine of rats fed and injected with relatively large quantities of the dye. Earlier Kinosita (1940a) had reported that the carcinogen was reduced in vivo to N,N-dimethyl-p-phenylene diamine and aniline and that these compounds were excreted in the urine, but no experimental data were presented. Stevenson et aE. (1942) were unable to isolate these compounds unless sodium hydrosulfite was added to the urine, and under these conditions the amines may have been formed through the reduction of small amounts of dye in the urine. Using a sensitive analytical method (E. C. Miller et aZ., 194913) it was found that approximately 50% of the ingested DAB was excreted in the urine as acid-hydrolyzable conjugated forms of p-phenylene diamine and p-aminophenol (J. A. Miller and
-
-
Liver protein-Am dye compounds Liver proteins
=Reaction for which evidence exists --+ =Hypothetical reactioii
-t
Metabolicderivatives
I!
-
H
HCHO
demethylatedproducts
H H \
H H
H
- \N/0\N/ - \N/0\N/ 1 - 1
Monophenyl amines
f
H
H
------
1 - 1
H
--------I
FIG.2. The present knowledge of the metabolism of DAB and its derivatives in the rat.
4
CH,
362
JAMES A. MILLER AND ELIZABETH C. MILLER
E. C. Miller, 1947). Small amounts of conjugated forms of N-methylp-phenylene diamine, aniline, and o-aminophenol were also excreted, but only traces of N,N-dimethyl-p-phenylene diamine could be detected. Approximately the same quantities of these monophenyl amines were also excreted in the urine when MAB or AB was fed, except that no methylated diamines could be detected in the urine of rats fed the latter dye. Following the ingestion of any of these diamines the chief excretory product was a conjugate of p-phenylene diamine, but appreciable amounts of conjugates of the N-methyl- or N,N-dimethyl-p-phenylene diamine were also excreted when these compounds were fed. In each case the overall recovery was approximately 70 %. After the administration of aniline large amounts of conjugated p-aminophenol and smaller quantities of conjugated o-aminophenol and aniline were found in the urine. When either p-aminophenol or o-aminophenol was fed, the only metabolite identified was a conjugate of the isomer fed. The preponderance of AB rather than the methylated dyes in vivo and the much smaller amount of N,N-dimethyl-p-phenylene diamine excreted by rats fed DAB than by those fed the diamine itself suggest that in vivo the carcinogen may be largely demethylated to AB prior to reduction of the azo linkage. However, in vitro DAB is reduced by rat liver slices (Kensler, 1947,1948; Kensler and Chu, 1950) or, more rapidly, by rat liver homogenates (Mueller and Miller, 1948, 1949, 1950), and in the latter case the dye destroyed can be stoichiometrically accounted for as N,N-dimethyl-p-phenylene diamine and aniline. Maximum reduction of the dye by liver homogenates was obtained only when the system was anaerobic and fortified with triphosphopyridine nucleotide, diphosphopyridine nucleotide, magnesium ions, and an oxidizable substrate. A requirement for riboflavin-adenine dinucleotide could be demonstrated following carbon dioxide treatment of the homogenate ; riboflavin monophosphate was inactive in this system. A riboflavin coenzyme was also implicated in the liver slice system since the ability of the slices to destroy the dye could be correlated with their riboflavin content (Kensler, 1947, 1948, 1949). As the amines formed by reductive cleavage of the dye have so far exhibited little or no carcinogenic activity (Kinosita, 1940a; Sugiura et al., 1945; J. A. Miller and Baumann, 1945b; F. R. White et al., 1948; Druckrey, 1950a,b), at least a part of the protective action of dietary riboflavin against carcinogenesis by DAB probably results from its participation in the cleavage of the carcinogen to relatively inactive products. With either the slice or homogenate technics kidney was about one-third as active as liver and the other tissues studied, including liver tumors induced by the dye, were essentially inactive. Both slices and homogenates of liver similarly reduced MAB, AB, certain N-sub-
T H E CARCINOGENIC AMINOAZO DYE8
363
stituted derivatives of AB, and the ring-methyl derivatives of DAB.
In general DAB was reduced more rapidly than the other azo dyes studied, and there was no correlation between the carcinogenicity of a dye and its ease of destruction in vitro by liver preparations. Although reduction of the dyes implies that hydraao derivatives are formed as intermediates, there is no evidence that these compounds undergo a ((benzidine” rearrangement in vivo (see Sec. V). C . Hydroxylation of the “Aniline” Ring. The occurrence of large quantities of conjugated p-aminophenol and smaller quantities of conjugated o-aminophenol in the urine following the ingestion of DAB indicates that a high percentage of the ingested dye is hydroxylated at one or more stages in its metabolism. That hydroxylation can follow cleavage was shown by the finding of large quantities of these amines in the urine following the administration of aniline (J. A. Miller and E. C. Miller, 1947). However, the excretion of small quantities of the 2’- and 4’-hydroxy derivatives of MAB and AB in the urine of rats fed either DAB or MAB and the finding of small quantities of 4’-hydroxy derivatives following the incubation of DAB or certain of its ring-methyl derivatives with liver slices (Kensler and Chu, 1950) or homogenates (Mueller and Miller, 1948) demonstrate that some hydroxylation does occur prior to reduction. D. N-Demethylation. The isolation of N,N‘-diacetyl-p-phenylene diamine by Stevenson et al. (1942) also proved that the N-methyl groups were removed at some stage in the metabolism of DAB. This reaction can take place subsequent to reductive cleavage, since conjugated forms of N-methyl-p-phenylene diamine and p-phenylene diamine were found in the urine of rats fed N,N-dimethyl-p-phenylene diamine (J. A. Miller and E. C. Miller, 1947). However, the presence of small quantities of MAB and larger amounts of AB in the tissues (J. A. Miller et al., 1945) and of low levels of hydroxy derivatives of AB and its monomethyl derivative in the urine of rats fed DAB indicates that some demethylation must precede reductive cleavage. In fact, it appears likely that this may be the reaction sequence undergone by a major share of the dye, since the same levels of AB were found in the blood and tissues of rats fed either A% or its N-methyl derivatives (J. A. Miller et al., 1945). This logic requires the assumption that the amount of the primary aminoazo dye in the blood is an index of the amount of this substance being formed from the methylated dyes. This appears to be the case since the amount of AB in the blood is essentially proportional t o the amount of dye ingested (J. A. Miller el al., 1946). The demethylation of DAB to MAB is a reversible process since the same amounts of both dyes are found in the livers of rats fed either compound. The loss of the
JAMES A. MILLER AND ELIZABETH C. MILLER
364
second methyl group appears to be essentially irreversible; neither methylated dye has been detected in the livers of rats fed AB (J. A. Miller el al., 1945; E. C. Miller and Baumann, 1946).* N-Demethylation of the ring-methyl derivatives of DAB also occurs in a similar fashion; in each case primary, secondary, and tertiary aminoazo dyes were found in the liver while only the primary aminoazo dye could be detected in the blood (J. A. Miller and Baumann, 1945b). Dealkylation also occurs when azo dyes bearing other N-substituents are fed (Kensler et al., 1947; J. A. Miller and E. C. Miller, 1948). N-Ethyl groups are removed with relative ease, and the levels of AB in the blood and liver are essentially the same whether DAB, ethylmethyl-AB, or diethyl-AB is fed. On the other hand, 8-hydroxyethyl and benzyl groups are removed with difficulty, and only traces of AB can be detected in the blood of rats fed 8-hydroxyethylmethyl-AB, benzylmethyl-AB, or di-8-hydroxyethyl-AB. The fate in vivo of the N-methyl groups of DAB and certain of its derivatives has been demonstrated by the use of C14-labeled dyes. In the first study of this type Boissonnas et al. (1949) fed 4-dimethyl-C14aminoaaobenzene to an immature female rat and found about 50 % of the isotope in the respired air as C140a. They could not detect any significant quantity of C14 in the body choline or in the urinary creatinine obtained from this rat and concluded that transmethylation of the methyl groups of DAB did not occur under the conditions of their experiment. Somewhat different results were obtained by E. C. Miller et al. (1952b) and MacDonald el al. (1952b) employing similarly labeled dyes in its 3’-methyl adult rats of both sexes. 4-Dimethyl-C14-aminoazobenzene, and 4’-methyl derivatives, and 3-methyl-4-monomethyl-C14-aminoazobenzene were administered by stomach tube in oil solution in single or multiple doses for periods of five hours to twenty-eight days. In agreement with the data of Boissonnas et al. 50 to 70% of the label from each dye was respired as C1*OZwithin forty-eight hours. However, in each case 20 to 40% of the isotope remaining in the body (about 2 to 4% of the administered radioactivity) could be accounted for in the N-methyl groups of the body and liver choline and in the p-carbon of the serine in the body and liver proteins. This distribution of isotopt#is similar to that obtained after the administration of C14-labeled formaldehyde or formate (Sakami, 1948; Siekevitz and Greenberg, 1949; Plaut et al., 1950; Siege1 and LaFaye, 1950). Hence, although some transmethylation in these experiments cannot be excluded, it appears likely that the activity in the choline methyls is due to the formation of these one-carbon compounds from the N-methyl groups of the dye. In the case of the a
See footnote 1 on page 366.
T H E CARCINOQENIC AMINOAZO DYES
365
3'-methyl derivative it was noted that severe deficiencies of folic acid or vitamin Blz reduced but did not abolish the incorporation of the isotope from the dye into the choline and serine. In an earlier study Jacobi and Baumann (1942) reported that the feeding of DAB prevented or lessened the severity of kidney hemorrhages in rats fed a diet deficient in labile methyl groups. Neither AB nor AAT, which lack the N-methyl groups, protected the rats. That these results can be explained by the utilization of the methyl groups from DAB now appears unlikely in view of the relatively low order of incorporation of C14 from the N-methyl groups of the dye into the methyl groups of choline. Further information on the fate of the N-methyl groups of these dyes has been obtained from the metabolism of 3-methyl-MAB in liver homogenates (Mueller and Miller, 1951). This dye was chosen since in vitro demethylation of the secondary aminoazo dyes occurs much more rapidly than with the tertiary aminoazo dyes. Further, the 3-methyl group almost completely hinders the reductive cleavage of the azo group, so that the demethylation can be studied independently of other reactions. It was found that oxygen, triphosphopyridine nucleotide, diphosphopyridine nucleotide, adenosine triphosphate, and magnesium ions were required for optimum demethylation activity. When the reaction was carried out in the presence of semicarbazide, formaldehyde and 3-methyl-AB were recovered in amounts accounting stoichiometrically for the 3-Me-MAB metabolized. Hence it appears very likely that the reaction proceeds through a monomethylol derivative. This result together with the observed fate of these methyl groups in vivo indicates that a methylol derivative is probably an intermediate in the N-demethylation of DAB and its derivatives. Of course, it is entirely possible that in vivo some of the methylol derivative is further metabolized to the N-formyl or even to the N-carboxyl derivative and that these in turn yield formic acid and carbon dioxide, respectively. These interrelationships are shown in Fig. 3. In recent studies mouse liver homogenates have been found to contain two t o three times as much of the enzyme system responsible for the N-demethylation of these dyes as rat liver homogenates. Furthermore, the activity of mouse liver homogenates can be increased about 250% and of rat liver homogenates about 50% by feeding the animals diets containing certain meat products such as tryptone, liver powder, or oxidized cholesterol for at least one week before they are killed. The active factor is not identical with any of the known vitamins or accessory growth factors, and studies still in progress have shown that a number of organic peroxides (such as ascsridole or the photoxide of 9,lO-dimethyl-
366
JAMES A. MILLER AND ELIZABETH C. MILLER
1,2-benzanthracene) produce the same effect when incorporated in the diet at a level of about 0.05% (J. A. Miller et al., 1951a; Brown et al., 1952). E. The Formation of Protein-Bound Dyes. Several years ago we noticed that the liver protein from rats fed DAB turned pink when suspended in acid solution and light yellow when suspended in alkaline or neutral solvents. Like many other aminoazo dyes DAB is a sensitive acid-base indicator, and this observation showed the presence of dye associated with the liver protein. Subsequent studies have shown that the dye is bound through chemical linkages to certain of the liver proteins, and we have suggested that this reaction may play a role in car-
-N"H3
!
I
I
[
] = nd demonstrated
FIQ.3. The observed and possible interrelationships in the oxidative demethylation of the N-methylated aminoaro dyes by the rat.
cinogenesis by the dye (E. C. Miller and J. A. Miller, 1947; E. C. Miller et al., 1949b). Many of the initial observations have since been confirmed by Taki and Miyaji (1950). The protein-bound dyes appear in the liver shortly after feeding of DAB is begun and continue t o accumulate until a maximum level is reached at about one month. Thereafter, even though the dye-feeding is continued, the level of bound dye drops slowly and by four months is only about half-maximal. There are a number of reasons for believing that these dyes are chemically bound to the liver protein. Thus, they cannot be released by prolonged extraction with boiling organic solvents, by extraction with hot trichloroacetic acid, or by dialysis. The dyes are released upon destruction of the protein by t r y p i n or alkali; acid hydrolysis has not been used since aminoazo dyes are not stable in hot mid for prolonged periods. While the liver protein containing the bound dyes dissolves quickly in hot strong alcoholic alkali, the bound dyes are extractable only at a rate parallel with the progressive hydrolysis of the protein. This is also true of tryptic hydrolysis of these preparations. Following alkaline hydrolysis, about 10% of the liberated dye can be shown to be a mixture of MAB and AB. The major share of the dye, however, has
367
THE CARCINOGENIC AMINOAZO DYES
strongly polar properties since it can only be extracted from the alkaline hydrolyaate with a highly polar solvent mixture such as ethyl etherethanol. The bound dyes do not appear to be bound to the nucleic acids present in the liver protein preparations. Following the liberation of all the detectable nucleic acids by heating the protein preparations in 5% trichloroacetic acid for fifteen minutes (Schneider, 1945a), all the dye is still found combined with the residual protein precipitate. Whether or not the phosphorus remaining in such precipitates (as phosphoprotein or residual nucleic acid?) is involved in the binding is not known. In any case, of course, the finding that the dyes are not bound to the major share of the nucleic acids does not exclude them from being bound to the protein moieties of nucleoproteins. The released polar dye from the dye-protein compound has a spectrum in acid which is characteristic of N-disubstituted aminoazo dyes (J. A. Miller et al., 1948). While the nature of the polar group of this dye is obscure there are some clues as t o where and how the binding occurs on the dye molecule. On reduction the polar dye yields aniline and an unidentified polar amine (E. C. Miller et al., 1949b). This indicates that the dye is bound t o the protein either through some substituent on the ring bearing the -N(CHa)Z group or through a derivative of this group. It is felt that the latter possibility is the most likely one. Thus, it has been found that only N-methylated aminoazo dyes become bound to any large extent in vivo. An N-ethyl group permits only a slight amount of binding. This is also the case with the completely demethylated metabolite, 4-aminoa~obenzene.~Two dyes are known which bear N-methyl groups but which are not bound to an appreciable extent. These dyes also bear other groups (N-benayl or N-/3-hydroxyethyl) which have been found to hinder the demethylation of these dyes in the body (J. A. Miller and E. C. Miller, 1948, 1952a). It is the authors’ tentative conclusion that a N-hydroxymethyl derivative of the dye, which is presumably formed during the oxidative removal of the N-methyl groups, is the metabolite which becomes bound to the protein. N-Hydroxymethyl groups are known to be highly reactive and form stable bonds with compounds which possess reactive hydrogens through the elimination of the elements of water (Fraenkel-Conrat and Olcott, 1948). If such Mannich bases were formed with a protein grouping such as in tyrosine or histidine
I I
which has a reactive -CH,
the alkali stable
>
N-CH2-C-
would be formed. On the other hand reactive 4
See footnote on page 356.
I
I
grouping
NH groups in the
368
>
JAMES A, MILLER AND ELIZABETH C. MILLER
protein would yield the alkali labile
N-CHz-N/
\ grouping. The
small amounts of MAB and AB released in the alkaline hydrolysis of the protein may be derived from dyes bound through the latter linkage, i.e., the N-hydroxymethyl derivatives of DAB and MAB, respectively. However, the bound MAB and AB could also be attached through amide linkages. On this hypothesis the polar dyes would have been attached to the protein through the alkali-stable grouping,
>
N-CH2-C--,
I I
and,
following alkaline hydrolysis, would still be combined with an amino acid residue (Fig. 4).
0-y o e v r p i A + ~ < - 1c -+
' - 1 t e i n
KN
tyr-
' N o r dye'
protein
Wmbm wh
altUl In .mly*Is
w; ~ o a c " ~ N r343
"nm-pdor dye"
FIQ.4. A possible mechanism for the formation of protein-bound dye from DAB.
The protein-bound dyes may be conveniently estimated by hydrolyzAfter ing the liver proteins in alcoholic KOH for twenty hours at 80'. extraction with ethanol-ethyl ether the solvent is removed and the residue dissolved in alcoholic HC1. The optical density of the pink solution is obtained and the concentration is expressed as E per unit weight. Conversion factors based on the reduction of the released dyes to give known amines can be used to calculate the molar quantity of protein-bound dye present. Salzberg et al. (1951) found with 3'-MeC"-DAB that the amount of radioactivity in the extract of released bound dye agreed with the levels of dye as determined by the colorimetric methods. However, additional radioactivity was found in the residual alkaline solution after removal of the liberated dye. This component was not detectable by the colorimetric methods used for the dye, and it amounted to almost 50% of the C1*activity found in the extractable fraction. However, at least part of this component may consist of unreleased dye since the foregoing conditions of analysis only liberate 80% of the dye found after ninety-two hours of hydrolysis. After the latter time marked destruction of dye occurs. Hence the present
THE CARCINOGENIC AMINOAZO DYES
369
colorimetric method probably determines somewhat less than 80% ’ of the dye present in the protein. Thus in the case of Salzberg et al. some radioactive dye would be expected to be present in the alkaline phase. It is difficdlt to detect this dye by acidifying the alkaline phase since interfering pigments from the protein hydrolysis are present in addition t o the large volumes and quantities of salts produced upon acidification. The rate of disappearance of the protein-bound dyes in vivo has been determined following the removal of the dye from the diet. The half-life of the combination has been found to be about four days. This is approximately the same order of magnitude as the average half-life of three to seven days observed for the liver proteins of the normal rat (Tarver, 1951). It is of further interest that unpublished observations from this laboratory indicate that the protein-bound dyes are not attacked by the liver enzyme system which rapidly reduces the free dyes. Hence it appears likely that the destruction of protein-bound dye i n vivo consists largely of the breakdown of the protein moiety. The continual formation of protein-bound dye and its subsequent destruction could involve the turnover of a substantial quantity of protein in the liver during the several months required for the formation of tumors. Although quantitatively less important than other reactions involving the dye, the binding of derivatives of DAB to the liver proteins may be of major importance in the carcinogenic process induced by this dye since a number of correlations can be made between the levels of bound dye found under various conditions and the probability of tumor formation (E. C. Miller and J. A. Miller, 1947; E. C. Miller et al., 194913). Since the dye is highly specific for the liver of the rat, it is of interest that the protein-bound dyes have been found only in the liver and, to a small extent, in the blood plasma of rats fed DAB. Likewise the high species specificity of the dye permits another correlation to be made. No protein-bound dye could be detected in the livers of guinea pigs, hamsters, rabbits, cotton rats, chipmunks, or chickens fed the dye. No one has succeeded in producing liver tumors in these species by feeding DAB. Mice are much less susceptible than rats to the carcinogenic action of DAB, and only low levels of protein-bound dye are found in the livers of mice fed the dye. Two dietary conditions are known which lower the level of proteinbound dye in the rat liver and which also reduce the carcinogenicity of the dye. These are the addition of certain polycyclic hydrocarbons (Fig. 5 ) or extra riboflavin to the diet (E. C. Miller and J. A. Miller, 1947; E. C. Miller et aZ., 1952a). Since merely lowering the dye intake of the rat will also produce these results (E. C. Miller et al., 1949b), such effects must be interpreted with caution. In the case of riboflavin
370
JAMES A. MILLER AND ELIZABETH C. MILLER
it is known that this vitamin functions in an enzyme system which reduces the dye to inactive amines (Mueller and Miller, 1950), and it seems probable that the hydrocarbons also increase the rate of metabolism of the dye so that less dye is available to initiate the carcinogenic process. A further correlation is the requirement of a N-methyl group for the formation of high levels of protein-bound dye as well as for high carcinogenic activity toward the rat liver. Thus, as discussed earlier, those compounds which either lack a N-methyl group or which have a N-substituent hindering the metabolism of the N-methyl group are either noncarcinogenic or only weakly carcinogenic and are converted to protein,035,
T u r n at /\054%
'
3'-Me-DAB
10/[4 months
3 ,015
n .
P
l 3'-Me-DAB O + r .0033 MC
O
x
0/15
,005 5
10
15
20
25
30
35
DAYS
FIG.5. The effect of 20-methylcholanthrene (MC) in the diet on the formation of protein-bound dye and liver tumors by 3'-Me-DAB.
bound forms to only a small extent. Similarly the noncarcinogenic 4'-hydroxyl derivatives of DAB, MAB, and AB are not bound to the liver protein. Another correlation between carcinogenic activity and protein-bound dye formation was found on analysis of the livers from rats fed the ring-methyl derivatives of DAB. These isomers vary in carcinogenic activity from 0 to 12, and there is an approximate inverse correlation between the times of dye-feeding required before the maximum levels of bound dye are found in the liver and the carcinogenicities of these derivatives (E. C. Miller et al., 1949b). One of the most intriguing observations is that the protein-bound dyes are not detectable in the tumors induced by DAB and its derivatives even when the dyes are fed continuously (E. C. Miller and J. A. Miller, 1947; Price et al., 1949~). This difference between liver and tumor appears to be due to a difference in the intrinsic properties of these tissues, although its interpretation is complicated by the finding that
THE CARCINOGENIC AMINOAZO DYES
371
liver tumors are supplied only with arterial blood (Breedis and Young, 1949) while the liver receives 60 to 80% of its blood from the portal system. Thus, if the dye is absorbed into the portal system, the amount of dye reaching the liver would probably be much greater than that reaching the tumor. Actually, however, the main route by which ingested dye reaches the liver is not known since the arterial and portal blood in dye-fed rats do not contain detectable amounts of DAB (J. A. Miller et al., 1945) and estimations of this dye in the lymph have not been made. This circulatory problem has been circumvented to a large extent in an experiment (J. A. Miller and E. C. Miller, 1952a) in which rats bearing small liver tumors induced by DAB were fed a dye-free diet for two weeks so that no protein-bound dye remained in the liver. These rats were then given daily subcutaneous injections of an oil solution of DAB for a two-week period. The dye absorbed from the injection site would reach the heart via the vena cava (without passing through the liver) and then pass into the arterial circulation with branches to both the liver and the tumor. Under these conditions appreciable levels of protein-bound dye were found in the liver tissue but again none could be detected in the tumors. Further evidence has come from studies on the electrophoretic properties of the soluble proteins in the livers and liver tumors from rats fed DAB. Each of the morphological fractions of the liver from rats fed dye contain some protein-bound dye, but the major share is found in the soluble proteins (Price et al., 1948, 194913, 1950). On further fractionation of the soluble proteins Sorof et al. (1951) found that 70 to 90% of the bound dye was present in slow-moving proteins which comprised only 7 t o 15% of the total soluble proteins. Of considerable interest is the further finding of Sorof and Cohen (1951) that the liver tumors contained only greatly reduced amounts of this particular protein fraction and that other unrelated tumors exhibited a similar deficiency. The authors feel that the foregoing facts on the protein-bound dyes offer support for the hypothesis that the dyes induce neoplastic changes through the gradual deletion of key proteins essential for the control of growth (see Sec. V). 6 . Alterations in Chemical Composition Following Ingestion of the Dyes
As one approach to determining the changes associated with the induction of liver tumors a number of investigators have compared the composition of normal liver with that of liver from rats treated with a carcinogen and/or the resulting liver tumors. Regardless of the carcinogen employed such studies are complicated by the lack of a uniform histological picture in either the precancerous liver or in many of the
372
JAMES A. MILLER AND ELIZABETH C. MILLER
induced tumors. Some investigators have used cytochemical techniques in which the content of a given constituent in individual cells or small groups of cells can be determined, and these studies are of particular value in determining the distribution of the constituent among various classes of cells. From the standpoint of studying the early changes associated with the carcinogenic reaction, however, they have the disadvantage of depending on the investigator’s judgment as to which cells are undergoing changes associated with potential malignancy and which are undergoing alterations not related t o malignancy. Other investigators have analyzed either the whole tissue or the morphological fractions prepbred from it by differential centrifugation. These studies give better averages of the overall changes than cytochemical analyses, but give no information concerning the uniformity of composition of the various cells of the liver or tumor. Furthermore, significant alterations occurring in only a small percentage of the cells may be masked by large numbers of unaltered cells or by changes of the opposite type in another group of cells. The difficulties in the latter type of study can be partially offset by adequate histological study, but for the best histochemical picture of carcinogenesis both types of investigations appear desirable. Several Japanese workers (Nakahara et al., 1936; Fujiwara et al., 1937; Kishi et al., 1937; Masayama et al., 1938) have made biochemical studies on the gross composition of normal liver, liver undergoing carcinogenesis, and transplantable and primary liver tumors induced by AAT or DAB. Most of these differences and changes are too numerous and unrelated to list here. Unfortunately, only abstracts of these articles were generally available to the present authors. In another approach to the general problem outlined above Price et al. (1948, 1949b,c, 1950) compared the intracellulm composition of normal liver and of liver tumors induced by DAB with the composition of liver from rats fed DAB, its ring methyl derivatives, or 4’-F-DAB for approximately one month. The ingestion of the carcinogenic dyes resulted in large alterations from normal in the contents of nucleic acids, protein, and riboflavin in certain of the fractions, while little change was usually found when the noncarcinogenic dyes were fed, and, in some cases, the extent of change was roughly proportional to the carcinogenicity of the dye fed. These alterations in intracellular composition were even more exaggerated in the hepatic tumors induced by DAB. Figures 6 t o 9 compare the results obtained for the livers from rats fed the basal diet, AB, DAB, or one of the C-methyl dyes with the composition of the tumors induced by DAB. Thus, ingestion of the active carcinogen DAB produced a small increase in the desoxypentosenucleic acid content of the nuclear fraction
THE CARCINOQENIC AMINOAZO DYES
373
(Fig. S), while the 4’-fluoro and 3’-methyl derivatives, which are twice as active, caused 24 % and 100% increases in the amount of desoxypentosenucleic acid. The 3’-methyl derivative also caused a doubling of the amount of nuclear protein. The levels of both desoxypentosenucleic acid and protein in the nuclear fraction of the tumor were even higher than those found in the livers of rats fed 3‘-Me-DAB for just one month. These high levels of desoxypentosenucleicacid in tumors induced by DAB NUCLEAR FRACTION
5.0
9 a
OESOXYPLNTOSENUGLL I0 AG I D
40
4.0
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2 c
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$ I-
n t
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Silo- * U o MA0 DA0
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*-YO- Tumor DAB
FIG. 6. Comparison of the levels of desoxypentosenucleic acid and protein in the nuclear fractions from the livers of rats fed various azo dyes and in tumors induced by DAB. The number above each bar indicates the relative carcinogenic activity of the dye fed.
had been observed earlier by Masayama and Yokoyama (1940), Davidson and Waymouth (1944), and Schneider (1945b). However, as shown by Price et al. (1950), Cunningham et al. (1950b), and Mark and Ris (1949), the desoxypentosenucleic acid content per nucleus was the same for normal liver, precancerous liver, and induced liver tumors so that the increased level of this nucleic acid in these tissues resulted from greatly increased nuclear concentrations rather than from changes in the amount per cell. I n general, when the noncarcinogenic or weakly carcinogenic dyes were fed, the riboflavin and protein contents of the large granules were as high as those found in the livers of rats fed the basal diet (Fig. 7). Ingestion of the more carcinogenic of the C-methyl series of dyes appreciably reduced the amounts of protein and riboflavin in the large granule fraction, and in the case of 3’-Me-DAB the levels were almost as low as
374
JAMES A. MILLER AND ELIZABETH C. MILLER LARGE GRANULES Rl8OFLAVIN
1.0
0 PROTEIN
T .O
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*-Ma- Tumor DAB
FIG. 7. Comparison of the levels of riboflavin and protein in the large granule fractions from the livers of rats fed various azo dyes and in tumors induced by DAB.
= 0
LARGE GRANULES
AB
*MaDAB
mr MA0
*-Mc DAB
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PENTOSENUCLEIC ACID PROTEIN
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FIG.8. Comparison of the levels of pentosenucleic acid and protein in the large granule fractions from the livers of rats fed various azo dyes and in tumors induced by DAB.
THE CARCINOGENIC AMINOAZO DYES
375
those found in the liver tumors. However, the strong carcinogen 4’-F-DAB decreased the protein and riboflavin contents of the large granules only slightly, and not in proportion to its carcinogenic activity when compared with the C-methyl dyes. All the aminoazo dyes that were studied caused some reduction in the pentosenucleic acid content of the large granules, but in general the largest reductions occurred when the most carcinogenic dyes were fed (Fig. 8); in the case of the livers from rats fed 3’-Me-DAB the content was very similar to that of tumor SUPERNATANT FLUID 0
1)WOI
AB
PENTOSENUCLEIC ACID PROTEIN
2-Y.DAB
*MIMA8
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3’%c Tumor DAB
FIG.9. Comparison of the levels of pentosenucleic acid and protein in the supernatant fluid fractions from the livers of rats fed various azo dyes and in tumors induced by DAB.
tissue. From histological study it appeared most likely that the changes in the levels of protein, riboflavin, and pentosenucleic acid in the large granule fraction were largely the result of changes in the numbers of the granules per parenchymal liver cell rather than of gross changes in the qualitative composition of these particles. However, ingestion of the noncarcinogenic dye 2-Me-DAB apparently caused both an increased number of large granules in the parenchymal liver cells and an altered composition as reflected by the pentosenucleic acid to protein ratio. The protein contents of the supernatant fluid fraction from the livers of rats fed the basal or dye-containing diets or from liver tumors were very similar (Fig. 9). Similarly, the pentosenucleic acid content of this fraction was unchanged by the ingestion of any of the dyes except 3’-Me-DAB. The amount of this nucleic acid in the supernatant frac-
376
JAMES A. MILLBR AND ELIZABETH C. MILLER
tion from tumor was twice that found in normal liver, with a resultant increase in the ratio of nucleic acid to protein. A similar increase in the nucleic acid to protein ratio of the cytoplasm of tumor cells induced by DAB as compared to normal cells was found by Stowell (1949) who determined the light absorption of individual cells at 257 and 275 mp. No significant alteration from normal was found in the cytoplasm of nonneoplastic cells from tumor-bearing livers. He also observed that the nucleic acid to protein ratio in the nucleoli was higher in tumor cells than in normal liver cells. Opie (1946) and Opie and Lavin (1946) noted that ingestion of DAB caused degenerative changes in the liver which were accompanied by a chromatolysis of cytoplasmic structures and a loss of pentosenucleic acid. This stage was succeeded by a focal regeneration which was characterized by a reaccumulation of pentosenucleic acid and which appeared to be involved in the neoplastic changes. The composition of the livers from mice which had ingested DAB showed some of the same variations from normal as were observed in the livers from dye-fed rats, while the composition of hamster liver was unaffected by the ingestion of the dye (Price et al., 1951). As noted earlier mice develop tumors only slowly, while hamsters are resistant to the carcinogenic action of DAB. I n another study Price and Laird (1950) compared the intracellular composition of normal liver and DAB-induced liver tumors with regenerating liver one to twenty-three days after partial hepatectomy. Nuclear counts were made and all the results were calculated in terms of the amount per cell. Each type of liver cell had its own characteristic composition, and the intracellular compositions of tumor and regenerating liver were not similar enough to suggest a common pattern for growth. In a study of the sequence of changes following the feeding of 3'-MeDAB Griffin et al. (1948) and Cunningham et al. (1950a,b) found the hepatic level of desoxypentosenucleic acid to be 5001, above normal after only two weeks of dye-feeding and to be twice the normal level by eight weeks. On a fresh weight basis liver tumors induced by this dye contained three times as much desoxypentosenucleic acid as normal liver. Furthermore, radioactive phosphorus was incorporated to a much greater extent into the desoxypentose nucleic acid in the livers of dye-fed rats or in the induced tumors than into the desoxypentosenucleic acid of normal rat liver (Griffin et al., 1951). There was a significant increase in the globulin content of the liver following ingestion of the dye, and as seen earlier by other investigators (Kensler et at., 1941; E. C. Miller et al., 1948; Price et al., 1949b, 1950) there was a progressive decrease in total hepatic riboflavin and of the pentosenucleic acid content of the large
THE CARCINOGENIC AMINOAZO DYES
377
granule fraction following dye-feeding. This group of investigators (Cook et al., 1949) also found a threefold increase in the serum yglobulin level and a 15 to 20% decrease in the serum albumin level when rats were fed 3I-Me-DAB for two to eight weeks. Similar observations on the globulin level in rats fed DAB were made by Hoch-Ligeti et al. (1949) ; however, these workers observed no change in the albumin level and noted that the double peaks of albumin in normal rat serum changed to a single peak during gross tumor development. Homogenates of the livers from rats fed carcinogenic azo dyes also show a resistance to heat coagulation (Griffin and Baumann, 1948a). The livers from rats fed DAB or 3’-Me-DAB and the tumors induced by these dyes have essentially the same overall amino acid composition as normal liver (Schweigert et al., 1949; Sauberlich and Baumann, 1951). However, the proteins from each of the morphological fractions of the liver tumors contained 25 to 41% less methionine and 15 to 44% more cystine than the proteins from the same fractions of normal liver. In general tumors have been found to contain only low levels of most vitamins, and in the case of the tumors induced by DAB the levels of riboflavin, vitamin Be, biotin, pantothenic acid, and nicotinic acid are about one-fourth to one-tenth of those in normal liver (Kensler et al., 1941; Pollack et al., 1942a,b; Taylor et al., 1942a,b; West and Woglom, 1942; E. C. Miller et al., 1948; Price et al., 1949a,c; Higgins et al., 1950). Ingestion of DAB for periods short of tumor formation also results in marked decreases in the hepatic levels of riboflavin, vitamin Be, and biotin (Kensler et al., 1940; E. C. Miller et al., 1948). The level of riboflavin in the liver also decreases when other dyes are fed, and the extent of the loss is roughly proportional to the carcinogenicity of the dye fed (E. C. Miller et al., 1948; Griffin and Baumann, 1946, 194813; Griffin et al., 1948). Some studies have also been made on the enzymatic composition of liver which is undergoing carcinogenesis. Thus Roskelly et al. (1943) and Hoch-Ligeti (1947) showed that slices from the livers of rats fed DAB underwent a progressive loss of succinoxidase activity as the feeding time increased and that liver tumors induced by the dye contained even less of the enzyme system. Similar results were obtained by Potter et al. (1950) and by Viollier (1950b) with tissue homogenates. Potter et al. (1950) also analyzed the livers of rats fed other azo dyes and found that the succinoxidase activity depended on the dye fed and could be correlated with the amount of large granule protein in the liver. On the other hand the amount of oxalacetic acid oxidase, which is low in liver tumors, was not appreciably lower in preneoplastic than in control livers.
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JAMES A. MILLER AND ELIZABETH C. MILLER
Gallico and Boretti (1948) found decreased levels of both succinic and malic dehydrogenases in the livers of rats fed AAT. The hepatic levels of glyoxalase (Cohen, 1945), lipase (Mark, 1950), xanthine oxidase (Westerfeld et al., 1950), arginase and histidase (Viollier, 1950a), choline oxidase (Viollier, 1950b), tributyrinase (Viollier and Waser, 1950), and the enzymes involved in the synthesis of citrulline and p-aminohippuric acid (Tung and Cohen, 1950) also decrease following the ingestion of DAB or 3’-Me-DAB. The levels of most of these enzyme systems are even lower in the induced liver tumors. On the other hand the acetylcholinesterase activity of liver and plasma (Viollier and Waser, 1950; Langemann and Kensler, 1951) and the alkaline phosphatase activity of liver (Woodard, 1943; Mellors and Sugiura, 1948; Pearson et al., 1950) increase following the ingestion of DAB or 3’-Me-DAB, and the levels of these enzymes are even higher in the tumors induced by these dyes. Zamecnik and his associates (1948, 1951) suggested that tumors induced by DAB had a faster rate of protein synthesis than normal rat liver, since the protein from tumor slices incubated with C14-labeled alanine contained approximately six times as much radioactivity as slices from normal liver incubated under the same conditions. The opposite conclusion might be inferred from studies on the in vivo incorporation of C14-labeled alanine or glycine into liver and tumor proteins (Zamecnik and Frantz, 1949; Griffin et al., 1950). Under these conditions the uptake of radioactivity into the tumor protein is slower than in the case of liver protein, but the maximum specific activities attained in each tissue are approximately equal. However, the latter studies were probably complicated by the differences in the blood supply of liver and liver tumors (Breedis and Young, 1949; Zamecnik et al., 1951), while the in vitro studies were complicated by differences in the amounts of endogenous substrates present in the slices from normal liver and liver tumor. In general the oxidative enzyme systems appear to be relatively deficient in the induced liver tumors as compared to normal liver (Potter, 1944, 1951), although the activity of lactic acid dehydrogenase is as high as in normal liver (Meister, 1950). On the other hand, the levels of certain phosphatases, nucleic acid depolymerases, cathepsins, desamidases, and nucleic acid desaminases are usually about as high in the liver tumors as in normal liver (Greenstein, 1947). For detailed discussions of theenzymatic composition and metabolism of the liver tumors induced by the aminoazo dyes the reader is referred to several recent reviews of this subject (Greenstein, 1947; Potter, 1944, 1951 ; Rusch and LePage, 1948; Zamecnik and Frantz, 1949; Zamecnik et al., 1951; Olson, 1951).
THE CARCINOGENIC AMINOAZO DYES
IV. STUDIESON
THE
370
HEPATO-CARCINOGENICITY OF OTHER Azo DYES
I. %',3-Dimethyl-.4-aminoazobenzene (o-Aminoazototuene, A A T ) and
Related Compounds Although the early observations on the carcinogenicity of AAT for rat liver (Sasaki and Yoshida, 1935; Shear, 1937) have been confirmed by other investigators (see Hartwell, 1941; Crabtree, 1949; J. A. Miller et al., 1949), its low carcinogenicity in this species as compared with DAB has discouraged extensive use. Considerably more study has been made on the carcinogenicity of AAT for mouse liver, since in this species it is more active and less toxic than DAB (see Shear, 1937; Hartwell, 1941; Law, 1941; Andervont and Edwards, 1943a; Andervont et al., 1944; Kirby, 1945a,b; Crabtree, 1949)., Early dietary studies showed that the activity of AAT was considerabIy diminished when either liver (Mori, 1941a), wheat or cod liver oil (Ando, 1941a,b) was included in the diet of rats, but the addition of cholesterol to the diet of mice was without effect (Baumann et al., 1940). Andervont and his associates (1942, 1943a, 1944) found that subcutaneous injection of oil solutions or glycerol suspensionsof AAT induced hepatomas in the mice of several strains and that the female mice of each strain were more susceptible than the male mice. A single subcutaneous injection of 2 mg. induced hepatomas in three of twenty-nine virgin female mice of the A strain and a single injection of 4 mg. induced liver tumors in seven of thirty-one mice. A single oral dose of 2 mg. was inactive in this experiment, but a 4-mg. oral dose elicited hepatomas in six of sixteen female mice (Andervont and Edwards, 194313; Andervont, 1947). In studying the effects of hormone stimulation Andervont and Dunn (1947) showed that intact females of the C strain were more susceptible to the induction of hepatomas than either castrated females or intact males. The susceptibility of male mice was considerably increased by castration, but castrate male or female mice bearing testosterone propionate pellets were almost as resistant as intact male mice. Andervont et al. (1942, 1943a,b, 1944, 1947) and Kirby (1945a) also found that the subcutaneous injection of AAT induced lung tumors, hemangioendotheliomas, and fibrosarcomas in many strains of mice. A number of compounds related t o AAT have been tested for carcinogenic activity in either rats or mice, but no extensive study has been made of the structural features required for activity. Crabtree (1949) prepared six isomeric aminoazotoluenes and fed them to both rats and mice. 2',3-Dimethyl-4-aminoazobenzene was the only isomer which
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JAMES A. MILLER AND ELIZABETH C. MILLER
induced gross hepatic tumors in rats although microscopic areas which appeared to be hepatomas were found in the livers of rats fed 2’,5-dimethyl-2-aminoazobenzene. These two compounds and 2,4’-dimethyl4-aminoazobenzene induced gross hepatic tumors in mice while 4’,5-dimethyl-2-aminoazobenzene,2,3‘-dimethyl-4-aminoazobenzene, and 3,4’-dimethyl-4-aminoazobenzene were essentially inactive. Kirby (1945a) also failed to induce tumors in the livers of mice by subcutaneous injection of 4’,5-dimethyl-2-aminoazobenzene,while Yoshida (cited by Shear, 1937) obtained no tumors when the compound was fed t o rats for fifteen months. N-Methylation of AAT did not alter its activity toward either rat or mouse liver (J. A. Miller et al., 1948; J. A. Miller and E. C. Miller, 1952a), but acetylation reduced its potency (Kinosita, 1940a). Law (1941) found liver tumors in two of fifty-four and two of twenty mice which received subcutaneous injections of 4‘-hydroxy-2,3’azotoluene or 2,3’-azotoluene, respectively; Otsuka and Nagao (cited by Cook et al., 1937) had previously reported that 2,3’-azotoluene was inactive for rat liver. Nagao (1940) observed liver damage but no tumors following the ingestion of 2‘,3-dimethyl-4-aminoazoxybenzeneby rats, and Kirby (1947a, 194830) and Otsuka (see Cook et al., 1937) were unable to induce hepatomas in mice by oral, topical, or subcutaneous application of diazoaminobenzene. 2. Azonaphthalene Series Because of the similarity in the carcinogenic action of 2,2’-azonaphthalene and some of the arninoazo dyes, the studies on this dye are included here even though it lacks the amino substituent characteristic of the other hepato-carcinogenic azo dyes. Thus, high incidences of cholangiomas and hepatomas were reported by Cook and his associates (1940) in mice given oral, topical, or subcutaneous applications, although it should be noted that these terms were used to designate new growth of bile duct and liver cells and not necessarily to denote true tumors. The isomeric 1,l’-azonaphthalene produced liver damage in only a few mice, whereas 1,2’-azonaphthalene and 4-amino-l,2’-azonaphthalene were inactive. In strong acid solution 2,2’-azonaphthalene after partial reduction undergoes a benzidine rearrangement to 2,2’-diamino-l, 1‘dinaphthyl, and this compound readily loses ammonia to give 3,4,5,6dibenzcarbazole. Boyland and Brues (1937) and Strong et al. (1938) had reported that the latter compound was carcinogenic for mouse liver, while Andervont and Edwards (1941) have mentioned only hepatic lesions. Although this series of reactions has not been demonstrated in vivo, Cook et al. suggested that 2,2‘-azonaphthalene might be carcinogenic through in vivo conversion to the carbazole. This postulation was
THE CARCINOGENIC AMINOAZO DYES
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strengthened by the reported inactivity of 1,2,7,8- and lJ215J6-dibenzcarbasole (Boyland and Brues, 1937) for the liver; these compounds would be formed if the inactive 1,l’-or lJ2’-aaonaphthalenes underwent a similar series of reactions in vivo, The carcinogenic activities of the and l12’-diamino-2,1’-dinaphcorresponding l,l’-diamino-2,2’-dinaphthyl thy1 were not determined. Neither the azonaphthalenes or 2,2’-diamino1,1’-dinaphthyl (Cook et al., 1940) nor 3,4,5,6-dibenzcarbazole (Boyland and Brues, 1937) induced hepatic tumors in rats. N-Methylation or N-ethylation of 3,4J5,6-dibenzcarbazole nearly abolished the hepatocarcinogenic activity of the compound, although these derivatives were both active toward the skin and subcutaneous tissue (Kirby and Peacock, 1946; Kirby, 1948a). 3. Trypan Blue Gillman and his associates (1949) have observed a marked hepatic response following the subcutaneous injection of trypan blue (sodium acid) into salt of o-tolidindisa~o-bis-l-amino-8-naphthol-3~6-disulfo~c rats a t weekly or biweekly intervals. In most of the animals the reaction was characterized by an extensive reticulosis which sometimes terminated in reticulum cell sarcoma of the liver. In an occasional rat the reticulosis was less extensive, and the connective tissue elements underwent malignant transformation to spindle-cell sarcoma. These observations are of particular importance since the induction of reticulum cell sarcomas by other means has not been reported. Cohen and Cohen (1951) have reported in a short note on the behavior of rats in which subcutaneous transplants of hepatomas induced by DAB grew and then regressed. When these rats were treated with total body irradiation by x-rays or with subcutaneous injections of trypan blue, all the tumors started growing again and grew progressively for the next four months until death. These rats, in contrast to the controls, had a high tendency toward bronchiectatic lesions, cysticercosis of the liver, and bartonella infection. Since cysticercosis is known to induce sarcoma of the liver (Bullock and Curtis, 1924) these investigators feel that the trypan blue may act by deranging some resistance mechanism and thus activating and disseminating latent spontaneous tumors as well as rendering the host susceptible to chronic infections such as bartonellosis and cysticercosis.
4. Commercial Food Dyes The synthetic food dyes are examples of the increasing number and variety of foreign compounds that humans are exposed to in modern society. Since the known carcinogens exhibit great differences in
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JAMES A. MILLER AND ELIZABETH C. MILLER
activity in different species, tests on technologically useful compounds for carcinogenicity in experimental animals can never adequately ascertain their activity in man. Man has been shown to be susceptible to the carcinogenic action of several chemicals and occasional or frequent contact with a variety of foreign compounds in the air, food, drugs, and contact articles may summate with intrinsic factors to increase the incidence of cancer. The use of natural organic coloring matters is subject to the same objection except in the case of compounds of known physiological value such as the carotenes. Since the demand for the use of synthetic and natural coloring and flavoring materials of no food value is unlikely t o diminish it appears to the authors that these materials should a t least be subjected to extensive tests for carcinogenicity in a variety of experimental animals. Only a few such tests have been reported. The U. S. Government through the Food and Drug Administration permits the use of nineteen different coal-tar dyes in foods, drugs, and cosmetics after individual batches of these dyes have passed toxicity tests (Jablonski, 1951). Ten of these dyes are azo dyes, and these include four oil-soluble dyes. Two of the latter dyes are o-aminoazo derivatives and are used for coloring fats and oils; they are F D and C Yellow No. 3 (Yellow AB, phenylazo-8-naphthylamine)and FD and C Yellow No. 4 (Yellow OB, o-tolyaso-0-naphthylamine). So far these dyes have been found to be noncarcinogenic in rats (Sugiura, 1942, 1946) and in mice (Badger et al., 1942), although they have some toxic effects in these species. Cook et al. (1940) also failed to find liver tumors in mice after the administration of a number of water-soluble azo dyes used as food colorings in Great Britain and in this country. However, in Great Britain, Kirby and Peacock (1949) found hepatomas in six of eighteen male and one of eighteen female mice injected subcutaneously with up to 150 mg. of benzeneazo-p-naphthol (Sudan I or “Oil Orange E”). One of eighteen female and none of sixteen male mice developed hepatomas after similar injections of “Oil Yellow HA” (formula not published). It is of interest that the other two oil-soluble F D and C dyes are close relatives of benzeneazo-b-naphthol. They are F D and C Red No. 32 (Oil Red XO, m-xylylazo-p-naphthol) and F D and C Orange No. 2 (Orange SS, o-tolylazo-/%naphthol). Samples of another dye, watersoluble and not an azo dye, F D and C Green No. 2 (Light Green S F Yellowish, disodium salt of dibenzyldiethyldiaminotriphenylcarbinol trisulfonic acid anhydride) have produced sarcomas after subcutaneous injection in rats (Schiller, 1937; Harris, 1947a). The use of the trivial name “butter yellow” for DAB should be discontinued for it is no longer an accurate description of the use of this
THE CARCINOQENIC AMINOAZO DYES
383
compound and since several of the oil-soluble dyes now in use in foods are known by this term in commercial circles.
V. ON
THE
MECHANISM OF Azo DYE CARCINOGENESIS 1. General Considerations
The fate of any foreign compound in the body is obviously dependent on how it is attacked by the enzymes of the cells. The fate of the host, on the other hand, will depend on the extent to which the presence of the foreign agent causes quantitative or qualitative alterations in the constituents and potentialities of the cells. Both the metabolism of the compound and the alterations in the host cells may occur more or less simultaneously. Depending on the nature of the cellular changes, such an altered cell might recover and go on unchanged, or the cell and its descendants might even be able to meet future attacks more easily. Or if certain irreversible changes occur, the cell might die. However, still other irreversible changes might occur which, though permitting the cell to live, would place it in a new relation to the rest of the organism. If the cells merely ceased t o perform special functions and did not multiply a t an abnormally high rate, they might escape notice unless they were so numerous that the whole organism suffered from a lack of functional tissue. If, however, the affected cells suddenly, or gradually in future cell generations, escaped from the growth controls of the organism, they might reproduce themselves fast enough to form gross tumors possessing various degrees of autonomy. It is likely that each of the above responses is t o be found in any tissue susceptible to a carcinogen, and the fate of any one cell may be determined by the quantity of the primary carcinogen (i.e., the derivative directly initiating the process) which reaches it. Thus, as discussed in the section on Histology, following the administration of the aeo dyes some cells appear histologically undamaged, others die, and still others have altered characteristics; and it is in these areas of altered cells that the tumors appear t o arise. Likewise the magnitude of the liver changes and the survival of the animals are functions of the dose of carcinogen administered. The nature of the fundamental changes which make cancer cells relatively free of the factors which control the growth of normal tissues has not been defined, but they appear to be both irreversible and heritable. From the present knowledge of the composition of cells and of the maintenance of cell types through successive generations, it appears almost certain that this type of change must involve either c e w proteins or nucleic acids or both. With most carcinogenic procems there is little basis a t present for deciding in which of these constituents
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JAMES A. MILLER AND ELIZABETH C. MILLER
the fundamental changes occur or in which part of the cell the crucial changes take place, but in the case of the a50 dyes the evidence points toward the importance of alterations in certain of the liver proteins. In studying the mechanism of chemical carcinogenesis there are a t least two major problems to be answered. The first is the identification of the compound or set of compounds which are directly involved in the initiation of the process. Thus, when a compound is administered, it may be metabolized to some derivative which is directly involved in the carcinogenic process and which could be properly considered as the primary carcinogen. Similarly, the identities of the initial sites of action of chemical carcinogens must be discovered before the whole process can be understood. Even a partial realization of these objectives should enable a more logical attack to be made on the mechanism of action of the carcinogen. These problems are analogous to those met in studies on vitamins, for the vitamins in the food must often be converted to coenzymes and the coenzymes must combine with specific proteins before the physiological activity of the vitamin is realized. The general property of chemical carcinogens that they do not need to be present in the tissue throughout the whole period of carcinogenesis should aid in the solution of these problems. 2. SpeciJic Hypotheses
A number of hypotheses have been proposed on the nature of the initial reactions involved in carcinogenesis with the azo dyes. These suggestions are outlined below. A. The Benzidine Rearrangement Hypothesis. Cook et al. (1940) have proposed that 2,2'-azonaphthalene is active in the mouse through conversion to 3,4,5,6-dibenzcarbazole. This conversion in vivo would, if analogous with the known in vitro reactions, involve a partial reduction to the hydrazo compound followed by a beneidine rearrangement to the corresponding diaminodinaphthyl and finally in a deaminative cyclization to the dibenzcarbazole (Fig. 10). However, the evidence that this actually occurs in vivo is incomplete (see above). A similar suggestion in the case of DAB has been made by Elson and his associates (1944, 1946), who have made use of the observations of Cook et al. on the azonaphthalenes as well as the work of Kensler et al. (1942a) on the toxicity of various diamines toward certain enzymes. Thus Elson and Warren (1944) found that the ingestion of azobenzene (a noncarcinogenic compound; Hartwell, 1941; Spitz et al., 1950) by rats led to the excretion in the urine of aniline and a substance which upon acid treatment was converted into 4,4'-diaminobiphenyl or benzidine. The authors assumed that the benzidine precursor was a derivative (ethereal sulfate?) of
T H E CARCINOGENIC AMINOAZO DYES
385
hydrasobenzene, and i t was suggested that a rearrangement of this precursor to benzidine might also take place in vivo. Elson and HochLigeti (1946) also considered it probable that DAB undergoes a similar partial reduction and rearrangement in vivo to yield 2,4’-diamino-5dimethylaminobiphenyl (Fig. 11) but they offered no direct supporting evidence. This compound, a substituted p-phenylene diamine, was found to be a potent inhibitor for certain enzymes and hence might be involved in the carcinogenic process induced by the parent dye. Direct and protracted tests of this rearrangement product for carcinogenic activity in the rat have yielded negative results (J. A. Miller and E. C. Miller, 1948; J. A. Miller et al., 1949). Positive evidence against the
FIQ. 10. The possible rearrangement products of 2,2’-azonaphthalene in the mouse (see Cook el al., 1940).
participation of a benzidine rearrangement in the carcinogenic process induced by DAB has been obtained through tests on the carcinogenicity of various mono- and polyfluoro derivatives of this dye (J. A. Miller et al., 1949, 1951b). These derivatives were chosen since the fluoro group is small and since the C-F bond is one of the strongest known. Thus 4’-F-DAB and 2-F-DAB proved t o be considerably more active than DAB and the 2‘-fluoro derivative had essentially the same activity as the parent dye. The 4’-flUOr0 substitution should have greatly reduced the carcinogenic activity and the 2’-fluoro and 2-fluoro compounds should have been less active if benaidine or semidine rearrangements occurred in the metabolism of the dye and were of decisive importance in the initiation of the carcinogenic process. More decisive positive evidence has been obtained in tests with 2’,4’-difluoro-DAB and 2’,4‘,6’-trifluoro-DAB. Each of these dyes has also proved to be more active than the parent dye. Neither of the two possible benzidine rearrangements and only one of the three possible semidine rearrange-
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JAMES A. MILLER AND ELIZABETH C. MILLER
ments could occur in the case of the trifluoro derivative (Fig. 11). The remaining semidine rearrangement would presumably be inhibited in the case of 2-F-DAB where one of the two ortho positions is unavailable for reaction. Yet this dye is twice as carcinogenic as DAB. Thus it is felt that conclusive evidence now exists against the participation of bensidine or semidine rearrangements in the carcinogenic process induced by 4-dimethylaminoasobenzene. It is of interest that the 3'-flUOrO derivative of this dye is also considerably more active than the unsubQN=O(CH&
12H
Q-&yWk J \
Benzidine Rearronpemento
Semidine Rearrangements
0 ' positions blocked in 2',~,6'-trifluoro-DAB
FIQ. 11. The possible rearrangement products of Pdimethylaminohydrazobensene.
stituted dye. Indeed, the data on the high activities of the four monofluoro and the two polyfluoro derivatives can be used to argue that of the four positions involved, i.e., 4',3',2', and 2, probably none are directly concerned in the carcinogenic process.K While the 3 position remains to be tested in this regard, the above data and the essentiality of the azo group and of at least one N-methyl group for carcinogenicity indicate that the initial carcinogenic reaction probably involves one or both of these groups and not any of the ring positions. B. The Split Product Hypothesis. A different approach was taken by Kensler and his associates (1942a) in an attempt to identify the primary carcinogen and the initial sites of action in carcinogenesis by DAB. These workers advanced the suggestion that it is not the parent dye itself which initiatea the carcinogenic process but its reduction or split product, N,N-dimethyl-p-phenylene diamine, which was found to inhibit 6
See footnote on page 358.
THE CARCINOGENIC AMINOAZO DYES
387
certain important glycolytic and oxidative enzymes (Kensler et al., 1942a,b; Potter, 1942; Potter and DuBois, 1943). In support of this concept Kensler et al. correlated the toxicities of various p-diamines toward a yeast zymake system with the reported carcinogenicities of the aminoazo dyes from which they might arise in vivo. However, their correlation has been shown to rest on just two of the dyes whose carcinogenicities were quoted and extensive further tests of this hypothesis have not provided it with any support (J. A. Miller and Baumann, 1945b; Sugiura et al., 1945; J. A. Miller and E. C. Miller, 1948; J. A. Miller et al., 1949). In particular, N,N-dimethyl-p-phenylene diamine has not been found to be carcinogenic in the rat even when fed at high levels for long times (Kinosita, 1937; Sugiura et al., 1945; J. A. Miller and Baumann, 1945b; F. R. White et al., 1948; J. A. Miller and E. C. Miller, 1948). Furthermore, while it seems very likely from in vitro studies with liver homogenates (Mueller and Miller, 1949, 1950) that this diamine is formed, at least to some extent, from the dye in vivo it was also found that riboflavin-adenine-dinucleotideis the prosthetic group of the enzyme which reductively cleaves the azo dye. Since high levels of dietary riboflavin greatly delay the carcinogenic action of DAB (see Sec. III,3) it seems unlikely that the diamine could be the primary carcinogen when it should be present in vivo in the greatest amount under these conditions. Potter (1942) and Kuhn and Beinert (1943) presented evidence a t variance with the conclusions of Kensler et al. (1942a,b) that the diamines act as enzyme inhibitors through oxidation to SH-reactive free radicals. Kuhn and Beinert also felt that the carcinogenicity of the aminoazo dyes might depend on the oxidation of the dyes to various p-quinones. Although the present authors have published much of the data that do not support the hypothesis of Kensler et al., we feel that their basic concept still has definite heuristic value. Thus it is possible that reduction of the protein-bound derivatives of DAB occurs in vivo to form protein-bound diamines which could then function as enzyme inhibitors. Likewise a partial reduction-oxidation system such as azo+hydrazo may participate in the carcinogenic process. The hydrazo form of the dye is merely a substituted form of N,N-dimethyl-p-phenylene diamine and might act as an enzyme inhibitor in a reaction which would oxidize the hydrazo form back to the parent azo dye. Similarly, the reactivities of the N-methyl groups, which are required for this carcinogenic process, may differ greatly in these two forms. The report by Kinosita (1940b) that 4-dimethylaminohydrazobenzene is not carcinogenic cannot be taken as conclusive since no data were given on the preparation of this hitherto unknown (and, according to Kinosita, unstable) compound. C . The Methyl Deficiency Hypothesis. Although it is not known
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JAMES A. MILLER AND ELIZABETH C. MILLER
whether there is any relationship between the induction of liver tumors in the rat by DAB and the choline deficiency-induced hepatomas described by the Alabama group (Copeland and Salmon, 1946; Engel et al., 1947), the properties of DAB and its metabolites make it possible t o consider a situation such as that depicted in Fig. 12. The removal of the methyl groups in an oxidized form and the presumed requirement for preformed methyl groups for remethylation could constitute a “leak” in the supply of available labile methyl groups in the liver. The inability of choline to protect against tumor formation and the failure of nicotinamide or glycocyamine to accelerate tumor formation by the dye do not suggest that this set of circumstances plays a significant role in the carcinogenic action of the dye (see Sec. 111,3), On the other hand, it is known that
’
- -DrI>diet
functional uses
[CH3-l
labile meth I POOf
MAB
I
C Hr;d: : I I I
I
end product
limited resynthesis
FIG.12. A possible mechanism for the production of a partial methyl deficiency in the rat liver by DAB and its metabolites.
DAB lowers the level of choline oxidase in the rat liver (Viollier, 1950b). This enzyme is necessary for transmethylation from choline, and as suggested by Kensler and Langemann (1951) a reduction in its level would be equivalent to a partial choline deficiency in the rat. Attempts to influence the activity of the dye by the products of this enzyme, such as betaine, would be of interest here. D. The Protein (or Enzyme) Deletion Hypothesis. The most recent suggestion concerning the mode of action of the aminoazo dyes has come from the authors’ laboratory (E. C. Miller and J. A. Miller, 1947; E. C. Miller et al., 1949a). This arose from the observation that the formation of liver tumors in rats fed DAB or any of its carcinogenic derivatives is preceded by the accumulation of protein-bound derivatives of the dye in the liver. As outlined in the section on Metabolism a number of correlations have been made between the presence or level of the bound dye and the probability of tumor formation under different conditions, and these correlations have indicated that the protein-bound dyes may bear a causal relationship to tumor induction by the dyes. Thus (1) under most conditions tumors are found only in the liver of the rat, and this is the only tissue in which the.protein-bound dyes can be detected. (2)
THE CARCINOGENIC AMINOAZO DYES
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Of the eight species tested only two, the rat and the mouse, have proved susceptible to carcinogenesis by DAB. Similarly, these are the only two species in which the protein-bound dyes can be detected. More bound dye is found in the liver of the more susceptible species, the rat, than in the liver of the mouse. (3) MAB, the only known carcinogenic metabolite of DAB, gives rise to the same level of bound dye as DAB, while the essentially noncarcinogenic metabolite AB yields only a low level of bound dye. Little or no bound dye has been detected fdlowing the administration of the noncarcinogenic hydroxylated aminoazo dyes. (4) When the extents of protein-binding of several of the ring-monomethyl derivatives of DAB are compared, it is found that the rapidity with which maximum protein-binding occurs can be directly correlated with the carcinogenicity of the dye concerned. (5) The levels of bound dye in the liver are lower when the dyes are fed in diets (such as those containing high levels of riboflavin or low levels of certain polycyclic hydrocarbons) which inhibit tumor induction by the dye. (6) In differential centrifugation experiments some bound dye has been found in each morphological fraction from the liver cells. However, over 50% of the bound dye in the liver is associated with the soluble proteins, and of this at least SO% is attached to a slow-moving fraction which accounts for only 15% of the soluble proteins. The bound dye is not found in the tumors produced by continuous feeding of the dye, and in the soluble proteins, at least, the amount of the electrophoretic component bound to the dye in the liver is greatly reduced in the tumor. Furthermore, evidence for similar carcinogen-protein complexes has been found in the case of two of the polycyclic hydrocarbons (E. C. Miller, 1951; Heidelberger and Weiss, 1951, Wiest and Heidelberger, 1952). While these studies are less complete than those on the azo dyes, they indicate that a similar reaction mechanism (i.e., formation of carcinogen-protein complexes) might operate in many or all cases of chemical carcinogenesis (E. C. Miller and J. A. Miller, 1952). From these correlations it has seemed reasonable to suggest a tentative hypothesis of carcinogenesis by DAB and related dyes. According t o this proposal the administered dye is metabolized to a derivative (the primary carcinogen, possibly a N-hydroxymethyl aminoaso dye) capable of combining with certain liver proteins (the initial sites of action). These liver proteins are considered to play key roles in the response of the cell to its intrinsic growth controls (e.g., competitive reactions) and to the extrinsic growth controls (e.g., hormones) exercised by the rest of the organism. In any case the binding of these proteins with the dye metabolite is thought to reduce or prevent the further synthesis of these proteins so that eventually, in subsequent generations, cells would result with
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JAMES A. MILLER AND ELIZABETH C. MILLER
less and finally none of the proteins originally attacked by the dye. Such cells could only respond to nutrition by continued growth and thus would be tumor cells, Although the centrifugation and electrophoresis data show that there is some specificity for the proteins attacked, not all the proteins thus affected need necessarily play a role in the carcinogenic reaction. The soluble proteins which bind the dye may not be the key proteins and may only serve as indicators of a similar reaction taking place with a protein in another part of the cell which is less amenable to study a t present. Since virus reproduction necessarily draws on the building blocks required by normal cells, it is clear that ultimately both a carcinogenic virus and a chemical carcinogen could produce the same result through altering the protein (enzyme) balance of the cell (see for example Potter, 1944, and Potter et al., 1950). Again, the destruction or partial inactivation of certain cell proteins by radiations could produce a similar state. Thus, the whole series of carcinogenic agents could be conceived as acting through the induction of abnormal protein patterns in a cell, The protein pattern leading to abnormal growth need not be identical in the induction of tumors by different carcinogens or even when different neoplastic cells are induced by a given carcinogen. But it must always leave the cell viable and free of many of the growth control systems. REFERENCES Andervont, H. B. 1947. J . Natl. Cancer Znst. 7 , 431-32. Andervont, H.B., and Dunn, T.B. 1947. J . Natl. Cancer Znst. 7 , 455-61. Andervont, H. B., and Edwards, J. E. 1941. J . Natl. Cancer Znst. 2, 139-49. Andervont, H. B., and Edwards, J. E. 1943a. J . Natl. Cancer Znst. 3,349-54. Andervont, H. B., and Edwards, J. E. 1943b. J . Natl. Cancer Inst. 3,355-58. Andervont, H. B., Grady, H. G., and Edwards, J. E. 1942. J . Natl. Cancer Znst. 3, 131-53. Andervont, H. B., White, J., and Edwards, J. E. 1944. J . Natl. Cancer Znst. 4, 583-86. Ando, T. 1941a. Gann 36,62-63. Ando, T. 1941b. Gann 86,20144. Antopol, W.,and Unna, K. 1942. Cancer Research 2, 694-96. Badger, G.M.,Cook, J. W., Hewett, C. L., Kennaway, E. L., Kennaway, N. M., and Martin, R. H. 1942. Proc. Roy. SOC.(London) Bl31, 170-82. Baumann, C. A. 1948. J . Am. Dietet. Assoc. 24,573-81. Baumann, C. A.,Rusch, H. P., Kline, B. E., and Jacobi, H. P. 1940. Am. J . Cancer 38, 76-80. Berman, C. 1951. Primary Carcinoma of the liver. Lewis, London. Boissonnae, R. A., Turner, R. A,, and du Vigneaud, V. 1949. J . Biol. Chem. 180, 1053-58. Boyland, E., and Brues, A. M. 1937. Proc. Roy. SOC.(London) Bl22, 429-41. Breedis, C., and Young, G. 1949. Federation Proc. 8, 351. Brock, N., Druckrey, H., and Hamperl, H. 1940. Z.Krebaforsch. 60,431-56.
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Brown, R. R., Miller, J. A., and Miller, E. C. 1952. Federation Roc. 11, 192. Bullock, F. D., and Curtis, M. R. 1924. J . Cancer Research 8, 446-81. Clayton, C. C., and Baumann, C. A. 1949. Cancer Research 9,575-82. &hen, A., and Cohen, L. 1951. Nature 167, 1063. Cohen, P. P. 1945. Cancer Research 6, 626-30. Cook, H. A., Griffin, A. C., and Luck, J. M. 1949. J . Biol. Chem. 177,373-81. Cook, J. W., Haslewood, G. A. D., Hewett, C. L., Hieger, I., Kennaway, E. L., and Mayneord, W. V. 1937. Am. J . Cancer 29, 219-59. Cook, J. W., Hewett, C. L., Kennaway, E. L., and Kennaway, N. M. 1940. Am. J. Cancer 40, 62-77. Copeland, D. H., and Salmon, W. D. 1946. Am. J . Path. 22, 1059-67. Cortell, R. 1947. Cancer Research 7, 158-61. Crabtree, H. G. 1949. Brit. J . Cancer 3, 387-98. Cunningham, L.,Griffin, A. C., and Luck, J. M. 1950a. Cancer Research 10, 194-99. Cunningham, L., Griffin, A. C., and Luck, J. M. 1950b. Cancer Research 10,211. Dalton, A. J., and Edwards, J. E. 1942. J . Natl. Cancer Inst. 3, 319-29. Davidson, J. N., and Waymouth, C. 1944. Biochem. J . 38,379-85. Day, P. L., Payne, L. D., and Dinning, J. S. 1950. Proc. SOC.Ezptl. Biol. Med. 74, 854-55. Druckrey, H. 1950a. Arch. klin. Chir. 264, 45-55. Druckrey, H. 1950b. Arch. exptl. Path. Pharmakol. 210, 137-58. Druckrey, H.,and Kupfmtiller, K. 1948. 2.Naturforsch. 31, 254-66. Edwards, J. E.,and White, J. 1941. J. Natl. Cancer Inst. 2, 157-83. Elson, L. A., and Hoch-Ligeti, C. 1946. Biochem. J . 40, 380-91. Elson, L. A., and Warren, F. L. 1944. Biochem. J . 38,217-20. Engel, R. W., Copeland, D. H., and Salmon, W. D. 1947. Ann. N . Y. Acad. Sci. 49,Art. 1, 49-67. Fischer, B. 1906. Milnch. med. Wochschr. 63, 2041-47. Fischer-Wasels, B. 1936. Centr. allgem. Path. u . path. Anat. 66, 359-60. Fraenkel-Conrat, H., and Olcott, H. S. 1948. J . Biol. Chem. 174, 827-43. Fujiwara, T., Nakahara, W., and Kishi, S. 1937. Gann 31,51-62;abstract in 1938, Am. J. Cancer 33, 283. Gallico, E., and Boretti, G. 1948. Tumori 22, 130-138;abstract in 1949, Cuncer 8, 564. Giese, J. E., Clayton, C. C., Miller, E. C., and Baumann, C. A. 1946. Cancer Research 6, 679-84. Giese, J. E., Miller, J. A., and Baumann, C. A. 1945. Cancer Research 6, 337-40. Gillman, J., Gillman, T., and Gilbert, C. 1949. 8.African J . Med. Sci. 14,21-83. Greenstein, J. P. 1947. Biochemistry of Cancer. Academic Press, New York. Griffin, A. C., and Baumann, C. A. 1946. Arch. Biochem. 11, 467-76. Griffi, A. C., and Baumann, C. A. 1948a. Cancer Research 8, 135-38. Griffi, A. C., and Baumann, C. A. 1948b. Cuncer Research 8,279-84. Griffin, A. C., Bloom, S., Cunningham, L., Teresi, J. D., and Luck, J. M. 1950. Cancer 3, 316-20. Griffin, A. C., Clayton, C. C., and Baumann, C. A. 1949. Cancer Research 9,82-87. Griffin, A. C., Cunningham, L., Brandt, E. L., and Kupske, 0. W. 1951. Cancer 4, 410-15. Griffi, A. C., Nye, W. N., Noda, L., and Luck, J. M. 1948. J . Biol. Chem. 176, 1225-35. Gyorgy, P., Poling, E. C., and Goldblatt, H. 1941, Proc. SOC.Exptl. Biol. Med. 47, 41-44.
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Gyorgy, P., Tomarelli, R., Ostergard, R. P., and Brown, J. B. 1942. J . Exptl. Med. 76, 413-20. Haddow, A., Harris, R. J., Kon, G. A. R., and Roe, E. M. F. 1948. Trans. Roy. SOC.(London) A241, 147-95. Harris, P. N. 1947a. Cancer Research 7, 36-36. Harris, P. N. 1947b. Cancer Research 7 , 178-79. Harris, P. N. 1949. Cancer Reaearch 9, 602. Harris, P. N., Krahl, M. E., and Clowes, G. H. A. 1947% Cancer Research 7,162-75. Harris, P. N., Krahl, M. E., and Clowes, G. H. A. 194713. Cancer Research 7 , 176-77. Hartwell, J. L. 1941. Survey of Compounds Which Have Been Tested for Carcinogenic Activity. U.S.Public Health Service, Washington, D.C. Heep, W. 1936. Frankfurt. 2.Path. 60, 48-62. Heidelberger, C., and Weiss, S. M. 1951. Cancer Research 11, 885-91. Higgins, H., Miller, J. A., Price, J. M., and Strong, F. M. 1950. Proc. SOC.Exptl. Biol, Med. 76, 462-65. Hoch-Ligeti, C. 1947. Cancer Research 7 , 148-57. Hoch-Ligeti, C. 1949. Brit. J. Cancer 8, 285-88. Hoch-Ligeti, C., Hoch, H., and Goodall, K. 1949. Brit. J . Cancer 8, 140-47. Jablonski, C. F. 1951. Chap. IX. Coloring Matters in Foods. In Jacobs, M. B. (Editor), Food and Food Products, 2nd Ed. Interscience Publishers, New York. Jacobi, H. P., and Baumann, C. A. 1942. Cancer Research 2, 175-80. Kensler, C. J. 1947. Ann. N . Y . Acad. Sci. 49, Art. 1, 29-40. Kensler, C. J. 1948. Cancer 1, 483-88. Kensler, C. J. 1949. J . Biol. Chem. 179, 1079-84. Kensler, C. J., and Chu, W. C. 1950. Arch. Biochem. 26, 66-73. Kensler, C. J., Dexter, S. O., and Rhoads, C. P. 1942a. Cancer Research 2, 1-10, Kensler, C. J., and Langemann, H. 1951. Cancer Research 11, 264. Kensler, C. J., Magill, J. W., and Sugiura, K. 1947. Cancer Research 7, 95-98. Kensler, C. J., Sugiura, K., and Rhoads, C. P. 1940. Science 91, 623. Kensler, C. J., Sugiura, K., Young, N. F., Halter, C. R., and Rhoads, C. P. 1941. Science 98,308-10. Kensler, C. J., Young, N. F., and Rhoads, C. P. 1942b. J . B i d . Chern. 148,465-72. Kinosita, R. 1936. Gann SO, 423-26. (In Japanese.) Kinosita, R. 1937. Jap. Path. SOC.Trans. 27, 665-727. Kinosita, R. 1940a. Yale J . B i d . and Med. 12, 287-300. Kinosita, R. 1940b. Gann 84, 165-67. Kirby, A. H. M. 1945a. Cancer Research 6, 673-82. Kirby, A. H. M. 1945b. Cancer Research 6, 683-96. Kirby, A. H. M. 1947a. Cancer Research 7 , 263-67. Kirby, A. H. M. 194713. Cancer Research 7 , 333-41. Kirby, A. H. M. 1948a. Biochem. J. 43, lv. Kirby, A. H. M. 194813. Brit. J . Cancer& 290-94. Kirby, A. H. M., and Peacock, P. R. 1946. Brit. J . Exptl. Path. 27, 179-89. Kirby, A. H. M., and Peacock, P. R. 1947. J . Path. Bact. I S , 1-18. Kirby, A. H. M., and Peacock, P. R. 1949. Glasgour Med. J . 80,364-72. Kishi, S., Fujiwara, T., and Nakahara, W. 1937. Gann 81, 1-11; abstract in 1938. Am. J . Cancer 88, 283. Kline, B. E., Miller, J. A., and Rusch, H. P. 1946. Cancer Research 6 , 641-43. Kline, B. E., Miller, J. A., Rusch, H. P., and Baumann, C. A. 1946a. Cancer Research 0, 1-4.
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Kline, B. E., Miller, J. A., Rusch, H. P., and Baumann, C. A. 1946b. C a n w Reaearch 6, 5-7. Kline, B. E., and Rusch, H. P. 1944. Cancer Research 4, 762-67. Kuhn, R., and Beinert, H. 1943. Ber. 76, 904-09. Kuhn, R., and Quadbeck, G. 1949. 2.Krebsfmsch. 66,242-45. Langemann, H., and Kensler, C. J. 1951. Cancer Research 11, 265. Langer, E. 1942. 2. Krebsforsch. 62, 443-54. Law, L. W. 1941. Cancer Research 1,397-401. Lowenhaupt, E. 1949. Cancer Research 9, 121-26. MacDonald, J. C., Miller, E. C., Miller, J. A., and Rusch, H. P. 1952a. Cancer Research 12, 50-54. MacDonald, J. C., Plescia, A. M., Miller, E. C., and Miller, J. A. 195213. Cancer Research 12, 280. Mark, D. D. 1950. Arch. Path. 49, 545-54. Mark, D. D., and Ris, H. 1949. Proc. SOC.Exptl. Biol. Med. 71, 727-29. Masayama, T., Iki, H., Yokoyama, T., and Hasimoto, H. 1938. Gann 32,303-06. Mwayama, T., and Yokoyama, T. 1940. Gann 34, 174-75. Meister, A. 1950. J. Natl. Cancer Inst. 10, 1263-71. Mellors, R. C., and Sugiura, K. 1948. Proc. SOC.Exptl. Biol. Med. 67, 242-46. Miller, E. C. 1951. Cancer Research 11, 100-08. Miller, E. C., and Baumann, C. A. 1946. Cancer Research 6, 289-95. Miller, E. C., Baumann, C. A., and Rusch, H. P. 1945. Cancer Research 6, 713-16. Miller, E. C., and Miller, J. A. 1947. Cancer Research 7 , 468-80. Miller, E. C., and Miller, J. A. 1952. Cancer Research 12, 547-56. Miller, E. C., Miller, J. A., and Brown, R. R. 1952a. Cancer Research 12,282-83. Miller, E. C., Miller, J. A., Kline, B. E., and Rusch, H. P. 1948. J. Exptl. Med. 88, 89-98. Miller, E. C., Miller, J. A., Sandin, R. B., and Brown, R. K. 1949a. Cancer Research 9, 504-09. Miller, E. C., Miller, J. A., Sapp, R. W., and Weber, G. M. l949b. Cancer Research 9, 336-43. Miller, E. C., Plescia, A. M., Miller, J. A,, and Heidelberger, C. 1952b. J. Biol. Chem. 196, 863-74. Miller, J. A. 1947. Ann. N . Y . Acad. Sci. 49, Art. 1, 19-28. Miller, J. A., and Baumann, C. A. 1945a. Cancer Research 6 , 157-61. Miller, J. A., and Baumann, C. A. 1945b. Cancer Research 6 , 227-34. Miller, J. A., Brown, R. R., Miller, E. C., and Mueller, G. C. 1951a. Cancer Research 11, 269. Miller, J. A., Kline, B. E., and Rusch, H. P. 1946. Cancer Research 6, 674-78. Miller, J. A., Kline, B. E., Rusch, H. P., and Baumann, C. A. 1944a. Cancer Research 4, 153-58. Miller, J. A., Kline, B. E., Rusch, H. P., and Baumann, C. A. 194413. Cancer Research 4, 756-61. Miller, J. A., and Miller, E. C. 1947. Cancer Research 7 , 39-41. Miller, J. A., and Miller, E. C. 1948. J. Ezptl. Med. 87, 139-56. Miller, J. A., and Miller, E. C. 1952a. Unpublished. Miller, J. A., and Miller, E. C. 1952b. Cancer Research 12, 283. Miller, J. A., Miller, E. C., and Baumann, C. A. 1945. Cancer Research 6, 162-68. Miller, J. A,, Miller, E. C., and Sapp, R. W. 1951b. Cancer Research 11, 269. Miller, J. A., Sapp, R. W., and Miller, E. C. 1948. J. Am. Chem. SOC.70,346&63. iller, J. A., Sapp, R. W., and Miller, E. C. 1949. Cancer Research 9, 652-60.
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Miller, W. L., Jr., and Baumann, C. A. 1951. Cancer Research 11, 634-39. Miner, D. L., Miller, J. A., Baumann, C. A., and Rusch, H. P. 1943. Cancer Research 8, 296-302. Mori, K. 194la. Gann 36, 106-19. Mori, K. 1941b. Gann 36, 121-25. Mueller, G. C., and Miller, J. A. 1948. J. Biol. Chem. 176, 535-44. Mueller, G. C., and Miller, J. A. 1949. J. Biol. Chem. 180, 1125-36. Mueller, G. C., and Miller, J. A. 1950. J. Biol. Chem. 186, 145-54. Mueller, G. C., and Miller, J. A. 1951. Cancer Research 11,271. Nagao, N. 1940. Gann 34, 13-19. Nagao, N. 1941a. Gann 36,8-20. Nagao, N. 1941b. Gann 36,280-82. (In Japanese.) Nakahara, W., Kishi, S., and Fujiwara, T. 1936. Gann SO, 499-508; abstract in 1936. Am. J. Cancer 28, 790. Olson, R. E. 1951. Cancer Research 11, 571-84. Opie, E. L. 1944a. J. Exptl. Med. 80, 219-30. Opie, E. L. 1944b. J. Exptl. Med. 80, 231-46. Opie, E. L. 1946. J. Exptl. Med. 84, 91-106. Opie, E. L. 1947a. J. Exptl. Med. 86, 339-46. Opie, E. L. 1947b. J. Exptl. Med. 86, 45-54. Opie, E. L., and Lavin, G. I. 1946. J. Ezptl. Med. 84, 107-12. Orr, J. W. 1940. J. Path. Bact. 60, 393-408. Orr, J. W., and Price, D. E. 1948. J. Path. Bact. 60, 461-69. Pearson, B., Novikoff, A. B., and Morrione, T. G. 1950. Cancer Research 10, 557-64. Plaut, G. W. E., Betheil, J. J., and Lardy, H. A. 1950. J . B i d . Chem. 184,795-805. Pollack, M. A., Taylor, A., Taylor, J., and Williams, R. J. 1942a. Cancer Research a, 739-43. Pollack, M. A., Taylor, A., Woods, A., Thompson, R. C., and Williams, R. J. 1942b. Cancer Research 2, 748-51. Potter, V. R. 1942. Cancer Research 2, 688-93. Potter, V. R. 1944. Advances in Enzymol. 4, 201-56. Potter, V. R. 1951. Cancer Research 11, 565-70. Potter, V. R., and DuBois, K. P. 1943. J. Gen. Physiol. 26, 391-404. Potter, V. R., Price, J. M., Miller, E. C., and Miller, J. A. 1950. Cancer Research 10, 28-35. Price, J. M., Harman, J. W., Miller, E. C., and Miller, J. A. 1952. Cancer Research 12, 192-200. Price, J. M., and Laird, A. K. 1950. Cancer Research 10, 650-58. Price, J. M., Miller, E. C., and Miller, J. A. 1948. J. Bio2. Chem. 173, 345-53. Price, J. M., Miller, E. C., and Miller, J. A. 1949a. Proc. SOC.Exptl. Biol. Med. 71, 575-78. Price, J. M., Miller, E. C., Miller, J. A., and Weber, G. M. 1949b. Cancer Research 9, 398-402. Price, J. M., Miller, E. C., Miller, J. A., and Weber, G. M. 1950. Cancer Research 10, 18-27. Price, J. M., Miller, J. A., and Miller, E. C. 1951. Cancer Research 11, 523-28. Price, J. M., Miller, J. A., Miller, E. C., and Weber, G. M. 1949c. Cancer Research 9,96-102. Richardson, H. L., and Borsos-Naohtnebel, E. 1951. Cancer Research 11, 398-403. Richardson, H. L., Stier, A. R., and Borsos-Nachtnebel, E. 1952. Cancer Research 12,356-61.
THE CARCINOQENIC AMINOAZO DYES
395
Roskelley, R. C., Mayer, N., Horwitt, B. N., and Salter, W. T. 1943. J . Clin. Invest. 22, 743-51. Rumsfeld, H. W., Jr., Miller, W. L., Jr., and Baumann, C. A. 1951. Cancer Research 11, 814-19. Rusch, H. P., Baumann, C. A., Miller, J. A,, and Kline, B. E. 1945. Experimental Liver Tumors. In Moulton, F. R. (Editor). Research Conference on Cancer, American Association for the Advancement of Science, Washington, D. C. Rusch, H. P., and LePage, G. A. 1948. Ann. Rev. Biochem. 17,471-94. Rusch, H. P., and Miller, J. A. 1948. Proc. SOC.Exptl. Biol. Med. 08, 140-43. Sakami, W. 1948. J. Biol. Chem. 170,995-96. Salaberg, D. A., Hane, S., and Griffin, A. C. 1951. Cancer Research 11, 276. Sasaki, T., and Yoshida, T. 1935. Virchow’s Arch. path. Anat. 296, 175-200. Sauberlich, H. E., and Baumann, C. A. 1951. Cancer Research 11, 67-71. Schiller, W. 1937. Am. J. Cancer S1, 486-90. Schmidt, M. B. 1924. Virchow’s Arch. path. Anal. 26S, 432-51. Schneider, W.C. 1945a. J. Biol. Chem. 101, 293-303. Schneider, W. C. 194513. Cancer Research 6, 717-21. Schweigert, B. S.,Guthneck, B. T., Price, J. M., Miller, J. A., and Miller, E. C. 1949. Proc. SOC.Exptl. Biol. Med. 72, 495-501. Shear, M. J. 1937. Am. J . Cancer 29, 269-84. Siegel, I., and Lafaye, J. 1950. Proc. SOC.Exptl. Biol. Med. 74, 620-23. Siekevite, P., and Greenberg, D. M. 1949. J . Biol. Chem. 180,845-56. Silverstone, H. 1948. Cancer Research 8, 301-08. Sorof, S.,and Cohen, P. P. 1951. Cancer Research 11, 376-82. Sorof, S., Cohen, P. P., Miller, E. C., and Miller, J. A. 1951. Cancer Research 11, 383-87. Spite, S., Maguigan, W. H., and Dobriner, K. 1950. Cancer 3, 789-803. Stevenson, E. S., Dobriner, K., and Rhoads, C. P. 1942. Cancer Research 2,160-67. Stowell, R. E. 1949. Cancer 2, 121-31. Strong, L. C.,Smith, G. M., and Gardner, W. U. 1938. Yale J . Biol. and Med. 10, 335-46. Sugiura, K. 1942. Proc. SOC.Exp. Biol. Med. 60,214-15. Sugiura, K. 1946. Proc. SOC.Exp. Biol. Med. 61,301-02. Sugiura, K. 1948. Cancer Research 8, 141-44. Sugiura, K. 1951. J. Nutrition 44, 345-60. Sugiura, K.,Halter, C. R., Kensler, C. J., and Rhoads, C. P. 1945. Cancer Research 6,235-38. Sugiura, K., and Rhoads, C. P. 1941. Cancer Research 1, 3-16. Taki, I., and Miyaji, T. 1950. Gann 41, 194-95. Tarver, H. 1951. Chap. XIII. The Metabolism OF Amino Acids and Proteins. In Greenberg, D. M. (Editor), Amino Acids and Proteins. Charles C. Thomas, Springfield, Ill. Taylor, A., Pollack, M. A,, Hofer, M. J., and Williams, R. J. 1942a. Cancer Research 2, 744-47. Taylor, A., Pollack, M. A., Hofer, M. J., and Williams, R. J. 1942b. Cancer Research 2, 752-54. Tung, T. C., and Cohen, P. P. 1950. Cancer Research 10,793-96. du Vigneaud, V., Spangler, J. M., Burk, D., Kensler, C. J., Sugiura, K., and Rhoads, C. P. 1942. Science 96, 174-76. Viollier, G. 1950a. Helv. Physiol. Acta 8, C34-C36. Viollier, G. 1950b. Helv. Physiol. Acta 8, C37-C39. Viollier, G., and Waser, P. 1950. Helv. Physiol. Acta 8, C39-C41.
396
JAMES A. MILLER AND ELIZABETH C. MILLER
West, P. M., and Woglom, W. H. 1942. Cancer Research 2, 324-31. Westerfeld, W. W., Richert, D. A., and Hilfinger, M. F. 1950. Cancer Research 10, 486-94.
White, F. R., Eschenbrenner, A. B., and White, J. 1948. Acta union intern. conlre cancer 6, 75-78. White, F. R., and White, J. 1946. J . Natl. Cancer Znst. 7 , 99-101. White, J., and Edwards, J. E. 1942% J . Natl. Cancer Znst. 2, 535-38. White, J., and Edwards, J. E. 1942b. J . Natl. Cancer Inst. 8, 43-59. Wiest, W. G., and Heidelberger, C. 1952. Cancer Research 12, 308. Wilson, R. H., De Eds, F., and Cox, A. J. 1941. Cancer Research 1, 595-608. Woglom, W. H. 1913. Studies in Cancer and Allied Subjects, Vol. 1. Columbia University Press, New York. Woodard, H. Q. 1943. Cancer Research 9, 159-63. Yoshida, T. 1933. Trans. Jap. Path. SOC.as, 636-38. (Cited by Shear, M. J. AT J . Cancer 20, 269-84.) Zamecnlk, P. C., and Frantz, I. D. 1949. Cold Spring Harbor Symposia Quant. Biol. 14, 199-208. Zamecnik, P. C., Frantz, I. D., Loftfield, R. B., and Stephenson, M. L. 1948. J . Biol. Chem. 176, 299-314. Zamecnik, P. C., Loftfield, R.B., Stephenson, M. L., and Steele, J. M. 1951. Cancer Research 11, 592-602.
The Chemistry of Cytotoxic Alkylating Agents W. C. J. ROSS* Chester Beatty Research Institute, Royal Cancer Hospital, London, England
CONTENTS
I. Introduction 11. PChloroethyl Sulfides (Sulfur Mustards) 1. Reactions in Water 2. Reactions with Anions 3. Reactions with Bases 4. Reactions with Proteins 6. Reactions with Nucleic Acids 111. 2-Chloroethylamines Aliphatic Derivatives (Aliphatic Nitrogen Mustards) 1. Reactions in Water 2. Reactions with Anions 3. Reactions with Bases 4. Reactions with Proteins 5. Reactions with Nucleic Acids Aromatic Derivatives (Aromatic Nitrogen Mustards) 1. Reactions in Water 2. Reactions with Anions 3. Reactions with Bases 4. Reactions with Proteins IV. 1,2-Epoxides 1. Reactions in Water 2. Reactions with Anions 3. Reactions with Bases 4. Reactions with Proteins 5. Reactions with Nucleic Acids V. Miscellaneous Agents VI. Discussion References
Page 397 399 399 402 406 408 410 41 1 41 1 41 1 415 416 418 419 419 419 425 427 429 429 429 432 434 434 435 436 437 440
I. INTRODUCTION In recent years a considerable amount of work has been devoted to the study of the chemotherapy of cancer using agents related t o the vesicant war gas, di-2-chloroethyl sulfide (mustard gas). The first agents to be used extensively were two so-called nitrogen mustards, methyldi-2chloroethylamine (HNJ and tri-2-chloroethylamine (HNa); there is a * British Empire Cancer Campaign Research Fellow. 397
398
W. C. J. ROBS
very extensive literature on the use of these compounds; references to review articles are given by Philips (1950). These agents are administered intravenously as their water-soluble hydrochlorides. A compound of similar type, 2-naphthyldi-2‘-ch1oroethylamineJhas received clinical trials in England (Matthews, 1950); it has an effect essentially similar to that of HNa but is slower acting and more easily controlled.* It also has the advantage of being effective when administered orally. Some measure of success has attended the use of these mustards in thetreatment of neoplastic diseases of the hematopoietic organs. No significant effect on well-established malignant growths a t other sites has yet been observed. It is, however, hoped that these compounds will find an application as an adjunct to surgical and radiation treatment since they may well be able to control the growth of smaller metastases which are inaccessible t o these other technics. Boyland (1948) has drawn attention to the similarity between the biological actions of x-irradiation and of the mustards and used the term “radiomimetic,” which had previously been employed by Dustin (1947), to describe the effects of the chemical agents. These actions include vesication, the delayed lethal effect due to hemoconcentration and leucopenia, the inducement of chromosomal abnormalities, the mutagenic action, the characteristic damage caused to the bone marrow, the bleaching of the hair at the site of action, the inhibition of numerous enzyme systems, the retarding action on the growth of certain neoplasms, and the ability to suppress antibody formation. References to the work published on these effects may be found in Boyland’s review and in an article by Philips (1950). This latter author points out that while the term “radiomimetic” is an attractive one it should be used with due regard for the differences between the effects produced by the mustards and radiation. In particular the types of chromosomal aberration induced appear to depend upon the agent used (Ford, 1948; Auerbach, 1949) and even to vary from one chemical agent to another (Loveless and Revell, 1949, 1950). Although prior to 1939 di-2-chloroethylsulfide was not regarded as particularly reactive under physiological conditions, work done since that time has shown that both sulfur and nitrogen mustards are capable of direct reaction with many functional groups in proteins (Banks et at., 1946; Fruton et al., 1946a; Herriott et at., 1946) and in nucleic acids (Elmore et al., 1948; Fruton et al., 1946b). The close relationship between the biological activity and the chemical reactivity of the aromatic
* Since this review was written several Italian workers have published the results of clinical trials using the Znaphthylamine derivative, for example, P. Introzzi, and M. Ninni, 1950, Haematobgica 84, 925-65.
CYTOTOXIC ALKYLATINQ AGENTS
399
derivatives of nitrogen mustard gas strongly suggests that such biological activity is due t o a chemical reaction rather than to a purely physical effect on the system (Haddow et al., 1948). The importance of the chemical reactivity of these agents has been amply demonstrated by the recent finding of very similar biological activity in the case of epoxides (Rapoport, 1947b, 1948; Loveless and Revell, 1949; Ross, 1950c), ethyleneimines (Lewis and Crossley, 1950; Burchenal et al., 1950), p-tolueneand methanesulfonic acid esters (Timmis, 1949, 1950), and methyl sulfate (Loveless, 1950), all of which are capable of reacting in a similar manner under mild conditions. In this review a survey will be made of the reactions of a group of radiomimetic agents all of which may be regarded as alkylating agents under physiological conditions of temperature and pH. It is proposed to consider each class of compound with special reference to its reactions under mild conditions and with few exceptions no reaction which takes place under more drastic conditions will be discussed. In the main section of this article the reactions of each agent with water, anions, bases, proteins, and nucleic acids, where these have been studied, will be described, and in the final section it will be shown that each type of compound can be regarded as an electrophilic reagent capable of reacting with nucleophilic groups present in biological systems. The functional groups of such systems will be discussed in the light of their potential nucleophilic capacity.
11.
2-CHLOROETHYL SULFIDES (SULFUR
MUSTARDS)
The reactions of di-Zchloroethyl sulfide have recently been reviewed by Ogston (1948a), Boursnell (1948), and Needham (1948). 1. Reactions in Water
Unlike ordinary alkyl halides, compounds with a halogen atom in the 8-position to a nitrogen or sulfur atom (I) are very reactive in polar solvents, tliough they have no outstanding reactivity in nonpolar solvents. The activating effect of the hetero atom can be ascribed t o its inductive effect, electron repelling in character, which will facilitate the removal of the halogen atom leaving the positively charged carbonium ion (11)
400
W. C. J. ROSS
Price and Wakefield (1947) have suggested that this carbonium ion must be stabilized by,[passing into the cyclic ethylene sulfonium structure (111) since it is able t o discriminate between the molecular species with which it might react. It is also possible to regard the tendency to form a cyclic structure as the driving force for the ionization: CHz / \ R.8 ............ CHa.*.*.Cl+ R.5
+ c1-
the reaction being an internal bimolecular one (Lord, 1946; Hirst, 1950). However, Price and Wakefield (1947) consider that the formation of the ion precedes cyclization, and though Ogston’s work threw no light on the chemical nature of the activated form, he was able to rule out the existence in reaction mixtures of significant concentrations of an internal sulfonium compound. Stein and Fruton (1946) also found no evidence for the accumulation of a cyclic form in solutions containing di-2-chloroethyl sulfide. The formation of such a cyclic ion will depend on whether the tendency of the lone pair of electrons on the sulfur atom to form a bond with the 8-carbon atom is sufficiently powerful to distort the valency angles of carbon or whether a more stable structure can be produced if the &carbon atom coordinates with an electron pair from an external source such as a water molecule. That it is possible for an inductive effect to modify the reaction at the P-carbon atom without the necessity of postulating a cyclic intermediary is shown by the relative ease with which esters of hydroxyethyl sulfides are hydrolyzed as compared with ethyl esters (Davis and Ross, 1950). Whatever the merits of the sulfoniurn ion theory it is possible to interpret the reactions of di-2-chloroethyl sulfide on the basis of a primary ionization t o yield a carbonium ion (11). With a water molecule in dilute solution the ion will react thus: R.S*CH&Hz+
+
In more concentrated solutions the sulfur atom in a second molecule of sulfide becomes an effective competitor for reaction with the ion (11), the hydroxyethyl sulfide being more likely to react than the chloroethyl sulfide on account of the greater availability of the electrons in the sulfur atom of the former substance, and a sulfonium ion is formed:
401
CYTOTOXIC ALKYLATING AGENTS
R.SGHzCHz+
+ :S
+/
\
R.S.CH2CHzS CHaCHnOH
\
R
03) CHzCHzOH
Mustard gas hydrolyzes in dilute ( <0.1%) aqueous solution to form thiodiglycol (V), equal amounts of hydrogen and chloride ion being eliminated (Stein et al., 1946b). The velocity constant for this hydrolysis is 0.012 mine-' at lo", 0.047 min.-l at 20°, and 0.21 min.-' at 30" (Ball et al., 1942), indicating that the energy of activation for the process is 17,500 calories per gram mol. As a result of more exact measurements Bartlett and Swain (1949) found a value of 0.15 min.-' a t 25'; Ogston (1941) records the figure 0.09 min.-' at this temperature. Ogston also found that the rate of reaction was independent of pH over the range 5-10 though it was very sensitive to changes in solvent composition; for example, at 25" the velocity constant in 50% alcohol is 0.018 min.-' and in 90% alcohol is 0.0003 min.-' The rate constant for the hydrolysis of the chlorohydrin (IV) in dilute solution a t 25" is 0.23 fin.-' (Ball et al., 1942); Bartlett and Swain (1949) record the value 0.26 min.-' at 25'. This increase in reaction rate following the replacement of a strongly electron attracting chlorine atom in di-2-chloroethyl sulfide by a hydroxyl group would have been anticipated in the light of the proposed reaction mechanism since the structural modification results in a greater electron availability a t the remaining chlorine atom. If di-chloroethyl sulfide and water are allowed to react in the proportions of 1:3 then very little acidity is developed, though appreciable amounts of chloride ion are eliminated; this indicates the formation of considerable quantities of sulfonium chlorides (e.g., VI). When the dihalide and water react together at 20" for twenty-four hours in the proportions 1:50, then 78% of hydrolysis (reaction a) occurs and 22% of sulfonium salt is formed (reaction b). On heating the reaction mixture to boiling point the sulfonium salts break down thus:
The extra acidity produced is a measure of the sulfonium halide originally present (Stein et al., 194613). The following scheme shows the products which have been isolated from the reaction of di-2-chloroethyl sulfide in water alone:
402 /CHzCHzCI
s\ CHzCHzCl
p
HIO
CHaCHaCl +/ CHZCHZOH CHzCHay\ I CHzCHzOH’ c1-
W. C. J. ROSS
,CHzCHzOH
s\CHzCHpCl (IV)
EIzo
+;.;I
/ CHzCHzOH
s\CHzCHzOH (V)
//ko
/CHzCHzOH CHzCHzOH
/
s\
s\CH~CH~~Y
~‘CH~CH~OH
+A
/ 4 0
a-
Q1,CHpCHzOH
CH~CH~A-CH~CH~OH /
s\ CH,CH,~+--CH~CH,OH
i
‘CH z c1-
~
~
z
~
~
(VU
The disulfonium salt (VI) can apparently dissociate under mild conditions for when it is kept at pH 7.6 in an aqueous bicarbonate buffer acidity is developed and if thiosulfate is present in the reaction mixture there is evidence for ester formation. (VI) has a relatively high toxicity and is one-tenth as effective as mustard gas in causing necrosis (Stein et aZ., 1946b). 1. Reactions with Anions The reactions of di-2-chloroethyl sulfide in solutions containing organic and inorganic anions have been studied most extensively by Moore et al. (1946), Ogston (1948b), and Ball et aZ. (1942). If a 2-chloroethyl sulfide is allowed to react in a solution containing an added anion then clearly this anion will compete with water and sulfide for reaction with the carbonium ion (11);the undissociated form of an acid not being nucleophilic does not react. The chloride ion eliminated from the halide will also be competing and in the resulting solution the following reactions will be taking place: R.S.CHzCHzOH +
R.S.CHaCHzC1
*R.S*CH~CHZSR’I H / . /
+ H+
(c)
(4
R-S*EzCHa-
+ c1-
A- kC1
R.S‘CHaCHzC1
(4
403
CYTOTOXIC ALKYLATINO AGENTS
In dilute solutions when the concentration of anion is considerably greater than that of the chloroethyl sulfide reactions (d) and (e) can generally be ignored and the rate of formation of ester (reaction f) divided by the rate of formation of hydroxy compound (reaction c) will be kA[A-]/k,[H20]. This ratio measures the relative affinitx of the anion for the carbonium ion as compared with the affinity of the water molecule. The reaction of 2-chloroethyl sulfides in aqueous solutions has been shown to be effectively of the S,l type (Ogston, 1948b), the rate of reaction being of first order with respect to halide and independent of added reagents but affected by changes in solvent composition and temperature. In dilute solutions and in the absence of added anions the rate of production of the carbonium ion will be practically identical with the rate of elimination of hydrogen ions, but in the presence of an anion the rate of elimination of hydrogen ions is retarded. This retardation is obviously proportional to the amount of ester formed since the carbonium ion is produced a t a steady rate. The difference between the amount of acid produced in water alone, Ho, and in the presence of added anion, Ha, which can be directly titrated, is a measure of the amount of ester formed. The amount of ester formed will be dependent on the concentration of anion and on the rate constant, thus we may write:
or
Amount of ester formed HO- Ha =-Ha Amount of hydroxy compound formed = FA Ho - H a HJA-1 k,[HzOI
kr
--~A[A-] k,[H~0]
This expression F A has been termed the “competition factor” of the anion by Ogston (1948a). High values of F A thus indicate a high affinity of the anion for the electrophilic carbonium ion; when the anion is present in a solution a t a concentration of l/FAin the absence of other reactants one-half of the chloroethyl sulfide will be converted into the corresponding ester. Ogston (1941, 1948b) and Ball et al. (1942) have determined the competition factors of a wide range of anions toward the carbonium ion derived from sulfur mustard gas and have remarked on the enormous variation in the values, some of which are shown in Table I. Ball et al. (1942) have discussed the influence of the structure of the anion on its competition factor. They point out that in an organic acid, R.C02H, the electron donating potentiality of the group R determines the dissociation constant of the acid and it might be expected that anions derived from acids with low dissociation constants, in which RC02would be strongly nucleophilic thus hindering the ionization of H, should
404
W. C. J. ROSS
TABLE I ~~~
Competition Factor F
Anion
PH
1.2 x 106 3.4 x 104 2.7 x 104
Ethane dithiophosphonate* Diethyldithiocarbamate* Thiosulfate* Phosphate* Chloride* Acetate* Laotatet Nitratet
8 8 8 8
75
7 7
21 10 1.1 0.2
-
* Ogaton (1948a).
t Ball ef ol. (1942).
have high competition factors. There is no general correlation between acid strength and the competition factor of the anion hence other factors are operative. TABLE I1
Competition Factor F A
Anion Adipate Glutarate Succinate Malonate Oxalate Maleate Fumarate Citraconate
COZ-*CHZCHZCH&H~*COBCO~-*CHZCHZCH~.COICOz-CHzCHn. COzCOz-.CHz*COz-
co~-~coz-
COZ-~CH= CHCOn- cis COZ-CH = CHCOz- trans COZ-CH = C(CH*)C02- trans
10.8 10.2 13.8 16.8 21.8 21.6 21.8 24.3
Table 11, taken from a report of Ball et aE. (1942), shows that the proximity of the second carboxyl group increases the value of FA, this effect falling off with distance although the conjugated system in maleic and fumaric acids can transmit the effect. They relate the high value of FA for sulfur containing anions to the presence of negatively charged sulfur atoms possessing unshared electron pairs and draw attention to the following series : -
-
:o:
: ..o : .. : ..s : ..o : :o:
Thiosulfate F = 27,000
Sulfite F = 1500
.. : ..o :*.
: ..o : ..s : ..o : : o.. : Sulfate F - 5
CYTOTOXIC ALEYLATINQ AQENTS
405
This clearly shows the effect of increasing the number of lone pairs on a sulfur atom in an anion. The carbonium ions derived from chloroethyl sulfides will react with the ionized forms of thiols, phenols, and thiophenols; aliphatic alcohols will not yield significant amounts of anion under physiological conditions and reaction with carbohydrates has not been observed. Stevens et al. (194th) found that the phenolic group in tyrosine (pK, 10) did not react at pH 6-8 but did so when the pH was raised as would be expected since under more alkaline conditions a higher proportion of the groups would pass into the phenolate ion form. High values for the competition factors of thiols and thiophenols have been recorded (Ogston, 1941, 1948b; Ball et al., 1942) as would have been anticipated from the presence of sulfur atoms possessing unshared electrons. It is, however, possible that the figures cited do not represent the true values at pH 7.5 because of a limitation of the method of determination. For example Ball et al. (1942) added a small excess of sodium hydroxide to a solution of mustard gas containing the thiol and followed the development of acidity by timing the change in color of an indicator. Though the pH of the solutions before each addition of alkali was undoubtedly 7.5, it can be calculated from their quantities that just after the addition of alkali the pH of the solution was 10.5. Since it has been established that only the anionic form of an acid or thiol can react with a carbonium ion (Ross, 1950a), it is clear that the pH of the solution is an all important factor and must be kept constant throughout the determination if variation is likely to cause a change in the proportion of anion present. For acids with pK, below 7 no appreciable error will be introduced by the aforementioned procedure but for thiophenols, for example, with pK, values in the range 7-10 then the relative amounts of substance in the reactive anionic form will constantly change throughout the determination and the value of F A found will necessarily be much higher than the true value in a solution buffered a t the stated pH. There can be no doubt that the F A value for an anion R.S- is very high, but an explanation of the finding that the mustards as a class are not inhibitors of -SH enzymes probably lies in the fact that relatively small proportions of the -SH groups present are in the reactive form at physiological pH. Competition factors of 1200 for cysteine and 1700 for cysteine ethyl ester at pH 7 are recorded by Ogston (1948b). As stated these values are probably too high but they do indicate high reactivity in the pH range 7-10. Stein et al. (1946a) and Boursnell et al. (1946a) have shown that di-2-chloroethyl sulfide reacts with cysteine under slightly alkaline conditions, which will, of course, favor reaction with the -SH group in the absence of other competitors, and the product (VII) has been isolated by the former workers.
406
W. C. J. ROSS
/ S \
x“’
CH&H&3CHi H*COsH CHzCHISCHzCH*COiH
I
NHa
VII
The object of the work on competition factors was to find a substance of sufficiently high reactive ability to be of therapeutic value; the aim being t o maintain a high concentration of the compound at the site of action of the mustard gas and thus protect the tissues. Generally this attempt at “competition therapy” was not successful. One explanation of this failure is, no doubt, to be found in the relative impermeability of cellular membranes to ionized as compared to un-ionized molecules. Thus the mustard gas molecule will be able to diffuse into the cell and there react while the substance of high competition factor-most of those tested in this way appear to have been sulfur-containing anions-cannot diffuse to any extent and are therefore unable to protect the system. 3. Reactions with Bases
The ability of mustard gas to react with amines has been recognized for a long time, but the conditions used by earlier workers could not be compared with those encountered in uivo. For example, Davies (1920) obtained thiazan (VIII, R = H) by the reaction of di-2-chloroethyl sulfide with alcoholic ammonia in a sealed tube; Lawson and Reid (1925) prepared a series of N-alkyl thiazans (VIII, R=alkyl) by refluxing an alcoholic solution of a primary amine containing sodium carbonate with the dihalide, and Ok&E (1934) similarly prepared N-phenylthiazan by reaction with aniline. Under these conditions secondary amines react to give derivatives of structure (IX) whilst prolonged treatment of tertiary amines, such as trimethylamine and pyridine, with di-2-chloroethyl sulfide affords diquaternary compounds (X) (Lawson and Reid, 1925). c1-
I
/CHzCHz \
S
‘CH2CH2
/N*R
(VIII)
/
S \
CHaCHaNRa CHaCHzNRi
(IX)
/CH~CH~&R~
S \
CH~CH&R~ I c1-
(X)
CYTOTOXIC ALKYLATING AGENTS
407
Cashmore and McCombie (1923) described the preparation of diglycinodiethyl sulfide (XI) by heating sulfur mustard gas with glycine in an alcoholic solution containing sodium acetate and carbonate while Boursnell et al. (1946a) obtained 1,Cthiazan-Cacetic acid (XII)under identical conditions, but they were unable to prepare any identifiable derivatives of amino acids under physiological conditions of pH and temperature.
/
S \
CHnCHiNHCHzCOzH CHzCHzNHCHzC02H
/CHzCHz \ 13 \ /N.CHzCozH CHzCHz
(XI)
(XII)
The reactions so far described can belregarded as bimolecular displacements of the chlorine atom not involving a free carbonium ion mechanism. Ball et al. (1942) have pointed out that though amines, in the form RNH2 but not in the cationic form RNHa+, are very effective nucleophilic reagents by virtue of the lone pair of electrons on the nitrogen atom and would therefore compete efficiently for reaction with carbonium ions derived from chloroethyl sulfides in aqueous media, many naturally occurring amines are too highly dissociated at physiological pH for any appreciable concentration of the reactive form to be present. Possible exceptions to this generalization are the terminal a-amino groups (pK,, 7.5-8.5) and the imidazole group of histidine (pK, 6-7) occurring in proteins. The amine groups in amino acids will be unreactive owing to internal salt formation. Thus the ability of amines to react under conditions likely to be encountered in vivo is limited to the less basic types. Moore et al. (1946) have demonstrated a reaction between di-2-chloroethyl sulfide and glycine, alanine, ly sine, glycylglycine, and benzoyllysineamide in water under slightly alkaline conditions by measuring the decrease in amino nitrogen (Van Slyke method) following treatment. These conditions would tend to increase the proportion of the amino groups in the reactive form; as would be expected, no reaction with the amino groups in glycine or glycylglycine could be observed under acid conditions. The 2-chloroethyl sulfide (XIII) CHz.S.CHzCHzC1
also reacts with various amino acids at pH 8 (Stein et al., 1946b) and, as in the case of mustard gas glycylglycine appears to react more completely than glycine. If the p H is raised to 9.5, then a more complete reaction
408
W. C. J. ROSS
with the amino acid is noted: this change would, of course, still further increase the proportion of the amino groups in the reactive form. The 2-Chloroethyl sulfide (XIII) reacts more extensively a t pH 8 with pyridine (pK, = 5.2) than with triethylamine (pK, = 10.8); this again is attributable to the higher proportion of the former base which is in the undissociated form. Ogston (1948b) has measured the competition factors of pyridine (54), trimethylamine (6.7), D-L-alanylalanine (IS), and D-L-leucylglycylglycine (19); the values cited here are nominally those at pH 7 but the method of estimation used (see p. 405) probably results in these figures being somewhat too high. The ability of the carbonium ion to react with the sulfur atom in a thio ether has already been indicated (p. 400) by the formation of sulfonium compounds during simple hydrolysis. Stein and Moore (1946) have demonstrated the reaction of methionine with di-2-chloroethyl sulfide under mild conditions to give (XIV) which breaks down on boiling in water for two hours to give (XV). A reaction was also established with the thio ether group in carbobenzoxymethionine amide.
iH8
S(CHzCH2 CHeCHn I
C1
Ha0 loOD2 hr.
S(CH2CHdCHs)i
WV)
(XV)
4. Reactions with Proteins A number of reviews of the reactions of di-2-chloroethyl sulfide with proteins have appeared recently (Herriott, 1947; Boursnell, 1948; Philips, 1950), and the reader is referred to these for an extensive bibliography. The effect of this agent on enzyme systems is outside the scope of this article but a recent review has been published (Needham, 1948). The extent to which different groups in proteins will react with the carbonium ion derived from a 2-chloroethyl sulfide will depend upon the conditions of the experiment under which the treatment is performed. Carboxyl groups (pK, 3-4) will react under most conditions in aqueous solutions since they will be present in the reactive anionic form in all except the most strongly acid solutions. Reaction with amino groups will be favored by more alkaline conditions as will reactions with thiol groups. The ratio of chloroethyl sulfide to protein will also be of importance in deciding whether a particular group is likely to react (see the discussion in the concluding section), for once the more reactive groups are covered, the less reactive centers can then react if there is still reagent available. It is therefore apparent that unless the pH is rigidly controlled and the ratio of chloroethyl sulfide to protein is kept down to that
CYTOTOXIC ALKYLATINO AGENTS
409
likely to be present in vim, the results will have little bearing on the reactions of mustard gas when administered to living tissues. The following brief summary of work done on the reactions of mustard gas with proteins must be considered with this reservation in mind. Herriott et a2. (1946) treated a number of proteins at pH 6 and found extensive reaction with carboxyl groups, but except in an isolated experiment on hexokinase there was no reaction with amino groups. Using an excess of mustard gas on kerateine at pH 7.4 Peters and Wakelin (1947) found that one-quarter of the combined vesicant was attached to -SH groups. Davis and Ross (1947) treated horse oxyhemoglobin and serum albumin a t pH 7.5 and 5.5; in the first case carboxyl and imidazole groups reacted but a t the more acid pH only carboxyl groups were involved. The action of mustard gas containing radioactive sulfur as a tracer element on proteins at pH 7.5-8 and a t 37" was studied by Banks et al. (1946), who found no evidence for reaction with amino groups and that reaction with -SH groups could only account for a small part of the combination of the vesicant with serum proteins. Pirie (1947) has suggested that the reaction with collagen in solutions kept a t neutrality by titration with alkali using neutral red as internal indicator involved carboxyl groups but not amine groups. Acid hydrolysis of insulin treated with labeled butyl 2-chloroethyl sulfide at pH 7.4 in a borate buffer yields N-(2-butylmercapto)ethylphenylalanine (Stevens et al., 194813). The chloroethyl sulfide had probably reacted with the terminal amino groups in a phenylalanine moiety of the intact protein. Carpenter et al. (1948) have shown that a large proportion of the vesicant residues bound to certain proteins could be removed by the action of alkali. Thus 67% of the residues attached to insulin treated with labeled butyl 2-chloroethyl sulfide were liberated by the action of 5 % sodium hydroxide at 0" in three hours, whereas under similar conditions the vesicanttreated protein component of tobacco mosaic virus lost 86% of the attached residues. These alkali labile linkages are almost certainly formed by the reaction of the chloroethyl compound with ionized acid groups in the protein. The general picture emerging from this short account is that the more nearly do the conditions approach those likely to be realized in the treatment of living tissues with mustard gas then the more significant does the reaction with carboxyl groups become, though there is always a small percentage of reaction a t other sites. In this connection it is interesting to note that Neukom (1949) reports that di-2-chloroethyl sulfide will cross-link molecules of pectic or alginic acids by reaction with the ionized carboxyl groups. An observation by Fleming et al. (1949) concerning the reaction of
410
W. C. J. ROSS
di-2-chloroethyl sulfide with proteins may throw some light upon the reason for the greater effectiveness of difunctional chloroethyl sulfides as cytotoxic agents. Mustard gas reacts with serum proteins in the presence of sodium phosphate at pH 8-9 giving complexes which must be formed by the reaction of one of the side chains with the protein and the other with the phosphate ion. These complexes give rise to strongly precipitating antisera when injected intravenously into rabbits. If the treatment of the protein is carried out at pH 8-9 in the absence of phosphate then the product does not produce serum precipitins. 6. Reactions with Nucleic Acids
The ability of the compounds of the mustard gas type to induce mutations, cause chromosome breakage, inhibit mitosis, and initiate malignant growth has naturally led to an examination of the reactions of these substances with nucleic acids. Philips (1950) has reviewed work dealing with the consequences of such reaction; the present account will deal with the more purely chemical aspects. Banks et al. (1946) found that under comparable conditions thymus nucleoprotein combined with more chloroethyl sulfide than did mixed human serum proteins. Berenblum and Schoental (1947) showed that thymus nucleoprotein was precipitated from solution following mustard gas treatment, the increased sulfur content of the precipitate indicated that combination had occurred. Electrometric titration of thymus nucleic acid which had been treated in 35% alcohol at pH 7 with an excess of di-2-chloroethyl sulfide suggested that primary and secondary phosphoryl groups and the amino groups of purine and pyrimidine components had reacted (Elmore et al., 1948). These results are in accord with expectation, since the phosphate groups will be present in the reactive anionic form and the amino groups (pK, 3-4) will be in the undissociated state. The extent of the reaction with purine and pyrimidine hydroxyl groups (pK. 9.5) in thymus nucleic acid and guanylic acid was unexpected, but this is possibly accounted for by the large excess of mustard gas used-it is unlikely that such groups would react under milder conditions. One of the two products obtained after treatment of thymus nucleic acid was more viscous than the untreated material, and in this product two phosphoryl groups were blocked by each molecule of di-2-chloroethyl sulfide which reacted. These results were considered as evidence in favor of the formation of intermolecular cross linkages. Butler and Smith (1950) allowed mustard gas to react with calf thymus nucleic acid in a bicarbonate buffer solution under conditions much milder than those used by Elmore et al. (1948). The treatment resulted in the disappearance of the structural viscosity, an effect com-
41 1
CYTOTOXIC ALKYLATINQ AQENTS
parable to that of x-irradiation. The results were consistent with a depolymerization of the nucleic acid molecule. Similar effects were obtained using the monofunctional 2-chloroethylethyl sulfide and 2-chloroethyl 2-hydroxyethyl sulfide; the former was about one-fifth as effective a degrading agent as di-Zchloroethyl sulfide. The nucleic acid moiety of tobacco mosaic virus treated with labeled butyl2-chloroethyl sulfide contained 5 % of the radioactivity acquired by the virus. Since the virus contains only 6% of nucleic acid, this has clearly competed effectively with the protein component for reaction with the halide. Only 33% of the vesicant residues in the treated nucleic acid were alkali labile as compared with 86% of the residues in the protein (Carpenter et al., 1948). Elmore et al. (1948) record that in vesicant-treated guanylic acid 0.65 equivalents of primary and secondary phosphoryl groups react as compared with 1.4 equivalents of amino and hydroxyl groups. They also observed extensive removal of vesicant residues when the pH was raised above 10. If, as seems likely, only the phosphate ester linkages are readily hydrolyzed, then about 32% of the combined vesicant will be alkali labile, a figure comparable with that found by Carpenter et al. (1948), see above,
111. 2-CHLOROETHYLAMINES ALIPHATIC DERIVATIVES (ALIPHATIC NITROGEN MUSTARDS)
1. Reactions in Water
Hanby et al. (1947) observe that the first transformation taking place when a 2-chloroethylamine (XVI) is dissolved in water is the formation of a carbonium ion (XVIII). In the absence of added reactants this ion can react in three ways: (1) with water, (2) with the liberated chloride ion to re-form the original compound, and (3) internally to form an ethyleneimonium ion (XIX).
(XVI)
(XVII)
.
(XVIII) Hb
.
R,N.CHZCHZOH
Cohen et al. (1948) suggest that the strongly polar molecule of 2-chloroethylamine tends to coil up as shown in the transition complex (XVII). Thermal activation of this complex will result in the rupture of the C-Cl bond giving rise t o the carbonium ion. This carbonium ion will have only a momentary existence, and in the absence of substances reacting more readily than water the need for sharing an electron pair can be most
W. C. J. ROSS
412
easily satisfied by cyclization as shown. The position of equilibrium between the carbonium ion and the cyclic ion in a series of chloroethylamines will depend upon the relative availability of the lone pair of electrons on the nitrogen atom, that is, upon the basicity of the amine. The dependence of the rate of cyclization upon basicity is shown in Table 111. The velocity constant for the initial stage of the hydrolysis TABLE I11 Amine EtzN*CHnCH&l EtN(CH&HnCl) p N(CHzCHnC1)s
PK.
k min.-l*
k min.-'t
8.82 6.57 4.39
0.145 0.128 0.073
0.202 0.085 0.0055
* Rata conatant for the initial cyclization in water at lKO (Cohcn et al., 1848).
t Rate constant for cyclisation in 2: 1 acetone-water at 26'
(Bartlett et al., 1Q47b).
of methyldi-2-chloroethylamine is 0.0023 min.-' at O", 0.024 min.-' at 15' (Cohen et al., 1948), and 0.096 min.-' at 25" (Hanby et al., 1947). The energy of activation for the process is 24,000 calories per gram molecule (Cohen et al., 1948). The nucleophilic character of the nitrogen atom is much greater than that of the sulfur atom, and therefore it is not surprising to find that ethyleneimonium ions have much greater stability than the postulated ethylenesulfonium ions, and derivatives of the former type have been isolated and well characterized (Golumbic et al., 1946a; Fruton and Bergmann, 1946). The stability of these cyclic onium ions is also shown by the fact that when, for example, methyldi-2-chloroethylaminereacts in water there is a rapid elimination of one equivalent of chloride ion but very little liberation of hydrogen ion (Golumbic et al., 1946a). The subsequent reaction of the cyclic ion with water or the chloride ion could involve either a unimolecular reaction (i), which implies ring opening before reaction, or a bimolecular reaction (ii), ring fission occurring at the approach of a reagent. With water the reactions would proceed as follows:
CYTOTOXIC ALEYLATING AGENTS
[
413
These two separate mechanisms have also been postulated for the opening of epoxide rings particularly in the case of the conjugate acids H--/[~~]-
which are similar to the ethyleneimonium ions
(Kadesch, 1946). The presence of the cyclic ion in fresh solutions of 2-chloroethylamines can be demonstrated by adding sodium thiosulfate and measuring the amount of reagent consumed after ten minutes (Golumbic et al., 1946a). The so-called instantaneous thiosulfate titer has been used as a measure of the concentration of such onium ions. A further proof of the existence of stabilized cyclic ions has been provided by S. D. Ross (1947) who showed that l-diethylamino-2-chloropropane (XX) gave the rearranged product, 2-diethylamino-propanol-1 (XXII), on hydrolysis with sodium hydroxide. This result is readily comprehended if the cyclic ion (XXI) is an intermediate in the reaction; ring fission takes place in a different direction from ring formation. EtzN.CHzCHC1
--D
I CHs
Etz& r2 \ CH.CH8
+ c1-
+ HzO
+
EtzN.CHCHzOH
I
CHr
In the above experiment a primary alcohol is formed by the opening of the substituted ethyleneimonium ring, and it is interesting to note that Kerwin et al. (1947) obtained the secondary alcohol, l-dibenzyl-amino2-butanol (XXV) when 2-dibenzylamino-l-chlorobutane(XXIII) was hydrolyzed with bicarbonate in aqueous alcohol. The cyclic ion (XXIV) is clearly involved in the reaction. CHz
+ (PhCHz)zN*CHCHzCl+ (PhCHz)zN+ < [ H c ~ z ~ ~(PhCHdzNCHzCHOH hHzCHn (XXIII)
+a-
AHzCH8 (XXIV)
(XXV)
I n unbuffered solutions-the concentration of amine being about 1%-the hydrolysis of methyldi-2-chloroethylamineceases when approximately one equivalent of hydrochloric acid has been produced (Golumbic et al., 1946a; Hanby et al., 1947) this is due to the stabilization of the chlorohydrin by salt formation (XXVI) for further cyclization cannot occur while the nitrogen atom carries a positive charge.
414
W.
C. J. ROSS
Thus in contrast to the 2-chloroethyl sulfides the rate of reaction of aliphatic nitrogen mustards will be dependent upon the p H of the solution. This point is of importance in the following connection: a t physiological pH an amine such as dimethyl-2-chloroethylamine(pK, 8.6) will be present in solution largely (90%) as the ammonium cation which cannot yield a carbonium ion; this clearly restricts the ability of the more basic chloroethylamines t o react with biological material in vivo (Davis et d.,1950). In buffered solutions the hydrolysis of methyldi-2-chloroethylamine can proceed to completion and as in the case of sulfur mustard gas dimeriaation is possible, both cyclic and linear dimers being produced with the nitrogen compounds. The sequence of reactions for methyldi2-chloroethylamine is shown below.
CH&$
Yn
Ha fC1CHnCHnCl /Ha0
CHaCHzOH CHa<
+
* /
cH";i"'cl C1CHaq-YHs CHzCHzOH CHz
CYTOTOXIC ALKYLATING AGENTS
415
The reactions of ethyldi-2-chloroethylamine and tri-Zchloroethylamine are very similar to those of the methyl derivative (HN2), but the ethyl compound has rather less tendency to form dimers, possibly because of steric factors (Bartlett et al., 1947a), and the trichloro derivative forms only small amounts of dimer in relatively concentrated solution (Golumbic et al., 1946b). 2. Reactions with Anions
When an aliphatic nitrogen mustard is allowed to react in a solution containing an anion a substituted aminoethyl ester is formed. The reaction may be regarded as a direct attack by the anion on the cyclic system (bimolecular reaction), or a reaction of the anion with a carbonium ion formed by the preliminary opening of the ethyleneimonium ion (unimolecular reaction cf. p. 412), or again reaction between the anion and the carbonium ion formed directly after the ionization of the chlorine atom (p. 411) may proceed without cyclization. The formation of a cyclic ion in such solutions will depend on the relative nucleophilic capacity of the anion. A powerful nucleophilic anion such as the thiosulfate ion can probably satisfy the need of the carbonium ion for sharing an electron pair more efficiently than the nitrogen atom. In the case of anions of lesser nucleophilic power such as those derived from organic acids, cyclization will precede ester formation even though eventually all the cyclic ion may be converted into ester since this is the more stable structure. Fruton et al. (1946a) allowed methyl- and ethyl-di-2-chloroethylamine to react in slightly alkaline solutions containing either sodium acetate or hippurate. They found no evidence for ester formation under these conditions and ascribed this to the known ease of hydrolysis of esters of methyldi-2-hydroxyethylamine (for example Cohen and Artsdalen, quoted by Fruton et al. (1946a), found that the propionate was readily hydrolyzed at pH 7.4). On the other hand tri-2-chloroethylamine reacted in solutions containing sodium acetate, hippurate, acetyldehydrophenylalanine, and acetyldehydrophenylalanyldehydrophenylalanine under similar conditions giving esters which were isolated and characterized in the case of the last two anions. The competition factors for anions in their reactions with aliphatic chloroethylamines have not been determined by the method which Ogston (1948b) used for the chloroethyl sulfides, but two other procedures have been employed for assessing their relative reactivities. In the first, Fruton et al. (1946a) allowed an aliphatic nitrogen mustard to react in a solution containing alanine at pH 7.5-8.0 and after a definite time the decrease in the amount of amino nitrogen present in the solution was
416
W. C. J. ROSS
measured by the Van Slyke method. This experiment was repeated with the extra addition of various anions and the decrease in amino nitrogen again measured. The results were expressed as the percentage decrease in the amount of alanine reacting when the extra reagent was added. Clearly the more effectively the anionic reagent competes with alanine for reaction with the mustard the greater the percentage decrease. In this way the reaction of methyl- and ethyl-di-2-chloroethylamineand tri-2-chloroethylamine with anions of the following acids was established : nicotinic acid (in this case reaction was probably mainly with the basic nitrogen atom) , hippuric acid, acetic acid, carbobenaoxy-L-glutamic acid, carbobenaoxy-L-aspartic acid, and DL-methionine. The same technique was also used to confirm reaction with the following anions of phosphoric acids : phosphate, pyrophosphate, glycerophosphate, fructose 1- and 6-phosphate, glucose 3- and 6-phosphate1 cytidine diphosphate, and adenosine triphosphate. The relative competing power of these phosphate anions showed considerable variation, fructose 6-phosphate being significantly more effective than any other. Since aliphatic hydroxyl groups would not be dissociated at the pH used in this method i t is not surprising that no reaction was detectable with desoxyribose. A second method used to estimate the extent of reaction with an anion was to determine the “instantaneous thiosulphate titer,” which measures ethyleneimonium ion concentration, of solutions of the chloroethylamine in a bicarbonate buffer a t various times in the absence and presence of added reagents. Any increase in the rate of disappearance of the ethyleneimonium ion over that in the control was considered due to reaction with the added substance. By this method slight acceleration of the rate was observed when sodium acetate or ammonium chloride was present. 3. Reactions with Bases
Prolonged heating of alkyldi-2-chloroethylamines or -bromoethylamines with an alcoholic solution of aniline results in the formation of Palkyl-1-phenylpiperaaines (Prelog and Stephan, 1935). The first extensive study of the reactions of these halides with basic groups in aqueous solutions under conditions akin to those in biological material was made by Fruton et al. (1946a) who examined the reaction of methyldi-, ethyldi-, and tri-2-chloroethylamine with various amino acids and peptides. They determined the extent of the reaction with the amino group by measuring the decrease in amino nitrogen (estimated by the Van Slyke method). At pH 8 the amino group in the following amino acids was alkylated: glycine, L-alanine, L-serine, DL-threonine, L-glutamic
CYTOTOXIC ALEYLATING AGENTS
417
acid, carginine, L-lysine, L-histidine, and 8-alanine. The pK. of the amino groups in these acids is in the range 9-10 and hence a significant proportion of the amino groups will be in the reactive nonionized form a t pH 8. At pH 9.5 an even greater proportion will be in the reactive form and it is, in fact, found that L-alanine reacts more completely a t this higher pH. The amino groups of L-phenylalanine, m-methionine, and L-tryptophan also react readily with the chloroethylamines a t pH 9.5. The following peptides react more completely than L-alanine at pH 8 ; L-tyrosineamide acetate, glycylglycine, L-leucylglycine, and L-leucylglycylglycine. This higher reactivity would again be expected since the a-amino groups in peptides have pK. in the range 7.5-8.5. The €-amino group in benzoyl-L-lysineamide does not react as extensively a t pH 8 as the various a-amino groups in amino acids; this is in accord with theory since the pK, of the €-amino group is 10.5 and a relatively smaller proportion is in the reactive form at pH 8. The compound (XXVIII) was isolated from the reaction product obtained when methyldi-2-chloroethylamine was added t o a solution containing phenylalanine at pH 9.5, it is formed by the condensation of two molecules of amino acid with one of chloroethylamine.
CHsN
/ \
iHaPh
CHzCHzNH HCOzH
CHZCH~NHCHCO~H AHZPh (XXVIII)
There are indications that at pH 8 alanine and methyldi-2-chloroethylamine react in equimolecular proportions and Fruton et al. (1946a) have suggested that this might be due t o the formation of a derivative of the dichlorocyclic dimer (XXVII) ; it seems equally probable that a piperazine derivative is formed. Chanutin and Gjessing (1946) obtained spectrophotometric evidence that tri-2-chloroethylamine reacted with adenine, guanine, xanthine, and uracil in a borate buffer kept adjusted between pH 8 and 10 by the addition of ammonium hydroxide. In the case of the last two bases reaction is also possible with enolic groups as well as with the nitrogen atoms in the cyclic structure. Gurin et al. (1947) investigated the reaction of methyldi-2-chloroethyl amine with various amines using the method already outlined in which the rate of disappearance of ethyleneimonium ion was measured either in the presence or the absence of base. They found that tertiary amines
418
W. C. J. ROSS
were generally more reactive than secondary amines; that replacement of an N-methyl group in a tertiary amine by a larger alkyl group reduced reactivity; that one carbinol (CH20H) or carboxymethyl (CH2C02H) group can replace a methyl group without loss of reactivity but more such groups have a de-activating effect; that compounds containing the nitrogen atom in a cyclic structure were very reactive toward the ethyleneimonium ion; and finally that the most effective reactants appeared to be those substances containing two or more tertiary nitrogen atoms separated by methylene groups. In particular they found that compounds of the hexamethylenetetramine type were highly reactive toward chloroethylamines. The high reactivity of hexamethylene tetramine has also been observed by Fruton et a2. (1946b). Fruton et al. (1946a) showed that the thioether group in methionine was unreactive towards methyl- and ethyldi-2-chloroethylaminebut there was a tendency t o form a sulfonium chloride with tri-2-chloroethylamine though this tendency was less marked than in the reaction with di-2chloroethyl sulfide. Fruton et aZ. (194613) found that thiodiglycol competed favorably with alanine for reaction with aliphatic chloroethylamines.
4. Reactions with Proteins Only a limited study has been made of the reactions of the aliphatic nitrogen mustards with proteins. Fruton et a2. (1946a) examined the reaction of an excess of methyldi-2-chloroethylamine with aqueous solutions of crystalline egg albumin and of gelatin. It was shown that if the reaction was allowed to proceed for twenty-four hours at 25" and at pH 7.5 then one-quarter of the amino groups in egg albumin and one-third of the amino-groups in gelatin were blocked. No measurements of the reaction with carboxyl or sulfhydryl groups were recorded. Barron et al. (1948) quoting unpublished work of Hellerman and Dixon, state that chloroethylamines react in weakly alkaline solutions with the -SH groups in denatured albumin. The reaction, under conditions not specified, is apparently a very rapid one since one-half of the sulfhydryl groups of the protein are involved within five minutes. Despite this high reactivity of the halogenated alkylamines towards the -SH groups, so-called -SH enzymes such as succinoxidase, adenosine triphosphatase, papain, D-amino acid oxidase, phosphoglyceraldehyde dehydrogenase, carboxylase, and transaminase are not affected by M/1000 solutions of aliphatic nitrogen mustards. The phosphokinases are the most sensitive toward nitrogen and sulfur mustards. Barron et aZ. (1948) regard the alkylchloroethylamines as structural inhibitors of enzyme systems with special characteristics because of the seemingly irreversible combination between the agents and the protein.
CYTOTOXIC ALKYLATING AGENTS
419
6. Reactions with Nucleic Acids
Gjessing and Chanutin (1946) established the depolymerizing action of methyldi- and tri-2-chloroethylamine on sodium thymonucleate which they associated with the chemical reactivity of the mustards since competing anions, particularly the thiosulfate ion, inhibited the effect. Evidence was also obtained for a chemical combination between tri-2chloroethylamine and nucleic acids from a study of the absorption spectrum of treated thymonucleate. Butler and his co-workers (Butler et al., 1950; Butler and Smith, 1950) showed that when dilute solutions of thymus deoxyribonucleic acid were treated with methyldi-2-chloroethyl amine a t pH 7-9 the characteristic structural viscosity of the acid was destroyed, an effect very reminiscent of the action of x-rays on this material. Preliminary evidence indicated that the product of the reaction between the mustard and the nucleic acid was markedly heterogeneous and that considerable lowering of the molecular weight had occurred (Conway et aZ., 1950). Dimethyl-2chloroethylamine did not exert any comparable effect on the viscosity. These workers have discussed the similarity of the effects of x-rays and of methyldi-2-chloroethylamine and di-2-chloroethyl sulfide on nucleic acids (see also Butler and Conway, 1950) and have suggested that an initial combination of one side chain of the mustard with the acid is later followed by the reaction of the second side chain with the same or another nucleic acid molecule resulting in a breaking down of the nucleic acid structure. A somewhat similar mechanism involving hydroxyl radicals has been postulated for the action of x-rays on nucleic acids; this conception has also been considered by Ross (1950b). The esterification of the phosphoric acid groups of nucleic acids by these alkylating agents would be expected to prevent the association of the nucleic acid with a basic protein and might lead to the dissociation of a nucleoprotein. The possibility that stilbamidine, a strong base which could compete with a histone for combination with phosphoric acid groups, exerts its cytotoxic effect by dissociating some essential cellular nucleoprotein has been considered by Kopac (1947). AROMATIC DERIVATIVES (AROMATIC NITROGEN MUSTARDS)
.'i Reactions in Water
The hydrolysis of aryldi-2-halogenoalkylamines in aqueous acetone has been studied by Ross (1949a), Everett and Ross (1949), and Davis et al. (1950). This solvent was used because of the low solubility of these compounds in water alone. The reaction has been shown to be of the
W. C. J. ROSS
420
sN1 type as judged by the following criteria of Hughes (1941). The hydrolyses exhibit unimolecular kinetics but the value of the rate coefficient gradually falls as the reaction proceeds due to the increasing importance of the back reaction whereby halogenoalkylamine (XXIX) is formed by the recombination of the carbonium ion (XXX) with the halide ion.
R,
(=W
/
. R, PICHt
recombination. ionisation
\
+
CH2CHrC1 (xxx)
;FH,CHI
R"\CHIcHrO~
R-
PCH,OH
\CH~CH,C~
The addition of anions other than the halide ion derived from the mustard causes an increase in the velocity of reaction, but the halide ion causes a considerable decrease in the rate by reversing the ionization step. If the carbonium ion is an intermediate in the reaction taking place in an aqueous solution then the relative amounts of ester and hydroxy compound formed should be independent of the halogen in the halogenoalkylamine; this has been found to be true for the chloro- and bromoethyl derivatives of 2-naphthylamine. In an sN1 reaction the rate determining step is the separation of the halide ion and it therefore follows that electron repelling substituents in R (XXIX) should facilitate this ionization and increase the reaction rate. The effect of substitution in the aromatic nucleus of NN-di-2-chloroethylaniline on the rate of hydrolysis is shown in Table IV. Electron repelling groups such as methoxy and methyl groups have an accelerating effect on the reaction while electron attracting groups like chloro, aldehydo, carbethoxy, and nitro groups have a deactivating effect. The order of this effect supports the proposed s N 1 mechanism : the alternative bimolecular (&2) mechanism would demand that the effect of these substituents was the reverse of that actually found (compare the study of the reaction rates of phenyl chloroethyl sulfides, Baddeley and Bennett, 1933). It was observed earlier that the formation of a cyclic ethyleneimonium ion from the carbonium ion produced by the initial ionization would depend upon the relative availability of the lone pair of electrons on the nitrogen atom. It is, of course, well known that aromatic amines are much weaker bases than their aliphatic counterparts, and it would be expected that the carbonium ions derived from arylhalogenoalkylamines would have much less tendency to cyclire. The stability of the derived ethyleneimonium ion, if formed, would be much lower than that of the aliphatic analogues. No evidence of the kind that has been cited to
CYTOTOXIC ALKYLATING AGENTS
42 1
TABLE IV Effect of Substituents on the Rate of Hydrolysis of Di-2-chloroethylanilines R.N (CHZCH&l) 8 Rate of Hydrolysis %*
R o-Anisyl
0-
Activity as a Tumor Growth Inhibitort
89
OCHa
83 68
38 m-Toluyl Phenyl
Q-
21
D-
20
12
ua=-
p-Chlorophenyl CI
p-Aldehydophenyl CHO /
p-CarbethoxyphenylCOzEt
9
-
2,4-Dinitrophenyl N O Z m -
<1 <1
*
The rate of hydrolysis given here and in subsequent tables refera to the percentage hydrolysis occurring when the substance is heated in 50 % aqueous acetone at 6B0 for one-half hour (Ross. 19498). t As measured against the growth of the transplanted Walker rat carcinoma (Haddow, 1950).
prove the existence of imonium ions in solutions of aliphatic chloroethylamines has been obtained in the case of the aromatic compounds. For example, during the hydrolysis of aryldi-2-halogenoalkylaminea, chloride ions and hydrogen ions are eliminated at equal rates (compare p. 412 where it is stated that only chloride ion is eliminated from the aliphatic derivatives a t the start of the reaction) ;this means that negligible amounts of tertiary nitrogen compounds, such as, imonium ions or piperazine dimers, are being formed during the reaction. The instan-
422
W. C . J. ROSS
taneous thiosulfate titer of solutions of the mustards has been used to detect ethyleneimonium ions, solutions containing aromatic nitrogen mustards do not rapidly consume thiosulfate. A further indication that cyclization of the carbonium ion does not occur is given by the finding that (XXXI) and (XXXII) hydrolyze without rearrangement (compare the results obtained by S. D. Ross (1947) with aliphatic derivatives,
&Ha
(XXXI)
(XXXII)
Attempts to obtain quaternary salts by the action of methyl iodide on a number of aryldi-2-chloroethylamineshave been unsuccessful as might have been anticipated from the low basicity, the pK. of NN-di-2-chloroethylaniline is 2.2, and the presence of two bulky chloroethyl groups. Internal quaternization, such as is required for ethyleneimonium ion formation, would also be expected to be difficult. It was realized, however, by Ross and his co-workers that the possibility of the transitory existence of the cyclic ions could not be entirely ruled out on the available evidence but that if these are formed they must be very short-lived. Another respect in which the aromatic nitrogen mustards differ from the aliphatic compounds is that in unbuffered solutions the hydrolysis is continuous, no arrest occurring a t approximately 50% reaction (p. 413). This is due to the fact that in the dilute solutions employed the pH of the reaction mixture is never lowered sufficiently to convert any significant proportion of the aromatic amine into the ammonium salt form (such as XXVI p. 414). Aryldi-2-halogenoalkylamines hydrolyze more slowly than the aliphatic derivatives; direct comparison cannot be made since the rates of hydrolysis of the aromatic compounds in water alone have not been measured. In 50% acetone the rate constant for the reaction of 2-naphthyldi-2-chloroethylamine is 0.00017 min.-' at 37" and 0.0055 min.-' at 66". The energy of activation for the hydrolysis of the simpler aromatic derivatives in 50% acetone is of the order of 24,000-26,000 calories per gram molecule (Ross, 1950d). The effect of varying the aromatic structure (R in XXIX) upon the ease of hydrolysis is shown in Table V. Variations in the chloroethyl side-chain have a profound effect on the rate of reaction as indicated in Table VI. The enhancing effect of the presence of the nitrogen atom on the reactivity of the chlorine atom is lost when this atom is present
423
CYTOTOXIC ALKYLATING AGENTS
TABLE V Effect of Varying the Ring System R on the Rate of Hydrolysis of Di-2-chloroethylarylamines R
Rate of Hydrolysis
a-
-~
Phenyl
-
20
o - X en y l 0 - b
31
-
-
12
C
H =C
p-Xenyl
D-D-
Stilbenyl U 1-Naphthyl
2-Naphthyl
2-Fluoren yl
I()$
H
1
(Lo, CHz
-D
-
18 50
15
26
9
11
in a 3-chloropropyl or a 6-chlorohexyl group. The effect of introducing a 2’-methyl substituent is very marked, and all 2-chloro-n-propyl derivatives hydrolyze relatively rapidly. The introduction of a 2’-chloromethyl group, on the other hand, decreases reactivity. Table VII shows the effect of varying the halogen on the rate of hydrolysis. There is an increase in reaction rate on passing from chlorine t o bromine, but in all examples of halogenoethyl compounds there is a surprising decrease when the bromine atom is replaced by an iodine
424
W. C. J. ROSS
TABLE VI Effect of Varying the Bide Chain on the Rate of Hydrolysis of Chloroalkylamines, R.NRI'
R'
R Phenyl
phisyl
Rate of Hydrolysis, %
2-Chloroethyl .CH &H &1 3-Chloropropyl .CH2CH2CH2Cl 6-Chlorohexyl .(CHl)&l 2-Chloropropyl .CH&HMeCl
20 1 (in 60 min.) <1 90
2-Chloro-l-cycbhexyl
98
0
/ CJ PChloroethyl CHzCHZC1 2-Hydroxy-3-Chloropropyl CH&HOHCHzCl 2,3-Dichloropropyl CH2CHC1CH&1
68 4
34
TABLE V I I Effect of Varying the Halogen on the Rate of Hydrolysis of Di-2-halogenoalkylamines R*NRz' R PNaphthyl 6- Methyl-2-naphthyl
2-Phenanthryl
3-Phenanthryl
Phenyl
R' CHzCHsCl CHZCHzBr *CHzCHzI *CHzCH&l .CH&HZBr CHzCHzI .CHzCHzCl .CHzCHsBr .CHzCHzI GHzCHzCl .CH,CHZBr *CH&HzI .CH&HzCHzCl CH~CHzCHzBr *CHtCH&HzI
Rate of Hydrolysis % 15 80 64 25 85 74 9 79 62 11 76 64 <1
9 23
atom. The high affinity of the iodide ion for the carbonium ion (XXX) may account for some of this decrease in rate since this affinity will result in a more rapid re-formation of the halide by the back reaction (p. 420), but calculations indicate that this effect cannot account for the extent of the lowering in reaction rate which actually occurs (Davis, 1951). The reaction of the iodo compounds is complex, there being evidence of the existence of a simultaneous unimolecular and bimolecular reaction
CYTOTOXIC ALKYLATINB ABENTS
425
mechanism. It is interesting t o note that in the case of the 3-halogenopropylamines where the reaction mechanism is almost certainly bimolecular the rate of hydrolysis is in the expected order C1 < Br < I. NN-Di-2-chloroethylaniline (XXXIII) is 20 % hydrolyzed under the standard conditions whereas the monochloro derivative (XXXIV) is 58 % hydrolyzed.
(XXXIII)
(XXXIV)
This difference in reaction rate is due to the fact that in the monochloro compound the whole of the electron repelling effect of the nitrogen atom is directed into the one side chain carrying the electron attracting chlorine atom. The electron attracting ability of the chlorine atom in compounds of this type is shown by the lowering of the basicity, which depends on the electron availability at the nitrogen atom, in NN-di-2-chloroethylaniline (XXXIII, pK, = 2.2) and in N-ethyl-N-2-chloroethylaniline (XXXIV, pK, = 3.5) as compared with diethylaniline (pK, = 5.84, all values measured in 50% ’ aqueous alcohol). 6. Reactions with Anions The relative reactivity of various anions with the carbonium ions derived from aryldi-2-chloroalkylamines has been determined by a method similar to that used by Ogston (1948b) in his study of the reactions of di-2-chloroethyl sulfide (Everett and Ross, 1949; Ross, 1949b). The reaction of the halide in aqueous solutions containing added anions can be represented for convenience by the simplified scheme:
+ H+
RC1 RA
If xE and xclrepresent the percentage of hydrogen and chloride ion respectively which is eliminated in a standard time, then it can be seen from the above that xHis a measure of the rate of formation of hydroxy compound whilst zcI - zH is a measure of the rate of formation of ester since zc1 measures the total amount of halide reacting. Thus: .c
Rate of formation of ROH -= Rate of formation of RA
k,[R][HzO] =- XH ZCl - zH kdkl [A-I
426
W. C. J. ROSS
This may be rewritten in the form:
This expression is identical with that used by Ogston to define the competition factor FA of an anion. These factors may be determined by allowing the chloroalkylamine to react in solutions containing known concentrations of anions and then estimating the amounts of hydrogen and chloride ion liberated. The competition factors for a range of organic and inorganic anions have been determined for reaction with 2-naphthyldi-2'-chloroethylamine in 50 % aqueous acetone solution. A selection of the values obtained are given in Table VIII. A comparison of these figures with those in Table I TABLE VIII Anion
Competition Factor in 50% Acetone
Thiosulfate Iodide Chloride Oxalate Bromide Acetate Lactate Fluoride Nitrate
1680 760 250 140 125 100 60 20 4 ~~~~~~
~
shows that the order of reactivity of these anions is the same for both types of chloroethyl compound. The actual values are generally higher in the case of reactions carried out in 50% aqueous acetone; this is partly due to the lower concentration of water molecules, a similar effect being observed in the reactions of epoxides (Ross, 1950~). The formation of esters under the conditions used to determine competition factors has been verified by the isolation and characterization of these derivatives in a number of instances. It has already been mentioned (p. 423) that the replacement of a hydrogen atom in the 2' position by a methyl group has an appreciably enhancing effect on the rate of hydrolysis; it also has the effect of considerably reducing the ability of the derived carbonium ion to react with anions. For example, the competition factor of the acetate ion for reaction with 2-naphthyldi-2'-chloroethylamine is three to four times the factor for reaction with 2-naphthyl-2'-chloro-propylamine. The effect of the methyl group is, no doubt, mainly due to its electron repelling
427
CYTOTOXIC ALKYLATING AGENTS
character which will act against the approach of a negatively charged anion but will not oppose the approach of a neutral water molecule. Some measure of steric hindrance to the larger acetate anion may also be operative. No appreciable ester formation occurs when an aryldi-2-chloroethylamine reacts in a solution containing acetic acid; this confirms the nonreactivity of undissociated carboxyl groups toward carbonium ions. This feature of the reactions of the halides is also demonstrated by the high reactivity of mercaptoethanol and thiophenol under conditions where significant concentrations of the ionized form of the sulfhydryl group may exist. However, in solutions of pH sufficiently low to greatly reduce the concentration of the ionic form of the thiol no reaction with the carbonium ion takes place. The reaction of aryldi-2-halogenoalkylamines in aqueous acetone solutions containing sulfide ions is a very convenient one for the synthesis of aryl-substituted thiazans (Ross, 1950d). 5. Reactions with Bases
Amino groups are nucleophilic by virtue of the lone pair of electrons on the nitrogen atom and would therefore be expected to react readily with carbonium ions. Such reaction has been demonstrated between the ions derived from aryldi-2-halogenoalkylaminesand primary, secondary, and tertiary amines (Ross, 1949~). The products formed when 2-naphthyldi-2’-chloroethylaminereacts with aniline, methylaniline, and dimethylaniline are shown in the following scheme: R,(CHzCHzNHPh CHzCHaNHPh (XXXVI)
R.~cHzCHzC1
PhNHMe
\
CHzCHzCl
\ phNM%
R. (2HzCHzNMePh CHzCHzNMePh (XXXVII)
c1R,$H~CHzj%;%ee i+/Ph ‘ale
CHzCHzN-Me j ‘Ph
c1-
(XxxIX)
Cl(XXXVIII)
W. C. J. ROSS
428
Reaction with aniline gives a high yield of the piperazine (XXXV). This reaction is a general one and can be used very conveniently for the preparation of NN-disubstituted piperazines (Davis and Ross, 1949). The dianilino compound (XXXVI) corresponding to the diglycino derivative (XI, p. 407) and the diphenylalanino derivative (XXVIII, p. 417) could not be detected in the reaction mixture; this is in accord with the finding of Boursnell et al. (19464 that the six-membered heterocyclic ring system is preferentially formed in such reactions. Two products were isolated from the reaction with methylaniline the bis-methylanilino derivative (XXXVII) and the cyclic quaternary compound (XXXVIII). The double quaternary salt (XXXIX) is the product obtained in the reaction with dimethylaniline. The monosubstituted intermediates in the formation of (XXXV), (XXXVII), and (XXXIX) respectively are (XL), (XLI), and (XLII). Cl-
/ R-N \
CHzCHaNHPh CHzCH2Cl
(XL)
/ R.N \
I I +/
CHnCHnNMePh R.N CHzCHnCl
(XLI)
/ \
Ph
CH2CH2N-Me
\
Me
CHaCHnCl
(XLII)
The anilino group in (XL)and the methylanilino group in (XLI) possess nitrogen atoms bearing unshared electron pairs the electron repelling characteristics of which might be expected to reinforce the effect of the nitrogen atom originally present on the ease of ionization of the second chlorine atom. As a result of this effect the presence of a primary or secondary amine increases the overall rate of reaction of the dihalide and since the reaction proceeds by an SN1mechanism this increase is independent of the amine concentration. On the other hand the intermediate (XLII) possesses a positively charged nitrogen atom on one side chain and this would be expected to reduce the rate of ionization of the second chlorine atom. This effect has been observed. The determination of the competition factors of primary amines has not been carried out but it has been established that the more basic amines react most readily; the following order of reactivity of some aromatic amines has been established: p-anisidine > p-toluidine > aniline > 2-naphthylamine > p-chloroaniline. The cationic form of an amine, R.NHs+, is not nucleophilic and would not be expected to react with a carbonium ion. This conclusion has been confirmed by experiment, for in a solution containing 2-naphthyldi2'-chloroethylamine, aniline, and sufficient nitric acid to convert the
CPTOTOXIC ALKYLATINQ AGENTS
429
aniline into its salt no reaction other than simple hydrolysis could be demonstrated. When glycine, alanine, or phenylalanine was present in unbuffered aqueous acetone solutions of NN-di-2-chloroethyl-p-anisidine, no reaction with the amino acid occurred, but when the ethyl ester of glycine was present 1-p-methoxyphenyl-PcarbethoxymethylpiperaEine(XLIII) was formed. CHzCHs
\
CHaO-N’
-
\ CHzCHz/NCHzCozEt (XLIII)
Thioethers react with the chloroethylamines giving sulfonium compounds. Thus evidence for sulfonium formation was obtained in the reaction of the above mentioned p-anisidine compound with phenyl methyl sulfide and also with dibutyl sulfide, the dialkyl sulfide giving the higher yield of sulfonium salt (Ross, 1950a).
4. Reactions with Proteins Preliminary work by Alexander and Fox (1951) has shown that NN-di-2-chloroethyl-p-anisidine is capable of bringing about a small measure of cross linking in wool as judged by changes in supercontraction. Following the reaction, which was carried out in a bicarbonate buffer solution (pH S), evidence was obtained for the alkylation of a proportion of the e-amino groups of the lysine and the phenolic groups in the tyrosine component. No estimate of the extent of the reaction with carboxyl groups was possible since the vesicant residues which were introduced titrated in the same pH range as these acid groups.
Iv. 1,2-EPOXIDES 1 . Reactions in Water
A 1,Zepoxide reacts with water to give a glycol, the reaction being effectively : R.CH-CHz
+ Ha0 ko RCHOH.CHnOH ---t
‘0’
This reaction proceeds slowly in water and the rate constant (ko) for this for ethylene oxide (R = H), spontaneous reaction at 20’ is 3.6 X 2.8 X lo-’ for glycidol (R = -CHzOH), and 9.7 X lo-’ sec-l for epichlorohydrin (R = C1CH2-) (Bronsted et al., 1929). The energy of activation for the spontaneous reaction of ethylene oxide with water is
430
W. C . J. ROSS
19,000 calories per gram molecule (Lichtenstein and Twigg, 1948). The hydration of epoxides is strongly catalyzed by acids, the velocity confor stants for the hydrogen ion catalyzed reaction (ha) being 5.33 X for glycidol, and 4.1 X set.-' for epiethylene oxide, 2.5 X chlorohydrin at 20' (Bronsted et al., 1929). The hydration is also catalyzed by bases. Lichtenstein and Twigg (1948) found that the velocity constant for the hydroxyl ion catalyzed reaction (boa) at 20' for ethylene oxide was 8.1 X lods sec.-l. These workers have pointed out in this reaction whereas in the hydrolythat the ratio koH/ha is about sis of esters and amides it is about lO+4. It has recently been suggested (Ross, 1950c) that whereas in the spontaneous and the base catalyzed reactions the water molecule or the hydroxyl ion attacks the partially polarized oxirane ring (XLIV) a t the carbon atom in the initial stage, in the acid catalyzed reaction the hydrogen ion attacks the oxygen atom. The mechanism may be formulated thus: SPONTANEOUS
R AH
H
R (XLIV)
--i
HO-+ HYDROXYL ION CATALYZED
HOH -,
\
d -CHs
R AH
\
AH
AH\
CHaOH
+k
I-+ + h
\
t oii: Ho...H...A......cH,.....oiiI HO AH CH,OH
HYDROGEN ION CATALYZED
The reactions ultimately involve the combined action of an acid and a base, in the Lowry-Bronsted sense, and are thus examples of the Lowry mechanism (Branch and Calvin, 1941). The suggestion that the spontaneous reaction initially involves nucleophilic attack on the carbon atom and the acid catalyzed reaction involves electrophilic attack on the oxygen atom is consistent with the effect of variations in the group R (in XLIV)
43 1
CYTOTOXIC ALKYLATINQ AGENTS
+
on the rate of reaction. When R is electron attracting, e.g., Rs'N.CH2or ClCHZ-, the electropositivity of the terminal carbon atom will increase and the rates of the spontaneous reactions will also increase: if R is electron repelling, e.g., R~N-CHZ-,CH,-, or CH2:CH-, these rates are reduced. Variations in the group R will have the opposite type of effect on the acid catalyzed reactions since electron attracting groups will reduce the electronegativity of the oxygen atom at which the initial attack of the hydrogen ion is made while electron-repelling groups will facilitate this attack. Ross (1950~)has shown that the predicted effects are observed. It is interesting t o note (Twigg, 1950) that as a consequence of the suggested mechanisms the ratio koa/ko will be independent of the nature of the group R whereas the ratios koa/ka and ko/ka Will be sensitive to the type of substituent, being increased by an electron attracting group. The values of ko, ka, and kOHfor the reaction at 37" of some monoepoxides and also of some diepoxides which exhibit considerable cytotoxic properties have been determined (Ross, 1950~). The available data are shown in Table IX.
TABLE IX Velocity Constants for the Reaction of Various Epoxides in Aqueous Solutions at 37" Epoxide
ko sec.-l
Glycidol Propylene oxide Epichlorohydrin Di-(2,3-epoxypropyl ether) 1,2-3,4Diepoxybutane 1,2-5,6-Diepoxyhexane 1,2-5,6-Diepoxyhexane (in 50% acetone)
1.9 x 2.2 X 5.1 x 1.9 x 1.9 x 6.4 x 0.64
x
10-6
10-6 10-6
10-6
lo-'* lo-'
ka
S€!C.-l
12.5 x 123 X 1.9 x 6.4 x 1.9 x 177 X
10-3 lo-* 10-3 10-3 10-8 lo-*
-
10-6
sec.-l
~ O E
2 . 3 x 10-4 2.8 X ~4.x 5 10-4 -
* This is the rate constant for the initial stages; the reaction gradually slows down in this case, but
in all the other reactions the coefficient remains constant throughout the course of the reaction.
It will be noted that the rate of reaction of 1,2-5,6 diepoxyhexane (XLV) is somewhat faster than would have been expected from its structure. This is probably connected with the formation of 2,5-di(hydroxymethy1)furan (XLVI) during the hydration (Wood and Wiggins, 1949; Wiggins and Wood, 1950). CHz
CHz
CHz
*
CHz
W. C. J. ROSS
432
The rate of hydration of this diepoxide in 50% aqueous acetone is approximately one-tenth of the rate in water alone. 2. Reactions with Anions
It has already been stated above that a nucleophilic group can displace the oxygen atom attached to the terminal carbon atom of a substituted ethylene oxide. Since most anions are strongly nucleophilic it is not surprising to find that they are capable of opening the oxirane ring; esters being formed thus: R AH HOH
\ A-cHI
-[
t A-
R
'
AH
HO...H...A......CH ,....A-
1-
R AH
HO-
+ HA
'CHnA
In the subsequent reaction of the displaced oxygen atom with a water molecule a hydroxyl ion is produced; this results in the solution of an epoxide containing anions becoming alkaline. Titration of this alkalinity affords a convenient method of estimating the extent of the reaction. Anions vary in their ability to react with an epoxide: Bronsted et al. (1929) found that the order of reactivity of a number of anions for reac> thiocyanate tion with epichlorohydrin was: iodide (2.3 X > bromide (1.4 X lo-*) > chloride (2.6 X > acetate (1.5 X (1.4 X > benzoate (1.2 X > formate (1.1 X sec.-l), the figures in brackets being the velocity constants for reaction with the anion at 20'. Ross (1950~)found the following order of reactivity for anions toward 1,2-3,4-diepoxybutane: thiosulphate (100) > iodide (97) > citrate (82.5) > chloride (80.3) > acetate (75.5) > benzoate (65.6) > tartrate (58.1) > oxalate (53.6) > formate (31.2) > nitrate (7.6); in this case the figures in brackets represent the percentage of the epoxide which is converted into ester under identical conditions of reaction. The order of reactivity of the anions is essentially the same in both cases and is comparable with the order of the competition factors of the same ions toward the mustard gas types of compound. The nitrate ion competes only feebly, and the formate ion is the least reactive of the organic ions. The high reactivity of the thiosulfate and iodide ions is common to both types of compound. The anions are clearly being placed in the order of their relative nucleophilic tendencies. The value of the velocity constant for reaction with glycidol is 5.7 X loFe set.-' for the chloride ion at 20' (Bronsted et al., 1929) and 2.3 X set.-' for the hydroxyl ion a t 37" (Ross, 1950~). Whilst these figures cannot be directly compared they do dispose of a statement by Bronsted et al. which has been accepted and discussed b y later workers
CYTOTOXIC ALKYLATING AGENTS
433
(Hammett, 1940; Bartlett et al., 194713; Grunwald and Winstein, 1948; Bartlett and Small, 1950) to the effect that the hydroxyl ion does not react with an epoxide a t a rate comparable with that of the chloride ion. The reaction of ethylene oxide and propylene oxide with the anionic forms of substituted phenols has been studied by Boyd and Marle (1914). Epoxides do not appear to react with aliphatic hydroxyl groups in neutral solution, but will do so if sufficient alkali is added. For example, sucrose, which does not react in purely aqueous solution, reacts readily in the presence of 0.25N-sodium hydroxide (LeMaistre and Seymour, 1948). The p H of such a solution is between 13 and 14 and since the pK of the hydroxyl groups in sucrose is 12.6, an appreciable proportion of these groups will be in the ionic form in the alkaline solution. Nevertheless, the reaction of epoxides with aliphatic hydroxyl groups is unlikely to be important under physiological conditions. Sulfur-containing anions are known to be powerfully nucleophilic and the reaction of an epoxide with the thiosulfate ion, described by Culvenor et al. (1949), has been utilized in a convenient rapid method for the estimation of oxirane oxygen (Ross, 1950~). The rate of reaction of various epoxides with the thiosulfate anion has also been shown to run parallel with their cytotoxic activities. Increasing substitution of the carbon atoms in the oxirane ring reduces this reaction rate and a t the same time reduces the toxicity of the compound and its capacity to inhibit the growth of a transplanted rat carcinoma (Ross, 1950~). Culvenor et al. (1949) have also shown that ethylene oxides react rapidly with sulfides, sulfites, and alkaline solutions of thiols. There are indications that in the same way that substitution on the @-carbonatom of a chloroethylamine reduces the ability of the compound to react with an anion (p. 426), substitution on the terminal carbon atom of an epoxide reduces its ability to react with an anion. Just as the reaction of an epoxide with water may be catalyzed by the presence of hydrogen ions so may the reaction with an anion. The intermediate conjugate acid (XLVII) reacts with the anion to give a hydroxy ester thus:
(XLVII)
However, the acid catalyzed reaction of most epoxides with anions is not important in the pH range 4-8.5 and so is of little significance in the reaction of these epoxides with biological material a t physiological pH.
W. C. J. ROBS
434
An exception may be the reaction of 1,2-5,6-diepoxyhexane which has a high value for the rate coefficient of the acid catalyzed reaction and so this reaction may become appreciable at pH 7.5 (see also below p. 438). 3. Reactions with Bases
As would be expected the nucleophilic alpino group reacts readily with the oxirane ring system. The stepwise formation of triethanolamine from ammonia demonstrates the types of product formed:
The reaction probably involves an attack by the amino group on the carbon atom of the epoxide followed by proton transfer from a water molecule to the oxygen atom (compare p. 430). This mechanism is in accord with the finding of Knorr (1899) that the reaction with amines proceeds much more readily in the presence of water and may not occur a t all under completely anhydrous conditions. Smith et al. (1946) have studied the reaction of different epoxides with ammonia and with primary, secondary, and tertiary amines. They find that the rates of reaction of amines with epichlorohydrin is greater than with ethylene oxide. As has been pointed out on p. 431 this is consistent with an initial nucleophilic attack on the terminal carbon atom. Smith et al. also find that the rates of reaction of an epoxide with substituted amines are in the following order, the most reactive being placed first: MesN
> MeZNH > MeNHz > NHs
this is the order of basicity of the amines and hence also the order of their nucleophilic capacities. Fraenkel-Conrat (1944) has observed that amine groups react preferentially in alkaline solution; this would be anticipated since the cationic form present in acid solutions, RNHS+, is not nucleophilic. Kiprianov (1926) has isolated a small proportion of the di(hydroxyethy1)amino acid (XLVIII) from the reaction of glycine ester with ethylene oxide but the lactone (XLIX) constitutes the major part of the product. (HOCHzCHz)~NCH,COgH
I /
HOCHzCHzNCHzCO
‘ 0
CHzCHz (XLVIII)
(XLIX)
4. Reactions with Proteins
Fraenkel-Conrat (1944) has shown that water-soluble epoxides such as ethylene oxide and propylene oxide readily react with crystalline egg
435
CYTOTOXIC ALKYLATINO AGENTS
albumin and with p-lactoglobulin under mild conditions. The isoelectric points of the protein derivatives were moved 1 to 3 pH units toward the alkaline side as compared with the starting material, and the new compounds were insoluble in the isoelectric region and more soluble on the acid side than on the alkaline side of the isoelectric point. These effects on the properties of the protein are consistent with the esterification of a high proportion of the carboxyl groups. Evidence was also obtained for the reaction of the epoxides, when present in large excess, with phenolic, primary amino, and sulfhydryl groups; the two former groups reacted more completely in alkaline than in acid solutions. No evidence for the further reaction of the newly introduced hydroxyalkyl groups with epoxide was obtained, and it was considered unlikely that aliphatic hydroxyl groups would react under the mild conditions used. The hydroxyalkyl group linkages were stated to be surprisingly resistant to both acid and alkaline hydrolysis though small proportions of the substituted carboxyl and amino groups were hydrolyzed. 1,2-5,6-Dianhydro-3,4-acetonemannitol (L, first prepared by Wiggins, 1946) reacts with the acidic side chains of the wool fiber giving a crosslinked structure (Speakman, 1948). Fibers treated with a 4% solution of this diepoxide a t pH 5 and at 50’ for twenty-four hours show an increased resistance to extension and a reduction in shrinkage in the milling process. CHz
‘o/
.
CH
.
CH
-
CH
A d
.
CH
*
CHz
‘d
‘C/ CH/, ‘CHa
&I It is of interest to note that the reaction of acidic groups in pectic and alginic acids with various epoxides has been demonstrated (Deuel, 1947) and one diepoxide, 1,2-3,4-diepoxybutane, has been shown to cross-link two alginic acid molecules by reaction with the carboxyl groups. 6. Reactions with Nucleic Acids Alexander (1951) has shown that propylene oxide and 1,2-4,5-diepoxypentane react with the phosphate groups in thymus nucleic acid. The reaction with the acid groups was assessed by measuring the uptake of a basic dye, methylene blue, before and after treatment with the agent. The amount of basic dye fixed by the nucleoprotein was practically restored to the initial value following the action of alkali due almost certainly to the hydrolysis of the phosphate ester linkages. The reaction of nucleic acid phosphate groups with the triaaine (LII), methyldi-2-
W. C. J. ROSS
436
chloroethylamine, dimethyl-2-chloroethylamine, and the bis-methanesulfonate (LV) has also been established by this method. An interesting observation was the finding that for equal amounts of esterification of the phosphate groups difunctional compounds were more effective in reducing the affinity of the nucleic acid for protamine than the corresponding monofunctional compounds. If the suggestion that these cytotoxic agents act by preventing the association of nucleic acid with a basic protein (p. 419) is valid then these results may well indicate a reason for the considerable difference in effectiveness between mono- and difunctional alkylating agents. Preliminary work by Butler (1950) has not revealed any significant viscosity changes following the treatment of solutions of thymus nucleic acid with 1,2-3,4-diepoxybutane or 1,2-5,6-diepoxyhexane.
V. MISCELLANEOUS AGENTS Ethyleneimine (LI) and a number of its derivatives, notably 2,4,6triethyleneimino-l,3,5-triazine (LII) and the diethylene derivative of hexamethylenurea (LIII), have recently been shown to have similar effects on cell division and upon the growth of animal tumors to those elicited by the mustard gas types (Buckley et al., 1950; Burchenal et aZ., 1950; Lewis and Crossley, 1950; Sugiure and Stock, 1950; Biesele et al., 1950; Rose et aZ., 1950; Haddow, 1950; Philips, 1950). CHz
CHz
CHz
CHI N.CO.NH(CH2)6NH.CO.N
\
\
N
N
CHz
(LIII)
Very little has been done as yet in studying the reactions of these imines under mild conditions in aqueous solution, but their reactions are probably similar to those of epoxides. For example, Braz and Skorodumov (1947) have shown that ethyleneimine reacts with diethylamine in the presence of water but not in its absence (compare p. 434). Ross (1950~) has established that the triazine (LII) reacts with anions and with water in a manner similar to that of the epoxides. The triazine was characterized by a very high value for the coefficient for the acid catalyzed reactions. This compound has been used as ti cross-linking agent in the
437
CYTOTOXIC ALKYLATINQ AQENTS
textile industry (Preston, 1949), since it will react with the wool fiber under mild conditions. Divinyl sulfone will produce typical “radiomimetic ” effects on the growing root tips of Vicia faba (Loveless, 1950). This sulfone can react with nucleophilic centers such as anions by a Lowry mechanism since the ethylenic double bond is polarized by the proximity of the electron attracting sulfoxide system: R O
HO-H
A
+ &&CH2
s-
+ R.SOzCHzCHzA
O
a+
+ OH-
+ A-
The reaction of the highly reactive thiosulfate ion with divinyl sulfone has been demonstrated by Stahman et al. (1946) and by Ross (1950~). Banks et al. (1946) consider that di-2-chloroethyl sulfone, which is converted into divinyl sulfone, and divinyl sulfone itself react mainly with amino and sulfhydryl groups in proteins, the reaction being of quite a different character from that of di-2-chloroethyl sulfide. Timmis (1949) has prepared p-toluenesulphonic esters of di-2hydroxyethylarylamines: two of these (LIV, R = H or C1) are active as tumor growth inhibitors. The reactions of these compounds will be exactly analogous to those of the halogenoethylarylamines (p. 419).
p-Toluenesulfonyl esters of glycols such as lJ3-propanediol and 2,4pentanediol are not biologically active, but activity has been demonstrated in the case of the dimethanesulfonyl ester of 1,4-butanediol (LV) (Timmis, 1950). This compound reacts with water and with the thiosulfate ion a t a rate comparable with that of the biologically active di(2,3-epoxypropyl)ether. The reactions of the dimethanesulfonyl esters are of the sN2 type; as in the case of the epoxides they involve nucleophilic displacements on carbon and the extent of the reaction is proportional to the concentration of the nucleophilic reagent (Ross, 1950d). VI. DISCUSSION The biologically active compounds discussed above fall into two groups in respect of their reactions in aqueous media. 2-Chloroethyl sulfides and amines react by an 8 N 1 mechanism while epoxides and sulfonyl esters of glycols react by an 812 mechanism. This difference in reaction mechanism has interesting consequences when the transforma-
438
W.
C. J. ROSS
tions of the agents in biological systems are considered. A feature of the S N 1 type of reaction is that the rate at which the reagent reacts is dependent upon the rate of ionization of the compound, which is determined by the medium and is largely independent of the nature and concentration of the groups with which reaction subsequently takes place. On the other hand in the s N 2 mechanism reaction only occurs on the approach of the reacting group. This difference may be represented quite simply thus: SN1 R X + R+ s N 2 ARX
+
+ A.***R*..*X XR+ + A-+ RA + RA 4-X-
The rate of combination of an agent which reacts by the second mechanism is entirely dependent upon the concentration of the group (A) which displaces group (X). This is probably of some importance in the reaction of the compounds with biological systems where the concentration of reacting centers will vary from site t o site. In particular, it will result in an increased amount of an agent which reacts by an s N 2 mechanism being involved in regions where there is a high concentration of groups capable of reacting. Another factor which will influence the amount of reaction at different sites is the hydrogen ion concentration of the environment. This is especially important in the case of those epoxides and ethyleneimines which have a high coefficient for the acid catalyzed reaction (see pp. 434,436). As has been stated, in the case of these compounds reaction is much more rapid under conditions of lower pH. This effect is obviously of importance when a compound is to be administered orally for clearly a compound with a high acid coefficient will be rapidly decomposed in the stomach where the pH is about 1. There is a possibility that this influence of pH upon reactivity may be exploited to obtain more selective action on tumor tissue. Stevens et al. (1950) have pointed out that cancerous tissue is often more acid than normal tissue on account of an increased production of lactic acid, and this acidity may be accentuated by injection of glucose, when the tumor fluids may reach pH 6.5 as compared with a pH of 7.5 in most normal tissues. They have demonstrated a selective deposition of sulfapyrazine, which is less soluble under acid conditions, in the tumor area in rats bearing the transplanted Walker carcinoma. It would seem worth while examining the possibility of obtaining an increased cytotoxic action in malignant growths by similar methods using appropriate epoxides or ethyleneimines. The definitions of the s N 1 and::&:! :mechanisms given above indicate that the group R in each type of compound has a tendency t o combine with a nucleophilic (electron-rich) center. In the case of the s N 1 mechanism this is due to the effective production of a carbonium ion and
CYTOTOXIC ALKYLATINO AGENTS
439
in the SN2prccess it is due to the displacement of a very weak nucleophilic group, e.g., the methanesulfonate ion, from the radical R by a more powerful nucleophilic group. All the compounds now under consideration can therefore be regarded as electrophilic reagents which will react with nucleophilic centers in biological systems. It now becomes of interest to decide which functional groups in such systems are nucleophilic under physiological conditions. The main types of nucleophilic groups likely to be encountered are ionized acid groups such as the anions of organic and inorganic acids (e.g., R C 0 2 - and
\
-P.O-),
/
ionized forms of hydroxy compounds (R-0-) and ionized
sulfhydryl groups (RaS-). Amines and thioethers are also nucleophilic on account of the unshared electron pair on the nitrogen or sulfur atom. Undissociated acid groups and the cationic form of amines (RNHd+) are not nucleophilic. When these facts are realized, it is possible to decide which functional groups are nucleophilic a t pH 7.5. Quite obviously the dissociation constant of a particular group will decide whether in the case of an acid it is present in the reactive anionic form or in the case of a base in the reactive undissociated form (e.g., as RNH2). It is convenient to illustrate this point by considering the groups present in two important biological materials, the proteins and nucleic acids. Table X shows the dissociation constants of the main groups likely to be involved, together with an estimate of the proportion of these groups which will be in the reactive form a t pH 7.5. A high percentage of the carboxyl groups in proteins and the phosphoryl groups in nucleic acids is reactive. The basic groups with which reaction is likely to occur are the terminal a-amino groups of peptide chains and the histidine amino group and also the aromatic type amino groups in the guanine, adenine, and cytosine components of nucleic acids. Relatively small proportions of phenolic, aliphatic hydroxyl, and thiol groups will be in the reactive anionic form a t pH 7.5. Some reaction can occur with undissociated water molecules and probably with undissociated alcoholic and phenolic groups but this will only be of importance if high concentrations of these groups are present in the system. Even in their reactive forms the various groups exhibit appreciable differences in their ability to react with an electrophilic center. This ability to react, which is a measure of the nucleophilic capacity, can be regarded as an “affinity” factor; it is this factor that is measured by Ogston (194813) in his determination of competition factors. It will be remembered (p. 403) that the competition factor of, say, an anion (FA)is defined as kA/lc,[HzO], this being a comparison of the rates a t which the
440
W. C. J. ROSS TABLE X pR. Values of Acidic and Basic Groups in Proteins and in Nucleic Acids
Group -
Proteins a-Carboxyl Carboxyl (aspartyl) Carboxyl (glutamyl) Phenolic hydroxyl (tyrosine) Sulfhydryl (cysteine) Imidazolium (histidine) a-Ammonium (terminal) +Ammonium (lysine) Guanidinium (arginine) Nucleic acids Primary phosphoryl Secondary phosphoryl Aromatic hydroxyl (uracil and thymine) (guanine) Sugar hydroxyl Aromatic amino (cytosine) (adenine) (guanine)
PKO
Fraction of the Groups in the Reactive Form at pH = 7.5 (f)
3.0-3.2 3 .O-4.7 4.4 10.4 10.8 5.6-7.O 7.6-8.4 9.4-10.6 11.6-12.6
0.9999 0.9999-0.999 0.999 0.001 0.0006 0.99-0.76 0.44-0.11 0.01-0.001 0.0001-0.00001
2.0 6.0 10.2 10.1 13 4.2 3.7 2.3
0.9999 0.96 0.002 0.0025 0.00001 0.999 0.999 0,9999
anion and a reference substance, water, react with the agent. Another factor which is important in deciding the extent to which a particular group will react is the concentration of that group in the system. This is especially important when the cytotoxic compound is administered to an animal in the relatively low dosage required t o inhibit tumor growth or to induce tumor formation, for under such conditions a small amount of reagent will be available to react with a large excess of reacting groups. The extent to which any particular type of group can compete for reaction with the electrophilic reagent a t pH 7.5 will be proportional to FA X f X e (where FA is the competition factor of the reactive form of A-this will be the anion in the case of an acid or a thiol and the free base in the case of an amine; f is the fraction of the groups which is in the reactive form a t pH 7.5; and c is the concentration of the particular group in the system). It may now be realized why the results obtained by allowing a large excess of radiomimetic agent to react with various compounds under conditions more acid or more alkaline than physiological pH can be misleading as an indication of the type of reaction likely to be encountered in vivo. In the first place, if a large excess of reagent is employed all
CYTOTOXIC ALKYLATING AGENTS
44 1
potential reacting groups will eventually combine for even in the case of groups with a low value off the combination of the reactive form will lead to the production of more of this form and ultimately the reaction will go to completion. Groups with high values of FA will react rapidly, but when these have combined the agent can then attack groups of lower nucleophilic capacity. Only if the reaction times are kept short can the information obtained by using large excesses of reagent be of value. Secondly, the pH of the solution has often been kept well on the alkaline side, particularly in reactions with the mustards where aciility would otherwise have developed. This procedure quite obviously leads to a far higher concentration of the reactive forms of amines and thiols; indeed, the higher reactivity of amines under alkaline conditions has often been observed (pp. 408, 417, 434). The relative inefficiency of the mustard gas type of compound as an inhibitor of the so-called -SH group of enzymes (Dixon and Needham, 1946) has surprised workers who have demonstrated a very high affinity of the thiol group for mustard gas used in large excess and/or in alkaline solution. Despite the very high value of the competition factor for the ionized thiol group, this high degree of combination would not be expected if limited amounts of reagent were available and the medium was kept at pH 7.5. The reaction of electrophilic reagents with mixed systems under acid conditions has not been studied to any extent, but as Olcott and Fraenkel-Conrat (1947) have pointed out under such conditions the thio ether linkage in proteins is one of the few groups likely to be involved. Consideration of the factors mentioned above suggests that the groups in proteins and nucleic acids most likely to combine with electrophilic reagents in viva are the acidic groups and certain amino groups of favorable dissociation constant. This conclusion appears to be supported by the work of Carpenter et al. (1948; pp. 409, 411) on the reaction of butyl 2-chloroethylsulfide with tobacco mosaic virus. Since compounds, such as diacid chlorides, diisocyanates, and diiodoacetyl compounds, which readily react in aqueous solutions with amino groups and thiols but not to any extent with acid groups, do not appear to be radiomimetic, obe is led t o the conclusion that the various cytotoxic agents owe their especial properties t o an ability to react with ionized acid groups under mild conditions. It is not, of course, suggested that these acids groups are necessarily those in nucleoproteins, the characteristic biological effects may be due to reaction with centers in essential enzymes, vitamins, hormones, or other growth factors. In view of the possible formation of esters by the reaction of the various cytotoxic agents with ionized acid groups, it was clearly of interest to study the relative stability of the ester linkages formed by
442
W. C. J. ROSS
different compounds. Davis and Ross (1950) prepared the acetate and benzoate corresponding t o several chloroethyl amines and sulfides and also certain epoxides and showed that these esters were more readily hydrolyzed under alkaline conditions than simple aliphatic esters. The rates of hydrolysis in alkaline solution do not necessarily bear any direct relationship to the rates at physiological pH; for example, the lability of hydroxy esters derived from epoxides is probably due to the assumption of the ionic form: CHaCHa0.CO.R
/’
-0
which will be present only in more strongly alkaline solution. Nevertheless it was thought that any acidic groups which reacted with the various agents would be only temporarily blocked. This view was expressed earlier by Peters (1947) who considered that regeneration of cell constituents attacked by mustard gas a t acidic groups was quite conceivable. Boursnell e2 al. (1946b) obtained further evidence for this statement when they showed that much of the mustard gas fixed in tissues had disappeared twelve hours after treatment. Until very recently it appeared necessary that there should be at least two alkylating groups in the molecule for a compound t o possess activity as a cytotoxic agent (compare Haddow et al., 1948). This led Goldacre et al. (1949) t o suggest that the two groups were required to permit the molecule to react at two distinct points, either on a single surface or upon two separate surfaces. This hypothesis was extended to suggest that a cross linkage might be formed between sister chromatids and that this would result in breakage of these chromatids during the process of cell division. The cytological picture of chromosome fragmentation and bridge formation was considered consistent with this theory of action. Rose et al. (1950) recognized that the mustards could function as cross-linking agents, but they did not consider that this property was a sufficient condition for biological activity. They suggest that only substances which can polymerize to give linear structures with reactive alkylating groups spaced along the axes are effective. Models showed that in such a polymer, e.g., (LVI) derived from a nitrogen mustard, the chloroethyl side chains appear on alternate sides of the chain a t distances apart which are multiples of 3.7 Angstrom units. Since this distance corresponds to the interpurine or interpyrimidine spacing in nucleic acids or to the interamino acid spacing in extended polypeptides, they considered that mitotic abnormalities might result from an attachment of the polymer at several points to the nucleic acid or protein chains of the chromatids.
443
CYTOTOXIC ALKYLATING AGENTS
Davis and Ross (1950) have pointed out that the formation of such polymers from aromatic nitrogen mustards is rather unlikely and that the polymer, if formed, would have unreactive side chains since the activating effect of the uncharged nitrogen atom on the halogen atom no longer operated. An alternative suggestion by Rose (1950) and his co-workers is more feasible, this is that one side chain of a mustard reacts as an alkylating agent with a center on the protein or nucleic acid chain and that subsequent polymerization occurs between molecules already in combination with this chain-they describe this as a type of “zipfastener ’’
n
(LVU
While the cross-linkage hypothesis was under consideration it became of interest t o decide to what extent the alkylating centers of a molecule could be separated while still retaining biological activity. It was known (see Kon and Roberts, 1950) that it was not essential for two chloroethyl groups to be attached to the same nitrogen atom, and these workers prepared a series of compounds of the general formula (LVII).
c:
Ar.N-(CH&-N.Ar ( H&,.Hal.
A
( H&,.Hal.
OlVII)
They concluded that biological activity is maintained when m = 2 or 3, but gradually falls off as the distance between t,he nitrogen atoms is further increased. Lengthening the halogenoalkyl side chain (e.g., to n = 3) leads to a complete loss of activity. Similar results have been obtained in a series of diepoxides of general formula (LVIII), prepared by Everett and Kon (1950).
(LVIII)
444
W. C. J. ROSS
Preliminary results indicate that in this series activity as an inhibitor of the growth of the transplanted Walker rat carcinoma falls off as n is increased (Haddow, 1950). It is of interest to note that in a series of chloroethyl sulfides of general formula (LIX) ClCHsCHsS(CH~)nSCHs*CHaCl (LIX)
Gasson et al. (1948) found that the vesicant power increased as n increased from 0-3 but then decreased as n increased from 4-10. Although it is diflicult t o draw any definite conclusions from these effects of chain lengthening since such ti modification will alter not only the distance between the alkylating centers but also certain physical properties of the molecule (e.g., lipoid solubility), the results do suggest that increasing this distance beyond a limiting value will reduce biological activity. Later work indicated (Ross, 1950c) that although two reactive centers in the molecule were required for activity these centers need not be of equal reactivity; for example, the effective 1,2-3,4diepoxy-2-methylbutane (LX) and N-2-chloroethyl-N-3-chloropropylaniline(LXII) (Kon and Roberts, 1950) both contained side chains of unequal reactivity.
CHiCHsCl CHsCHiCHiCl &XI11
CH&HsCH,Cl CHzCHnCHnCl (LXIII)
The compounds (LXI) and (LXIII) which contain two of the groups of lower reactivity in (LX) and (LXII) are not biologically active. These results suggested that only one group of high chemical reactivity was required-this would anchor the molecule to a receptor, facilitating the reaction of the second group with another center. The most recent studies have shown that certain monofunctional alkylating agents are radiomimetic in the sense that they will induce mutations, produce chromosome abnormalities, and inhibit the growth of transplanted tumors. Ethyleneimine and 2,4-dimethoxy-6-ethyleneimino-1,3,5-triazine (LXIV) cause cytological abnormalities in the dividing myelocytes in the bone marrow and elicit radiation-like damage in
CYTOTOXIC ALKYLATING AGENTS
445
the cells of the onion root tip. The triadne (LXIV) inhibits the growth of the mouse sarcoma 180. The monoepoxide, glycidol (LXV), evokes nuclear lesions in tissue cultures of mammalian cells (Philips, 1950; Biesele et al., 1950).
(LXIV)
(LXV)
Auerbach and Moser (1950) have shown that 2-chloroethyl-2-hydroxyethyl sulfide and butyl-2-chloroethyl sulfide are mutagenic, inducing sex-linked lethals in Drosophila. Stevens and Mylroie (1950)demonstrated the mutagenic action of butyl-2-chloroethyl sulfide, phenyl-2chloroethyl sulfide and diethyl-2-chloroethylamine on deficient mutant strains of Neurospora crassa, the effectiveness of the last-named compound on the same material has also been established by Jensen, Kirk, and Westergaard (1950). Loveless and Ross (1950) report that ethyl-2chloroethyl sulfide, dimethyl-2-chloroethylamine,ethyleneimine, ethylene oxide, and dimethyl sulfate produce chromosome breakage in plant material. Ethylene oxide has also been shown by Rapoport (1948)and by Bird (1950)to be mutagenic for Drosophila and the mutagenic activity of ethyleneimine was reported by Rapoport (1947a).
CLXVI)
N-(2,4-Dinitrophenyl)-ethyleneimine(LXVI) inhibits the growth of the transplanted Walker rat carcinoma (Rose, 1950; Haddow, 1950). It is of interest to recall that Butler and Smith (1950)found that ethyl-2chloroethyl sulfide was able to degrade nucleic acid in a manner similar to that of x-irradiation. It is thus now apparent that polyfunctional activity is not essential for the production of radiation-like effects in dividing cells. It is equally unnecessary t o postulate a cross linking or a polymerization mechanism for the production of chromosome aberrations. Nevertheless, it would still appear that difunctional compounds are more effective than the corresponding monofunctional agent and these mechanisms may account for the higher activity. Philips (1950)points out that a monoethyleneimine derivative has to be administered at 50 to 100 times the dose level
446
W. C. J. ROSS
of a polyethyleneimine in order to produce a comparable effect on hematopoietic organs or tumor tissue. Loveless (1950)has found that effects in plant material are elicited only by concentrations of monofunctional compounds, chloroethyl amines and sulfides and also epoxides, which are some fifty times that required of bifunctional analogues. The failure of earlier workers to detect cytotoxic effects of monofunctional compounds on tumor tissue in vivo may well have been that higher concentrations of such compounds would have been required than could be achieved in the body. Another point is that the monofunctional nitrogen mustard analogue, dimethyl-2-chloroethylamine,will not be particularly reactive toward nucleophilic centers at pH 7.5. This complication arises because the compound is a relatively strong base and at pH 7.5 about 90% of the amine will be present as an unreactive ammonium cation and also it will have a greater tendency to react to give a quaternary salt (p. 414) (Davis et al., 1950). It was the lack of biological activity of this amine that led earlier workers to believe that two reactive side chains were essential. Philips (1950) and Biesele et al. (1950) believe that cytotoxic action is dependent upon the presence in the compound, or its active intermediate, of an unstable three-membered heterocyclic ring system, but Loveless and Ross (1950)consider that the ability to react with nucleophilic centers effectively through a carbonium ion mechanism is a more general feature of this group of radiomimetic agents. The ability to react through a nucleophilic displacement on a carbon atom appears to cover the cases of the methane sulfonyl compound (p. 437) and dimethyl sulfate which cannot form three-membered heterocyclic ring systems. Although in the light of the later work it does not appear essential to have two or more functional groups in the molecule of a radiomimetic compound, one may conclude with Philips that duplication of reactive centers does increase the selective action of the compound toward proliferating cells. The outstanding feature of the group of cytotoxic agents discussed in this review is their ability to function as alkylating agents under the mild conditions arising in living tissues. REFERENCES Alexander, P. 1951. Personal communication. Alexander, P., and Fox, M. 1951. Personal communication. Auerbach, C. 1949. Heriditus, Suppl. Vol., 12847. Auerbach, C., and Moser, H. 1950. Nature 166, 1019-20. Baddeley, G., and Bennett, G. M. 1933. J. Chem. Sac. 261-68. Ball, E. G., Doering, W. E., and Linetead, R. P. 1942. OSRD Formal Report No. 1094, December 9. Banks, T. E., Boursnell, J. C., Francis, G . E., Hopwood, F. L., and Wormall, A. 1946. Biochem. J. 40, 745-56.
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Neukom, H. 1949. Mitteilung aus dem Agrikulturchemischen Institut E. T. H., Zurich. Ogston, A. G. 1941. Report to the Ministry of Supply (London) by Peters, No. 34. Ogston, A. G. 1948a. Biochem. SOC.Symposia 2, 2-7. Ogston, A. G. 1948b. Trans. Faraday SOC.44, 45-52. Ok&E,A. 1934. Chem. Listy 28, 227. Olcott, H. A., and Fraenkel-Conrat, H. 1947. Chem. Reus. 41, 151-97. Peters, R. A. 1947. Nature 169, 149-51. Peters, R. A., and Wakelin, R. W. 1947. Biochem. J. 41, 550-55. Philips, F. S. 1950. J. Pharmacol. Exptl. Therap. 99, 281-323. Pirie, A. 1947. Biochem. J. 41, 185-90. Prelog, V., and Stephan, V. 1935. Coll. Czech. Chem. Comm. 7 , 93-102. Preston, J. M. 1949. Fibre Science, The Textile Institute, Manchester, p. 266. Price, C. C., and Wakefield, L. D. 1947. J . Org. Chem. 12, 232-37. Rapoport, I. A. 1947a. Bull. Exptl. Biol. Med. U.S.S.R. 23, 3. Rapoport, I. A. 1947b. J. Gen. Biol. (U.S.S.R.) 8, 359-79. Rapoport, I. A. 1948. Doklady Akad. Nauk. S.S.S.R. 60, 469-72. Rose, F. L. 1950. Personal communication. Rose, F. L., Hendry, J. A., Walpole, A. L. 1950. Nature 166, 993-96. Ross, S. D. 1947. J. Am. Chem. SOC.69, 2982-83. Ross, W. C. J. 1949a. J. Chem. SOC.183-91. Ross, W. C. J. 1949b. J. Chem. SOC.2589-96. Ross, W. C. J. 1949c. J. Chem. Soc. 2824-31. Ross, W. C. J. 1950a. J. Chem. SOC.815-18. Ross, W. C. J. 1950b. Nature 166, 808-09. Ross, W. C. J. 1950c. J . Chem. SOC.2257-72. Ross, W. C. J. 1950d. Unpublished work. Smith, L., Mattson, S., and Anderson, S. 1946. Kgl. Fysiograf. Sallskap. Lund, Handl. 42, No. 7, 1-18. Speakman, J. B. 1948. Proceedings of the Swedish Institute for Textile Research, No. 7. Stahmann, M. A., Golumbic, C., Stein, W. H., and Fruton, J. S. 1946. J. Org. C h m . 11, 719-35. Stein, W. H., and Fruton, J. S. 1946. J . Org. Chem. 11, 686-91. Stein, W. H., Fruton, J. S., and Bergmann, M. 1946b. J . Org. Chem. 11, 692-703. Stein, W. H., and Moore, S. 1946. J . Org. Chem. 11, 681-85. Stein, W. H., Moore, S., and Bergmann, M. 1946a. J. Org. Chem. 11, 664-74. Stevens, C. D., Quinlin, P. M., Meinken, M. A., and Kock, A. M. 1950. Science 112, 661. Stevens, C. M., McKennis, H., and du Vigneaud, V. 1948a. J. Am. Chem. SOC.70, 2556-59. Stevens, C. M., and Mylroie, A, 1950. Nature 166, 1019. Stevens, C. M., Wood, J. L., Rachele, J. R., and du Vigneaud, V. 194813. J, Am. C h m . SOC.70, 2554-56. Sugiura, K., and Stock, C. C. 1950. Cancer Research 10, 244. Timmis, G. M. 1949. Ann. Rept. Brit. Emp. Cancer Camp. No. 27, p. 43. Timmis, G. M. 1950. Personal communication. Twigg, G. H. 1950. Personal communication. Wiggins, L. F. 1946. J. Chem. SOC.384-88. Wiggins, L. F., and Wood, D. J. C. 1950. J. Chem. SOC.1566-75. Wood, D. J. C., and Wiggins, L. F. 1949 Nature 164, 402-03.
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Nutrition in Relation to Cancer ALBERT TANNENBAUM AND HERBERT SILVERSTONE Department of Cancer Research, Medical Research Institute, Michael Reese Hospital, Chicago, Illinois CONTENTS Introduction I. Some General Considerations 11. Genesis of Tumors A. Influence of Caloric Intake 1. General Consequences of Calorie Restriction 2. Factors That Modify Extent of Inhibition a. Degree of Restriction b. Composition of Restricted Diet c. Potency of Carcinogenic Stimulus d. Kind of Tumor 3. Mode of Action of Caloric Restriction a. Duration of Restriction b. Stage of Carcinogenesis Where Action Occurs c. Significance of Body Weight d. Hormonal Factors e. Mitotic Activity of Tissue f. Generality of Caloric Influence B. Influence of Proportions of Dietary Components 1. Fat a. General Effects on Tumor Genesis b. Factors that Modify the Fat Effect c. Mode of Action of Dietary Fat 2. Protein a. Proportions of Protein Supporting Normal Body Weight b. Deficient Proportions of Protein 3. Vitamins a. Vitamin Deficiencies b. Vitamin Levels and Aeo Dye Liver Tumors c. Tumors Induced by Vitamin Deficiency d. Effects Independent of Calorie Intake and Body Weight 4. Minerals 111. Growth of Tumors 1. Caloric Intake 2. Fat 3. Protein a. Variations in Dietary Protein Only 45 1
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b. Mode of Action of Protein Restriction.. . . . . . . . . . . . . . . . . . . . . . . . . . . c. Protein Restriction Combined with Other Treatment. . . . . . . . . . . . . . . .............................................. 4. Vitamins.. . . . . IV. Nutritional State and Cancer in Man.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Body Weight and Cancer Incidence.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Dietary Deficiencies and Cancer Production.. . . . . . . . . . . . . . . . . . . . . . . . a. Thyroid Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Pharyngeal Cancer.. . . . . . . . . ... ... ........ c. Liver Cancer.. . . . . . . . . . . . . . ....................... V. Conclusions and Commentary.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Summary of Present Knowledge., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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b. Growth of Tumors in Animals.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. Cancer in Man.
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4. Future Developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
496 497
References.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
INTRODUCTION When living cells are subjected to carcinogenic influences they may undergo changes that finally result in a growing neoplasm. The energy and substance for the development of the first cancer cells are derived principally from the animal; the new cell type increases in number by assimilating nutrients from the host. It may be expected then that the diet and the nutritional state of the host influence the formation and growth of tumors. The existence, nature, and limits of the influence are the topic of this review. Despite gaps in our knowledge, and facts that do not easily fit into a simple viewpoint, sufficient data are available for a systematic approach. Inasmuch as this is the first volume of Advances i n Cancer Research, a relatively complete and possibly historical review of the relation of nutrition to cancer is desirable. As it happens, however, the main advances have been achieved during the past two decades, and we will center the discussion around the work of this period. It has been necessary to leave out many isolated findings; even the subjects taken up could not be treated exhaustively in discussion or bibliography. Perhaps these omissions will be corrected in reviews of smaller segments of the field in future volumes of Advances. The reader interested in other viewpoints and in a more extensive bibliography will find the following reviews and books helpful: Caspari, 1938; Waterman, 1938; Stern and Willheim, 1943; Morris, 1945; Rusch et al., 1945a; Greenstein, 1947; Tannenbaum, 1947; King et al., 1947; and Baumann, 1948. The subject matter is presented in five sections. The first is limited
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to some general and methodological considerations which may facilitate an understanding of the problems encountered. The second and third parts deal respectively with the influence of diet on the genesis and the growth of neoplasms; the significance of the level of caloric intake and the proportions of dietary fat, protein, vitamins, and minerals are discussed. Investigations on the action of crude materials and extracts are not included, since exact compositions and effective components are generally not known. The studies suggesting that nutrition plays a role in the formation of cancer in man are reviewed in the fourth section. Finally, an attempt is made to summarize the present knowledge. Liberties are taken in correlating it into a reasonable whole and in pointing out profitable areas for future work in this particular branch of cancer research.
I. SOMEGENERALCONSIDERATIONS The question as to whether and how a particular dietary change affects the origin or growth of a neoplasm immediately calls attention to experimental materials and procedures. These often determine the success or failure of an investigation and should be considered in evaluating the results. If neoplasms were not a large group of somewhat unrelated diseases, and if metabolic interrelationships were not so complex, experimental design and interpretation would be simpler. As it is, however, the complicating factors often weaken or deny the validity of the results of an experiment. Obvious features such as numbers of animals, duration of an experiment, and influence of deaths from other causes need not be discussed. On the other hand, there are a few general aspects of research and interpretation that might well be mentioned here. It is not intended t o supply an exhaustive list but only to take up some points that experience, sometimes unpleasant, has shown to be important. Expressedly or implicitly they provide criteria used in this review in evaluating the reported findings. They also illustrate the difficulties encountered and the possibilities for differences in technics and interpretations among those investigating the influence of diet on the cancer process. (1) Neoplasm. This term encompasses a group of pathologic entities varying in etiology, site, and kind of cell involved. Moreover, the process may be influenced by experimental procedures during any stage, from the initial host-carcinogen relationship to the large growing tumor. A dietary change might have diverse eff ects upon different carcinogens. Inasmuch as cells and tissues are not alike in metabolic pattern and in nutritive [requirements, it is not anticipated that neoplasms, arising from manifold cell types and growing in different sites, would
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be affected to the same extent or even in the same way by a particular alteration in diet. Even such characteristics as rate of growth and degree of malignancy are concerned in the response of the tumor to treatment. Much is gained by utilizing several kinds of tumors in a nutritional study. The dissimilarities as well as the uniform results provide a basis for more secure systematization and generalization. (9) Genesis and growth. The genesis of a neoplasm (also termed origin or formation) refers to the transformation of one or a few normal cells into cancer cells. It is distinct from the subsequent cellular proliferation or growth of the tumor. Not only in nutrition studies but in others as well, an experimental procedure may affect carcinogenesis in one way, tumor growth in another. Consequently, it is advantageous to consider separately the influence of diet on genesis and growth, rather than on the process as a whole. Not only is a more refined and correct interpretation possible, but also the information relevant to genesis may suggest preventive measures while that on growth might be applied to therapeutic procedures. (3) Potency of carcinogenic stimulus. The amount and potency of carcinogen, whether administered or of unknown or endogenous origin, determines the frequency of occurrence, the rate of formation, and possibly the malignancy of the resulting neoplasms. If the potency of the stimulus is of a high order, a moderately effective nutritional alteration may appear to be without influence. That is, a high “steam roller” dose of carcinogen may override the influence of an experimental procedure whose effect is obvious a t more “physiological” levels of carcinogen. Undoubtedly, negative findings have sometimes resulted because there was not a proper balance between carcinogenic potency and the other factors of the experiment. In this respect carcinogens are like other pharmacologic agents; doses must be within a range that permits recognition of modifying influences. (4) Characteristics of the diet. In planning nutritional studies, decisions must be made as to the nature and composition of the control dietand how best to insure that the experimental dietary change is actually and specifically attained. The rations may be made up of relatively purified, known components (synthetic diets) or of a mixture of natural foods (as in commercial diets). There is no special advantage of one over the other, provided the particular dietary modification being studied can be achieved with the same precision. It is generally easier to alter a diet composed of simple, known components; on the other hand, rations made up of natural foods are probably more “ complete.” At any rate, the composition of the rations should be specified, as well as the magnitude of the nutritional alteration and its relation to the diet as a whole.
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This permits adequacy to be judged and effects on tumors to be interpreted in terms of caloric content and proportions of components-and facilitates repetition and extension of the studies by other investigators. (5) Diet and the nutritional state, After choosing the experimental rations to be given the animals, there is still the question of what happens “within the body.’’ For it is the amount of food consumed and its effect upon the nutritional state of the host that determine the influence upon tumor formation and tumor growth. The caloric content of the ration as determined in the bomb calorimeter, and its composition as indicated by diet construction or analysis, may or may not represent what is available to the animal. The simplest example is a deficient diet which alters the appetite, resulting in a reduced intake. More subtle deviations occur through changes in digestibility and absorption, specific dynamic action, and synthesis or utilization of essential nutrients, particularly vitamins, by intestinal bacteria. All these factors can result in a nutritional state that in no way resembles that expected of a particular diet. And it is the nutritional state, not the diet, that influencea the neoplastic process! An alteration in diet may produce a distinct effect on a neoplasm. Can it be attributed specifically to the factor being studied or is it due to an indirect, secondary change-reproducible by a variety of measures having a common denominator? For example, some procedures that inhibit tumor formation produce voluntary restriction of food intake and resultant loss of body weight. And we now know that these themselves may strikingly hinder the formation of neoplasms. Perhaps the reduced caloric intake, not the particular experimental procedure, is responsible for the inhibition of tumor formation in these instances. Other situations could be cited where the findings are distinct but the interpretations questionable. It is the purpose here, however, t o emph* size that a dietary change can have many effects on the nutritional state of the host and t o stress the importance of trying to recognize which.of these might be responsible for the observed findings. Obviously, intimate metabolic reactions and mechanisms involved in these processes are not understood a t this time. However, we have advanced from the stage where only the experimental procedure and final result concern US. Between these lies the fertile field of understanding.
11. GENESISOF TUMORS The adequacy of a diet is conventionally described by the quantity and the relative amounts of essential foodstuffs-protein (or amino acids), lipids, carbohydrates, vitamins, and minerals. So far as metabolism is concerned, these are highly interdependent. The total amount of
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food, measured by its digestible caloric value, significantly influences the assimilation and interconversions of the components. Conversely, the proportion of one dietary constituent may modify the utilisation of the others and also the energy available to the animal. Nevertheless, mainly because of the regulation shown by the complex organism in the face of changes in its external environment, including its food supply, it is possible to investigate the influence of alterations in amounts of specific dietary factors. The interpretations, however, must always be qualified by the known and presumed interactions, particularly if large variations are being studied. The convenient dichotomy of quantity and quality suggests division of this section into (1) the influence of caloric intake and (2) the influence of the proportions of dietary components. A. INFLUENCE OF CALORIC INTAKE
There are no objective bases for definition of an optimal or normal caloric consumption. When food intake is unrestricted, mice, rats, domestic animals, and even man may become quite obese. Thus, under certain conditions, ad libitum food consumption may be supernormal. Only in this way have we a suggestion of the influence of high caloric intakes since there are no studies on carcinogenesis dealing with supervoluntary feeding uncomplicated by other treatment. In the main, the investigations on the effects of caloric intake have been concerned with restriction of the experimental animals to between one-half and twothirds of the rations eaten by their controls.
I. General Consequences of Calorie Restriction Caloric intake has been shown to influence the genesis of virtually all the mouse tumors that have been studied. Numerous and extensive investigations have established the fact that among mice chronically restricted in caloric intake the incidence of tumors is decreased and the time a t which the tumors appear is delayed. The impressive inhibitory action of long-term caloric restriction on the formation of a variety of tumors is summarixed in Table I. The neoplasms are of spontaneous origin or induced by known carcinogens, and occur in a variety of organs and tissues. In the mouse, eight tumor types of diverse nature are known to be affected by calorie restriction. Only a few studies have dealt with neoplasms of the rat, and these indicate that the formation of lymphosarcoma (Saxton, 1941) and induced mammary carcinoma (Dunning et al., 1949) is hindered. The reduced tumor incidence is not the consequence of any untoward
457
NUTRITION IN RELATION TO CANCER
TABLE I Results of Investigations on the Inhibitory Effect of Caloric Restriction on the ' Genesis of Tumors in Mice
Type of Tumor
DuraKind of tiont of Dietary Restric- Study tion* (weeks)
Mammary U carcinoma, C F spontaneous C F C C U Hepatoma, spontaneous C
+ +
Lung adenoma, primary
U U U U
U Leukemia, spontaneous C F Leukemia, induced by carcinogen U Skin tumors, induced by U carcinogen C C C Skin tumors, C induced by C UV light
+
Sarcoma, induced by carcinogen
*
- -
U C C C
80 78 96
Formation of Tumors: Control/Restricted InciStrain dencet Mean of (per Time! Mouse cent) (weeks)
References
64 58
dba CsH CsH dba CsH CJI
40/2 67/0 100/20 54/0 58/0 44/0
62
CaH
64/0
96 54 52 70
ABC Swiss Swiss A (males)
52/27 48/8 30/5 50/30
110
Ak
65/10
Tannenbaum, 1940a Visscher et al., 1942 White et al., 1944 Tannenbaum, 1942a Tannenbaum, 1945b - Tannenbaum and Silverstone, 1949c - Tannenbaum and Silverstone, 1949c - Tannenbaum, 1940a - Tannenbaum, 1942a - Tannenbaum, 1942a - Larsen and Heston, 1945 39/62 Saxton et al., 1944
55
dba
96/35
11/34 White et al., 1944
100
77 44 60 46 25 39 39 42 60 38 23
ABC 44/19 Swiss 62/40 dba 65/22 dba 100/71 Rockland 82/18 C 87/7 C 63/24 ABC CWBI dba C
47/15 74/44 16/2 85/70
63/71 41/35/64 74/47/-
28/55 20/27 28/38 18/39
Tannenbaum, 1940a Tannenbaum, 1942a Tannenbaum, 1942a Tannenbaum, 1945a - Boutwell et al., 1949a 33/37 Rusch et al., 1945c 33/33 Rusch et al., 1945c 26/32 24/28 30/15/17
+
Tannenbaum, 1942a Tannenbaum, 1942a Unpublished Rusch et al., 1945b
U underfeeding (all components of diet restricted); C F = only carbohydrate and fat restricted; C only carbohydrate restricted (caloric restriction per 80). t For spontaneous tumors this indicates age of mice at end of experiment; for induoed tumors it La the interval between 1st treatment with carcinogen and end of the experiment. 1Proportion of animals which developed tumors during course of experiment (numbers of miee were sufficient for statistical significance of observed differences). 8 Some of the figures in thia column were eatimated from summarizing tablea or curves in the indicated references.
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ALBERT TANNENBAUM AND HERBERT SILVERSTONE
factors such as general debility or premature deaths among animals on the low-calorie diets. The restricted mice are active, sleek, and healthy; almost invariably their life span is increased, even when compared with the nontumor controls (Tannenbaum, 1942a;Ball et al., 1947). Indeed, since the classic work of MacCay el al. (1935,1939) it is an accepted fact that moderate degrees of caloric restriction prolong life and retard the onset of senescence. The influence of caloric restriction on the formation of tumors seems to have considerable consistency and generality. As more neoplasms are studied, however, exceptions may be found. Already there are two instances that require consideration. For the hepatic tumor induced in rats by feeding carcinogenic azo dyes, indirect evidence has been interpreted as suggesting that restriction of calories might not be inhibitory (Clayton and Baumann, 1949). An approach with fewer confounding factors is needed to clarify this point. King and associates (1949)have reported that the incidence of adrenal adenomas, arising subsequent to ovariectomy of CsH strain mice, was not reduced by caloric restriction. Since all the ovariectomized mice developed the adrenal tumors, the carcinogenic stimulus must have been of high intensity. It will be demonstrated later that the influence of caloric restriction can be masked by massive doses of carcinogen. If less potent tumorigenic conditions were designed, onset of adenomata in the restricted mice might be delayed, or even the incidence reduced. 2. Factors That Modify Extent of Inhibilion
The actual extent of the calorie effect is dependent on several factors, notably, the kind of tumor, the potency of the carcinogen, the degree of restriction imposed, and the composition of the restricted diet. These influences are additive, and the net influence is related to the experimental conditions. With one type of tumor, a low dose of carcinogen, and a 30 to 50% reduction in caloric intake, formation of neoplasms may be completely blocked. On the other hand, with another tumor, a high dose of carcinogen and only a small reduction in calories, inhibition may not be recognized. a. Degree of restriction. In the initial studies on the influence of caloric intake the restricted animals were given from one-half to twothirds the calories ingested by the full-fed controls. This degree of underfeeding is practicable and allows relatively good health and longevity. Many physiological functions are repressed at this level, however, particularly those concerned with reproduction. To ascertain whether or not less drastic treatment also inhibits the genesis of neoplasms, the influence of caloric intake a t graded levels was determined. These
NUTRITION IN RELATION TO CANCER
459
studies were performed with the induced skin tumor, the spontaneous mammary carcinoma, and the benign hepatoma (Tannenbaum, 1945a, 1945b; Tannenbaum and Silverstone, 1949c; Boutwell et al., 1949a). Carcinogenesis was found to be affected by even small degrees of caloric restriction, and the magnitude of the inhibition was dependent on the extent of the restriction. On an arithmetically scaled graph a complete relationship (covering a 0 to 100 per cent range of tumor incidence) may be pictured as an
i;/[ .-
/Moderate Dose
Low Dose
20
, / /
I
60
70
80
90
100
Mean Daily Caloric I n t a k e
(as percent of a d - l i b i t u r n i n t a k e ) FIG. 1. Idealized relation between degree of caloric restriction and tumor incidence: curves that can be obtained with low, moderate, or high carcinogenic doses.
J-shaped curve with the upper arm longer than the lower, and the s t e e p est slope at the 50 per cent level. Of course under actual experimental conditions either the upper or lower arm of the curve might be missing, depending on whether a small or large carcinogenic stimulus was employed (Fig. 1). The influence of caloric intake on the formation of neoplasms is evidenced not only in comparative incidence, but in rate of appearance as well. I n one experiment the dose of carcinogen was so large that nearly all mice developed tumors; nevertheless, the mean latent period of formation increased with decreasing caloric intake (Fig. 2). The relationship between tumor incidence and degree of calorie restriction seems to be of the type often found in pharmacological studies:
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ALBERT TANNENBAUM AND HERBERT SILVERSTONE
the per cent of animals with tumors, expressed in probits,* is a straight line function of the logarithm of the caloric intake (Fig. 3). It offers no support to the possibility that there is a critical level or threshold of caloric supply essential for the genesis of tumors. Rather, it suggests that the energy value of the ration is only one of many factors in carcinogenesis.
8.1
I
8.9 9.6 10.3 11.0 11.7 12.8
I
14,3
Mean Daily Caloric Intake FIQ.2. Relationship between the degree of caloric restriction and the mean time of appearance of benzpyrene-inducedneoplasms of the skin. Shown for both carcinomas and total skin tumors (Tannenbaum, 1945a).
b. Composition of restricted diet. Experiments concerned with restriction of food intake have been performed in several ways: (1) The restricted diet was made identical in composition with the control diet, all components being restricted proportionately; (2) the restricted ration contained the same amounts and kinds of protein, vitamins, and minerals as the control, but the amounts of fat and carbohydrate were reduced; (3) the restricted diet was limited only in carbohydrate content. The first procedure has been described as “underfeeding,” the third as ‘ I caloric restriction per se.” Designating a decrease in carbohydrate as caloric restriction represents a simplification. Certainly, as the diet * Probits are the transformationof the percentage incidence to areas of the normal
distribution curve, these areas being measured in standard deviation units.
NUTRITION I N RlLATION TO CANCER
46 1
is diminished in amount by the removal of carbohydrate, there must occur increased diversion of fat and protein to meet energy requirements. Nevertheless, limiting the carbohydrate content offers the best approximation to “caloric restriction per se.” All three modes of limiting caloric intake affect the formation of tumors, but there are quantitative differences. Apparently the nature of the components of the restricted diet modifies the magnitude of the aJ -
g 95-
v, 90.n CI
2
.c1
c aJ
70-
2 50-
2
6 30u I= aJ
U .0
c 1050
a
I-
~~
60
70
80
90
100
Mean Daily Caloric I n t a k e (Log. Scale) ( a s p e r c e n t of a d - l i b i t u r n i n t a k e )
FIQ.3. Linear relationship between tumor incidence on a probit scale and degree of caloric restriction on a logarithmic scale. The straight lines satisfactorily fit the
data, taken from the authors’ experiments: A, spontaneous mammary carcinoma; B, spontaneous hepatoma; C, induced skin tumors. The statistical procedures are standard (Fisher and Yates, 1943).
inhibitory action. For instance, with restricted diets of equicaloric value, high fat rations may almost prevent the low-calorie effect observed with low fat rations (Tannenbaum, 1945b). The influence of specific dietary components is discussed later, but it can be stated now that the composition of the diet, as well as its energy value, plays a role in the carcinogenic process. c. Potency of carcinogenic stimulus. Moderately large doses of cutaneously applied carcinogen may evoke tumors in nearly all mice regardless of the level of caloric intake. Under such conditions caloric restriction does not evidence any restraining effect if only the incidences of animals with tumors are considered. However, the caloric influence
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ALBERT TANNENBAUM AND HERBERT SILVERSTONE
is still mirrored by both the delayed appearance (increased latent period) and decreased number of lesions per animal (Tannenbaum and Silverstone, 1947). It seems likely that even these signs of inhibition might be obliterated if a sufficiently massive amount of carcinogen were used. The steam-roller action of large doses could be anticipated, as a feature common to pharmacologic experimentation. Along these lines, we regard the findings of White and White (1944a) with the spontaneous mammary carcinoma, and those of King and co-workers (1949) with the postcastrational adrenal adenoma, as instances where a high carcinogenic stimulus tended t o mask the low-calorie effect. d. Kind of tumor. Sufficient data are presented in Table I t o indicate that the inhibitory influence of caloric restriction is less striking for some tumors of the mouse than for others. Without exposition of the actual experimental conditions, it is seen that the formation of sarcomas and skin tumors induced by hydrocarbons is least affected; primary lung tumors and leukemias occupy an intermediate position; and spontaneous mammary carcinomas and benign hepatomas are most inhibited. These differences are the consequence of several factors, probably interrelated. Among them are: the potency of the carcinogenic stimulus and the resultant intrinsic vigor of the tumor (and its genesis) ; the time interval over which this stimulus operates; and the effect of the altered nutritional state on the carcinogen itself, and on the tissue in which the tumor arises. The authors have speculated as to how these factors produce the differential response of various tumor types. The arguments and conjectures are not being detailed, however, since more facts, not discussion, are needed. 3. Mode of Action of Caloric Restriction
The magnitude of carcinogenic inhibition brought about by limitation of caloric intake-and the fact that so many tumor types respond-has compelled interest in the question of mechanism. Some factors and influences that may alter the extent of the caloric effect have already been discussed. There remain, however, certain biological facts and a few hypotheses that bear on the way this simple nutritional alteration operates. Obviously, these are mere beginnings, but they may eventuate in a better understanding of the mechanism not only of caloric restriction, but of carcinogenesis itself. a. Duration of restriction. In most studies concerned with the influence of low-calorie diets, the experimental mice were underfed continuously throughout the period of observation, ranging from a few months in some instances to about two years in others. There are only a few experiments that give information as to the minimum period of restriction
463
NUTRITION I N RELATION TO CANCER
necessary to produce a caloric effect. Clearly, this is not a specified time applicable to every set of experimental conditions and mode of carcinogenesis. Even a few weeks or months may result in some delay in carcinogenesis, and it is probable that the extent of the inhibitory effect is correlated with duration of restriction. That it must be relatively continuous in order to be effective is suggested by an experiment in which twice-weekly fasts for twenty-four hours, with ad libitum feeding between fasts, had no inhibitory influence on the formation of mammary tumors (Tannenbaum and Silverstone, 1950). b. Stage of carcinogenesis where action occurs. In experiments with the spontaneous mammary carcinoma in strain dba mice, it was observed that institution of caloric restriction a t 2, 5, or 9 months of age, respectively, produced relatively the same degree of tumor inhibition (Tannenbaum, 1942a). This suggested that limiting the caloric intake would have an effect if begun at any time before tumors appeared. A study with strain C3H mice, which develop multiple mammary carcinomas, supports this inference. Each mouse was fed ad libitum until its first tumor appeared; subsequent caloric restriction resulted in a striking reduction in the incidence of second and third tumors (unpublished). The above experiments suggested that chronic restriction of calories probably acted, not during carcinogenic stimulation, but in some later part of the cancer process. This was tested in a study in which skin tumors induced by a carcinogenic hydrocarbon were utilized, because the period of treatment with the carcinogen could be better controlled (Tannenbaum, 1944a). Four equivalent groups of mice were employed, and all received the same amount of 3,4-benzpyrene to the skin; administration was discontinued before any tumors appeared. Thus the experiment was made up of two arbitrary intervals: the first, during which carcinogen was applied; and the second, during which tumors appeared. Four different sequences of a high- and a low-calorie ration were fed, as indicated in Table 11. The results were interpreted as showing that caloric restriction might have had a slight influence in the period of TABLE I1 Incidence of Induced Skin Tumors in Relation to Period of Caloric Restriction
Group H H HL L H L L
Diet during Period of Diet during Period Carcinogen Application of Tumor Appearance (10 weeks) (52 weeks) High calorie High calorie Low calorie Low calorie
High calorie Low calorie High calorie Low calorie
Tumor Incidence (per cent) 69 31 55 24
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ALBERT TANNENBAUM AND HERBERT SILVERSTONE
carcinogen application, but that the major inhibitory action occurred during the period in which tumors were emerging. The concept that carcinogenesis proceeds through several sequential stages has arisen from the investigations of many workers, most prominently Rous and Berenblum. Although there are differences in points of view and nomenclature, it is generally agreed that at least two phases can be distinguished : (a) a stage variously designated as initiation, inception, or latency, in which the carcinogen acts upon normal cells to render them biased toward the cancerous transformation; these initial changes are limited but self-perpetuating; (b) a stage of development or promotion, in which the initiated or biased cells, under favoring conditions, develop into cancer cells. This ends carcinogenesis; the growth of the tumor begins with the active proliferation of the primary cancer cells. The following references are representative : Rous and Kidd, 1941; MacKenzie and ROUS,1941; Berenblum, 1941; Mottram, 1944; Tannenbaum, 194413; Kline and Rusch, 1944; Berenblum and Shubik, 1949; Friedewald and ROUS,1950. It is not within our province t o discuss in detail this important contribution to the understanding of carcinogenesis, but rather to emphasize that the work on nutrition is in agreement with these concepts and has expanded them. Specifically, caloric restriction appears to have its main influence on the developmental stage of carcinogenesis. c. Significance of body weight. In seeking information that might elucidate the mode of action of limited caloric intake, the concomitant phenomena of lower metabolic turnover and reduced growth or body weight were considered. The possible significance of low body weight, in the formation of the spontaneous mammary tumor, was examined in studies in which sodium fluoride or dinitrophenol was incorporated into the diet, or the mice were kept in a cold room a t about 50°F. Compared with the controls, the mice fed sodium fluoride consumed about 10 per cent less food; the animals of the other two groups ate about 10 per cent more than the controls-all that was offered them. All three procedures resulted in a significant retardation in body growth and a striking reduction in tumor incidence (Tannenbaum and Silverstone, 194913). Under similar conditions of food intake, the feeding of either sodium fluoride or dinitrophenol also resulted in reducing body weight and the incidence of spontaneous lung adenomas. It was postulated that it was not the level of caloric intake or total metabolic turnover, but rather the body weight level a t which a balance was struck between caloric intake and utili~ationthat might be a factor in the origin of some tumor types. This possibility was strengthened by the results of studies in which mice were fed thyroid extract (Silverstone
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and Tannenbaum, 1949). They consumed 40 to 50 per cent more food, yet weighed about 10 per cent less than the controls. This impressive augmentation of caloric intake and metabolic activity did not produce a large effect upon the formation of skin tumors or sarcomas induced by 3:4 benzpyrene. In these experiments the formation of tumors was more closely related to the average body weights of the groups. That the overall weight of an animal has importance is indicated by another experiment in which mice were intermittently fasted-twice a week for twenty-four hours with ad libitum feeding on the other days. They ate the same amount of food on a per week basis, grew as well, and had the same incidence of spontaneous mammary tumors as the controls (Tannenbaum and Silverstone, 1950). However, there are other observations that do not support the idea that the level of body weight is a major factor in the development of neoplasms. Two hundred and fifty strain CaH female mice were permitted to eat ad libitum of an adequate stock diet. The maximum weights attained by the mice ranged from 22 to 42 g. Yet there was no correlation between body weight and incidence or time of appearance of spontaneous mammary cancer (unpublished). I n other experiments, moreover, feeding of sodium fluoride or dinitrophenol failed to hinder the formation of induced skin tumors and sarcomas, although body weight was markedly reduced (Tannenbaum and Silverstone, 194913). Nevertheless, the weight of evidence available a t this time indicates that the inhibitory action of caloric restriction, and possibly some other experimental procedures, may be correlated with the accompanying limitation of body weight. At present, we prefer to look upon low caloric intake and low body weight as interconnected, without attempting to regard either as being more intimately related to the tumor-hindering action of calorie restriction. It is in this sense that the combined terms are used in the review. It might be well to mention here that there are special situations in which the experimental group weighs less yet develops more tumors than the controls. This can occur where the experimental diet differentially increases both the effective tissue dose and the toxicity of the carcinogen. d. Hormonal factors. In mice and rats given one-half to two-thirds the average ad libitum caloric intake there is a sequence of changes, notably in the ovaries, uteri, and mammae, that simulate those following hypophysectomy (Mulinos and Pomerantz, 1940). This so-called pseudohypophysectomy has been proposed as an explanation of the influence of caloric restriction on the formation of spontaneous mammary cancer. The action is presumed to be dual, involving a decrease in estrogen production and a diminished response of mammary tissue t o
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estrogen (Huseby et al., 1945). No attempt has been made to extend this hypothesis to other tumor types. I n contrast to this suggestion of pituitary insufficiency-arereports that caloric restriction may actually augment the output of adrenocorticotropic hormone (Boutwell et al., 1948; Sayers, 1950). This is indicated by a relative increase in adrenal size, a marked involution of lymphoid tissue (notably thymus), and an apparent increase in glyconeogenesis. Boutwell and associates have suggested that perhaps the changes in adrenal function may explain the tumor-inhibiting action of caloric restriction. e. Mitotic activity of tissue. Bullough (1950) has postulated that the developmental stage of carcinogenesis is influenced by the mean mitotic activity of the tissue; by inference this includes mitosis of the latent cancer cells. It was observed, for example, that mitotic activity was definitely inhibited by caloric restriction, in proportion to the degree of the limitation (Bullough and Eisa, 1950). These and other experiments led him to propound that a limiting factor in cell division is the amount of carbohydrate and carbohydrate intermediates available for the energy requirements of mitosis. The mitotic activity hypothesis is compatible with all that is known with regard to caloric restriction. f. Generality of caloric influence. Chronic caloric restriction results in many changes: in the absolute and relative weights of organs and tissues; in proportions of tissue and body fluid constituents; in mitotic activity; in hormone production; and in metabolism. Presumably there is a restricted supply of nutrients a t the loci of tumorigenesis. Which of these or other alterations are responsible for the influence on carcinogenesis? Whatever the answer, it must account for the generality of the action and its ultimate effectiveness in a wide variety of tissues and cell types. B. INFLUENCE O F PROPORTIONS OF DIETARY COMPONENTS
Each of the classes of dietary components, proteins, carbohydrates, fats, vitamins, and minerals, is constituted of a variety of substances. Proteins are similar on the basis of nitrogen present in peptide bonds, but differ widely in component amino acids and in physical and biological properties. Lipids consist of glycerides of various fatty acids and a host of other esters. Vitamins bear no chemical resemblance to one another, being classified together as organic substances needed in small amounts. Minerals, of course, are grouped only on the basis of being inorganic. It is a t once obvious that experiments on the influence of varying the components of diet can therefore yield only particular data. Rational generalization on the basis of collateral knowledge is necessary to give
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the results broader and useful significance. For example, suppose that varying the proportion of casein (as the sole source of protein) is found t o influence the incidence of tumors. It is not at all certain that exactly similar results would be obtained if lactalbumin, gelatin, or a mixed source, were employed as the dietary protein. Or suppose that experiments had been conducted utilizing partially hydrogenated cottonseed oil (easily available and commonly used) as the dietary fat. The results might differ if animal fats, completely hydrogenated oils, highly unsaturated oils, or pure synthetic triglycerides, were used instead. Such facts are tacitly understood in nutritional research and are of importance when interpreting the results obtained with a particular member of a class. The practical design of experiments involving different proportions of nutritional components poses another problem. Alterations in diet often lead t o changes in body weight relative to that of the control animals. This may be consequent to a voluntary increase or decrease in caloric intake, or determined by an influence on the metabolism of the animal. Inasmuch as the genesis of tumors can be modified by a change in caloric intake and body weight, it becomes necessary t o determine, either by logic or experimentation, whether the observed effects on tumor formation are due to the experimental modification or to the accompanying caloric and body weight changes. Investigators have tried to meet this problem by such technics as paired feeding or force-feeding, or by giving to all animals equicaloric quantities of diets a t a reduced level, below that consumed by those with the poorest appetite. Even under these conditions, however, differences in absorption, composition, interconversions, and excretion may effect notable differences in body weight. For this reason, and because the animal’s weight is an excellent index of usable calories, it has sometimes been found expedient to adjust calorie intake so that control and experimental animals have equal average body weights. Of course, even this does not necessarily result in comparable tissue weights. If deemed helpful-and whenever possible-more than one technic should be utilized. In this way the confounding factors may be resolved and a decision obtained as t o whether the dietary alteration itself, or the resulting caloric or weight changes, is responsible for the effect on carcinogenesis.
1. Fat Under this heading we intend to discuss principally the effects of dietary neutral fats. It is recognized that the investigations with f a t actually utilized either butter, lard, partially hydrogenated vegetable oils, etc., but in general the influences were dependent on their neutral
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fat content. The many reports on the significance of dietary phospholipids and of cholesterol are omitted since the results are not convincing. In early studies, fat enriched diets were fed without considering their greater energy value. When low- and high-fat rations were fed ad libitum, the latter were usually consumed in calorically larger amounts because of their compactness. Probably the observed effects on carcinogenesis were the resultant of both increased fat and increased calories. In more recent investigations the caloric intakes have been equalized, permitting better evaluation of the significance of dietary fat. The discussions that follow are based on equicaloric comparisons, although in fact this has not always been achieved with exactness. a. General egects on tumor genesis. When the proportion of dietary fat is increased from low levels (about 2 per cent) to moderate or high levels, the response is related to the tumor type. With some neoplasms, genesis is enhanced; with others there is either no effect or slight inhibition. Skin tumors induced in mice, by tarring, by carcinogenic hydrocarbons, or by ultraviolet light, have been most frequently used in investigating the influence of fat enriched diets. Many different kinds of fats have been employed, of animal or vegetable origin, of different fatty acid constitution and degrees of saturation. I n all these studies tumors appeared in greater incidence and at an earlier average time in the mice on the high-fat rations (Watson and Mellanby, 1930; Baumann et aZ., 1939; Baumann and Rusch, 1939; Jacobi and Baumann, 1940; Lavik and Baumann, 1941, 1943; Tannenbaum, 194213, 1944c, 1945b; Rusch et al., 1945c; Boutwell el al., 1949a). The genesis of the spontaneous mammary carcinoma also is significantly augmented in mice fed high-fat diets. Tumors appear earlier and in greater numbers (Tannenbaum, 1942b, 1945b; Silverstone and Tannenbaum, 1950). There is suggestive evidence that the same is true for mammary tumors induced in rats by implantation of stilbestrol (Dunning et al., 1949). The rate of formation of the spontaneous benign hepatoma of strain C8H mice is slightly accelerated by dietary fat enrichment (Silverstone and Tannenbaum, 1951a). The influence of this procedure on hepatic tumors induced in the rat by p-dimethylaminoazobenzene is not clear, although the weight of evidence suggests that here, too, enhancement occurs (Opie, 1944; Miller et al., 1944b; Kline et al., 1946; Silverstone, 1948). Special features related to this tumor will be discussed later. In contrast to the promoting influence of fat-enriched diets on the formation of spontaneous mammary carcinoma and induced skin tumors, and probably hepatic neoplasms, are the negative results with the sarcoma
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induced by subcutaneous injections of carcinogenic hydrocarbons (Baumann et al., 1939; Tannenbaum, 194213;Lavik and Baumann, 1943; Rusch et al., 1945b),the primary lung adenoma (Tannenbaum, 1942b) and the spontaneous and induced leukemia of mice (Lawrason and Kirschbaum, 1944). It has been reported that an increase in dietary fat decreased the incidence of ocular orbit tumors occurring in rats fed acetylaminofluorene (Engel, 1951). Where the genesis of a tumor is augmented by fat enrichment of the diet, the effect can be demonstrated a t many levels of calorie intake. With both the mammary carcinoma and induced skin tumor, augmentation was observed, for example, not only a t near ad libitum levels (about 12 Calories daily), but with restricted diets as well (about 7 Calories daily) (Tannenbaum, 1945b; Boutwell et al., 1949a). b. Factors that modify the fat efect. Some of the tumors that respond to changes in dietary fat content have been studied with the purpose of ascertaining factors that may modify the magnitude of the effect. These are the degree of fat enrichment, the potency of the carcinogenic stimulus, and the action on the carcinogen itself. (1) Degree of fat enrichment. I n experiments with the spontaneous mammary carcinoma the rate of tumor formation, as measured by both incidence and average time of appearance, increased with increasing levels of dietary fat (Silverstone and Tannenbaum, 1950). The response was not arithmetically proportional to the fat content of the diet; an increase from 2 to about 8 per cent resulted in as great an augmentation as that consequent to an increase from 8 to about 26 per cent. Furthermore, there appeared to be a definite plateauing or maximal effect with diets containing about 16 per cent fat. Consistent with these findings is the observation that a diet containing 27 per cent fat stimulated the formation of induced skin tumors to about the same degree as one containing 61 per cent fat (Boutwell et al., 1949a). The available data indicate that within limits the incidence of spontaneous mammary and induced skin tumors follows a doseresponse relationship to the proportion of dietary fat. ( d ) Potency of carcinogenic stimulus. When a moderate dose of carcinogen was employed to induce skin tumors, fat enrichment of the diet resulted in both increased incidence and accelerated onset. Practically all animals, whether on low- or high-fat diets, developed neoplasms when the dose of carcinogen was large. Nevertheless, the augmenting influence of fat enrichment was still evidenced by the significantly earlier appearance of the tumors (Tannenbaum and Silverstone, 1947). (3) E$ects o n carcinogen. It was briefly stated in the section concerned with general observations that a change in the diet might have
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profound influences on the carcinogen itself. For instance it might alter the effective tissue dose of the carcinogen. How such a situation might arise and affect the results and interpretation of an experiment is now discussed. I n studies on skin carcinogenesis, the high-fat ration might be constructed or fed in such manner that the skin of the animal became greasy or oily through external contact. There would follow a striking augmentation, considerably in excess of the enhancement that occurs in the absence of such external contamination (Watson and Mellanby, 1930). The mere application of oil to the skin during the period of carcinogen administration in itself results in an increased tumor incidence. This occurs not only with skin tumors induced by chemical carcinogens, in which case greater solubility and absorption into the skin might be evoked as an explanation, but also with skin tumors induced by ultraviolet light (Rusch et al., 1939). We believe it necessary to distinguish between this nondietary action of fat which influences the effective dosage of carcinogen in the initiatory stage of skin carcinogenesis, and the dietary action of fat which appears to operate principally during the developmental stage (see below), and is of relatively small magnitude (Lavik and Baumann, 1943; Tannenbaum, 1944~). c. Mode of action of dietary fat. ( 1 ) Stage of carcinogenesis where action occurs. It was previously pointed out that caloric restriction exerted its main effect in the developmental stage of carcinogenesis. Similarly, experiments with induced skin tumors have demonstrated that fat enrichment of the diet augments tumor formation during the developmental stage (after the limited period of carcinogen application). Little influence resulted from feeding high fat rations in the period of carcinogen application only-during the initiatory stage (Lavik and Baumann, 1941; Tannenbaum, 1944~). ( 2 ) Metabolic influence. The same qualitative effects on carcinogenesis are elicited by the various fats employed in altering the diet, except in the case of the azo dye induced liver tumor of the rat (discussed in next paragraph). The fats are mainly triglycerides of fatty acids along with a small percentage of other substances. Glycerol and the nonsaponifiable fraction from cottonseed oil had little influence on the induction of skin tumors. On the other hand, the reconstituted triglycerides (after removal of nonsaponifhble matter) were as stimulatory as the partially hydrogenated cottonseed oil from which they were obtained. Corn oil, coconut oil, lard, ethyl laurate (Lavik and Baumann, 1941, 1943) or butter (Watson and Mellanby, 1930), were also effective. Most likely the fatty acid moiety is responsible for the enhancing action of dietary fat on skin and mammary tumors. There are no conclusive
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data on the influence of metabolic products of fat or of short chain fatty acids; the only experiments in which these were added in amounts sufficient for exhibition of a response were performed with induced sarcoma, a tumor that is relatively unaffected by dietary fat. The relation of fat enrichment of the diet to hepatic tumors induced in the rat by feeding p-dimethylaminoazobenzene is of special interest. It has been reported that the genesis of these tumors is accelerated by rations with moderate or high fat content as compared with rations containing little fat, but this has not been found consistently. With this neoplasm, however, the nature of the fat is important (Miller et al., 1944a, 1944b; Kline et al., 1946). Tumors developed less rapidly in rats consuming rations containing olive oil than in those given corn oil, while hydrogenated coconut oil retarded tumor formation significantly. This latter result is not attributable to a deficiency of essential unsaturated fatty acids. Increasing the corn oil content from 5 per cent to 20 per cent of the diet strikingly enhanced tumor formation, but replacing the 5 per cent corn oil with 20 per cent partially hydrogenated cottonseed oil or 20 per cent lard produced no appreciable augmentation. This diverse action of different fats has not been shown for other tumors and despite excellent investigations has not yet been explained in terms of the chemical, physical, or biological properties of the several fats. I n an attempt to explain the augmenting action of fat enriched rations, attention has been called to the following phenomenon: Increasing the fat content of a diet results in an increased efficiency of energy utilization (Forbes et al., 1946a, 1946b). This metabolic characteristic has been advanced as the factor possibly responsible for augmenting the incidence of induced skin tumors inasmuch as this “saving” of net body energy might be regarded as equivalent to an increase in caloric intake (Boutwell e l al., 1949a). However, there are two points which negate this suggestion (Silverstone and Tannenbaum, 1950). First, the magnitude of the increase in net body energy can account for only a small part of the observed augmentation of tumor formation. Second, if the fat effect were mediated through the increased net body energy gain it would be reasonable to expect fat enriched diets to enhance the formation of all types of tumors influenced by the level of caloric intake; this is not the case. In fact, the converse idea, that the influence of caloric restriction is mediated through the depletion of cellular and tissue fat consequent to a restricted food intake, could better be supported. What are the net conclusions from all the above considerations as to how isocaloric dietary fat enrichment affects carcinogenesis? The tissue dosage of carcinogen and thus the initiatory stage may be modified. On the other hand, the alterations in fat content of the tissues in which the
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carcinogen is acting, the local nutritional state, and the changes in net body energy, may all influence the developmental stage. The variable response of different types of neoplasms may depend on the extent to which these separate factors are involved. 2. Protein
So far as the animal is concerned the usefulness of dietary protein depends not only on the amount but also on the nutritional quality. Briefly, adequate protein may be considered as that which supplies the kind and amount of amino acids essential to normal physiology and growth. Rations that fall short of this standard are inadequate. The point at which diets are designated as adequate or deficient is of course somewhat arbitrary. When otherwise adequate semipurified rations are given ad libitum to young adult mice, varying the proportion of dietary casein between 9 and 45 per cent has little influence on body weight. Actually, the mice ingesting the higher protein rations may tend to be somewhat smaller because of a voluntary decrease in caloric intake. At isocaloric levels, the mice on diets containing 18 per cent casein are about 10 per cent heavier, on the average, than those on the 9 per cent casein ration; further increases up to 45 per cent casein have no great influence on body weight. Thus mice can maintain normal body weight if allowed free access to a 9 per cent casein ration, even though the efficiency of utilization is aomewhat decreased. Under the same conditions, mice fed lower proportions of casein do not grow well. We have therefore accepted proportions of casein of 9 per cent or over as being adequate for adult mice. The plans, results, and interpretations of the investigations concerned with dietary protein direct the separation of the discussion into: the consequences of varying the proportions within limits adequate for growth; and the effects of protein-deficient diets. a. Proportions of protein supporting normal body weight. (1) Tumors not affected. The incidence and rate of appearance of 3,4-benzpyreneinduced skin tumors did not vary significantly among groups of mice which were fed ad libitum semipurified diets containing 9, 18, 27, 36, or 45 per cent casein (Tannenbaum and Silverstone, 1949a). With the same series of rations, the rate of formation of spontaneous mammary carcinoma in strain CsHfemales also was found to be unaffected by the level of dietary protein. In another experiment in which isocaloric diets were employed, mice on 9 per cent casein weighed somewhat less and formed mammary tumors a t a slightly slower rate as compared with those on 18 or 36 per cent casein. The induction of sarcomas by carcinogenic
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hydrocarbons was not modified by an increase in the proportion of dietary casein from 18 to 32 per cent (Tannenbaum and Silverstone, 19490,) and from 13 t o 26 per cent in diets fed ad libitum, or from 20 to 40 per cent in calorie restricted rations (Rusch et al., 194513). Thus with three distinct types of tumors, carcinogenesis was uninfluenced by varying the proportion of casein between 9 and 45 per cent. Perhaps under other experimental conditions, particularly a less potent carcinogenic stimulus, small but significant effects would be exhibited. However, the results obtained might be expected since most of the tissues of the animal were not markedly affected (either in siae or protein content) by variations within this range. Notable exceptions are the kidney mass and protein content of the liver, which increase with increasing dietary protein, even within these limits. This “sensitivity” of the liver to changes in protein supply serves as an introduction to the following discussion. (2) Tumors of the liver. The rate of formation of the spontaneously occurring benign hepatoma in mice given 18,27,36, or 45 per cent dietary casein was of about the same order. However, on 9 per cent casein rations there was a striking inhibition of hepatoma formation. This result has been observed with two strains of mice and in females as well as males. Moreover, the reduced incidence on 9 per cent casein occurred with diets fed ad libitum or isocalorically, and also in experiments in which caloric intakes were controlled so as to maintain equivalent body weights among the several groups (Tannenbaum and Silverstone, 1949a; Silverstone and Tannenbaum, 1951b). Addition of 9 per cent of gelatin (an inadequate protein) to the 9 per cent casein ration, did not significantly enhance hepatoma formation. In contrast, supplementing the 9 per cent casein with methionine and cystine increased the incidence of hepatomas to that observed among mice on 18 per cent casein. This might be interpreted as indicating that the sulfur-containing amino acids play a specific role in the genesis of hepatomas. However, there is a more likely explanation. The proportion of sulfur-containing amino acids in casein is a limiting factor in its nutritive value, and its biological potentiality is raised by supplementing with methionine or cystine. I n fact, adding 0.2 per cent methionine to a ration containing 9 per cent casein produces a diet nutritionally equivalent to one with 12 to 14 per cent casein. It is likely that the augmenting effect on hepatoma formation was due to the increased “balanced ” protein, not the supplementary sulfur-containing amino acids per se. The induction of malignant liver neoplasms in rats fed carcinogenic azo dyes is dependent on a number of dietary features, one of these being the proportion of protein. Increasing the dietary protein or improving
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the biological value of casein by supplementation with sulfur-containing amino acids has been reported to be either without influence or more often inhibitory for genesis of these neoplasms (Kensler et al., 1941; Miller et al., 1941; Rusch et al., 1945a; Harris et al., 1947; Silverstone, 1948). The outcome, however, is dependent on other features of the ration. Most significant perhaps is the amount of dietary riboflavin and the resultant concentration of riboflavin in the liver, the latter being enhanced by high dietary protein (Sarett and Perlzweig, 1943; Czaczkes and Guggenheim, 1946) and depressed on ingestion of carcinogenic dyes (Kensler et al., 1940; Griffin and Baumann, 1946). “It is doubtful whether the beneficial effects of dietary protein in rats fed azo dyes involves the critical carcinogenic reaction. More probably they represent merely another example of the ability of dietary protein to increase the resistance of animals to such diverse toxic agents as benzene, chloroform, arsphenamine, selenium, and the carcinogenic hydrocarbons ” (Griffin et al., 1949). The divergent effects of protein on the two neoplasms described-of the same organ-emphasizes that the response to a particular dietary change may vary with the kind of tumor. Raising the casein from 9 to 18 per cent enhances the development of the spontaneous benign hepatoma in mice; a similar change either inhibits or does not modify the genesis of dye-induced malignant liver tumors in rats. b. Deficient proportions of protein. In certain sublines of strain dba mice, leukemia can be induced by cutaneous application of methylcholanthrene. The production of this type of leukemia was significantly inhibited when the mice were on diets containing only 4 or 5 per cent casein, which are deficient in cystine (J. White et al., 1947). If these diets were supplemented with cystine, the induction of leukemia proceeded at its maximal rate. In contrast, rations deficient in lysine or tryptophan had little influence, except for a slight retardation in time of appearance of the disease (Table 111). The divergence of the effects observed with the respective deficiencies prompted White and associates to conclude that “cystine played a role in the development of leukemia not associated with its properties as an essential amino acid for growth but with some other attribute not yet determined.” It was pointed out, however, that among the mice on the low casein diet (without the cystine supplement) sclerosis of the aorta occurred in about one-half the animals. Since these latter died (some without leukemia) a t about the average time of appearance of the leukemia,‘ the results are somewhat obscured. A diet containing 18 per cent gliadia (lysine-deficient) or one with 4 per cent casein (cystine-deficient) effected a considerable suppression of
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the formation of spontaneous mammary carcinoma in strain C,H mice (White and Andervont, 1943; White and White, 194413). On the gliadin ration, the incidence was only 25 per cent, on the low casein ration, zero; in contrast, tumors appeared in nearly 100 per cent of the controls. The mice fed the gliadin ration averaged 21 g. in body weight; those given the low casein ration, only 13 g. It seems likely that nearly identical effects on tumor formation would have been obtained if the mice had been maintained a t these body weight levels through caloric (carbohydrate) restriction alone. TABLE I11 Effect of Diet on the Induction of Leukemia by Methylcholanthrene
Diet * 1. Cystine deficient Cystine deficient 0.5 % bcystine 2. Lysine deficient Lysine deficient 0.62% L-lysine 3. Tryptophan-deficient Tryptophan-deficient 0.1% L-tryptophan
+ +
* The
+
Body Mice Developing Weight? Leukemia1 Change Incidence Mean Latent (grams) (per cent) Period (days) -0.2 +4.5
+0.4
+ 6 .2
0
+ 5 .3
55 92 90 90 85
88
113 97 124 110 136 91
diets were semisynthetic rations with the following protein sources: 1. cystine deficient,
5 % casein; 2. lysine deficient, 18 % gliadin; 3. tryptophan deficient, 3 % casein, 7 % peroxide-treated oasein, and 0.28 % methionine.
t Mean change in body weight of
the mice during first sixty days of experiment. Praotically all the animals died with leukemia except in the group fed the cystine-deficient diet. Here there were a number of deaths with aortic sclerosis, many without leukemia, a t an average latent period of 121 days. f There were 38 to 40 mice initially in each experimental group.
The influence of 5 per cent casein rations, with and without supplementary cystine, on the formation of hepatic tumors induced by azo dyes, has also been studied. The food intake and consequently the dose of carcinogen was adequately controlled by paired feeding. All the animals developed liver neoplasms, but they appeared much later in the rats given the cystine supplement (White and White, 1946). Formation of spontaneous lung adenoma was greater in mice on a 4 per cent casein ration supplemented with cystine than in those on the unsupplemented ration. Food intakes were ad libitum, and the latter ate less and weighed less. However, when the food intakes were maintained at isocaloric levels, there was no significant difference in tumor incidence (Larsen and Heston, 1945). Investigations utilizing amounts and proportions of protein that result in diminished metabolism and growth of the host present two
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difficulties in the evaluation of effects on carcinogenesis. One has already been discussed in detail-the significance of differing caloric intakes. The other is related to the manner in which the deficient protein diet is altered to prepare the control diet. Sometimes this is done by supplementation with the deficient amino acid. It is obvious that this measure results in rations that differ not only in proportions of the particular amino acid, but also in the proportions of balanced, adequate protein. The question arises, therefore, whether an observed change in the formation of tumors is due t o the specific amino acid or to the resultant increase in adequate protein. In many instances the latter appears to be the correct explanation. Therefore, the conclusions of some of these investigations-that particular amino acids play a specific role in the genesis of particular neoplasms-require reexamination. It is concluded that modification of carcinogenesis through a change in the proportion of dietary protein, within limits that permit relatively good growth and body weight, has so far been unequivocally demonstrated for tumors of the liver only. The spontaneous mammary carcinoma, induced skin tumor, and induced sarcoma apparently are not responsive. As previously mentioned, this may be related to the fact that alterations in dietary protein are reflected in the composition of the liver, but much less so in that of most extrahepatic tissues. Probably the observed effects on hepatic tumors are related to amounts of protein in the liver that are marginal with respect to optimal health and function of the organ. Perhaps the establishment of similar critical conditions in other tissues, with carefully controlled caloric intake and body weight, might also reveal the influence of protein deficiency on the intimate changes leading to the formation of tumors in these sites. 3. Vitamins
Vitamins are so important to the nutritional state of the host, that they are brought to mind whenever growth processes are being considered. Cancer is no exception. Consequently, numerous attempts have been made to link carcinogenesis with both deficiencies and ahundances of these food components. It is therefore disconcerting that vitamin deprivations usually result in lower food intake and body weight and a shortened life span, events which in themselves either modify the onset of neoplasms or do not permit the completion of the process. These complications may interfere with the intent of a study and render the interpretation unconvincing. The early investigations have been extensively reviewed (Stern and Willheim, 1943; Burk and Winsler, 1944; Morris, 1947). We have
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chosen a few of these for illustration and will discuss the more recent contributions. In the main, however, the purpose has been to attempt an appraisal of the subject. Only the B vitamins are considered, since there are no conclusive data that the others have noteworthy effects. a. Vitamin de$ciencies. The influence of a partial riboflavin deprivation on the formation of spontaneous mammary carcinoma in CaH virgin female mice has been studied (Morris, 1947). The groups on the deficient diets developed significantly fewer tumors than those on the riboflavin-supplemented rations. However, the food consumption and body weights of the depleted mice were also lower, and to an extent that could account for much of the reduction in tumor incidence. That adequate nutrition is necessary for the genesis of tumors, as a general rule, is indicated by the following investigation. A semipurified ration was designed to contain levels of B vitamins just sufficient to maintain young mature rats a t their initial weight. Addition of a carcinogenic dose of p-dimethylaminoazobenzene (butter yellow) t o the diet caused the rats to lose weight; some died, and no liver tumors were found in survivors that had ingested the diet for as long as six months. Under these conditions of multiple vitamin deficiency, even a low level of riboflavin, known to favor the genesis of azo dye liver tumors, failed to evoke them (Miner et al., 1943). b. Vitamin levels and azo dye liver tumors. In contrast to the foregoing are the studies indicating that, with an otherwise adequate diet, low levels of riboflavin enhance the development of liver tumors in rats fed azo dyes (Kensler et al., 1940, 1941). The investigations of the University of Wisconsin group, including a survey of 34 diets, demonstrated that dietary supplements rich in both protein and B vitamins, particularly riboflavin, inhibited tumor formation (Miller et al., 1941). Protein is thought to have little direct effect, acting rather by altering the concentration of riboflavin in the liver. The significance of the concentration of riboflavin actually attained in the liver-not the amount in the diet-as a key factor in the genesis of liver tumors induced by means of azo dyes, is now established (Miller, 1947; Kensler, 1947; Griffin and Baumann, 1948). Low levels enhance, high levels inhibit tumor formation: There has even been progress as to the mechanism of action. Riboflavin appears to be a constituent of a coenzyme in the systems that break down dimethylaminoazobenzene and related compounds (Kensler, 1948; Mueller and Miller, 1950). This implies that riboflavin acts on the initiatory stage of carcinogenesis by modifying the effective dose of carcinogen. The consequence of varying the amount of riboflavin in $he diet is not of the same order for the various azo dyes (Giese et al., 1946); and an influence on the induction of
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hepatic tumors in rats fed a different type of carcinogen, acetylaminofluorene, seems to be absent or negligible (Harris, 1947). It should be mentioned here that dietary alterations might also affect the genesis of these neoplasms during the developmental stage. After a limited period of feeding p-dimethylaminoazobenzene in a brown rice diet, rats were continued on dye-free rations of brown rice, brown rice supplemented with yeast, or brown rice supplemented with milk powder. The incidences of liver neoplasms were significantly lower in the rats given the yeast or milk powder (Sugiura, 1944, 1951). Of the other B vitamins only biotin and pyridoxine appear to affect appreciably the rate of formation of azo-dye-induced hepatic tumors. Relative deficiencies of either of these vitamins hinder the genesis of the neoplasms (Burk et al., 1943; Miner et aE., 1943; Kline et al., 1945; E. C. Miller et al., 1945; Miller, 1947). The history and findings of the dietary studies, including vitamin alterations, on liver tumors induced in rats fed p-dimethylaminoazobenzene, have been more extensively reviewed elsewhere (Rusch et aE., 1945a; Opie, 1947). c. Tumors induced by vitamin deficiency. Up to this point in the review, experiments on diet and the nutritional state of the host as modifiers of carcinogenesis have been presented. There are two areas of investigation, however, suggesting that certain dietary deprivations may indeed be initiators of carcinogenesis-produce tumors without the administration of a known carcinogen. It has been recognized for some time that rats ingesting inadequate rations, deficient in vitamin A particularly, develop papillomatosis of the forestomach. The lesions may not be true neoplasia but rather an exaggerated example of the hyperplasia, hyperkeratinization, and metaplasia of epithelial tissues found in the vitamin A-deficient state (Passey et al., 1935; Fridericia et al., 1940). More recently it was reported that liver tumors develop in rats severely deficient in choline. The strain of rat used in these studies has a high requirement for choline, and the diets were so depleted that periodic administration of choline was necessary to prevent the death of the animals. The authors stated that “the choline deficient rats were in reasonably good condition for several months. Following this they gradually became unthrifty in appearance. There was marked loss of hair, muscular weakness, drowsiness, and lethargy ” (Copeland and Salmon, 1946; Engel et aE., 1947). A variety of pathological lesions was found, some obviously inflammatory. The state of the liver is of greatest ipterest. Severe degenerative and advanced cirrhotic changes were found in all animals which were on
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the choline-deficient diet for a t least eight months. Almost half the rats had hepatomas-these were not uniform in histologic appearance, and many were benign. This finding poses the important question as to whether the nodules are regenerating masses of liver cells in a severely damaged organ or true neoplasia. If it be the latter, do not these results support the often expressed view that tumors arise not by stimulation of normal cells, but as an adaptation to specific kinds of tissue injury? It is already known that cancer can be produced by physical, chemical, or parasitic agents. May severe dietary deficiencies, under special circumstances, also be carcinogenic? d. Effects independent of caloric intake and body weight. Several investigations have been carried out, in the past few years, on the influence of varying the level of B vitamins from what might be considered barely adequate for growth to a few times the optimal amounts. The studies utilized several tumor types, and control, or a t least appraisal, of caloric intake and body weight was accomplished. I n one such experiment the effect of multiple B vitamins, in low, control, and excess amounts, on the incidence of spontaneous lung adenoma in strain A mice was tested. Differences in tumor formation occurred, but these could easily be attributed to the differences in deaths from causes other than cancer (Taylor and Williams, 1945). Under similar dietary conditions the formation of skin tumors induced in mice with 3,4-benzpyrene was studied (Boutwell et al., 1949b). Ten of the B vitamins were varied as a group from levels estimated to be just adequate for maintenance to those considerably above the optimal. In other experiments, individual, pairs, and sets of B vitamins were given in minimal amounts, along with adequate quantities of the rest of the members. All diets were fed isocalorically. There were no significant differences in skin tumor formation, except for a somewhat lower incidence in the group fed low levels of all B vitamins. The mice of this group were reported to have shown some evidence of pyridoxine deficiency. Previously, the same laboratory reported an inhibition of skin tumor induction in mice fed a pyridoxine-deficient diet; caloric intakes and average body weights were controlled (Kline et al., 1943). We also have investigated the influence of B vitamins as a group, a t maintenance levels and in threefold or ninefold greater amounts, on the induced skin tumor (unpublished). Final incidences of neoplasms were of the same order in the three groups although the rate of formation was possibly slower in the mice of the low vitamin group. The same dietary factors were employed in experiments on the spontaneous mammary carcinoma of dba mice. The animals fed the low
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B vitamin diets weighed approximately 10 per cent less than those receiving three or nine times the minimal amounts. There were no noteworthy differences in the incidence of neoplasms, although here again they appeared in the groups on the low vitamin intake at a somewhat later average tihe (unpublished). With the spontaneous hepatoma of CsH mice, minimal levels of riboflavin or of B vitamins as a group had no important influence on rate of formation. Hepatomas developed in somewhat fewer of these animals, compared with controls, but the result is explicable on the basis of slightly lower average body weight (Silverstone and Tannenbaum, 1951a; unpublished). Analysis of the findings on the influence of varying the level of B vitamins suggests that wide changes in dietary content, in the range above minimal needs, have little effect on carcinogenesis. At least, this appears to be valid for the induced skin tumor and the spontaneous mammary, lung, and liver tumors of the mouse. As deficiency levels are approached there may be some inhibition of carcinogenesis, but in these instances caloric intake and body weight changes may be the major cause of the altered response. Except for the liver tumors induced by a50 dyes or choline deficiency, the levels of dietary vitamins have not been shown to affect significantly the genesis of tumors. Further work along these lines may prove fruitful, but only if the mistakes of the past are avoided, and the delicacy of the problem is recognized.
4. Minerals Several inorganic substances are implicated in the genesis of tumors. Clinical and experimental evidence reveal the carcinogenic action of arsenic, beryllium, chromates, and radium and other radioactive substances; however, none of these is a normal dietary constituent. There is no evidence that the formation of neoplasms is influenced by altering the proportions of inorganic components natural to the diet. The literature contains many contradictory reports; and the best studies, in the main, conclude that there are no important effects (Shear, 1933; Stern and Willheim, 1943). In recent investigations with the induced skin tumor, the spontaneous benign hepatoma, and the spontaneous mammary carcinoma, mice were fed semipurified diets containing a complete salt mixture at levels of 2, 4, or 8 per cent; a salt content of 4 per cent may be considered as normal. With all three tumor types there were no significant differences in carcinogenesis. The small effects observed could be attributed to the differences in caloric intake and body weight (unpublished).
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111. GROWTHOF TUMORS The neoplastic process is composed of a series of stages. Those comprising genesis have already been discussed; those grouped under the term “growth” are the subject of this section. For experimental consideration, the growth of a neoplasm can be divided into: 1) establishment in the host of the first few neoplastic cells, whether these developed in situ or were contained in fragments transplanted from another tumor; and 2) growth proper, that is, multiplication of the tumor cells to result in an increase in mass. Sufficiently distinct t o merit separate consideration is the spread of the tumor through invasion and release of metastastic emboli; this problem has been largely neglected in nutritional studies. The fact that this section on tumor growth is shorter than the foregoing discussion on genesis is no measure of their relative significance or of the number of investigations in these two areas. Actually, it seems a fair guess that more studies have dealt with growth. Disproportionate space has been allotted these subjects in the present review because changes in the nutritional state have definite and often striking influence on the genesis of tumors. In contrast, the studies concerned with the growth of tumors commonly reveal smaller effects or none at all; and for some diet components there is only a host of cloudy and contradictory reports. 1. Caloric Intake
Among the earliest studies on nutrition in relation to cancer are those indicating that underfeeding retards the growth of transplanted tumors (Moreschi, 1909; ROUS,1914; Sugiura and Benedict, 1926; Bischoff et al., 1935). Later this was shown to be a consequence of restriction in calories; i.e., it could be produced by reduction of only the carbohydrate (or fat) content of the diet (Bischoff and Long, 1938). I n some of these studies, there was a clear separation between establishment and growth of the tumors. When mice bearing mammary carcinoma were transferred to a low calorie ration, the growth rate of the tumors was significantly diminished; some even ceased growing and others became smaller, but complete regression was only rarely observed (Rous, 1914; Tannenbaum, 19404. The life span of the mice was increased to a small extent. In similar studies with sarcomas that had been previously induced in full-fed mice, caloric restriction hindered tumor growth but did not prolong life (unpublished). Mice inoculated with certain strains of leukemic cells lived longer, on the average, when underfed; however, with another strain of leukemia
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the average length of life was unaffected or possibly shortened (Flory et al., 1943). The underfeeding was begun the day after inoculation, and it is uncertain whether the restriction delayed the establishment of the leukemia or inhibited subsequent proliferation. Underfeeding or caloric restriction does not appear to be a successful means of controlling the growth of tumors. Not only is the neoplasm affected, but at the same time the normal tissues of the host diminish. It is questionable whether the life of the animal can be significantly or consistently lengthened. 2. Fat Variation in the proportion of dietary fat does not seem to influence the growth of tumors. Establiihment and growth of transplanted mouse and rat carcinomas and sarcomas were unaffected by fat enrichment of the diet (Sugiura and Benedict, 1930; Baumann et al., 1939). Sarcomas induced in mice on high fat diets grew a t the same rate as those induced in animals on a low fa t diet (Baumann et al., 1939; Tannenbaum, 1942b). The mean growth rate of spontaneous mammary carcinomas, arising in animals ingesting a semipurified diet containing 24 per cent fat, was the same as that in animals on a diet in which the fat was reduced to 2 per cent by equicaloric substitution of carbohydrate. Furthermore, the interval between recognition of the tumor and death of the mouse (survival time) was not correlated with the proportion of dietary fat-2, 4, 8, 16, or 24 per cent (Silverstone and Tannenbaum, 1950). 3. Protein
Numerous studies on dietary protein have succeeded in illustrating the vigorous ability of neoplasms to become established and grow, even in animals that are experiencing severe depletion. However, as with restriction of calories, protein deprivation decreases the rate at which these processes proceed. A more recent development has been the demonstration that variations in protein intake can modify the action of nondietary procedures. a, Variations in dietary protein only. The imposition of drastic dietary restriction of protein significantly hinders the establishment and growth of tumors. Spontaneous mammary carcinoma increased in size less rapidly in animals fed diets low in lysine or in cystine and methionine (Voegtlin and Thompson, 1936; Voegtlin and Maver, 1936; Voegtlin et al., 1936; Morris and Voegtlin, 1940). Kocher (1944) found that lysine-deficient diets produced only transient slowing of tumor growth; or, if the deficiency was instituted when the tumors were as large as 25 mm. in diameter, there was no effect. Although the results were
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discussed in terms of amino acid deficiencies, they can also be interpreted in terms of protein deprivation. In a well-designed study with a transplantable mammary adenocarcinoma, mice were fed a diet practically free of protein; the animals remained in continuous negative nitrogen balance and lost considerable weight during the experiment (White and Belkin, 1945). One week after institution of protein restriction the mice were inoculated with a mammary adenocarcinoma. The establishment of the tumors was not prevented by the protein deprivation. The rate of growth, however, was retarded but still was remarkably rapid-74 per cent of that in the controls-when one considers that the mice were consuming an almost protein-free diet. The final mean volume of the tumors in the depleted animals was nearly half that of the control tumors. A similar experiment with the Walker rat tumor 256 has been reported by Green and associates (1950). In this study the establishment of the implants was definitely delayed in rats protein-depleted by means of a diet containing only 1 per cent casein. Once the tumors became palpable, however, they increased in volume at about the same relative rate as those in control rats on a 22 per cent casein diet. Of course, since they became established later, the absolute rate of growth was less, and the tumors were not as large. Moderate dietary deficiency of protein has only a small effect on the growth of neoplasms. This has been shown in almost identical studies from two laboratories. Rats maintained on an otherwise adequate ration containing 5 per cent casein weighed only one-sixth less than those fed a 20 per cent casein diet. Correspondingly, the transplanted Walker 256 tumors in the protein-restricted animals attained weights about onesixth or one-twelfth less than those of the controls (Green and Lushbaugh, 1949; Devik et al., 1950). Essentially the same observations were made with another neoplasm, the rat hepatoma 31 (Voegtlin and Thompson, 1949). Inasmuch as diets containing as little as 5 per cent casein have only a small retarding effect upon the growth of tumors, one would expect that rations with higher proportions of casein would not be inhibitory. Actually, mice themselves grow well and at about the same rate when fed diets containing between 9 and 45 per cent casein. In mice consuming rations with casein contents within these ranges, spontaneous mammary carcinomas enlarged a t rates independent of protein intake; nor were there any effects upon the survival times and incidences of metastases (Tannenbaum and Silverstone, 1949a). Comparably, the growth rate of induced sarcomas in mice was not modified by varying the proportion of protein within the same limits (Rusch et al., 1945b; Tannenbaum and
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Silverstone, 1949a). Transplantable Walker tumor 256 grew as well in rats on a 10 per cent protein ration as in those on a 20 per cent protein intake (Elson and Haddow, 1947). I n summary, the response of tumor growth to alterations in dietary protein parallels the response of the host. Wide variations that have little effect upon the host, have no perceptible effect upon the neoplasm. Only with protein deprivation serious enough to cause striking loss of body weight is there significant retardation of the rate of establishment and rate of growth of tumors. b. Mode of action of protein restriction. Algire and Chalkley (1945) have demonstrated that growth of transplanted tumors does not become evident until completion of the primary vascularization process, on or about the fifth day. Complementary observations indicate that the initial inflammatory reaction to the implant is followed by development of a primitive connective tissue network into which the neoplastic cells migrate (Devik et al., 1950). In animals ingesting rations adequate in protein (for example, 20 per cent casein) the ingrowth of capillaries and organization of a connective tissue “capsule” is evident by the fourth to seventh day-establishment of the tumor is completed. A comparison of tumor implants in rats on 5 per cent casein with those in animals on 20 per cent casein revealed that the initial inflammatory reaction was greater on the low protein diet; on the other hand, the ingrowth of capillaries and the organiration of the connective tissue within and around the implant was delayed. These detailed findings indicate that one definite consequence of low dietary protein is delayed establishment of implants. There are no data as to whether these effects are specific or also follow other kinds of deficiency in dietary components or calories. Once established, even in animals markedly depleted in protein, a tumor increases in size and weight. It is remarkable that growth of the neoplasm proceeds while the host may be in continuous negative nitrogen balance. Apparently the protein of the tumor can be derived almost entirely from the host This fact should not lead to the oversimplification that the tumor has a specific affinity for protein only. Neoplasms grow, although at a reduced rate, in animals undergoing a variety of dietary deficiencies; such animals may be losing fat, vitamins, and minerals, as well as protein, yet these essential cellular constituents are accumulated in the growing neoplasm. Rather than regarding increase in protein as a special feature of tumors, it can be considered as only one aspect of their ability to grow under adverse circumstances. c . Protein restriction combined with other treatment. When 1,2,5,6dibennanthracene or other carcinogenic hydrocarbons were injected
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intraperitoneally in fairly large doses, the growth of tumors was inhibited (Haddow, 1935). More recently it has been shown that this action is highly dependent on the proportion of protein in the diet (Elson and Haddow, 1947). Rats were maintained on diets containing 20, 10, or 5 per cent protein for two weeks and then implanted with Walker tumor 256; the following day, half the rats on each protein level were injected with 50 mg. of dibenzanthracene. During the following thirteen days the tumors of the untreated rats grew to about the same average size; in other words, unrelated to the proportion of dietary protein. In contrast, the mean find tumor weights decreased with decreasing protein intake among the animals given dibenzanthracene ;the most pronounced inhibitory effect of the hydrocarbon was obtained with the rats on the 5 per cent protein diet. Likewise, Green and Lushbaugh (1949) found that intraperitoneal injections of 44imethylaminostilbene, beginning four days after the subcutaneous implantation of the Walker tumor 256, retarded tumor growth. This treatment was much more effective in the rats given a diet containing only 5 per cent casein than in those on a 22 per cent casein ration. The reports concerned with the influence of dietary protein on the response of tumors to x-radiation are of considerable interest (Elson and Lamerton, 1949; Devik el al., 1950). These studies were performed with Walker tumor 256 transplanted to rats on 5 per cent or 20 per cent protein diets. Irradiation of the tumor bed was begun six days after implantation. In the animals receiving the 5 per cent casein diet, the transplants first grew slowly and then more rapidly, ultimately causing the death of most of the animals; however, 15 per cent of the neoplasms completely regressed. Among the rats on the 20 per cent casein ration, on the other hand, the tumors grew well for the first few days, then began to shrink in size; in 90 per cent of the animals the neoplasms regressed completely. These three studies intimate synergistic action of protein restriction and the nondietary procedures. They also point out a potentially fruitful field for investigation. It should be noted, however, that in the described experiments the nondietary treatments were instituted at one, four, or six days, respectively, probably before the implants were fully established in the animals on the low protein diet. Thus, at least part of the differential effects might have occurred through action on tumor establishment.
4. Vitamins Critical examination of the numerous investigations on the influence of vitamins, in deficient and excess amounts, on the growth of neoplasms
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reveals relatively few instances of clearly demonstrated effects. On perusal of reviews on this subject, one is impressed by the number of studies, diversity of results, and the inherent complicating factors (Stern and Willheim, 1943; Burk and Winzler, 1944; Morris, 1947). Various multiple and individual vitamin deficiencies undoubtedly hinder the growth of tumors; but in only a few instances can any specificity be attached to the results. Recently, there has been improvement in the experimental approach; and the development of the antivitamin analogue technique has supplied a new and useful tool. The following few examples of current research are representative. Illustrating the problems encountered in studying the influence of dietary deficiencies on tumor growth are Morris’ (1947) experiments on thiamine deprivation in mice bearing spontaneous mammary carcinomas. When diets were fed ad libitum, the mice on the low-thiamine ration ate less food and lost weight in comparison with those on the thiaminesupplemented ration. As might be anticipated tumor growth was retarded. However, if the food intake of the control mice was restricted to that of the thiamine-deficient animals, the rates of tumor growth were essentially the same. Unexpected were the results of an experiment in which thiamine-deficient and thiamine-supplemented rations were force-fed in equal amounts, presumably at about the level that would be voluntarily ingested by the controls: The depleted mice showed no relative loss in body weight, and their tumors grew a t an apparently accelerated rate in comparison with those of the thiamine-supplemented animals. The findings of this last experiment are in agreement with those obtained by DobrovolskaIa-Zavadskaia (1945), who reported that daily subcutaneous injection of thiamine hindered the growth of spontaneous mammary carcinoma in mice. The influence of various vitamin deprivations on the growth of established mouse lymphosarcoma C3H-ED transplants was examined by Stoerck and Emerson (1949). The deficiencies were induced by the use of antagonistic analogues. Those of thiamine, niacin, and folk acid had no effect. However, production of deficiencies in either pyridoxine or riboflavin caused apparently complete regression of the tumors. Correction of the pyridoxine deficiency resulted in reappearance and growth of the tumors, whereas no recurrence was observed following relief of riboflavin deficiency. The establishment and possibly the growth of Rous chicken sarcoma was markedly inhibited by folic acid deficiency. In contrast, restriction of other vitamins did not influence tumor growth except when the chicks were depleted to dangerous levels (Little et al., 1948).
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Folic acid antagonists have a marked inhibitory effect on some lines of transmissible mouse leukemia, presumably through the induced folk acid deficiency. It has been pointed out that in most studies on mouse leukemia the antagonists were administered very shortly after inoculation of the leukemia cells. Possibly then, interference occurs in the tumorhost integration or establishment (Kirschbaum el al., 1950). In studies with four different transplanted leukemias neither aminopterin nor amethopterin prolonged the life span of the host if treatment was delayed until eight days after inoculation. Nevertheless, there must be some influence on the multiplication of leukemic cells; unquestionable support for this is the often dramatic effect.in children with acute leukemia. There is an interesting report on the consequences of ascorbic acid deficiency on the growth of a previously established sarcoma in the guinea pig. After institution of a scorbutogenic diet tumor growth slowed apprec'ably, even before any changes were noted in food consumption or body weight. The neoplasms attained a weight only about one-fourth those of the control pigs. A greatly altered stroma and decrease in collagen of the tumors in the scorbutic animals were regarded as significant (Robertson et al., 1949). The growth of several kinds of tumors is suppressed by administration of 8-azaguanine. In studies with the mammary adenocarcinoma 755 in strain C67 Black mice, it was found that the effect could be greatly enhanced by injection of other substances, begun four to fourteen days after tumor implantation (Shapiro and Gellhorn, 1951). Some of the observed synergisms might have been anticipated; for instance, desoxypyridoxine and 7-methylpteroylglutamic acid (in doses that had no consistent effect when administered alone) intensified the retardation. Against expectation was the observation that similar extension of the azaguanine inhibition generally followed administration of vitamin Bla or folic acid. The effects could not be attributed to obvious toxicity to the animal. In the main, our general remarks in the last paragraph under " Genesis of Tumors-Vitamins '' apply also to growth. Unfortunately, the increment to our knowledge through research on vitamins in relation to tumor growth is not commensurate with the time and funds expended. Perhaps, as more is learned about both the cancer process and the science of nutrition, studies in this area will become more definitive.
STATEAND CANCERIN MAN IV. NUTRITIONAL
mars
Our knowledge of the role of diet in the genesis and growth of has come mainly from investigations with animals. However, there are
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statistical and clinical studies on man relevant to the significance of the nutritional state, Some of these are in agreement with the findings with animals; others might well provide a basis for further animal experimentation. 1. Body Weight and Cancer Incidence
The genesis of many types of tumors is arrestingly curtailed in mice that are calorie-restricted or underweight. Does this phenomenon have its counterpart in human beings? This question cannot be answered readily by experimentation, but statistical and clinical studies throw light on the subject. For some years, insurance companies have been interested in the connection between various social and biological factors and the relative frequencies of the principal causes of death. Relevant to this review is the correlation between body weight and cancer mortality. The general methods of approach and the validity of these studies are acceptable (Tannenbaum, 1940b, 1947). Although the statistics relate to cancer mortality, the latter corresponds roughly to cancer incidence. In this regard, it is important to emphasirie that the insured were classified according to weight at issue of policy, many years before they were diagnosed as having cancer. Six of the insurance studies and the results of one extensive questionnaire on dietary habits have been summarized in a review (Tannenbaum, 1940b). A brief outline of the findings is presented in Table IV. They imply that individuals who overeat and are overweight when past middle age are more likely to die of cancer than persons of average weight or less. In one study the cancer mortality per 100,000 insured persons 25 per cent or more overweight, normal weight, and 15 per cent or more underweight, were 143, 111, and 95, respectively. A difference of 50 per cent in cancer mortality between overweight and underweight individuals! Inasmuch as this relationship between cancer incidence and body weight is strongly supported by controlled animal experimentation, it seems reasonable to expect that the avoidance of overweight would result in the prevention of a considerable number of cancers in man, or at least in the delay of their time of appearance. Furthermore, the dietary control need not be drastic since both the animal studies and the insurance statistics reveal a dose-response relationship. Even moderate continued caloric restriction or control of body weight deters the development of neoplasms. These remarks apply only to prevention of the genesis of tumors and not to the treatment of cancers once they have formed. Present evidence holds no hope that calorie restriction or lowering of body weight is a practical means of controlling the growth of an established cancer.
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TABLE IV Statistical Studies on Body Weight and Cancer Mortality Reference
Source of Material
Conclusions
Actuarial Society of America Contributing Insurance Cancer mortality increases and Association of Life Companies with increasing body weight Insurance Directors, 1913 Cancer mortality increases Union Central Life Dublin, 1929 with increasing body weight Insurance Company Metropolitan Life A high percentage of those Dublin, 1929 Insurance Company dying from cancer are overweight Actuarial Society of America Contributing Insurance No clear relationship between cancer mortality and and Association of Life Companies body weight Insurance Directors, 1932 Questionnaires of Overnutrition is unusually Hoffman, 1937 common in histories of hospitalized patients cancerous patients with cancer Cancer mortality increases Metropolitan Life Dublin and Marks, 1938, with increasing body weight Insurance Company 1939 (true for some sites but not for all) Hunter, 1939 New York Life Cancer group has a higher Insurance Company average weight than control group (true for some sites, not for all)
2. Dietary Dejiciencies and Cancer Production
The differences in the dietary of various races, coupled with clinical and pathologic data on the relative frequency of various types of neoplasms, have led to impressions that some tumor types in man might be related to dietary deficiencies. The evidence and reasoning with regard to the pathogenesis of these neoplasms are circumstantial. Before accepting these oft-repeated claims, it might be well to await further clinical study and experimental proof. a. Thyroid cancer. The presence of nodular goiter appears to influence the occurrence of cancer of the thyroid. BBrard and Dunet, cited by Wegelin (1928), found that in endemic goitrous regions from 2.5 to 4.0 per cent of all malignant tumors arose in the thyroid, whereas in relatively goiter-free areas the frequency was only 0.4 to 0.5 per cent. Wegelin himself commented upon the much greater incidence of thyroid cancer in necropsies in Berne, the center of a goitrous region, in comparison with that reported from Vienna, Prague, and Berlin. Statistics obtained from both operations and autopsies indicate that these tumors
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occur about ten times more frequently in Switzerland than in the United States. That nontoxic nodular goiter is a precursor of malignancy is also suggested by studies on individuals within this country (Ward, 1944; Cole et al., 1945). Inasmuch as goiter occurs with much greater frequency in regions where there is an iodine deficiency of the soil, drinking water, and foodstuffs, it is reasoned that a chronic iodine deficiency may be a factor in the genesis of thyroid cancer. Perhaps the iodine deficiency sensitizes the thyroid to goitrogens and even potential thyroid carcinogens. b. Pharyngeal cancer. Women of those areas of Sweden and Finland within the Arctic Circle are reputedly prone to develop carcinoma of the pharynx, oral cavity, and esophagus. Individuals with such tumors often have a history of Plummer-Vinson syndrome, characterized by anemia, achlorhydria, and atrophy of the mucous membranes; later, hyperkeratoses of the oral and pharyngeal mucosa develop (Ahlbom, 1936; Adair, 1947). Ahlbom found this syndrome in 80 of 123 women with cancer of the mouth, pharynx, or esophagus. Might it be related to an iron- and vitamindeficient dietary-reindeer meat and fish, with few green vegetablea-supposedly common to the inhabitants of the region? c. Liver cancer. The frequency of primary cancer of the liver, as a proportion of all neoplasia, is high in some regions of the world, low in others. Among natives of southeastern Asia-China, Japan, Java, and Sumatra-the relative incidences range between 7 and 41 per cent, and even higher among young male African Negroes (Bantus) employed as gold miners (Bonne, 1937; Berman, 1940; Gilbert and Gillman, 1944). In contrast, the disease accounts for only about 1 per cent of all cancers among Caucasians, and is also low among Negroes living in Europe and the United States (Pack and LeFevre, 1930; Berman, 1940; Kennaway, 1944). Specific racial susceptibility, endemic infections, and faulty nutrition have been suspected as probable etiologic factors of liver cancer among those groups generally afflicted. The dietary theory has attracted interest, particularly since cirrhosis of the liver is also common among the population prone to cancer of the liver (Berman, 1941; Gilbert and Gillman, 1944). Attention is thus focused on the rather general opinion that hepatic cirrhosis in man is frequently associated with primary cancer of that organ. There is little experimental evidence for a causal dependence. Liver cancers have been produced without cirrhosis either as a precursor or as an associated condition. On the other hand, severe liver damage, including cirrhosis, has been produced in rats by feeding cornmeal mush and sour milk, the principal food of the Bantus, and yet no tumors were found (Gillman, 1944; Gillman et al., 1945).
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More recently, it has been reported that primary cancer of the liver may be initiated by the ingestion of certain crude substances. Rats fed chilli peppers (Hoch-Ligeti, 1951) or alkaloids of Senecio jacobaea (Cook et al., 1950) developed tumors of the liver. These substances, while not considered normal dietary components, are nevertheless used by some of the populations revealing the abnormally high incidence of liver cancer. By analogy with the influence of diet on hepatocarcinogenesis through azo dyes, poor dietaries might favor the development of liver cancer among individuals ingesting such crude “ carcinogens.” The entire subject is illuminated in a recent monograph by Berman (1951). He suggests that liver cancer in man depends on a sequence of events, a combination of environmental circumstances. Malnutrition, by injuring the liver, may make it more vulnerable to a variety of infections and toxic substances that have carcinogenic potentialities. This reasoning also may apply to the neoplasms discussed above-of the pharynx and the thyroid.
V. CONCLUSIONS AND COMMENTARY I n the preceding sections we have attempted to organize the available knowledge on the significance of diet in the cancer process. Important contributions may have been omitted inadvertently. More often, however, reports have not been included because of their uncertain meaning due to one or more of the following: the omission of details revealing the composition of the diets, the very small numbers of animals utilized, the lack of data on food intake and body weights, the stormy course of the experiment including many nontumor deaths early in the study, or questionable results. Many interesting experiments concerned with the effects of animal tissues, organ extracts, and other relatively crude materials have not been taken up because of insufficient information as to their composition and the components responsible for the effects noted, and for lack of space. Nevertheless, a reasonably large number of facts have been accumulated, allowing a better degree of comprehension and systematization than possible a decade or two ago. It is intended in this section to summarize current knowledge, to discuss some implications of the findings, and the directions future work might take; and finally, to make some speculative and provocative comments as to the role that nutrition plays in the genesis of neoplasms. I . Summary of Present Knowledge a. Genesis of tumors in animals. It has been demonstrated that the origin of neoplasms depends in part on the nutritional state of the host. A noteworthy example is the inhibition of the formation of tumors that
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is brought about by dietary restriction of calories. Many diverse types of neoplasms respond to caloric deprivation by a reduction in tumor incidence and a delay in appearance: spontaneous mammary carcinoma, skin tumors induced by carcinogenic hydrocarbons or ultraviolet light, induced sarcoma, spontaneous lung adenoma, spontaneous hepatoma, and spontaneous and induced leukemia-all of the mouse; and lymphosarcoma and induced mammary carcinoma of rats. In fact, all tumor types tested extensively have been affected in this way. For completeness it should be mentioned that in two instances a low-calorie effect has not been found, but this may be due to masking by special factors of carcinogenesis. The magnitude of the inhibition of tumorigenesis by caloric restriction is related to the extent of the deprivation and the composition of the restricted diet. In addition, the repression occurs mainly during the developmental stage; the initiatory stage seems little affected. In contrast t o the arresting and relatively consistent influence of caloric restriction are the quite diverse effects of fat enrichment of the diet. The genesis of the spontaneous mammary carcinoma and induced skin tumor is somewhat enhanced by high fat diets. Hepatic neoplasms, spontaneous and induced, also tend to form more readily when the animals ingest these diets, Contrariwise, no noteworthy effect has been observed with the induced sarcoma, primary lung adenoma, and spontaneous and induced leukemia. Where high fat rations favor carcinogenesis, there is evidence that the influence occurs in the developmental stage; and its intensity is dependent on the degree of fat enrichment. Altering the proportion of dietary protein-within limits (9 to 45 per cent casein) that support relatively normal growth and weight of the mouse-influences the genesis of some tumor types but not that of others. The spontaneous mammary carcinoma and the induced skin tumor are not significantly affected, On the other hand, formation of the spontaneous hepatoma of the mouse is strikingly enhanced by changing the proportion of protein from 9 to 18 per cent, whereas a similar increase in dietary protein appears to hinder the formation of liver tumors induced by azo dyes in the rat. It has been reported that diets deJicient in total protein or essential amino acids suppressed the genesis of certain tumors. Increasing the proportion of B vitamins as a group, from levels considered minimal for good growth to those several times greater than optimal, exerts no significant influence on the formation of spontaneous mammary or liver tumors and of induced skin tumors of the mouse. Diets deJicient in vitamins generally retard the genesis of neoplasms, but the question arises as to whether the actions are specific or are mediated through the associated lower caloric intake and body weight. A clearly defined action of dietary vitamins on carcinogenesis is that
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of riboflavin on hepatic tumors induced in rats by certain azo dyes. Low proportions of the vitamin, or dietary alterations which result in a low level of hepatic riboflavin, enhance the formation of this neoplasm. Another interesting finding is the development of liver tumors in rats chronically deprived of choline, without the administration of a known carcinogen. Varying the level of a complete salt mixture, between reasonable limits of “normal” intake, has no recognizable effect on the genesis of spontaneous mammary carcinoma and benign hepatoma and induced skin tumors. b. Growth of tumors in animals. Dietary alterations and the nutritional state of the host influence the growth of neoplasms but less impressively than they affect the genesis. With transplanted tumors it should be kept in mind that the nutritional state may affect the establishment of the implant as well as its subsequent growth. Experiments designed to study these separately will yield the most definitive results. A tumor grows even while the animal is losing weight, incfact, at the expense of the host’s normal tissues. Consequently, although caloric restriction inhibits the growth of a neoplasm (it also causes the host to lose weight) the life span of the animal is not appreciably lengthened. Variations in the proportions of dietary fat, protein, vitamins, or minerals, within the limits that are necessary for relatively normal growth and body weight of the host, have not been found to modify tumor growth. On the other hand, protein or vitamin deficiencies may inhibit the rate of growth of neoplasms, but the retardation may be mediated in large part through voluntary restriction of food intake and consequent loss of body weight. From the presently available evidence, it seems that no specific tumor-controlling influence has been demonstrated for diets deficient in fats, proteins, and minerals. For the most part this is also true for vitamins, but there are a few instances in which specific vitamin deficiencies appear to inhibit particular tumor types. c. Cancer in man. Clinical and statistical findings suggest that the genesis of some kinds of tumors in man is partly dependent on the nutritional state, that is, whether the individual is overweight or underweight. Other aspects of the dietary, particularly deficiencies, conceivably play a role in the development of some neoplasms. However, there is no indication that controlling the nutritional state is a practical means of arresting the growth of tumors. 2. Implications
The increased interest and work of the past two decades have resulted
in a broadened recognition of the relationship of nutrition to cancer. A
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good many of the obvious experiments have been performed so that we now know, perhaps only generally and crudely, the significance of the level of caloric intake and of the proportions of dietary fat, protein, vitamins, and salts, in the genesis and growth of certain tumors. One cannot predict beforehand the importance of the isolated facts and correlations that have been unearthed. In the future they may or may not be integrated with information from other fields to furnish practical working tools. One significant implication is the following: the inhibitory influence of calorie restriction and lower body weight also has connotations for many nonnutritional investigations in cancer research. Restriction of calories or deprivation of particular essential components of the diet may be a planned part of a study, but can occur without the intention or knowledge of the investigator. For example, animals subjected to chemotherapeutic agents (hormones, antagonists, chemicals, etc.) or t o irradiation may reduce their food intake or develop an increased need for particular dietary essentials. Intercurrent infections can cause diarrhea or loss of appetite. These and other circumstances could bring on subnormal food consumption, altered metabolism, or lower body weight. Should any of the above conditions, recognized or not, become an unplanned factor of a cancer experiment, the genesis or growth of tumors might be retarded. It then becomes necessary to ascertain whether the results are directly related to the procedure under investigation, or are due to the accompanying side effects of undernutrition and decreased body weight. Of course, caloric restriction and low body weight are not the common denominators of all inhibitory effects, but for more exact interpretation the nutritional state of the animals should be known. 3. Speculations on Nutrition in Relation to Carcinogenesis
Considering the large variety of diseases that are classified together as neoplasms, it is not expected that they would all respond to a particular dietary change in the same manner. Actually, with some diet components there has been a striking conformity of effects. In the case of others, equally impressive diversity exists. At the present time it is difficult to determine how much of this disparity is dependent on real differences in the biology and biochemistry of the tumors themselves. Certainly there are suggestions, mainly untested, that part of the observed differences may be attributed to differences in factors of the experiments, such as the nature of the carcinogen, its potency, and how it reaches the site of action. Another determinant may be the varying effects of the particular dietary alteration on the respective tissues being acted on by the carcinogens.
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The uncommon and diverse responses of tumors of the liver to experimental nutritional procedures is an arresting problem for a reviewer attempting to systematize and generalize. The anatomy and manifold functions of this organ dispose it t o multiple types of reactions. Its special blood supply (portal vein) and property as a depot result in an intracellular composition that fluctuates widely with diet changes. Moreover, it is very susceptible to the action of toxic agents and necrotizing conditions. Certain carcinogens may be formed in the organ, while others, some deliberately administered, reach it indirectly. When more is known about the various sets of events that induce hepatic neoplasms and how a particular set is modified by a change in the nutritional state of the host (and liver), many of the seemingly divergent effects may fit into a more consistent pattern. The knowledge of agents that induce tumors is extensive, but our understanding of the way they act upon the cells and the cell environment to result in neoplasms is meager. A dietary change, and the consequent alteration in the nutritional state of the host, may influence carcinogenesis in the following ways: (1)by modifying the solubility, rate of metabolism, metabolic products, or amount of the carcinogen reaching the target tissues (the effective tissue dosage of actual carcinogen); (2) by modifying the susceptibility of the target cells to tumor-initiating action; and (3) by modifying the development of the initiated, biased cells. These latter two influences may involve not only cells but their environment: cell surface, ground substance, stroma, and blood supply. Stated otherwise, nutritional effects may be mediated through changes in the dose of effective carcinogen, the initiatory stage, or the developmental stage. For any particular nutritional modification none, one, or more than one of these possibilities exists. The influence may be determined systemically or locally, but the end result must be a t the actual site of tumor genesis. What is the relative significance of the three ways, stated above, in which modifications of the nutritive state can affect the genesis of tumors? There is no single answer. In the origin of some kinds of neoplasms striking changes may be produced because the nutritional alteration modifies the amount of actual carcinogen reaching the site of action. Such situations may not be uncommon. It is likely that for many tumor types, dietary changes that do not seriously affect the health of the host only slightly modify the susceptibility of the target cells to the carcinogen. The initiation of neoplasms is probably influenced to only a small extent under these conditions. However, in the origin of some tumors, dietary deficiencies may play an important role in modifying and sensitizing the target cells so that they become more vulnerable to
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carcinogens. From evidence now available, we are of the opinion that for most tumors nutritional alterations have their main effects during the developmental stage of carcinogenesis. Whether these involve energy only or are intimately bound up with essential cellular constituents is only conjecture at this time. This review has been concerned with the influence of known and relatively welldefined components of diets. However, natural foods contain a number of constituents which have been given little attention in nutrition and cancer research because they aoparently are not dietary essentials. In addition, there must be others yet undetected. Perhaps among these unregarded substances are some with carcinogenic activity; and others that potentiate or oppose the action of carcinogens. There is danger here that findings may be coupled with suggestions and guesses to build up concepts which by pyramided repetition become accepted. That is not the purpose of the reviewers, who have great respect for facts and recognize the pitfalls of speculation.
4. Future Developments We have no intention of making predictions, but believe it might be helpful to state our general views as to potentially fruitful fields of nutrition-cancer research. In fact, these are some of the problems of current interest to us. Although the subject has been probed and examined in a general way, there are many specific dietary components that have not been investigated. Hunch and logic suggest that most of such studies would not yield startling results, but the information would help to complete the overall picture. Another area for study, hardly begun, is the relationship of nutrition to the spread, establishment, and growth of metastases. In the section concerned with neoplastic growth, reference has been made to a few investigations in which dietary alterations were combined with a second experimental procedure. Two-factor approaches of this kind intimate that combined measures may have synergistic influences on the genesis and growth of tumors. Recently there has been a revival of interest in social and environmental factors as causes of cancer. Chronic malnutrition may be one of these, possibly not acting alone but preparing the soil for greater vulnerability to infectious and toxic carcinogenic agents. Other nutritive states, alone or in combination with special factors, may be implicated. As suggestive clinical and experimental evidence regarding carcinogenesis comes to our attention, it should be extended by vigorous clinical studies of the nutritive state and pathogenesis. Investigations in this area may result in revealing the nature of the essential conditions
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of some types of human cancer. Already there are indications that a search for carcinogenic compounds in human dietary regimens might be worthwhile. Early nutrition-cancer research had to be content with fact finding, and this has been accomplished to a satisfactory degree. More and more now, attention is being turned to studying the mode of action of those diet alterations which affect tumor genesis. This work may proceed to a better understanding of carcinogenesis itself. During the period, years or decades, that it takes to solve substantially a problem in medical research, perspective is both biased and clouded. It is often hampered by understandable high hopes and by unsubstantiated sensational claims. Nevertheless, an appraisal of the many solid facts unearthed by nutrition-cancer research and the present trend toward study of mechanisms leads one to conclude that the field is contributing t o the comprehension of the cancer process.
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Plasma Proteins in Cancer RICHARD J. WINZLER Department of Biological Chemistry, University of Illinois College of Medicine, Chicago, 111. CONTENTS
I. Some Methods of Study of Plasma Proteins
Salt Fractionation Electrophoresis Fractionation with Alcohol at Low Temperature and Ionic Strength Other Methods 11. Alterations of Plasma Proteins in Neoplastic Disease 1. Albumin A. General Considerations B. Thermal Coagulation Procedures C. Polarographic Serum Test D. Binding of Anionic Dyes E. Methylene Blue Reduction 2. Alpha Globulins 3. Especially Soluble and Stable Proteins A. Polypeptidemia B. Polarographic Filtrate Test C. Mucoprotein D. Albumin A 4. Beta Globulins 5. Fibrinogen A. Sedimentation Rate B. Heat Turbidity Test 6. Gamma Globulins A. Amounts and Immunity B. Flocculation with Lipoidal “Antigens ” C. Multiple Myeloma 7. Changes in Protein Stability to Precipitating Agents 111. Plasma Enzymes and Inhibitors 1. Enzymes A. Acid and Alkaline Phosphatases B. Aldolase c. D-Peptidases D. Carcinolysis E. Plasmin F. Fuchs’ Test G. Abwehrferment 2. Enzyme Inhibitors A. Inhibition of Proteolytic Eneymes 1. 2. 3. 4.
SO3
Page 506 506 507 511 “13 513 514 514 516 518 518 519 520 521 522 522 523 524 524 524 525 525 525 525 526 527 528 529 529 529 530 530 530 531 53 1 532 532 532
RICHARD J. WINZLER
504
Page B. Inhibitions of Hyaluronidase . . . . . . . . . . . . . . . ..................... 534 534 C. Inhibitors of Oxidiiing Enzymes.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Protein-Bound Carbohydrate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535 1. Total Polysaccharide. ........................ 535 2. Tryptophan-Acid ........................................ 537 .. . . . . . . . . . . . . 538 3. Diphenylamine R V. Discussion.. . . . . . . . . . . . . . . . . . . . . . . . 538 References . . . . . . . . . . . . . . . . . . . . . . . . . . . 539
One of the enigmas of the cancer problem is the mechanism by which neoplastic disease may kill a host when no apparent interference with a vital structure is evident. That cancer produces systemic effects in the host is well known, but there is little information on the profound changes in physiology which must be a t the basis of such systemic effects. It has also been recognized that there are abnormalities in the protein composition of the blood. However, the nature of the changes in the plasma proteins and their physiological and pathological bases are still largely unanswered problems. There has been a growing knowledge of the numbers and properties of the plasma proteins. Yet we are only beginning to understand something of the source, the significance, and the metabolic activity of these agents. It is presumptive indeed, to attempt a discussion of the relation of plasma proteins to cancer when so little is yet known about fundamental aspects of plasma proteins in the normal individual. There may be, however, some value in reviewing what is known about the relationship of plasma proteins to malignant disease. It is in this humble vein that this discussion is written. In this respect this discussion may be considered a supplement to the reviews by Toennies (1947), Huggins (1949), and Gutman (1948). Some aspects of the problem of the serodiagnosis of cancer are related to changes in plasma proteins and will be briefly considered. In Table I are listed some of the changes in plasma proteins which TABLE I Some Changea in Plmma Proteins Associated with Neoplastic Disease in Humans Abnormality in Cancer Decreased albumin content Increased alpha-1 globulin content Increased alpha-2 globulin content Increased fibrinogen content
Page No. 507, 514, Table I1
Sample References
Mider et al. (1950) Seibert et al. (1947) 508, 520, Table I1 Mider el al. (1950) Seibert el al. (1947) 508, 520, Table I1 Mider et al. (1950) Seibert et al. (1947) 508, 524, Table I1 Mider et al. (1950)
PLASMA PROTEINS IN CANCER
505
TABLE I (Continued) Abnormality in Cancer Decreased amounts of polarographically determinable- SS i r -SH groups Decreased amounts of free SH groups determined amperometrically Decreased “reducing power” when heated with methylene blue Decreased tendency toward thermal coagulation Decreased amount of iodoacetate required to prevent thermal coagulation Increased tendency of plasma to become turbid on being heated Reduced ability to bind anionic dyes
Page No.
Sample References
518, Table IV BrdiEka (1937, 1939) 517, Table IV Schoenbach et al. (1950, 1951) 519, Table IV Savignac et al. (1945) Black (1947a,b) 516, Table IV Glass (1951); Huggins et al. (1949b) 516, Table I V Huggins et at. (1949a,b)
Black et al. (1948) 525 518, Table IV Huggins et al. (1949a) Westphal et al. (1951) 535, Table VI Shetlar et al. (1949, 1950) Seibert et al., (1947, 1948)
Increased total protein-bound carbohydrate content Increased total protein-bound hexosamine 523, 536, Table V Increased mucoprotein content Increased amounts of highly soluble 522, 523, Table V and stable proteins determined polarographically or chemically Decreased tendency to gel upon treatment with lactic acid Increased aldolase content
528, Table V 530
Increased acid or alkaline phospha tase content (metastatic cancer in bone)
529
Increased activity of inhibitors of hyaluronidase Increased activity of inhibitors of proteolytic enzymes Increased tendency toward flocculation by lipoidal “antigens”
534
Decreased amount of heat coagulable soluble 11 albumins ” Increased erythrocyte sedimentation rate
532 526
524 525
,
West and Clarke (1938) Winzler and Smyth (1948) Wolff (1921); Winzler and Burk (1944) Hahn (1921); BrdiEka (193913) Kopaczewski (1935, 1937) Warburg and Christian (1943) Sibley and Lehninger (1949) Gutman and Gutman (1938) Kay (1929, 1930) Huggins and Baker (1951) Hakanson and Glick (1948) Cliffton (1949, 1950) West el al. (1949a, 1951c) Shaw-Mac Kenzie (1922) Gruskin (1929) Penn et al., (1950) Kahn (1925) Gram (1922); Gilligan et al. (1950)
506
RICHARD J. WINZLER
have a t one time or another been studied in connection with neoplastic disease in humans. Some of these involve well-characterized changes in concentration or in activity of specific proteins and rest on considerable fundamental research effort. Others, regrettably, have been studied entirely from an applied point of view, and little effort has been made to discover the fundamental bases of the changes noted. Almost without exception the alterations listed have no specificity with regard to generalized cancer. Table I emphasizes the fact that advanced neoplastic disease is associated with a multitude of changes in the amounts and activities of a large number of plasma proteins, or in phenomena associated with their presence. Many of these abnormalities have been discussed in previous review articles on serodiagnosis of cancer (Bing and Marangos, 1934; Davidsohn, 1936; Woodhouse, 1940; Stern and Willheim, 1943; Maver, 1944; Homburger, 1950; and Sobotka, 1951).
I. SOMEMETHODSOF STUDYOF PLASMA PROTEINS A number of the proteins in plasma are present in rather large amounts and may be designated as the major components. The amounts of some of these proteins or of related groups have been determined by a number of methods, chief among which are fractional precipitation by neutral salts, fractional precipitation by ethanol a t low ionic strength and low temperature, and electrophoretic analysis. The application of these methods to the study of the plasma proteins in disease has been discussed in an excellent review by Gutman (1948). 1. Salt Fractionation One of the most extensively used methods for the study of plasma proteins has been by fractional salting out with ammonium sulfate or sodium sulfate. A considerable amount of the work up to 1948 on the procedure and its significance has been discussed by Gutman (1948) and by Edsall (1947). The bulk of the information that has been gathered on plasma protein fractions in cancer and in other pathological states has been obtained by the method of Howe (1921) using sodium sulfate fractionation. In this procedure, albumin is defined as the protein left in solution at 21.5% sodium sulfate, and the globulins are designated as the proteins insoluble a t this salt concentration. This separation is based on protein solubilitysalt concentration curves obtained with plasma in which a critical concentration zone between 21 and 22% sodium sulfate produced little increase in precipitated protein. Further fractionation of the globulins into euglobulin and pseudoglobulin was developed by Howe (1921) based
PLASMA PROTEINS IN CANCER
507
on inflections in the salt concentration-protein solubility curves a t 14 95 and 16.9% sodium sulfate. It is now recognized that the Howe procedure gives protein fractions which are electrophoretically inhomogeneous, with albumin values higher than those determined electrophoretically (Tiselius, 1937b ; Luetscher, 1940; Gutman et al., 1941; Svensson, 1941; Taylor and Keys, 1943; Dole, 1944; Majoor, 1946, 1947; Milne, 1947; Petermann et al., 1947; Martin and Morris, 1949; Jager et al., 1950). The albumin fraction contains considerable quantity of alpha and beta globulins, and may give albumin values that are 20 to 40% higher than values obtained electrophoretically. This may be particularly marked in pathological samples where albumin is low and the alpha globulins are raised. The distribution of the plasma proteins as measured by the Howe method shows a characteristic trend toward lowered albumin values and normal or even elevated globulin values in a large number of diseases including neoplasia. A number of other procedures for the separation of albumin and globulin by salt fractionation have been developed which are improvements over the Howe method, e.g., 26% sodium sulfate (Milne, 1947; Majoor, 1946, 1947), 26.9% sodium sulfite (Cohn and Wolfson, 1947, 1948), saturated magnesium sulfate (Popjak and McCarthy, 1946), 21.5% sodium sulfate in the presence of ether (Kingsley, 1940). 6. Electrophoresis
The development of moving boundary electrophoretic methods by Tiselius (19374 has resulted in considerable advance in knowledge of the plasma proteins. This procedure makes use of the rate of migration of proteins in an electrical field, the boundaries between components being visualized from changes in refractive index as a function of the distance from the starting point. The hope that plasma electrophoretic patterns might be characteristic of specific diseases, and thus be of great diagnostic significance, has not been generally supported by the large amount of work that has now been carried out. A large number of apparently unrelated pathological conditions result in very similar abnormalities in electrophoretic patterns of plasma. However, the electrophoretic method applied to plasma has given a great deal of accurate information on the changes in levels of albumin, of alpha, beta, and gamma globulins, of fibrinogen, and of the occasional presence of abnormal proteins in plasma in various diseases. The application of electrophoresis to the study of plasma proteins has been reviewed by Stern and Reiner (1946), by Luetscher (1947), and by Gutman (1948). Typical electrophoretic patterns obtained with normal and cancer
508
RICHARD J. WINZLER
serum in pH 8.5 veronal buffer a t ionic strength 0.1 are shown in Fig. 1. The lowered albumin and raised alpha globulin and fibrinogen levels in cancer plasma shown in Fig. 1 are in general agreement with the observations of numerous investigators who have made this comparison (Longsworth el al., 1939; Luetscher, 1941; Seibert el al., 1947; Petermann and Hogness, 1948a; Petermann el al., 1948; Dillard el al., 1949; Mider et al., 1950; Schoenbach et al., 1951). I n the most extensive of these studies Mider, Alling, and Morton (1950) have studied electrophoretically the distribution of plasma proteins in 258 patients with uncomplicated neoplastic disease under the most commonly employed electrophoretic conditions (pH 8.5 veronal buffer
FIQ.1. Electrophoresisof serum at pH 8.5 in veronal buffer at ionio strength 0.1. Ascending boundary. Left pattern normal, right pattern mammary carcinoma. (Courtesy of Dr. J. W. Mehl.)
a t ionic strength 0.10 (Longsworth, 1942)). Some of the data obtained by Mider e l al. (1950) is shown in Table 11. The data show a highly significant trend toward hypoalbuminemia, and equally significant increases in alpha-1 globulin, in alpha-2 globulin, and in fibrinogen as the severity of the disease progresses. Definite but less significant increases in beta and gamma globulins occurred. The increase in the total globulin was less than the decrease in albumin, resulting in a slight but definite tendency toward hypoproteinemia in the cancer patients. These changes, however, are not sufficiently characteristic of neoplastic disease to distinguish it from other ailments. One of the more important contributions of the electrophoretic method to the study of plasma proteins has been t o identify or characteril;e plasma components separated by other methods. The demonstration of the heterogeneity of the Howe fractions, for example, rests to a great extent on the presence of a number of electrophoretic components
cd I?
TABLE I1 Electrophoretic Components in Plasma of Normal Individuals and Cancer Patients (From Mider et al., 1950)
Normal adulta Cancer Advanced cancer 1
Standard deviation.
Total Protein g. %
Albumin g. %
6.83 f .07' 6.60 f .04 6.55 .14
4.04 k .05 2.94 k .04 2.38 .05
+
+
Alpha-1 Globulin g. %
Alpha-2 Globulin g. %
Beta Globulin g. %
0.38 f .01
0.66 f .02 0.90 .01 1.05 +_ .05-
0.76 k .02 0.89 f .01 0.99 rt .05
0.53 k .01 0.65 f .03
b-
m
Fibrinogen B- % 0.31
.02
0.58 k .01 0.82 +_ .09
Gamma Globulin g.
%
F
%
a
0.66 f .03 0.75 4 .02 0.82 f .04
ii
P
510
RICHARD J. WINZLER
in each of these fractions (Tiselius, 1937b; Gutman et al., 1941; Svensson, 1941; Dole, 1944; Majoor, 1946, 1947; Milne, 1947; Petermann et al., 1947). The designation, identity, and homogeneity of the various fractions obtained by the low temperature fractionation methods developed by Cohn and his associates (Cohn et al., 1946, 1950; Edsall, 1947), also rests to a large extent upon their electrophoretic behavior. It has become a fairly well-standardized procedure to carry out electrophoretic examination of plasma in veronal buffer a t pH 8.5 at ionic
Alb
I
t
-
t
FIQ.2. Electrophoresisof serum at pH 4.5 in acetate buffer at ionic strength 0.1. Descending boundary. Right pattern normal, left pattern mammary carcinoma. (Courtesy of Dr. J. W. Mehl.)
strength 0.1 following the demonstration of Longsworth (1942) that favorable resolution of the plasma proteins occurred under these conditions. The advantages of standardization of conditions are obvious, but the possibility that additional information may be obtained by carrying out electrophoresis at other pH values should be emphasized. Thus Petermann and Hogness (1948b) demonstrated an acid component with an isoelectric point lower than p H 4 which occurred in increased amounts in the plasma of patients with gastric carcinoma. Mehl et al. (1949a, 1950) using acetate buffer a t pH 4.5 showed that this acid component is identical with a plasma mucoprotein isolated by Winder et al. (1948) and by Weimer et al. (1950). An additional acidic component (designated M-2) was demonstrable in pathological sera examined electrophoretically a t pH 4.5 (Mehl et al., 194Ya, 1950). Figure 2 shows patterns from serum from a normal individual and a patient with cancer examined under these conditions. Miller et al. (1950) have made an extensive study of the electrophoretic patterns obtained with plasma over the pH range 3.0 to 11.4. These
PLASMA PROTEINS IN CANCER
511
studies may prove especially important when extended to pathological sera. The cost of the equipment and the time required for individual runs has prevented routine application of electrophoresis t o clinical problems. However, it now seems possible that electrophoresis on filter paper strips may provide a rapid and convenient method for determination of the electrophoretic components of serum. Proteins from 0.01 t o 0.1 ml. of serum separate into distinct electrophoretic components on filter paper wet with buffer across which an electrical potential is applied (e.g., Cremer and Tiselius, 1950; Durram, 1950; Turba and Enenkel, 1950; Grassman, 1951). The proteins may be fixed and stained at the end of the run or may be extracted and determined chemically. Awapara et al. (unpublished) have determined the relative amounts of albumin, alpha-1 globulin, alpha-2 globulin, beta globulin, and gamma globulin of sera from normal individuals and cancer patients by this method and have found the characteristic decrease in albumin and increase of alpha-1 and alpha-2 globulins already discussed.
3. Fractionation with Alcohol at Low Temperature and Ionic Strength E. J. Cohn and his associates have developed during the course of the last few years several methods for the separation of plasma proteins, using alcohol at low temperature and low ionic strength. This work has been almost entirely confined to the isolation and characterization of proteins present in normal human plasma, and little application has yet been made to the study of plasma from patients with various pathological conditions. This very extensive program has been reviewed in a number of papers (Cohn, 1941, 1948; Edsall, 1947; Oncley, 1950). About thirty protein components of plasma have been separated and partially characterized by the Harvard Group (Table 111). Recently, a modified procedure (method 10) involving fractional extraction, protein-protein and protein-heavy metal interactions has been developed (Cohn et al., 1950). This procedure is of special interest here since it lends itself to rapid analytical or preparative procedures on a small scale. Method 10 has been modified to the use of filtration instead of centrifugation (Lever et al., 1951), and the distribution of protein, of cholesterol, of phospholipid, and of protein-bound carbohydrate in several fractions has been carried out with normal plasma. There is little doubt that extension of this work to pathological plasmas will be of utmost value in future studies of the plasma proteins in cancer and other pathological states. Pillemer and Hutchinson (1945) have used methanol (42.5 per cent at 0°C. and pH 6.7 to 6.9) to separate albumin and globulin, the albumin
5 12
RICHARD J. WINZLER
TABLE I11 Protein Components of Human Plasma and Certain of Their Chemical Properties and Interactions'
Protein Component
Estimated % of Plasma Proteins
orl-Acid glycoprotein Caeruloplasmin Choline esterase a 1 - B i i b i n globulin Serum albumins Mercaptalbumin or TGlycoproteins c u r Mucoproteins Fibrinogen Cold insoluble globulin Antihemophilic globulin al-Lipoproteins &-Lipoproteins
Sedimen- Approx. Isotation Constant electric Sz0, W Point
0.5
-
0.005 0.05 52 (34) 1.2 0.5 4 0.15
-
3. 5.
&-Lipid-poor euglobulins Bl-Metal combining protein Isoagglutinins 82-Globulins
3 (0.03) 3
r-Globulins
11
Accelerator globulin Prothrombin Heparin complement Plasminogen Plasmin inhibitor Hypertensinogen Iodoproteins Complement component CI1 C'2 Amylase Alkaline phosphatase Peptidase &Glucuronidase aTProtein &Protein 1
-3 . 6 -
4.6
9 9 9
-
5 7 7 20 5.0
7
3.0 4.4 4.5 4.7 4.9
< 5.3
-
-
5 . 8 Iron & copper 6 . 3 Incompatible red cells 6.3 {7.3 6.3 Antigens
-
-
Prothrombin Calcium & thromboplastin Heparin Streptokinase Plasmin Tennin
-
Antigen-antibody complex
-
-
0.1 0.05
From Onoley (1960) and Cohn at d. (1960).
-
-
-
2.9
6.
-
5 . 2 Steroids & carotenoids 5 . 4 Steroids & carotenoids 5.5
-
-
Copper Choline esters Bilirubin Fatty acids-bile salts - Dyes, drugs & mercury 4 . 9 Carbohydrates & barium 4 . 9 Carbohydrates & barium < 5 . 3 Thrombin
-
4
Specific Chemical Reactions
-
-
-
-
Starch Phosphate esters c l e wylglycylglycine 8-Glucuronides Barium
-
PLABMA PROTEINS I N CANCER
513
globulin ratios being lower than those determined by the Howe method and corresponding closely to those obtained electrophoretically. Using this method Nitsche and Cohen (1947) have found subnormal albumin levels in patients with leukemia or Hodgkins disease.
4. Other Methods The ultracentrifugal study of plasma proteins has been of considerable value in evaluation of homogeneity and molecular weights of partially purified protein preparations. Three or four major ultracentrifugal components are discernible when normal or pathological human serum is examined (McFarlane, 1935; Pedersen, 1945; and Oncley et al., 1947). Too little work has been carried out to determine whether abnormal ultracentrifugal patterns occur with any frequency in the plasma of patients with neoplastic disease. The great specificity and sensitivity of immunological methods for the detection and identification of specific proteins can be expected to be of considerable value in future work on plasma proteins in health and disease. Especially useful in this connection has been the development of quantitative immunological procedures for the determination of specific proteins through the preparation of antibodies to these proteins in rabbits and subsequently determining the nitrogen content of antigenantibody precipitates (Heidelberger, 1939; Kabat, 1943; Treffers, 1944 ; and Jager et al., 1950). Studies of plasma proteins which have quantitatable biological activities (other than the antibodies mentioned above) may also be used to characterize differences in blood between normal and pathological states. The activity of specific plasma enzymes (Homburger, 1950) and the activity of inhibitors of enzyme action (Winzler, 1950) may be significantly altered in neoplastic disease. The presence of certain proteins which are characterized by chemical tags may also be altered in the plasma of cancer patients. Examples of this type are the polysaccharide-containing proteins of plasma, the levels of which rise markedly in cancer and in other pathological states (see later discussion). Many changes in the physicochemical characteristics of plasma in relation to malignant disease have been shown t o occur. Such changes as decreased tendency to coagulate by treatment with heat or acid and decreased ability to bind anionic dyes will be discussed in later sections.
11. ALTERATIONS OF PLASMA PROTEINS IN NEOPLASTIC DISEASE It has been brought out in the preceding discussion that there are abnormalities in the absolute and relative amounts of the major protein
514
RICHARD J. WINZLER
components of plasma of patients with neoplastic disease. There is, in addition, considerable evidence that even more pronounced alterations in amount or kind of minor plasma components may be associated with this disease. Many of these studies have been stimulated by the desire to develop serodiagnostic procedures for detection of cancer. While some success in this direction has frequently been reported, extensions of such studies have generally led to the conclusion that the changes are not characteristic of cancer, but are shared with other wasting diseases and infections. Nonetheless, the demonstration that such alterations and abnormalities occur regularly in neoplastic disease is sufficient reason for the pursuit of their fundamental physiological and biochemical bases. In the following discussion a few of the procedures which have been proposed for the serodiagnosis of cancer will be considered, as well as some of the observations which have been studied for their biological interest alone. It is the purpose of this discussion to consider the procedures from the point of view of their biochemical significance, rather than from the point of view of serodiagnosis of cancer. Some of the diagnostic procedures have been reviewed by Homburger (1950), Sobotka (1951), Maver (1944), Stern and Willheim (1943), Woodhouse (1940), Davidsohn (1938), Bing and Marangos (1934), and Kahn (1925).
1. Albumin A. General Considerations. The general decrease in plasma albumin in malignant disease is well established by both salt fractionation and electrophoretic methods. The possibility exists that this decrease is the result of a negative nitrogen balance which is especially reflected in albumin metabolism. However, it has been demonstrated that low serum albumin levels may persiet under conditions where an overall positive nitrogen balance is maintained (e.g. , Homburger and Young, 1948; Mider et al., 1948). This demonstration, however, would not dispose of the possibility that the growth of the tumor and the consequent withdrawal of nitrogen from the body pools may result in a net loss of protein to the patient, in spite of an overall nitrogen gain in tumor plus host. It seems likely, however, that the serum albumin level is reduced more severely in cancer than are the other protein stores of the body. The physiological basis of the reduced serum albumin in malignant disease is, thus, a, pressing problem. An hepatic dysfunction induced in some unknown manner by the presence of a neoplasm may be the basis for the lowered serum albumin levels in cancer patients. That the liver is the major site of albumin synthesis seems well established (Madden and Whipple, 1940). Abels et al. (1943), in reviewing the work of the Memorial Hospital group on
PLASMA PROTEINS IN CANCER
515
metabolic abnormalities in patients with cancer of the gastrointestinal tract, reported lowered serum albumin levels in a large proportion of their patients and showed that this lowered albumin level bore no relation to the degree of dietary deficiency or to loss of blood by internal bleeding. Using several criteria for adequacy of liver function in control individuals and in patients with gastrointestinal cancer, they showed that hepatic function was defective in a large proportion of the cancer patients in comparison with individuals with atrophic gastritis or oral leukoplakia. This suggested that defective protein fabrication in the liver of the cancer patients was a major cause of hypoalbuminemia. In support of this suggestion, Ariel et al. (1943) and Ariel (1949) found that the administration of glycine t o patients with gastrointestinal cancer resulted in a significantly higher and more persistent level of free amino acid in the blood than was the case with normal controls, an observation which suggested that the synthesis of protein from free amino acid might be reduced in the cancer patients. On the other hand Norberg and Greenberg (1951) found that the rate of uptake of C14-labeledglycine into the plasma proteins of mice was increased above normal rates in mice bearing transplanted tumors. If the suggestion is substantiated that hepatic dysfunction is associated with cancer of various types, the mechanism by which such an influence might be brought about would become a topic of considerable importance. It is reasonably well established that tumors may have effects on enzyme systems remote from the site of the tumor. Thus Greenstein et al. (1941) have shown that the liver catalase activity of rats and mice bearing transplanted tumors is much decreased from the control levels and that removal of the tumor results in the rapid return of liver catalase activity to normal. Recently an agent has been isolated from tumor tissue which caused significant decrease in liver catalase activity when injected into normal mice (Nakahara and Fukuoka, 1949; and Greenfield and Meister, 1951). Recently Madden (1950) has shown that the presence of turpentine abscesses in dogs increased the urinary nitrogen and sulfate output by 40 to 50% but had little influence on the incorporation of Ss6-labeled methionine into the tissue or plasma proteins. This might suggest that a negative nitrogen balance and hypoalbuminemia is more closely related to increased protein breakdown than to decreased protein synthesis. Another possibility that may be at the basis of the low serum albumin levels in malignant disease could be an increased lability of the serum albumin in this disease. Such a suggestion has been made, for example, to explain the abnormal alkali lability of serum from dogs with experimental pneumonia (Crossley et al., 1941).
RICHARD J. WINZLER
516
A number of procedures have been proposed for detection of cancer or for following the development of the disease which appear t o be based on quantitative or qualitative changes in the serum albumin of patients with neoplastic disease. Some of these procedures are briefly considered in the following paragraphs. B. Thermal Coagulation Procedures. Glass (1940, 1950a, 1950b, 1951) and Huggins and his associates (1949, 1949a, 1949b, 1950, 1951) have demonstrated that serum from cancer patients is frequently less coagulable upon exposure t o heat than is normal serum and have pointed out that the determination of thermal coagulability of serum may be a very useful procedure for evaluation of the clinical state of patients and that it may serve as a useful diagnostic aid for detection of neoplastic disease. Glass determined the coagulation temperature and used this as a measure of coagulability (whereas Huggins et al. determined the least coagulable concentration of serum as the end point (Table IV)). TABLE IV Summary of Some Differences between Normal and Pathological Serum Depending upon Albumin Procedure Lowest Clottable protein concentration' Iodoacetate index' Sulfhydryl content' Polarographic serum testa Reduction times Binding of phenol red9 1
Normal Serum
Cancer Serum
1.33 f ,169 1.72 f .2 10.8 f .76 6.43 k 2.36 63.9 f .7 36.4 f 1.9 36.6 26.0 8.6 f .1 12.3 f .7 67.07 5.79 44.61 f 11.91
*
Other
Referenoe
1.39 f ,178 Huggina et ol. (1949b) 10.38 f ,918 Huggins et 02. (l949b) 22.3 f 7.6' Scboenbach et al. (1961) 26.88 Rusch et Ol. (1940) 10.2 f .3 Blaok (1947) Huggina et al. (19498)
-
Grams protein/lM) ml.
* Standard deviation.
Miacellaneoua diseases. pM iodoacetate/g. protein. 6 pM SH/100 ml. serum. 0 Wave height in millimeters. 7 Miacellaneoua states with abnormal A/G ratios. * Minutes. 9 pg. PSP bound by 1 ml. serum g. of albumin per 100 ml. serum a
.
Both procedures appear to give comparable results. Huggins et al. (l949,1949a), and Jensen st al. (1950) have extended the thermal coagulability test to the determination of the amount of iodoacetic acid which will prevent serum from thermal coagulation (iodoacetate index = micromoles iodoacetate/g. protein). The effect of iodoacetate on thermal coagulation is presumably by virtue of its reaction with protein-bound sulfhydryl groups. A considerable decrease in the iodoacetate index was found in the serum of cancer patients in comparison to normal individuals
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(Table IV). Ponder (1950) has developed a turbidometric measurement of the heat coagulability of small amounts of serum. A number of investigators, including the originators of the methods, have pointed out that decrease in thermal coagulability and the iodoacetate index of serum is not specific for neoplastic disease and is of limited value for the diagnosis of cancer (Cliffton, 1949; Bodansky and McInnes, 1950; Boyd, 1950; Dvoskin and La Due, 1950; Homburger et al., 1950; Kiefer et al., 1950; Pollack and Leonard, 1950; West and Keye, 1950; Gilligan et al., 1950; Finnegan et al., 1950; Ellerbrook, 1950; Henry et al., 1951; Ellerbrook et al., 1951b). The extent to which thermal coagulation is dependent upon specific protein fractions has as yet not been unequivocally established. It does seem clear, however, that the thermal coagulation of serum is intimately associated with its content of protein-bound sulfhydryl groups. A diminished sulfhydryl content of cancer serum was strongly indicated by the experiments of Waldschmidt-Leitz (1938), of Purr and Russel (1934), and of Meyer-Heck (1939) in which the reactivation of partially inactivated glyoxalase or papain by serum from cancer patients was less than with normal serum. Since approximately 80% of the sulfhydryl content of serum proteins is contained in the albumin (Weissman et al., 1950), this fraction would be implicated as the major factor in thermal coagulation of serum. Moreover, the thermal coagulation of serum was shown experimentally by Huggins et al. (1950) to be related primarily to the level of Berum albumin. These authors indicated however that a t any given level of albumin, the serum of cancer patients coagulated less readily than normal, and felt that a qualitative change in albumin was indicated. It is of special interest in this connection that purified albumin isolated from normal serum and from serum of cancer patients by the low temperature alcohol method (method 6 of Cohn et al., 194.6) showed no difference in iodoacetate index, although the serum from the same patients showed marked differences in this index (Huggins et al., 1949b), and it was suggested that substances associated with the serum albumin affecting its thermal coagulation were removed during the fractionation procedure. In this connection Schoenbach et al. (1950, 1951) showed by amperometric titration that the sulfhydryl content of serum of patients with cancer or with certain other diseases was markedly reduced from the normal levels (Table IV) and reported that this reduction could only partially be accounted for on the basis of reduced albumin content. This they attributed to some sort of qualitative change in the serum albumin, since the sulfhydryl content per gram of serum albumin was below the normal level.
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Ellerbrook (1950) also showed that the least coagulable serum concentration and the iodoacetate index, when corrected for the reduced albumin content, still showed values which were abnormal, again supporting some sort of qualitative change independent of albumin concentration. C. Polarographic Serum Test. When proteins are examined polarographically in ammoniacal buffer in the presence of cobalt ion, there appears a characteristic “double wave” on the current-voltage curves. This double wave is a result of the catalytic reduction of hydrogen ion by a cobalt-sulfhydryl complex and is given only by proteins which contain reactive or “unmasked” cystine or cysteine. BrdiEka (1937, 1939a,b) noted t,hat cancer serum gave a lower polarographic serum test than did normal serum (Table IV) an observation confirmed by a number of other investigators (Tropp, 1938; Meyer-Heck, 1939; WaldschmidtLeitz and Mayer, 1939; Rusch et al., 1940; Robinson, 1948). The subnormal albumin content of the serum of cancer patients appears to be the major basis for the polarographic serum test. Rusch et al. (1940) showed that the polarographic serum waves were closely proportional to the serum albumin concentration. The distinction between BrdiEka’s polarographic serum test and his polarographic filtrate test (BrdiEka, 193913) is sometimes overlooked. The polarographic seruw test depends upon reactive sulfhydryl and disulfide groups in the total serum proteins, whereas the filtrate test depends upon these groups in the filtrates of alkali-treated serum deproteinated with sulfosalicylic acid (see later discussion). Muller and Davis (1945,1947) have recently determined the ratio of the two determinations to give a ‘(protein index” which is independent of the temperature and of the properties of the particular dropping mercury electrode employed. D. Binding of Anionic Dyes. Certain proteins have the capacity to form reversible complexes with anionic dyes. Grollman (1925) and a number of observations have indicated that this capacity is reduced in the serum of cancer patients as compared to normal serum (Bennhold, 1932; Ehrstrom, 1937; Huggins et al., 1949a; Westphal et al., 1950, 1951 ; Huggins and Baker, 1951). The fundamental aspects of the binding of anions by proteins have recently been reviewed by Klota (1949), Scatchard (1949), Goldstein (1949), and Armstrong (1950). Different plasma proteins have very different capacities to bind anionic dyes, albumin being by far the most effective (Klotz, 1949; Huggins et al., 1949a; Westphal et al., 1950). The formation of protein-dye complex depends primarily upon the presence of free epsilon amino groups of lysine in accessible sites on the protein (Klotz, 1949). Methods employed for studying dye binding capacity of normal and pathological sera include the penetration of
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unbound dye into gelatin (Bennhold, 1932), the adsorption of unbound dye with charcoal (Ehrstrom, 1937; Gottlieb and Ludwig, 1937), equilibrium dialysis (Huggins et al., 1949b); and chromatography (Westphal et al., 1950, 1951). In the most recent work on this subject a significantly subnormal ability of cancer serum to bind phenol red (Huggins et al., 1949a) or azorubin (Westphal et al., 1950, 1951) has been noted (Table IV). The reduction in dye binding capacity, however, was not limited to neoplastic disease. The amount of phenol red bound by serum was dependent primarily upon its albumin concentration. However, the amount of dye bound per milligram of albumin in serum from cancer patients was significantly less than in normal serum, suggesting an intrinsic difference between the albumins in the plasma of normal individuals and of cancer patients (see also Ehrstrom, 1937). However, when electrophoretically homogeneous albumin was isolated from normal serum and from cancerous serum by a low temperature ethanolic procedure (method 6 of Cohn et al., 1946), no difference in dye binding between the two were apparent (Huggins et al., 1949a). The explanation of subnormal dye binding by cancerous serum, thus, is not yet clear. E. Methylene Blue Reduction. Savignac et al. (1945) and Black (1947a,b, 1948) have reported that the time required for serum or plasma of cancer patients t o reduce methylene blue under defined conditions of dye concentration and alkalinity is increased over that obtained with normal plasma. This phenomenon, they found, was not limited to cancer, but was sufficiently well correlated with malignant disease to suggest that it might have diagnostic use. Measurements of methylene blue reduction time of serum carried out in a number of laboratories using Black’s method (Stadie, 1948; Henry et al., 1951; Eriksen et al., 1951a) have only roughly correlated with malignant disease. The increased methylene blue reduction time of serum is not specific for neoplastic disease and is therefore of limited use in cancer diagnosis. Black, Kleiner, and Bolker (1948) and Stettner et al. (1948) combined the methylene blue reduction test with the heat turbidity test (see later) and claimed that the accuracy of diagnosis of malignant disease was improved. The use of this combination of tests by other workers (Henry et al., 1951), however, did not significantly improve the specificity of the tests. Savignac et al. (1945) and Black (1947a,b) advanced the hypothesis that the methylene blue reduction time depended upon sulfhydryl groups in serum albumin and that the increased reduction time observed with plasma from cancer patients was a result of an abnormality in the sulfurcontaining amino acids of albumin. Stadie (1948) pointed out that heating proteins in alkaline solution converts a large part of their cystine and
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cysteine sulfur t o sulfide ion, which is capable of reducing methylene blue. Subsequently Black (1948) reported that the increase in the methylene blue reduction times obtained with abnormal sera did not reflect differences in total potential reducing groups but appeared to be associated with a slower liberation of these groups in the proteins of the abnormal serum upon exposure to heat. It is perhaps regrettable that more attention has not been paid to the control of pH in carrying out the Black test, since the rate of liberation of sulfide ion from the so-called labile sulfur of proteins is very sensitive t o pH changes. It cannot be considered established that the methylene blue reduction time depends primarily on serum albumin, nor is it certain that the difference between normal and abnormal serum is associated with differences in this serum component. However, the fact that albumin is especially rich in “labile sulfur” and that serum albumin levels fall in cancer may be construed as evidence in favor of the view that serum albumin is primarily concerned in the methylene blue reduction test. From the foregoing discussion it appears that differences in thermal coagulation, polarographic activity in the presence of cobalt, dye binding, and reducing power between normal and pathological plasma appear t o be closely related to the serum albumin concentration. In addition there is suggestive evidence that qualitative differences between the albumins may also be involved. Hughes (1947, 1949) has shown that serum albumin contains a component containing one free sulfhydryl group per mole of protein (mercoptalbumin) and also contains a component lacking free sulfhydryl groups. About two-thirds of the normal serum albumin is mercoptalbumin (Hughes, 1947, 1949; Weissman et al., 1950). The attractive possibility that the relative amount of sulfhydryl containing albumin might be reduced in the serum albumin of cancer patients was not substantiated in the work of Huggins et al. (1949a), who found about two-thirds of a mole of sulfhydryl per mole of serum albumin isolated both from normal individuals and from patients with malignant disease. Human serum albumin has also been shown to be separable into different electrophoretic components at pH 8 (Hoch-Ligetti and Hoch, 1948) and at pH 4 to 4.5 (Luetscher, 1939,1940; Sharp et al., 1942; Miller et al., 1950). There is yet no evidence indicating whether the relative amounts of the electrophoretically separable albumins in serum from patients with neoplastic disease are abnormal. 2. Alpha Globulins
The serum levels of alpha-1 and alpha-2 globulins, determined electrophoretically, may increase significantly in neoplastic disease as has already been pointed out, but little can yet be said about the physiological
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significance of these observations. Each of these alpha globulin fractions contains a number of proteins which have the same mobility in an electric field at pH 8.5. Thus, from Table 111, there are a t least three components each in the alpha-1 and alpha-2 globulin fractions. Until detailed studies by such procedures as method 10 of the Harvard Group (Cohn et al., 1950; Lever et al., 1951) are applied to pathological plasma, the significance of the increase in the alpha globulins in neoplastic disease cannot be evaluated in terms of their individual components. An increase in the alpha globulins of plasma rather consistently accompanies acute febrile and inflammatory conditions, as well as cancer. Longsworth, Shedlovsky, and McInnes (1939) and Shedlovsky and Scudder (1942) pointed out the frequent association of raised alphaglobulin levels with inflammation and tissue destruction. The plasma alpha globulins levels may reflect the activity of generalized processes such as tissue destruction or replacement. However, much more information is needed on the sites of formation and the metabolic turnover rates of the individual protein components in health and disease before this thesis can be accepted. One component of the alpha-1 globulin fraction which rises markedly in neoplastic disease and which has been isolated and characterized as a mucoprotein (Winzler and Smyth, 1948; Winzler et al., 1948; Weimer et al., 1950; Smith et al., 1950) will be discussed in the section on especially soluble and stable proteins. Little is known about the specific components of the alpha-2 globulin fraction which increase in malignant disease. The plasma alpha-2 globulin contains at least two proteins which are rich in carbohydrate, and part of the increase in protein-bound polysaccharide noted in neoplastic disease may be associated with increases in one or both of these components. Seibert et al. (1947), for example, noted a close correlation between the serum polysaccharide levels and the amount of alpha-2 globulin in the serum of normal individuals and patients with tuberculosis or cancer (see section on serum polysaccharides). 3. Especially Soluble and Stable Proteins
A number of observations on sera of normal individuals and of cancer patients have indicated that the latter contain increased amounts of very soluble and stable proteins or protein derivatives. Some of these appear to be alpha globulins electrophoretically and are found with the albumin on fractionation by the Howe procedure. Some of the more thoroughly investigated of these soluble protein are briefly considered in the following paragraphs.
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A. Polypeptidemia. Plasma or serum contains proteins or their split products which are precipitated by tungstic or phosphotungstic acids but not by weaker precipitating conditions such as trichloroacetic acid (at high concentration), sulfosalicylic acid, perchloric acid, and heat coagulation. A number of investigators have assayed this fraction by determining the difference in the nitrogen or tyrosine content of phosphotungstic acid and of 20% trichloroacetic acid filtrates of serum or plasma (Hahn, 1921; Wolff, 1921; Goiffon and Spaey, 1934; Reding, 1936, 1938; Larizza, 1937; Godfried, 1939; Winzler and Burk, 1944). Trichloroacetic acid filtrates from normal serum or plasma contain about 5 mg. % more nitrogen and 2 mg. % more tyrosine than do tungstic acid filtrates, these differences being considerably increased in serum or plasma from patients with neoplastic or certain other disease (see Table V). The differences were considered to represent polypeptide nitrogen or tyrosine precipitated by phosphotungstic acid but left in solution by trichloroacetic acid. TABLE V Summary of Some Differences between Normal and Pathological Plasma Depending on Soluble and Stable Proteins Normal Polarographic filtrate test’ Mucoprotein Nitrogen Index of Polypeptidemia’
Cancer
Other
Reference
1 .O 2 . 4 rt .3* 1 . 5 f .2* BrdiEka et al. (1939) 2 . 7 f . 5 6 . 1 f 1 . 3 7 . 2 f . 8 Winzler and Smyth (1948) 3.7 f . 3 6.5 . 6 5 . 9 f .5 Winzler (unpublished)
mm. wave length for abnormal serum, ratio mm. wave length for normal serum Standard deviation. 8 Miscellaneous diseases. 4 Mg. mucoprotein tyrosine %. 6 Difference between nitrogen oontent of sulfaaalicylic acid and phosphotungstic acid filtrates of serum. Mg. %.
B. Polarographic Filtrate Test. BrdiEka has proposed a second nonspecific polarographic test for cancer based on the determination of high molecular weight, cystine-containing substances in filtrates of serum deproteinated with sulfosalicylic acid (BrdiEka et al., 1939; BrdiEka, 193913). The determination depends, as does the polarographic serum test, upon the catalytic reduction of hydrogen ion by a sulfhydryl-cobalt complex giving a characteristic “wave ” on a current-voltage curve. Sulfosalicylic acid filtrates of the sera of cancer patients gave significantly higher polarographic currents than did filtrates from normal sera (Table V). BrdiEka et al. (1939) attributed the increase in the polarographic filtrate test in pathological sera t o protein split products liberated as a
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result of the activation of Abderhalden’s “Abwehrferment ’’ (see later). BrdiEka recognized that the polarographic filtrate test was not a specific test for malignant disease, but felt that it was a useful diagnostic aid. A number of investigators have confirmed BrdiEka’s observations (Albers, 1940; Schmidt, 1940; Winzler and Burk, 1944). One of the objections to the polarographic method is the lack of absolute units by which results can be compared without reference to arbitrary normal sera studied simultaneously. Recently, Muller and Davis (1945, 1947) have carried out BrdiEka’s serum and filtrate tests in parallel and expressed the results as a ratio of the filtrate wave to the serum wave, this ratio being designated the “protein index.” Since in abnormal serum the filtrate wave rises and the serum wave falls, differences between normal and abnormal sera are accentuated by the “protein index.” Of special value is the fact that the use of this ratio minimizes the influence of temperature and eliminates differences between different capillaries and instruments, permitting the expression of polarographic data in units reproducible in different laboratories. C. Mucoprotein. Winder and Burk (1944) showed that “polypeptidemia” and the polarographic filtrate test were given by substances in blood which had the solubility and stability properties of proteoses. The responsible fraction was isolated from sulfosalicylic acid filtrates of normal rat blood. Further work with normal human plasma (Winzler et al., 1948) showed that this material had the properties and composition of mucoprotein in agreement with previous observations made by Mayer (1942). The solubility of this fraction in 0.6 M perchloric acid and its precipitation by phosphotungstic acid provided a chemical method for its estimation in plasma or serum (Winzler et al., 1948). Patients with neoplastic disease, pneumonia, tuberculosis, rheumatic fever, and other conditions have significantly higher plasma mucoprotein levels than do normal individuals (Table V), (Simkin et al., 1949; Winzler and Smyth, 1948; Greenspan et al., 1951; Ellerbrook, 1950; Kelley et al., 1950, 1951; Henry et al., 1951; Jager et al., 1951). The major component responsible for the mucoprotein determination migrates with alpha-1 globulin a t pH 8.5, and has been shown (Weimer and Winzler unpublished) to be a major constituent of the seromucoid preparation of Bywaters (1909) and of Rimington (1940) as well as of the “mucoid-lihnliche substanz” of Mayer (1942). It seems very likely that it is also identical with Schmid’s (1950) small acid glycoprotein (Weimer and Winzler, unpublished ; Schmid, personal communication). Although this component migrates with the alpha-1 globulins at pH 8.5, its very acid isoelectric point permits its demonstration in untreated plasma at pH 4 t o 4.5 (Petermann and Hogness, 1948b; Mehl et al., 1949a; Mehl and Golden, 1950). The
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significance of the acidic alpha-1 mucoprotein and of its increase in cancer is not yet known, nor is there any information on where this particular protein is synthesizied. D. Albumin A . A decrease in especially soluble “albumins” in the plasma of patients with malignant disease is the basis of the Kahn albumin A determinations (Kahn, 1925, 1930; Goldschmidt and Kahn, 1929; Hanke and Kahn, 1941; Hanke et al., 1944). Kahn’s procedure involves precipitation of the plasma proteins with 37.15 % ammonium sulfate, and testing the filtrate for soluble “albumins” by heat coagulation. This fraction is distinct from those responsible for the polypeptidemia, the polarographic filtrate or the mucoprotein determinations, in that it is heat coagulable, and its concentration is reduced in the plasma of cancer patients. A number of investigators (e.g., Tinozzi, 1927; Sievers, 1931, 1932; Lothammer and Pistofidis, 1937; Hinsberg et al., 1939) have confirmed the general aspects of Kahn’s work on albumin A, and have indicated that the decrease in this fraction is not at all specific for malignant disease. The nature of albumin A cannot be inferred from the meager information on its properties,
4. Beta Globulins At least four beta globulins have been demonstrated in plasma by low temperature-alcohol fractionation procedures (Table 111). The beta globulins are, in part a t least, associated with certain of the plasma lipids, and changes in the beta globulin levels are most frequently seen in association with accumulation of lipids and lipoproteins in the blood (e.g., Kunkel and Ahrens, 1949). A beta globulin is, for example, the serum component which gives the thymol turbidity test (Cohen and Thompson, 1947). Not enough work on the fractionation of the beta globulins has yet been done to assess the significance of the frequent rise in the serum beta globulin level in neoplastic disease. 5. Fibrinogen
The increase in plasma fibrinogen content which is often associated with cancer (e.g., Walton, 1933; Mider et al., 1950; Ellerbrook, 1950; Smith et al., 1951) is by no means characteristic of this disease, above normal levels being prominent in acute infections, nephrosis, pregnancy, after x-ray radiation and various injuries. Normal and pathological variations in plasma fibrinogen levels have been reviewed by Ham and Curtis (1938) and by Gram (1922). The factors affecting fibrinogen synthesis and breakdown are largely unexplored. There is good evidence that fibrinogen synthesis occurs in the liver. It is interesting to note that the argument that liver dysfunc-
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tion may be the cause of the lowered plasma albumin levels in cancer patients cannot be used to explain the increased plasma fibrinogen noted in a good proportion of these patients. A. Sedimentation Rate. An increased erythrocyte sedimentation rate is frequently associated with neoplastic disease (e.g., Gram, 1922; Walton, 1933; Ham and Curtis, 1938; Moeschlin, 1944; Glass, 1950a; Finnegan et al., 1950; Gilligan et al., 1950). Since the plasma fibrinogen level has been recognized as an important factor influencing the sedimentation rate (e.g., Gray and Mitchell, 1942), this correlation is not surprising. Above-normal fibrinogen levels are not specific indications of neoplastic disease, and a similar nonspecificity is shown by the increased sedimentation rate associated with this disease. B. Heat Turbidity Test. Black, Kleiner, and Bolker (1948) showed that plasma from cancer patients tended to become more turbid on short exposure to heat than did normal plasma. The effect was dependent primarily upon fibrinogen, since serum did not show any increase in turbidity during the ten-second exposure t o boiling water involved in the test. The heat turbidity values paralleled the fibrinogen content of the plasma. The nonspecificity of increased fibrinogen levels limits the usefulness of the heat turbidity test for cancer diagnosis (Erickson et al., 1951b; Henry et al., 1951). By combining the heat turbidity test with the methylene blue reduction test already described, Black, Kleiner, and Bolker (1948) ; Black and Speer (1950); and Stettner et al. (1948) felt that the accuracy of diagnosis of malignant disease was significantly increased. However, Henry et al. (1951) did not find that the combination of tests materially improved the differentiation between neoplastic and other diseases. 6 . Gamma Globulins
A. Amounts and Immunity. There appears t o be no consistent change in the amounts of gamma globulins (electrophoretically determined) in neoplastic diseases (Mider et al., 1950). The gamma globulin may be reduced in late cancer, possibly due to decreased protein availability. Parfentjev and associates (Parfentjev and Duran-Reynals, 1951 ; Parfentjev et al., 1951) have, in fact, noted a pronounced reduction in the amount of euglobulin and of two naturally present immune bodies in tumor-bearing chickens and humans, and Wharton et al. (1951) noted a decreased antibody content of mice bearing transplanted tumors. Since the gamma globulin fraction contains a major proportion of the specific antibodies of the blood (e.g., Tiselius and Kabat, 1939; Enders, 1944) i t would appear that specific antibodie! are not formed in any quantity in response t o the presence of spontaneous neoplasia. This generalization
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probably does not apply to transplanted or virus induced tumors. For example, there is a sharp rise in the gamma globulin of chicken serum with the spontaneous regression of fowl lymphomas, the chickens being subsequently immune to implantation of the tumors (Krejci et al., 1948; Sanders et at., 1944). An immunity of rats or rabbits to transplanted tumors after tumor regressions has frequently been noted (e.g., Pearce and Brown, 1923; Besredka and Gross, 1938; Ostwald and Kuttelwascher, 1940; Cheever and Janeway, 1941; Gross, 1943; Harris, 1943). Friedewald and Kidd (1941) have found that an antibody against extracts of the Va carcinoma in rabbits may be demonstrated in the serum of rabbits bearing this transplanted tumor. The presence of Brown-Pearce carcinoma results in an increase in the complement fixation titer of rabbit serum titrated against extracts of the tumor (Cheever, 1940; Kidd, 1940; Appel et al., 1942; MacKenzie and Kidd, 1945; Thornton et al., 1950). Kidd (1944) also found that the transplantability of Brown-Pearce tumor was inhibited by incubating explants with serum containing the complement fixing antibody. The immunological aspects of neoplastic disease up to 1942 have been thoroughly considered by Stern and Willheim (1943). The hope that there might be changes in the antibody makeup of the plasma in neoplastic disease rests in good part upon the possible presence of abnormal substances in the tumor which would cause sensitization of the host. Evidence for such autogenous antigens in spontaneous cancer is largely lacking although Mann and Welker (1940, 1943, 1946) have advanced some evidence that specific proteins may be detected immunologically in human cancer tissue. B. Flocculation with Lipoidal Antigens.” Immunological reactions have been at the basis of a number of useful diagnostic (and therapeutic) procedures in a variety of diseases. Considerable effort has naturally been made to extend this approach to neoplastic disease. However, the fundamental basis upon which such immunological reactions rest i.e., the existence of antigens and antibodies specific for neoplastic disease, has not been established. A number of serological tests for cancer have been based on flocculation reactions of serum with various lipoidal “antigens,” greater flocculation generally being observed with cancer than with normal serum. Some examples of such flocculation with lipoidal “antigens” are found in papers by Ascoli and Izar (1910); Shaw-MacKenzie (1922); Kahn (1923, 1924) ; Fry (1925, 1926) ; Gruskin (1929) ; Weiss (1932) ;Lehmann-Facius (1932, 1936); Landau and German (1932). Repetition of these procedures by other investigators (see ,Stern and Willheim, 1943) has generally led to the conclusion that the distinction between serum from
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normal individuals or patients with neoplastic or other disease lacks the specificity required for successful serodiagnosis of cancer. One of these test procedures (Gruskin, 1929) has recently been reexamined in considerable detail by Holmgren et al. (1951) and is worthy of mention since it illustrates the unsuspected difficulties that may be involved in such flocculation reactions. Gruskin’s procedure is based on the hypothesis that a foreign protein of embryonic character is present in malignant tissue and is also present in plasma and in fixed tissue cells. An alcohol-soluble “antigenic ” material was extracted from embryonic liver, and the serum t o be tested carefully layered over the “antigen.” Cancer serum formed characteristic floccules, whereas the control sera did not. In the reinvestigation by Holmgren et al. (1951), the principal agent bringing about the flocculation was shown t o be the alcohol used in the “antigen” preparations, and differences between normal and cancer sera were related to the rate of precipitation and the physical form 0: the protein precipitate at the alcohol-serum interface. There is considerable doubt as to whether immunological factors are involved in this or in other flocculation tests. The seroflocculation reaction for cancer recently described by Penn and his associates (Penn, 1950; Hall et al., 1950) is based on the hypothesis that endogenous nonsaponifiable lipids may become altered, carcinogenic, and essentially foreign substances with antigenic capacities. In the Penn procedure an “antigen ” prepared from the nonsaponifiable lipids of liver from patients with neoplastic disease is shaken with serum. The “antigen” with normal serum results in a fine persistent turbidity, whereas with cancer serum there is a rapid flocculation of the lipid and a clearing of the preparation. Preliminary reports (Hall et al., 1950) indicate a remarkable accuracy for this procedure, the number of erroneous diagnoses being relatively rare. Although this procedure differs from the older flocculation reactions in employing a nonsaponifiable “antigen,” it is not established that the phenomenon has an immunological basis. Fontaine et al. (1949) prepared a protein “antigen” from urine of cancer patients and, after heating the antigen with copper sulfate, found that more pronounced precipitation was produced with serum from cancer patients than with normal serum or serum from patients with various other diseases. As with the lipoidal antigens previously discussed, the antigenic nature of this urinary protein must be regarded with some skepticism until the reaction is shown to have an immunological basis. C. Multiple Myeloma. In multiple myeloma and in certain leukemoid diseases, the presence of exceedingly large amounts of abnormal beta or gamma globulin (more frequently the latter) may be demonstrated in serum by electrophoretic or salt fraction methods (Perlzweig
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et al., 1928; Gutman et al., 1941; Longsworth et al., 1939; Kekwick, 1940; Moore et al., 1943; Malmros and Blix, 194G; Adams et al., 1949; Pearsall
and Chanutin, 1949; Rundels et al., 1950; Schoenbach et al., 1951). High globulin values are frequently obtained by the Howe fractionation procedure. There has been, however, no consistency in the appearance of the abnormal globulin in any particular Howe fraction, the protein being in some cases associated with the pseudoglobulin and sometimes with the euglobulin fractions. In the low-temperature fraction method of Cohn et al. (1946) the abnormal globulin is usually found in fractions I1 and I11 but may also be found in fraction I (Pearsall and Chanutin, 1949). Similar inconsistencies have been observed in the electrophoretic studies where the abnormal protein may have mobilities varying mainly between those of the beta and the gamma globulins (Adams, Alling, and Lawrence, 1949). An interesting observation by Mehl and Golden (1950) is that the abnormal globulin may appear homogeneous at pH 8.5 but form two distinct components a t pH 4.5. It appears very probable that the hyperglobulinemia of multiple myeloma is due to the presence of large amounts of Bence-Jones or related abnormal proteins in the plasma. However, in some instances the abnormal globulins do not appear t o be typical Bence-Jones proteins. The present status of this work has been excellently reviewed by Gutman (1948) and by Adams, Alling, and Lawrence (1949). At the present time there is no definite evidence as to the site of formation of the abnormal globulins in multiple myeloma, although it seems likely that the protein is formed in the bone marrow lesions.
7. Changes in Protein Stability to Precipitating Agents A number of diagnostic tests for neoplastic disease have been proposed which are based on changes in behavior of the plasma proteins with various precipitating agents. Too little is known about the chemistry of most of these reactions to permit anything but speculation as to their fundamental basis. Some of these procedures may be based on such protective colloidal phenomena as have been studied by Munro (1944). A few examples of this sort of procedure are listed below, but are not discussed since their relation to specific proteins has not been established. In the lactogelification reaction of Kopaczewski (1935, 1936, 1937) serum from cancer patients forms a gel more rapidly upon the addition of lactic acid than does normal serum. The precipitation of serum proteins by vanadic and acetic acids follows a different dilution pattern in normal and cancer sera (Bendien test, Cronin-Lowe, 1933). Weltmann (1930) heated diluted serum in the presence of progressively decreasing calcium chloride concentration and found that the precipitation pattern was altered in cancer serum. Dilutions of normal serum treated with the cationic
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detergent, Bradosol (beta-phenoxyethyldimethyldodecyl ammonium bromide) formed a characteristic precipitation pattern which was markedly altered when serum from patients with neoplastic or other disease was employed (Mayer and Eisman, 1951; Bronfin et al., 1951).
111. PLASMA ENZYMES AND INHIBITORS The enzymatic and enzyme-inhibiting components of plasma are present in amounts too small to be determined by electrophoretic or isolation methods, and consequently alterations in their amount can be determined only through measurement of their activities. Although the physiological significance of most of these agents in normal blood is not known, studies of their activities under pathological conditions may yield information pertaining t o the normal physiological significance. Of special interest is the possibility that specific enzymes may escape from tumors into the blood stream and yield significant increases in their circulating levels more or less directly. Such activity alterations may, perhaps, be more specific for cancer than changes which measure the systemic response of the host to various diseases. 1 . Enzymes
Although a number of enzymes have been demonstrated in plasma, only a few have been shown t o have any consistent relationship to neoplastic disease. Some of those that have appeared a t one time or another to be of special interest are briefly considered in the following paragraphs. A. Acid and Alkaline Phosphatases. The relation of acid phosphatase to prostatic cancer with metastases to the bone marrow, lymph nodes, or liver is perhaps one of the most prominent instances of an enzyme with important diagnostic value in malignant disease. Normally present in relative low amounts, the serum acid phosphatase may rise to very high levels in patients with metastatic prostatic cancer. The work of Gutman and Gutman (1938), Barringer and Woodward (1938), Huggins and Hodges (1941), Gutman (1942), Herbert (1946), Dillard et al. (1949), and Huggins and Baker (1951) are a few examples of the application of serum acid phosphatase determinations t o diagnosis, therapy, and prognosis of metastatic cancer of the prostate. Since this enzyme is especially concentrated in prostatic tissue, it seems very probable that its presence in blood in abnormally large amounts in patients with metastatic prostatic cancer represents a direct contribution by the malignant tissue. Increases in the alkaline phosphatase content of serum may occur in hyperplastic disturbances of bone, as was shown by Kay (1929, 1930). The increased level of this enzyme appears to be due to the increased
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activity of the osteoplastic cells either because of primary osteogenic tumors being the stimulation of the osteoblastic activity produced by the presence of metastatic tumors in the bone (Huggins and Hodges, 1941; Huggins and Baker, 1951). Roche et al. (1946, 1947) have reported that low concentrations of zinc ion usually had an inhibitory effect upon the serum alkaline phosphatase of cancer patients, whereas it usually increased the activity of this enzyme in the serum of normal individuals and patients with other diseases. This work could not be substantiated in recent carefully conducted work (Bodansky and Blumenfeld, 1949; Fishman et al., 1949; and Ellerbrook et al., 1951a). B. AZdolase. Warburg and Christian (1943) observed that the glycolytic enzyme aldolase (zymohexase) was significantly increased in the serum of tumor-bearing animals. Significant increases were not observed in the serum of cancer patients, presumably because the tumor mass was too small a proportion of the total body weight. Recently Sibley and Lehninger (1949) modified and simplified the method for the determination of this enzyme and confirmed the work of Warburg and Christian. In a large proportion of tumor-bearing animals the aldolase level was'significantly above normal, but in only 20 out of 104 cases of human cancer was there a significant increase in the serum level of this enzyme. Particularly interesting, however, was the observation that general cachexia, infection, or pregnancy did not lead to increased serum aldolase levels, perhaps indicating that aldolase escapes from tumor into the blood stream. An increase in circulating aldolase would thus represent a direct contribution of the tumor rather than a secondary reaction of the host. C. D-Peptidases. The presence of D-peptidases in abnormally high amounts in the blood of cancer patients was claimed by WaldschmidtLeitz and co-workers (1940a,b). These observations have been challenged by a number of workers (Bayerle and Podloucky, 1940a,b,c; Herken and Erxleben, 1940; Euler et al., 1940; Maver et al., 1941; Berger et al., 1941; Ahlstrom e l al., 1942). On the basis of present evidence it does not appear that the D-peptidase activity of serum has any particular relationship to neoplastic disease. D. Carcinolysis. The Freund-Kaminer test for malignancy (Freund and Kaminer, 1910a,b; Kaminer, 1933) was based on the relative abilities of normal and pathological wra to bring about the in uitro cytolysis of cancer cells, usually prepared from liver metastases. Normal serum was more effective than serum from cancer patients in lysing the cancer cells. In spite of the extensive literature on the carcinolytic reaction, it is not clear whether differences between normal and pathological sera are due
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to alterations in enzyme content or to the presence of lytic inhibitors in the cancer serum. Stern and Willheim (1937) and Waterman (1931) have provided some evidence that the enzyme involved in the carcinolytic reaction is a Lipase. The literature on the carcinolytic reaction has been extensively reviewed in the monograph by Stern and Willheim (1943). E. Plasmin. The presence of proteolytic enzyme in normal plasma has been well established (e.g., Christensen and MacLeod, 1945; MacFarlane and Biggs, 1948), although considerable confusion has resulted from differences in terminology and methodology employed by various workers, the necessity of activation of the enzyme, and the presence of a t least one serum inhibitor of proteolytic enzymes. It seems very likely that the enzymes known as plasmin, fibrinolysin, serum protease, and serum tryptase are identical. This enzyme, if the identity is assumed, occurs in the plasma in an inactive form (plasminogen) and becomes activated when the plasma is treated with chloroform or with streptokinase. Quantitative measurement of this enzyme in serum is possible only after separation from the protease inhibitor which also occurs in serum. Dillard and Chanutin (1949) have studied the activities of plasmin (after separation from the inhibitor and activation with chloroform), of proteolysin (a spontaneously active proteolytic enzyme after separation from the inhibitor), and of trypsin inhibitor (see later) in the plasma of normal individuals, of cancer patients, and of patients with a miscellaneous group of diseases. Significant increases over the normal levels in all three principles were found both in the cancer and in the miscellaneous disease groups. Changes in the spontaneously active protease were the most marked and were also the most sensitive t o the clinical improvement following surgery. Dillard and Chanutin (1949) suggested that the proteolysin concentration may reflect the presence of a tissue product which stimulates the formation of the enzyme, or which induces the activation of plasminogen, and attributes its presence t o necrosis and inflammatory processes accompanied by elevated temperatures. An activator of fibrinolysin or plasmin (fibrinokinase) has indeed been shown to occur in animal tissues (e.g., Astrup et al., 1950; Tagnon and Pallade, 1950). F. Fuchs’ Test. A procedure for cancer diagnosis which has had wide discussion is the so-called Fuchs’ test (Fuchs, 1926, 1936). In this procedure chloroform-treated serum is incubated with fibrin or with dried trichloroacetic acid precipitated serum, the increase in nonprotein nitrogen being determined as a measure of proteolysis. The claim was made that fibrin from the blood of a patient with cancer was not split by proteases in the serum of other cancer patients, but was split by the serum
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proteases from normal individuals or from patients with other diseases. The converse was also claimed. Fuchs’ results have been supported by the work of some investigators (e.g., Bing and Marangos, 1934; Woodhouse, 1937; Wright and Wolf, 1939) and refuted by others (e.g., Hinsberg, 1940). Fuchs held the view that specific immune bodies were associated with the fibrin substrate and protected it from hydrolysis by proteolytic enzymes of the homologous type of serum. It seems more likely, however, that the differences observed by Fuchs were due t o the interrelations of plasmin, plasminogen, proteolysin, and the proteolytic inhibitor activities of serum, an interrelationship which requires considerable fundamental research before application to abnormal plasma is likely to be very fruitful. G . Abwehrferment. Abderhalden (1936, 1937, 1941, 1946) has studied the occurrence of a defensive enzyme designated “ abwehrferment ” in the plasma, this enzyme allegedly having the prbperty of breaking down specific substances which induced its formation. In the application to serodiagnosis of cancer, protein preparations from neoplastic tissues are employed, these presumably being more readily attacked by proteolytic enzymes in plasma of cancer patients than by those enzymes in normal plasma. A voluminous literature (reviewed by Stern and Willheim, 1943) has accumulated on this subject. The fundamental basis upon which this test rests, i.e., the production of enzymes with specific activity toward proteins not normally present, certainly needs more experimental foundation. There is also the inherent conclusion that there may be circulating in the blood stream an almost unlimited number of proteolytic enzymes, all different with respect to specificity toward their substrates. Again, any evaluation of the fundamental basis of the test must take into consideration its relation to plasmin, proteolysin, and the proteolytic enzyme inhibitors of serum. 2. E n z p e Inhibitors
Plasma has been shown to contain a number of factors which inhibit the action of certain enzymes. The activity of a number of these enzyme inhibitors may rise markedly in pathological states including neoplastic disease (Winzler, 1950). A. Inhibition of Proteolytic Enzymes. The most extensively studied of the serum enzyme inhibitors has been concerned with the inhibition of the action of trypsin. The early work of Brieger and Trebing (1908) showed that the plasma of cancer patients contained supernormal amounts of a very powerful inhibitor of trypsin-like enzymes. There
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has been a continued interest in this type of inhibitor with a number of recent publications bearing on the subject (Grob, 1943, 1949; Kaplan, 1946; MacFarlane and Biggs, 1948; Clark et al., 1948; Guest et al., 1948; Cliffton, 1949, 1950; Dillard and Chanutin, 1949; Duthie and Lorenz, 1949a; Loomis et al., 1949; Wells et al., 1949; Waldvogel and Schmitt, 1950; Waldvogel et al., 1949; Cliffton and Young, 1950; Lewis and Ferguson, 1950; Ungar and Damgaard, 1951). This inhibitor is a remarkably potent one, 0.003 ml. of normal serum containing enough inhibitor to inhibit by 50% of the action of 0.02 mg. of crystalline trypsin (Clark et al., 1948). Serum from patients with neoplastic disease is even more effective. There is no evidence which would indicate that the agent inhibiting trypsin is different from the one inhibiting plasmin. The inhibitor appears in the albumin fraction of serum or plasma by both salt and electrophoretic separations, and its heat lability and nondialyzability indicate that it is a protein in nature. Beloff (1946) has shown that the proteolytic enzyme of skin was powerfully inhibited by plasma, the inhibitor being in the very soluble albumin fraction. Further work will be necessary t o establish Beloff’s contention that this inhibitor of skin protease is different from the plasma trypsin inhibitor. West et al. (1949a,b, 1951a,b,c) and Tauber (1950) have studied the inhibition of chymotrypsin by human serum and have shown that the activity of the chymotrypsin inhibitor rises markedly in cancer and in a number of other pathological conditions. The possible identity of serum chymotrypsin inhibitor with the serum trypsin and fibrinolysin inhibitors has not yet been investigated. If further work proves that these proteolytic enzymes are all inhibited by the same plasma agent, a remarkable degree of nonspecificity would be indicated. The great potency bf proteolytic enzyme inhibitors and their increase in activity in pathological states suggest that the factors may have important physiological roles. Cliffton (1950) has indicated that increases in this factor are not related to tissue necrosis. In addition to the above proteolytic enzyme inhibitor or inhibitors, West et al. (1949a,b, 1951a,b,c) have shown that serum contains an inhibitor of rennin activity, this inhibitor being less potent than the chymotrypsin inhibitor. The chymotrypsin and rennin inhibitor levels were followed in individual cancer and leukemia patients given various types of therapy. It was noted that these two inhibitors exhibit characteristic changes in activity during clinical improvement of disease or with its reactivation. Effectiveness of therapy in individual cases could be correlated with the balance between the rennin and chymotrypsin inhibitors (West et al., 1949a,b, 1951a,b,c; Ellis and West, 1951). The two inhibitors frequently responded in opposite directions to therapy indicating that they were separate agents.
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Still another proteolytic enByme inhibitor in serum has been studied by Duthie and Lorenz (1949b), who have shown that the inhibition of bacterial gelatinases by serum is brought about by an agent which has a different solubility in ammonium sulfate, a lesser stability, and a different rate of reaction with the enzyme than does the serum trypsin inhibitor. B. Inhibitions of Hyaluronidase. At least two hyaluronidase inhibitors have been demonstrated in serum, these differing in their heat lability, in their specificity toward hyaluronidase from different sources, and in their tendency toward abnormal levels in different pathological conditions. One of these appears to be a neutralizing antibody for streptococcal antigens and is associated with the gamma globulins (Moore and Harris, 1949). Another hyaluronidase inhibitor is a normal nonspecific constituent of plasma. The activity of this inhibitor rises significantly in cancer and in a number of pathological conditions (Dorfman et al., 1948; Fulton et at., 1948; Hakanson and Glick, 1948; Glick, 1950; Kiriluk et al., 1950). Glick (1950) has recently reviewed the behavior of this serum hyaluronidase inhibitor in health and disease. The nature of the nonspecific hyaluronidase inhibitor has been extensively studied by Glick and his associates, who have found that it is heat labile and presumably protein, that it occurs in the albumin fraction of plasma fractionated electrophoretically (Glick and Moore, 1948; Moore and Harris, 1949), that it bears no relationship t o plasma mucoprotein (Glick et al., 1949), and that it may be a heparin-lipoprotein complex originating in tissues rich in mast cells (Glick and Sylven, 1951). C . Inhibitors of Oxidizing Enzymes. Hirschfeld et al. (1946) reported that serum from cancer patients inhibited the oxidation of tyrosine by potato tyrosinase more effectively than normal serum and suggested that the change was a rather specific manifestation of neoplastic growth. Subsequently Marx (1949) and Stadie et al. (1947) found that such differences as were found were due to an increase in the induction period obtained in the presence of cancer sera. Shacter and Shimkin (1949) investigated the catecholase-inhibiting activity of normal serum and serum from cancer patients and observed that the induction time was significantly longer in the presence of normal than of pathological sera. This relationship, which is the reverse of the situation observed by Hirschfeld et al. (1946) was ascribed to the lowered sulfhydryl content of the sera of patients with cancer and other conditions. The experiments of Shacter and Shimkin would appear to be still another indication pointing toward decreased sulfhydryl content of the serum of patients with neoplastic or other types of disease.
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IV. PROTEIN-BOUND CARBOHYDRATE 1. Total Polysaccharide
For a long time it has been recognized that a considerable amount of carbohydrate is associated with the serum proteins, the amount being somewhat in excess of the blood glucose levels. In a number of pathological conditions, including cancer, the amount of serum protein-bound carbohydrate may rise from normal values of about 100 mg. % ' to as high as 200 mg. % (Bierry et al., 1921; Lustig and Langer, 1931; Hinsberg and Merten, 1938; Lustig and Nassau, 1941; Novak and Lustig, 1947; Seibert TABLE VI Summary of Some Differences in Normal and Pathological Sera Depending on Protein-Bound Polysaccharide Normal Serum Nonglucosamine polysaccharide1 Glucosaminel
Tr yptophan-perchloric acid reaction' Diphenylamine reactions
Cancer Serum
Other Serum
Reference
111. f 9.32 171. f 32.5 149. f 29.18 Shetlar et al. (1949) 69 f 5.2 95 f 17.6 89 f 18.18 Shetlar et al. (1949) 60 106 1466 Seibert el al. (1948) 0.337 0.555 0.623' Niazi & State (1948)
Img. %. Standard deviation. 8 Non-neoplastic pathology. 4 Klett units. Advanced tuberculosis. 6 Optical denaity units. 7 Inflammations. 2
and Atno, 1946; Seibert et al., 1947, 1948; Shetlar, Erwin, and Everett, 1950; Shetlar et al., 1948, 1949, 1950). Similar increases in the proteinbound hexosamine are also noted in the plasma of cancer patients (Nilsson, 1937; West and Clark, 1938; Shetlar el al., 1949) some illustrative data is given in Table VI. The protein-bound carbohydrate is distributed among a number of proteins of plasma (Table 111),albumin being low and alpha globulin relatively high in carbohydrate content (Blix et al., 1941; Seibert et al., 1948). The protein-bound carbohydrate appears to consist of approximately equimolecular amounts of galactose, mannose, and glucosamine (Rimington, 1929, 1931, 1940; Friedmann, 1949; Waldron and Woodhouse, 1950).
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As has already been pointed out, one of the alpha-1 carbohydrate containing proteins has been isolated in a purified state (Weimer et al., 1950; Smith et al., 1950; Schmid, 1950). This mucoprotein has been shown (Weimer and Winzler, unpublished) to be a major constituent of the seromucoid preparations of Bywaters (1909) and Rimington (1940), Staub and Rimington (1948), as well as of the “mucoidlihnliche substanz” of Mayer (1942). It seems very likely that it is identical with the small acid glycoprotein described by Schmid (1950) (Schmid, personal communication; Weimer and Winzler, unpublished), and appears to be mucoprotein in nature (Meyer, 1945; Stacey, 1946). This component contains 16% hexose and 12% hexosamine and accounts for about 10% of the total serum polysaccharide (Winzler et al., 1948). Its concentration may rise several fold above normal in the plasma of cancer patients (Winzler and Smyth, 1948). This mucoprotein is isolated with the serum albumin fraction by most chemical separations, and therefore would account, in part at least, for the increase in the carbohydrate content of this fraction in pathological conditions (Novak and Lustig, 1947; Shetlar, Erwin, and Everett, 1950; Shetlar et al., 1950; Blix et al., 1941). Little is known about the behavior of other polysaccharide-containing proteins of plasm? in disease. It seems likely however, that the levels of a number of the carbohydrate-rich proteins in plasma rise in a variety of pathological states, including cancer. The levels of the different polysaccharidecontaining proteins may vary in a differential manner in different diseases. Seibert et al. (1947, 1948) showed that, in several wasting diseases, including tuberculosis, sarcoidosis and cancer, there was a parallel rise in the alpha-2 globulin and in serum polysaccharide, suggesting that the major serum polysaccharide increase was associated with the alpha-2 globulin fraction. Considerable interest and importance is concerned with the source of the protein-bound carbohydrate. One possibility is a local origin a t the site of the lesion which results in elevated levels. Seibert et al. (1947, 1948) suggested that the rise in alpha-2 globulin and serum polysaccharide was indicative of tissue destruction, a conclusion in accord with that of Longsworth el al. (1939) and Shedlovsky and Scudder (1942). Gersh and Catchpole (1949), Catchpole (1950), and Pirani and Catchpole (1951) have pointed out that the polysaccharide-containing proteins of serum may arise from the depolymerization of glycoprotein constituents in the ground substance of connective tissue in the region of certain lesions. Lustig and Nassau (1941) found that the carbohydrate content of the proteins of tubercular pleural effusions and cantharidin blisters frequently exceeded that of the serum proteins, suggesting that relatively carbohydrate-rich proteins entered the exudates from the tissues. Previously,
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Lustig et al. (1937) had reported that the carbohydrate-content of the proteins of the venous blood frequently exceeded that of the arterial blood, again suggesting that carbohydrate-rich proteins were entering the blood from the tissues. Shetlar and his associates have studied the serum polysaccharides in normal individuals (1948) and in patients with cancer or other pathological conditions (Shetlar et al., 1949, 1950), a large proportion of whom showed abnormally high levels. Similar high serum polysaccharide levels were noted in tumor-bearing rats (Shetlar et al., 1950). These investigators suggested that an increase in serum polysaccharide level was associated with tissue proliferation rather than with tissue destruction per se (Shetlar et al., 1949). Werner (1949) showed that the serum glucosamine level rose significantly in rabbits bled repeatedly and that damage to the liver produced by administration of phosphorus or benzene prevented this increase. The data suggested that the carbohydrate-rich plasma proteins were produced in the liver under pathological conditions and that a subfraction of the alpha or beta globulins was especially concerned in this increase. Although conclusive evidence is thus still lacking with respect t o the origin and significance of the increased plasma mucoprotein and polysaccharide concentrations in cancer, the general increase of these components in a wide variety of diseases suggests that the increase represents a systemic effect of the tumor and that the components may not be formed in the lesion itself. It is clear from the many conditions in which increases in serum polysaccharides have been noted that such determinations are not likely to be of much use for the diagnosis of cancer. The possibility remains, however, that specific polysaccharide-containing proteins, present in relatively minor amounts, may be shown to be more directly associated with malignant disease than is the total serum polysaccharide level. 2. Tryptophan-Acid Reaction
An interesting observation was made by Seibert et al. (1948) in which it was noted that serum heated with tryptophan and perchloric acid developed a color with an absorption spectrum characteristic of fructose and not of other hexoses, although free or bound fructose could not be demonstrated in the serum. The amount of the color developed in this “ tryptophan-acid reaction’’ was significantly increased in the plasma of patients with tuberculosis or cancer. Riegel and Beatty (1950), Shetlar et al. (1949), Israel et al. (1949), and Weisbrod (1950), have also observed an increase in the tryptophan-acid reaction in the serum of patients with
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neoplastic disease. The nature of the substance, presumably carbohydrate, responsible for the reaction merits further study. 3. Diphenytamine Reaction
Niazi and State (1948) and Ayala, Moore, and Hess (1951) have determined the purple color produced when the diphenylamine reaction (Dische, 1929, 1930) is applied to hot trichloroacetic acid extracts of human serum from patients with cancer, rheumatic fever, or other conditions had significantly higher levels of the reactive substance than did normal serum. No information is available on the precise nature of the chromogenic substance, although it would appear t o be carbohydrate in nature. V. DISCUSSION
It is clear that many abnormalities occur in the plasma proteins in patients with neoplastic disease. The nature of most of these abnormalities and their physiological significance are largely unknown. Elucidation of these factors will await a great deal of fundamental work on the normal protein components. Studies of abnormalities may, however, be of great use in clarifying the normal physiology of particular proteins. The amount and kind of the plasma proteins reflect the activities of the various tissues and organs of the body, and the interplay between these tissues resulting in a steady state level of each constituent. Since neoplasms may have effects on tissues remote from the site of the tumor, the plasma changes associated with this disease clearly may have a highly complicated etiology. From the work that has thus far been carried out, it would appear that a large number of pathological conditions lead to very similar abnormalities in the plasma protein picture. This suggests that most of the abnormalities considered in previous pages are associated with systemic changes in the host elicited by the neoplasm, rather than a direct effect of the tumor. Most attention has thus far naturally been focused on those proteins present in the largest amounts. It would seem very possible, however, that the most pronounced and perhaps specific changes associated with neoplastic disease may reside in those proteins present in very small amounts or essentially absent in plasma of normal individuals. Again, any attempt to pursue this approach must be associated with an extensive study of the characterization of the minor plasma proteins. In addition to studies of the amounts of the various plasma proteins, considerable information may ultimately be obtained by investigation of the metabolic activities of the major and minor proteins using isotopic techniques. Studies on the turnover of individual proteins in the plasma
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of normal individuals and of patients with neoplastic or other disease may be expected to yield very significant data on the fundamental causes of increased or decreased amounts of particular proteins. It is perhaps worthy of emphasis to note that it will scarcely be adequate to study the relative metabolic activity of the gross fractions obtainable by electrophoretic, ethanolic, or salt fractionation, since the most pronounced changes in a minor protein may be completely masked by contaminating major fractions. It is to be expected with some confidence that continuing improvements in fractionating techniques will permit the separation of plasma proteins into more and more homogeneous fractions. Perhaps one of the most pressing problems relating t o the plasma proteins is the elucidation of their source and their fundamental biological significance. Considerable fundamental work has already been carried out with respect to the source of a number of the major components, and with the significance of some of the proteins associated with blood coagulation. Much remains, however, in elucidating these points in connection with the minor plasma, proteins, and especially in disease states. The hope that specific abnormalities in plasma proteins might be of diagnostic significance in detection of cancer has not been substantiated by the work that has been reviewed in the previous discussion. Significant abnormalities are, indeed, associated with this disease, but thus far no specificity has been apparent. REFERENCES Abderhalden, E. 1936. Fermentforsehung 16, 245-50. Abderhalden, E. 1937. Ergeb. Enzymjorsch. 6, 189-200. Abderhalden, E. 1941. Abwehrfermente (Die Abderhaldenische Reaktion). Steinkopf, Dresden and Leipzig. Abderhalden, E. 1946. Schweiz. med. Wochnschr. 76, 47. Abels, J. C., Ariel, I., Rekers, P. E., Pack, G. T., and Rhoads, C. P. 1943. Arch. Surg. 46, 844-60. Adams, W. S., Alling, E. L., and Lawrence, J. S. 1949. Am. J. Med. 6, 141-61. Ahlatrom, L., Euler, H. V., and Hogberg, B. 1942. 2.physiol. Chem. 273, 129-57. Albers, D. 1940. Biochem. 2.306, 236-44. Appel, M., Saphir, O., Janota, M., and Straus, A. A. 1942. Cancer Research 2, 576-78. Ariel, I. M. 1949. Surg. Gynecol. Obstet. 88, 185-95. Ariel, I., Jones, F., Pack, G . T., and Rhoads, C. P. 1943. Ann. Surg. 117, 740-47. Armstrong, 5. H. 1950. Symposium on Nutrition 2. Plasma Proteins, C. C. Thomas and Co., pp. 22-61. Aacoli, M., and Izar, G. 1910. Miinch. med. Wochnschr. 67, 403-05; 954-56; 2 129-3 1. Astrup, T., Crockston, J., and MacIntyre, A. 1950. Acta Physiol. Scand. 21, 238-49. Ayala, W., Moore, L. V., and Hess, E. L. 1951. J . Clin. Invest. 90, 781-85.
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Barringer, B. S., and Woodward, H. Q. 1938. Trana. Am. Asaoc. Genito-Urin. Surgeuna 31,363-69. Bayerle, H., and Podloucky, F. H. 1940a. Biochem. 2. 304, 259-65. Bayerle, H., and Podloucky, F. H. 1940b. 2.phyajol. Chem. 264, 189-95. Bayerle, H., and Podloucky, F. H. 1940~. 2.Krebaforach. 60, 220-29. Beloff, A. 1946. Biochem. J. 40, 108-15. Bennhold, H. 1932. Ergeb. inn. Med. Kinderheilk. 42, 273-375. Berger, J., Johnson, M. J., and Baumann, C. A. 1941. J. Biol. Chem. 137, 389-95 Besredka, A,, and Gross, L. 1938. Ann. Inat. Paateur 60,5-12. Bierry, H., Rathery, F., and Levina, M. 1921. Compt. rend. 173, 56-57. Bing, M., and Marangos, G. 1934. Beitr. klin. Chir. 160, 417-44. Black, M. M. 1947a. Cancer Research 7, 321-25. Black, M. M. 194713. Cancer Reaearch 7, 592-94. Black, M. M. 1948. Science 108, 540-41. Black, M. M., Kleiner, I. S., and Bolker, H. 1948. Cancer Reaearch 8, 79-82. Black, M. M., and Speer, F. D. 1950. Am. J. Clin. Path. 20, 446-53. Blix, G., Tiselius, A,, and Svensson, H. 1941. J. Biol. Chem. 137, 485-94. Bodansky, O., and Blumenfeld, 0. 1949. Proc. SOC.Exptl. Biol. Med. 70,546-47. Bodansky, O., and McInnes, G. F. 1950. Cancer 3, 1-14. Boyd, L. J. 1950. N. Y. Slate J . Med. 60, 2167-72. BrdiEka, R. 1937. Nature 139, 1020-21. BrdiEka, R. 1939a. Acla radiol. et cancerol. Bohem. el Morav. 2, 7-16. BrdiEka, R. 1939b. Klin. Wochnachr. 18,305-08. BrdiEka, R., Novak, F. V., and Klumpar, J. 1939. Acla radiol. el cancerol. Bohem. el Morav. 2, 27-41. Brieger, L., and Trebing, J. 1908. Berlin. klin. Wochnachr. 46, 1041-44; 1349-51; 2260-6 1. Bronfin, G. J., Hart, R. W., Liebler, J. B., and Goldner, M. S. 1951. Proc. Soc. Exptl. Biol. Med. 77, 456-58. Bywaters, H. W. 1909. Biochem. 2. 16, 322-43. Catchpole, H. R. 1950. Proc. SOC.Exptl. Biol. Med. 76, 221-23. Cheever, F. S. 1940. Proc. SOC.Exptl. Biol. Med. 46, 517-22. Cheever, F. S., and Janeway, C. A. 1941. Cancer Research 1, 23-27. Christensen, L. R., and MacLeod, C. M. 1945. J. Gen. Phyaiol. 28, 559-83. Clark, D. G. C., Cliffton, E. E., and Newton, B. L. 1948. Proc. SOC.Exptl. Biol. Med. 69, 276-79. Cliffton, E. E. 1949. J. Nall. Cancer Znat. 10, 719-23. Cliffton, E. E. 1950. J. Natl. Cancer Znat. 11,33-50. Cliffton, E. E., and Young, L. E. 1950. Cancer 3, 488-92. Cohen, P. P., and Thompson, F. L. 1947. J. Lab. Clin. Med. 32, 475-80. Cohn, C., and Wolfson, W. Q . 1947. J. Lab. Clin. Med. 32, 1203-07. C o b , C., and Wolfson, W. Q . 1948. J . Lab. Clin. Med. 33, 367-70. Cohn, E. J. 1941. Chem. Revs. 28,395-417. Cohn, E. J. 1948. Blood 8,471-85. Cohn, E. J., Gurd, F. R. N., Surgenor, D. M., Barnes, B. A., Brown, R. K., Derouaux, G., Gillespie, J. M., Kahnt, F. W., Lever, W. F., Liu, C. H., Mittelman, D., Mouton, R. F., Schmid, K., and Uroma, E. 1950. J. Am. Chem. SOC.72, 465-74. Cohn, E. J., Oncley, J. L., Strong, L. E., Hughes, W. L., and Armstrong, S. H. 1946. J. Am. Chem.SOC.68, 459-75. Cremer, H. D., and Tiselius, A. 1950. Biochem. 2.380, 273-283.
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Wells, B. B., Marvin, H. N., and Waldvogel, M.
Washington, D.C.
548
RICHARD J. WINZLER
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Author Index Numbers in parentheses are references and are included to assist in locating references in which the authors”names are not mentioned in the text. Numbers in italics indicate the pages on which references are listed at the end of the article.
A
Andrew, V. W., 252, 271 Andrew, W., 66, 96, 99 Andrewes, C. H., 156,160, 246, 247, 255, 262, 264, 866, 267, 268 Anson, M. L., 398, 409, 448 Antopol, w., 347, 390 Apelgot, S., 328, 336 Apolant, H., 104, 117, 127, 160, 164 Appel, M., 526, 639 Argus, M. F., 293, 336 Ariel, I., 514, 515, 639 Armstead, E. B., 505, 508, 516, 517, 520, 528, 646, 647 Armstrong, E. C., 124, 160, 215(300), 218(320), 230, 831 Armstrong, M., 116, 121, 141, 160 Armstrong, S. H., 510, 517, 518, 519,
Abderhalden, E., 532, 639 Abels, J. C . , 514, 639 Adair, F. E., 490, 4.97 Adams, M. H., 256, 266 Adams, W. S., 528, 639 Ahlbom, H. E., 490, 497 AhlstrBm, L., 314, 315, 334,630,639, 641 Ahlstrom, C . G., 67, 98 Ahrens, E. H., 524, 643 Aiston, S.,252, 266 Aitken, H. A. A., 259, 266 Albers, D., 523, 639 Albert, S., 202 (158), 227 Alexander, P., 429, 435, 446 Algire, G. H., 484, 4.97 Allen, E., 119, 121, 135, 169, 164, 207 639,640 Arnesen, K., 146, 163 (205, 208), 210, 213(265), 288, 889 Alling, E. L., 504, 508,509, 528, 639, 644 Aron, M., 527, 641 Altman, S., 52, 64 Ascoli, M., 526, 639 Amies, C. R., 249,252, 256, 259, 260, 262, Astbury, W. T . , 136, 138, 139, 157, 169 Astrup, T., 531, 639 966,269 Athais, M., 195(93, 94), 212(93, 94), 826 Anderson, R. S., 256, 267 Atno, J., 504, 505, 508, 521, 535, 536, 646 Anderson, S., 434, 449 Au, M. H., 74, 100 Anderson, T. F., 253, 266 Auerbach, C., 398, 445, 446 Anderson, W., 50, 64 Andervont, H. B., 105, 106,107, 108,109, Austin, M. L., 157, 170 111, 112, 113, 114, 115, 116, 117, Ayala, W., 538, 639 121, 122, 124, 125, 126, 128, 129, 130, Ayengar, A. R. Gopal, 72, 98 132, 133, 134, 135, 136, 140, 142, 143, B 144, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 159, 16.9, 160, 168, 163, 166, 166, 170, 182(22), 195, Back, A., 333, 334 198(107), 204 (87), 205, 213 (275), Bacon, R. L., 221(357, 358), 838 215(291, 301), 217(275, 291, 317), Baddeley, G.,420, 446 220, 883, 886, 230, 291, 344, 379, Badger, G. M., 4, 31, 35, 36, 48, 49, 64, 280, 334, 382, 390 380, 390, 475, 601 Bagg, H. J., 112, 120, 148, 160 Ando, T., 379, 390 549
550
AUTHOR INDEX
Baker, J. W., 17, 64 Beatty, P. R., 537, 646 Baker, R., 505, 516, 518, 529, 530, 645 Begg, A. M., 136, 166, 259, 266 Baker, S. L., 258, 259, 266 Beinert, H., 387, S9S Baldock, G. R., 32, 42, 64 Belding, T. C., 263, 266 Ball, Z. B., 106, 108, 110, 117, 128, 129, Belkin, M., 483, 601 132, 134, 135, 136, 137, 138, 139, Bellamy, A. W., 527, 64.2 160, 162, 166, 171, 214(287), 280, Beloff, A., 533, 640 401, 402, 403, 404, 405, 407, 446, Bendich, A., 319, ,934 457, 458, 466, 497, 499, 601 Benditt, E. P., 483, 498 Bandoin, N., 530, 646 Benedict, S. R., 250, 270, 481, 482, 600 Bang, F. B., 258, 266 Bennett, G. M., 420, 446 Banks, T. E., 398, 409,410, 437,446 Bennett, L. L., Jr., 323, 331, 332, SSd, Banyen, D., 64, 88, 99 SS6, SS7 Bardet, J., 139, 164 Bennhold, H., 518, 519, 640 Barlow, H., 117, 168 Bennison, B. E., 144, 146, 160, 168 Barnes, B. A., 510,511,512,521,640,6~~ Benotti, J., 645 Barnes, L., 458, 499 Berenblum, I., 91, 99, 410, 447, 464, 497 Barnes, R., 458, 497 Berg, N. O., 67, 98 Barnes, R. H., 136, 171, 457, 601 Berger, J., 530, 640 Barnes, W. A., 106, 135, 160 Berger, M., 328, 3.96 Barnum, C. P., 117, 132, 134, 135, 136, Bergman, H. C., 523, 646 137, 138, 139, 143, 160, 249, 269, Bergmann, M., 398, 401, 402, 405, 407, 314, 325, 334 412, 413, 414, 415, 416, 417, 418, Baron, H., 519, 525, 646 444449 Barrett, M. K,, 135, 160 Berkman, S., 517, 519, 523, 525, 642 Barringer, B. S., 529, 640 Berman, C., 351, 390, 490, 491, 497 Barron, E. 8. G., 249, 266, 268, 418, 447 Bernhard, W., 245, 255, 269 Bartlett, G. R., 401, 418, 447 Berry, G. P., 248, $370 Bartlett, P. D., 433, 447 Berthier, G., 16, 30, 31, 32, 33, 42, 43, 44, Barton, A. D., 304, $84 64, 66, 66 Bashford, E. F., 104, 117, 160 Besredka, A., 526, 640 Bauer, D. J., 250, 266 Baumann, C. A,, 63, 100, 341, 343, 346, Betheil, J. J., 364, 394 Biddulph, C., 189(48, 52), 224 347,348, 349,350,353,354,355,356, 357,359,360, 362,363,364,365,371, Bierry, H., 535, 640 377,379,387, S90,$91, S92, 39Sl S94, Biesele, J. J., 72, 74, 95, 99, 436, 445, 446, 447 896, 452, 457, 458,468, 469, 470, 471, 474, 477,478,479,482,497,4998,499, Biesele, M. M., 72, 74, 99 Biggs, R., 531, 533, 644 600, 530, 640 Billingham, R. E., 66, 99 Baumann, E. J., 256, 268 Bing, M., 506, 514, 532, 640 Baumberger, J. P., 74, 94, 99 Bird, M., 445, 447 Bawden, F. C., 247, 866 Bischoff, F., 120, 160, 204(177, 178), 205, Bayerle, H., 530, 640 827, 481, 497 Beach, J. Y., 6, 15, 66 Biskind, G. R., 185, 186(35), 192(35), Beadle, G. W., 109, 160 196, 197(101),224, 226 Beard, D., 251, 253, 254, 255, 256, 258, 263, 266, 869, 270, 271 Biskind, G. S., 185, 224 Beard, J. W., 137, 160, 233, 251, 253, 254, Biskind, M. S., 185, 186(35), 192(35), 255, 256, 257, 258, 263, 266, 267, ,969, 196, 197(101),224, 226 870, 871 Bissell, A. D., 213(272), 299
AUTHOR INDEX Bittner, J. J., 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 122, 123, 124, 125, 126, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 141, 142, 143, 144, 145, 146, 148, 151, 152, 153, 154, 158, 160,161, 162, 164, 166, 166, 167, 169, 170, 171, 183(27), 200(125), 212, 213 (255, 276), 214(286), 215(292, 293), 217(316), 218(276, 318, 319, 322), 223, 226, 229, 230, 231 Black, A., 471, 498 Black, M. M., 505, 516, 519, 520, 525, 640 Blakemore, F., 150, 162 Bleyer, L., 235, 236, 267 Blix, G., 528, 535, 536, 640, 644 Bloch, H. S., 302, 334 Bloom, S., 302, 307, 336, 378, 391 Blumenfeld, O., 530, 640 Blumenthal, H. T., 121, 168, 212, 929 Bodansky, O., 517, 530,640 Boddaert, J., 188, 195, 196(91, 97), 205, 924, 226 Bohnel, E., 252, 266 Boissonnas, R. A., 290, 334, 364, 390 Bolker, H., 505, 519, 525, 640 Bonbme, R., 45, 66 Bonne, C., 490, 497 Bonser, G. M., 106, 122, 123, 124, 126, 155,160,162, 195,197(86), 213(271), 215(300), 226, 829, 230 Boon, M. C., 135, 164, 190(59), 191, 193(67), 194(67), 204 (185), 205 (185), 206(185), 224, 227, 457, 600 Boretti, G., 377, 391 Borgess, P. R. F., 107, 162 Borsook, H., 304, 307, 334 Borsos-Nachtnebel, E., 345,346,351,394 Boursnell, J. C., 398, 399, 405, 407, 408, 409, 410, 428, 437, 442, 446, 447 Boutwell, R. K., 108, 162, 457, 459, 466, 468, 469, 471, 479, 497 Boyd, D. R., 433,447 Boyd, L. J., 505, 516, 517, 640, 642 Boyland, E., 42, 46, 49,64, 207(210), 228, 280,288,289,334,336,358,380,381, 390, 398, 447 Brambel, F. W. R., 190, 924 Branch, G. E. K., 430, 447
551
Brandt, E. L., 317, 336, 376, 391 Brataler, J. W., 471, 498 Braa, G. I., 436, 447 Brdi6ka, R., 505, 518, 522, 640 Breedis, C., 346, 371, 378, 390 Brewer, J. I., 174(3), 223 Brewer, P., 280, 336 Bridgers, W. H., 257, 267 Brieger, L., 532, 640 Briggs, V. C., 198(107), 226 Brock, N., 345, 346, 390 Brockland, I., 517, 525, 641 Brockway, L. O., 6, 15, 66 Bronsted, J. N., 429, 430, 432, 447 Bronfin, G. J., 529, 640 Brown, A., 513, 646 Brown, D. M., 507, 513, 521, 536, 643, 646 Brown, E., 108, 164 Brown, G. B., 319, 320, 334, 336 Brown, H., 523, 643 Brown, J. B., 349, 391 Brown, M., 206(193), 207(203), 227, 228 Brown, R. D., 16, 48, 49, 64 Brown, R. K., 354, 388, 393, 510, 511, 512, 521, 640, 644 Brown, R. R., 351, 366, 369, 391, 393 Brown, W. G., 7, 27, 66 Brown, W. H., 526,646 Browning, H. C., 126, 162 Bruce, W. F., 45, 64 Brues, A. M., 275, 312, 319, 334, 358, 380, 381, 390 Brumfield, H. P., 252, 266 Brunschwig, A., 213(272), 229 Brush, M. K., 108, 162, 457, 459, 466, 468, 469, 471,479,497 Bryan, W. R., 106, 107, 110, 123, 132, 134, 135, 136, 137, 141, 144, 150,169, 160, 161, 166, 170, 201(139), 202 (130), 213(261), 226, 229, 240, 241, 242, 255, 257, 259, 266, 270, 284, 331, 332, 334, 337 Buchanan, J. M., 318, 334 Buck, P., 527, 641 Buckley, S. M., 436, 445, 446, 447 Bulliard, H., 73, 100 Bullock, F. D., 209(228), 228, 381, 391 Bullough, H. F., 63, 99 Bullough, W. S., 63, 99, 466, 497, 498
552
AUTHOR INDEX
Bunster, E., 189(50), 224 Burch, J. C., 208(215, 216), 228 Burchenal, J. H., 399, 436, 445, 446, 447 Burford, T. H., 219(332), ,93f Burk, D., 347, 396, 476, 478, 486, 498, 505, 522, 523, 648 Burkitt, F. C., 47, 64 Burmester, B. R., 263, 266 Burnet, F. M., 152, 168 Burnett, W. T., 203(160), 227 Burns, E. L., 210, 213(270, 273, 274), 220, 221(347), 229, 230, 231, 232 Burns, I. L., 121, 122, 168 BUITOWB, H., 104, 119, 162, 194(82), 195, 207(u)9, 210), 216(312), 219(330), 226, 227, 228, 231
Burrows, R., 174(2), 201(2), 219(2), 223 Busch, H., 302, 336 Butler, G. C., 409, 448 Butler, J. A. V., 410, 419, 436, 445, 447 Butterworth, J. S., 190((50),192(66), 224 Buu-Hoi, N. P., 7, 38, 47, 64, 66,. 293, 328,336 Buxton, L., 146, 163 Byran, R. S., 505, 535, 537, 646 Byrnes, E. W., 216, $30 Byrnes, W. W., 189(49),224 Bywaters, H. W., 523, 536, 6.40 C
Caldwell, A. L., 81, 100 Calnan, D., 240, 241, 266, 270 Calvin, M., 274, 276, 279, 334, 430, 447 Cambel, Perihan, 65, 67, 99 Campbell, H. W., 504,505,508,521,535, 536, 646 Carmichael, N., 117, 141, 171 Carpenter, F. H., 409, 411, 441, 447 Carpenter, G. E., 133, 166 Carr, C., 121, 166 Carr, J. G., 236, 237, 238, 240, 242, 246, 247,249,252,254, 255, 257, 258, 259, 260, 262, 266, ,966, 268 Carrel, A., 241, 243, 244, ,966 Carroll, D. M., 221(355), 232 Carruthers, C., 62, 63, 65, 74, 75, 76, 77, 81, 83, 84, 85, 88, 93, 95, 98, 99, 100, 101
Carter, C. E., 319, 337
Casas, C. B., 108, 121, 131, 16.9, 164, 166, 188(43), 214(288), 224, 230, 458, 462, @9 Cashmore, A. E., 407, 447 Caspari, W., 452, 498 Catchpole, H. R., 536, 640, 641, 646 Chaikoff, I. L., 203(161, 162), 227, 279, 313, 314, 334, 338 Chalkley, H. W., 484, 497 Chalvet, O., 47, 66 Chambers, L. A., 250, 253, 266 Chambers, R., 93, 99 Chamorro, A., 216, 230 Chang, C. H., 192(64), 224 Chanutin, A., 417, 419, 4.47, 448, 508, 528, 529, 531, 533, 641, 646 Chargaff, E., 310, 334 Cheever, F. S., 526, 640 Cheutin, A., 293, 336 Chipps, H. D., 530, 641 Chitre, R. G., 108, 166 Christensen, L. R., 531, 640 Christian, W., 505, 530, 647 Christopher, G. L., 515, 641 Chu, W. C., 358, 362, 363, 392 Clark, D. G. C., 533, 640 Clarke, D. H., 535, 647 Clarke, G. J., 204(177, 178), 205, 227' Claude, A., 137, 157, 162, 242, 245, 246, 249,250,251,252, 253,254, 255, 256, 257, 259, 267, 968, 269,270 Clayton, C. C., 343, 347, 348, 349, 350, 391, 458, 474, 477, 498 Clemmesen, J., 478, 498 Clemmesen, S.,478, 498 Cleveland, A. S., 516, 517, 643 Clifton, E. F., 135, 162 Cliffton,E. E., 193(72),194(72),224, 505, 517, 525, 533, 640, 646 Cloudman, A. M., 105, 115,135,162, 167 Clowea, G. H. A,, 81, 100, 347, 348, 392, 474,499 Cohen, A,, 381, 391 Cohen, B., 411, 412, 447 Cohen, J. A., 442, 4.47 Cohen, L., 381,392 Cohen, P. P., 371,378,381, 391, 396, 513, 524,640, 646 Cohn, C., 507, 640
AUTHOR INDEX
553
Cohn, E. J., 510, 511, 512, 517, 519, 521, Culvenor, C. C. J., 433, 447 Cunningham, L., 302, 307, 317, 336,373, 528, 640 Cohn, W. E., 312, 319, 334 376, 378, 391 Cunningham, R. S., 208(215, 216), 228 Cole, R. K., 106, 135, 160, 164 Curtis, F. C., 524, 642 Cole, W. H., 490, 498 Collins, V. J., 211(233), 228 Curtis, M. R., 201(147), 209(228), 216, Collip, J. B., 201(144), 202, 203(144, 220(335), 221(147), 226, 228, 230, 231, 381, 391, 456, 468, 498 163), 216, 226, 227 Coman, D. R., 93, 99 Czaczkes, J. W., 474, 498 Conway, B. E., 419, 447 Cook, H. A., 377, 391 D Cook, J. W., 280, S3g,359, 380, 381, 382, Dahl-Iversen, E., 208(219, 220), 228 384, 385, 390, 391, 491, 498 Dalton, A. J., 108, 166, 163, 168, 198 Coolen, M. L., 123, 168 Cooper, G. R., 253, 269, 520, 646 (107), 226, 345, 391, 487, 600 Cooper, 2. K., 64, 66, 68, 69, 70, 95, Daly, B. M., 533, 642 99, loo, 101 Damgaard, E., 533, 647 Dann, T. B., 200(127), 266 Copeland, D. H., 388, 391, 478, 498 Danysz, S., 123, 167 Cori, C. F., 120, 16'6, 212(250), 229 Darlington, C. D., 157, 162, 264, 266 Cornil, L., 530, 646 Dascomb, H. E., 248, 670 Cornman, I., 332, 536 Cortell, R., 345, 346, 391 Dauben, W. G., 280, 283, 284, 336 Costerousse, O., 328, 356 Daudel, P., 7, 25, 38, 47, 64, 66, 293, 328, Cottral, G. E., 263, 266 336 Daudel, R., 6, 7, 15, 16, 20, 25, 33, 38, Cottrell, T. L., 23, 66 47, 49, 50, 51, 64, 66,66, 328, 336 Coulson, C. A., 8, 20, 23, 24, 25, 26, 27, Davidsohn, I., 506, 514, 641 29, 30, 31, 33, 40, 46, 47, 64, 66 Cowdry, E. V., 62, 63, 65, 66, 70, 71, 72, Davidson, I., 135, 162 73, 74, 76, 81, 87, 92, 94, 95, 96, 98, Davidson, J. N., 309, 310, 3S6, 373, 391 99,100,101 Davies, M. C., 252, 669 Cox, A. J., 354, 396 Davies, W., 406, 433, 447 Davis, J. S., 518, 523, 646 Cox, E. C., 29, 66 Cox, H. R., 252, 266, 269 Davis, J. W., 433, 447 Crabtree, C. E., 221(353), ,232 Davis, S. B., 409, 447 Crabtree, H. G., 379, 391 Davis, W., 400, 409, 414, 419, 424, 428, 442, 443, 446, 447 Craig, D. P., 15, 16, 24, 66 Craigie, J., 136, 166 Day, P. L., 347, 348, 391 Cramer, W., 63, 66, 67, 73, 74, 90, 96, 99, Deansley, R., 202, 627 Deasy, C. L., 307, 334 100, 101, 120, 162, 201, 226 Crandall, D. I., 417, 448 de Bruyn, W. M., 244, 266,266 Crawford, V. A., 17, 27, 28,66 De Eds, F., 354, 396 Deihl, D. G., 259,266 Cremer, H. D., 511, 640 de Jongh, S. E., 122, 166, 189(54), 212, Crockston, J., 531, 639 Cronin-Lowe, E., 528, 641 224, 229 De Leon,H. P., 185(34), 186(34), 923 Crossen, R. J., 210, 229 Delluva, A. M., 318, 334, 417, 44.8 Crossett, A. D., Jr., 217(313), 231 Crossley, M. L., 399, 436, 447, 448, 515, Delrue, G., 527,628,646 Denton, R. W., 527, 64.9 641 De Ome, K. B., 106, 107, 110, 112,137, Crowell, M. F., 458, 499 Csaky, T. Z., 263, 266 166
554
AUTHOR INDEX
Deringer, M. K., 106, 107, 109, 111, 113,
121, 126, 130, 153, 155, 157, 159, 163, 166, 213(275), 217(275), 218 (323), 230,231 Derouaux, G., 510, 511, 512, 521, 640 Des Ligneris, M. J. A., 261, 262, 867 Desruisseaux, G., 530, 646 Deuel, H., 435, 447 Devik, F., 483, 484, 485, 498 Devor, A. W., 510, 521, 523, 536, 648 Dewar, M. J. S., 6, 11, 12, 15, 16, 24, 66 DeWitt Fox, J., 234, 267 Dexter, S . O., 384, 386, 387, 392 Dick, W., 23, 66 Dickie, M. M., 201(130), 226 Diddle, A. W., 213(265), 289 Dillard, G. H. L., 508, 529, 531, 533, 6-41 Dillon, E. S., 528, 646 Dillon, M. L., 528, 646 Dinning, J. S., 347, 348, 391 Dippel, R. V., 157, 1'70 Dirksen, A. J., 516, 518, 646 Dische, Z., 538, 641 Dixon, M., 441, 442, 447 Dixon, T. F., 157, 163 Dmochowski, L., 106, 107, 108, 110, 112, 113, 116, 117, 118, 120, 123, 124, 125, 126, 132, 133, 134, 135, 136, 138, 139, 140, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 157,163, 169, 204 (184), 205, 218 (325, 327) , 220 (339), 887, 231, 249, 250,252, 260, 262,267 Dobriner, K., 288,336,359,360,363,384, 396 Dobrovolska'La-ZavadskaSa,N., 486, 498 Doering, W. E., 401, 402, 403, 404, 405, 407, 446 Doerr, R., 235, 236, 267 Dole, V. P., 507, 510, 641 Doljanski, L., 239, 244, 262, 267, 269 Doniach, I., 64, 99 Dorfman, R. I., 120, 163, 199(121), 226, 534, 641 Dotti, L. B., 180(18), 223 Dougherty, T. F., 204(176, 179), 205,227 Dowdy, A. H., 527, 642 Drill, V. A., 194(79), 286 Druckrey, H., 343, 345, 346, 353, 362, 690, 691 Dry, F. W., 65, 99
Dublin, L. I., 489, 498 Dubnik, C. S., 108, 162, 163, 168 Duboff, G., 534, 642 Du Bois, K. P., 387, 394 DU B U ~ , H. G., 157,163, i r i , 278 Duffy, E., 491, 498 Dulaney, A. D., 146, 163 Dunlap, C. E., 281, 336 Dunn, J. B., 221 (356), 838 Dunn, T. B., 218(32!), 231, 379, 390 Dunn, Th. B., 104, 106, 124, 125, 126,
132, 133, 148, 149, 150, 151, 152, 153, 154, 155, 169,163, 164, 166 Dunn, W. J., 136, 168 Dunning, W. F., 201(147, 148), 202, 216, 220(335), 221(147), 286, 230, 231, 456, 468, 498 Duran-Reynals, F., 156, 164, 235, 236, 237, 238, 239, 243, 250, 252, 255, 259, 264, 267, 869, 870, 525, 646 Durram, E. L., 511, 641 Dustin, P., Jr., 398, 447 Duthie, E. S., 533, 534, 641 du Vigneaud, V., 290,334, 347, 364, 390, 396, 405, 409, 411, 441, 447, 449, 478, 498 Dvoskin, S., 517, 641 Dworecki, I. J., 505, 516, 648
E Eaves, G., 139, 157, 169 Ebeling, A. H., 243, 266 Eckert, E. A., 263, 866 Eddy, M. S., 134, 143, 150, 166 Edsall, J. T., 506, 510, 511, 641 Edwards, J . E., 198(107), 226, 343, 344,
346, 346,348,379, 380,390, 391, $96
Eggers, H. E., 260, 269 Ehrlich, P., 104, 117, 164 Ehrstrom, M. C., 518, 519, 641 Eidson, M., 337 Eisa, E. A., 466, 498 Eisen, M. J., 216, 230 Eisman, P. C., 529, 644 Eitelman, E. S., 510, 520, 524, 525, 6.64 Ekert, B., 293, 336 Elford, W. J., 255, 267 Ellerbrook, L. D., 517, 518, 519, 523, 524, 525, 526, 530, 641, 647
AUTHOR INDEX
555
Elliot, D. W., 135, 162 Ellis, F. W., 533, 641 Ellis, G. H., 458, 499 Elmore, D. T., 398, 410, 411, 447 Elson, L. A., 65, 100, 358, 384, 385, 391, 483, 484, 485, 498 Elwyn, D., 319, 323, 536 Emerson, G. A., 486, 600 Emmel, V. M., 221(354), 232 Enders, J. F., 525, 641 Enenkel, H. J., 511, 647 Engel, R. W., 388, 391, 469, 478, 4.98 Engelbreth-Holm, J., 123, 164, 241, 258, 263, 267, 270, 271 Engelman, M., 319, 336 Engle, E. T., 185, 207(207), 223, 228 Entenman, C., 279, 313, 338 Erf, L. A., 310, 311, 337 Erickson, J. O., 520, 646 Eriksen, N., 519, 525, 641 E r s t , T., 536, 644 Ernstene, A. C., 641 Erwin, C. P., 505, 535, 536, 537, 646 Erxleben, H., 530, 642 Eschenbrenner, A. B., 124,168,218(324), 231, 353, 355, 356, 362, 387, 306 Euler, H. von, 314, 315, 834, 336, 530,
Finnegan, J. V., 517, 525, 641 Firminger, H. I., 69, 99 Fischer, A., 243, 244, 267 Fischer, B., 341, 591 Fischer-Wasels, B., 341, 391 Fisher, J. C., 92, 93, 100 Fisher, R. A., 461, 498 Fishler, M. C., 279, 313, S38 Fishman, W. H., 530, 641 Fisk, A. A., 207(212), 208(212), 228 Flaks, J., 200(124), 226 Fleming, D. S., 409, 448 Flon, M., 293, 336 Flory, C. M., 482, 498 Folley, S. J., 212(248, 249), 229 Fontaine, R., 527, 641 Forbes, E. B., 471, 498 Forbes, T. R., 217(315), 231 Ford, C. E., 398, 448 Foster, G. L., 278, 536 Foster, J. V., 505, 535, 537, 646 Foulds, L., 148, 149, 151, 154, 164, 217 (314), 219(329), 231, 233, 246, 249, 260, 262, 263, 267 Fox, M., 429, 446 Fraenkel, E., 235, 236, 246, 255, 256, 267 Fraenkel-Conrat, H., 367, 391, 434, 441,
Evans, C. A., 116, 141, 161 Evans, H. M., 185,194(76,77), 198(105), 203(165, 166), 206(105, 198, 199), 223, 225, 227, 228 Evans, R., 66, 95, 99 Everett, J. L., 414, 419, 425, 443, 446,
Francis, G. E., 398, 405, 407, 409, 410, 428, 437, 442, 446, 447 Frankel, S., 77, 78, 82, 96, 100 Franklin, H. C., 64, 95, 99 Frants, I. D., 305, 338, 378, 396 Frantz, M., 121, 131, 164, 167, 188(43), 198(108, 109, 110), 199(110), 224, 226 Frederiksen, O., 258, 267 French, C. E., 471, 498 Freund, E., 530, 641 Fridericia, L. S., 478, 498 Friedberg, F., 301, 337 Friedman, 0. M., 330, 337 Friedmann, B., 284, 337 Friedmann, R., 535, 641 Friedwald, W. F., 247, 256, 267, 464, 498, 526, 641 Fruton, J. A., 398, 402, 407, 415, 416, 417,418, 448 Fruton, J. S., 400, 401, 402, 407, 412, 413, 437, 448, 449
639, 641
47,448
Everett, M. R., 505, 535, 536, 537, 646 Ewing, J., 117, 164
F Falk, H. L., 31, 66 Farber, E., 306, 336 Fekete, E., 105, 106, 112, 113, 115, 120, 121, 127,130, 131, 132, 133, 164, 172, 198(111, 112, 113, 114), 199, 214 (281), 226, 226, 250 Felsher, R., 524,646 Ferguson, J. H., 533, 6.44 Fevold, H. L., 207(206), 268 Findlay, G. M., 239, 263, 267 Finkelstein, H., 257, 267
448,449
556
AUTHOR INDEX
Fry, H. J. B.,526, 641 Fuchs, H.J., 531, 641 Fujiwara, T.,372,391,392,394 Fukuoka, F., 515,646 Fuller, R.H.,108, 164 Fulton, J. K.,534, 641 Furet, 8. S.,319,320,396 Furtado Dias, M. T., 195(94), 212(93, 94), 996 Furth, J., 106, 135, 147, 164, 166, 186, 190(58, 59, 60), 191, 193(67, 68, 69, 70), 194(67, 69),203(160), 204(185), !205(185), 206(58, 185), 884, 887, 260, 262, 263, 267, 268, 457, 482, 498,600
Furth, 0. B.,190(58), 191, 206(58), 224 Fuson, R.C.,413,448 G
Gaarenstroom, J. H., 189(54), 284 Gaines, J. A., 191, 192(65), 884 Gallico, E.,378, 391 Galloway, J., 259, 268 Gens, P.,189(53), 197, 294 Gant, J. C., 505, 519,646 Gard, S., 248, 249, 267 Gardner, R. F., 249,250,268 Gardner, W.U., 119, 120, 121, 123, 127, 128,131,135,148,155,169,163,164, 174(1, ll), 179(11), 180(11, 15, 16, 19), 185(33), 186, 187, 188(33), 190 (55), 191(62), 192(62, 63), 194(81), 195, 196(91, 97, 98),197(102a, 103), 198(104), 199, 201, 202(150, 1551, 204(87, 174, 175, 176), 205, 206(63, 192), 207(176), 208(214), 209(229, 230), 210, 211(229, 232, 233, 234), 213(260, 265, 266), 214(279, 280, 282), 215(15), 216(309), 218(149), 219(282), 221(348, 352, 354), 223, 224, 886, 226, 358,380,396 Gasson, E. J., 444,4.4.ff Gay, O., 185(34), 186(34), 223 Gedigk, P.,506, 518, 519,647 Geiser, R. C., 996 Geisse, N. C., 487,49G Geist, 8.H., 191, 192(65), 224 Gellhorn, A.,487,600 George, C.,533,644
Geren, B. B., 70, 99 German, W.M.,526,643 Gersh, I., 536, 641 Geschickter, C. F., 215(295), 216, 230, 527,528, 646 Gessler, A. E.,139, 158,164, 166 Giese, J. E.,343,347,349, 356, 391,477, 498 Gilbert, C.,381,391,490, 498 Gilbert, L. A., 419,447 Gillespie, H.B., 319,336 Gillespie, J. M., 510, 511, 512, 521, 640 Gilligan, D.R.,505, 517, 525, 641 Gillman, J., 381, 391, 490,498 Gillman, T.,381,991,490,498 Ginzton, L. C.,210,229 Ginaton, L.L.,123,169 Giroud, A.,73, 100 Gjessing, E. C., 417,419,447,448 Glass, G. B. J., 505,516, 525,641, 648 Glick, D., 132, 164, 249, 269, 505, 523, 534, 648, 643 Glucksmann, A,, 88, 100 Godfried, E.G., 522,648 Gogek, C.J., 281,336 Goiffon, R.,522,648 Goldacre, R. J., 442,448 Goldberg, R.C., 203(161, 162),887 Goldblatt, H.,348, 391 Golden, F., 510, 523,528, 644 Golden, J. R., 185, 283 Goldhaber, G., 262, 269 Goldner, M.S.,529,640 Goldschmidt, S.,524, 642 Goldsmith, Y.,146, 163 Goldstein, A.,518, 648 Goltz, H.L., 110, 171 Golub, 0.J., 252, 871 Golumbic, C., 398, 412, 413, 415, 418, 437,444 449 Good, R. A., 523,534,642,643 Good, 'f. A., 132, 164 Goodall, K.,377, 39W Gorbman, A.,202(159),887 Gorer, P.A., 115,135,145,164,166,167, 220(341), 231 Gottlieb, H.,519,648 Gottschalk, R.G., 246,260,262,263,264, 867
557
AUTHOR INDEX
Grady, H. G., 120, 128, 135, 170, 195, 204(87), 205, 226, 379, 390 Graff, A. M., 319, 336 Graff, M. M., 523, 642 Graff, S., 133, 136, 138, 140, 166,319,336 Graffi, A., 67, 100 Gram, H. C., 505, 524, 525, 642 Grassmann, W., 511, 642 Gray, S. J., 525, 642 Green, C. V., 120, 164 Green, R. G., 106, 116, 132, 136, 141, 142, 144, 145, 146, 156, 161, 166, 171 Green, J. W., 483, 485, 498 Greenberg, D. M., 298, 301, 302, 306, 307, 336, 336, 337, 364, 396, 515, 646 Greene, H. S. N., 201(133, 134, 135), 207(211, 212, 133, 134)) 208(211, 212), 211(211), 213(135, 264), 296, 228, 629, 239, 270 Greene, R. R., 174(3), 185(37), 186(37), 223, 924 Greenfield, R. E., 515, 642 Greenspan, E. M., 221(355), 232, 523, 642 Greenstein, J. P., 78, 87, 100, 108, 166, 170,275,336,340,341,378,391,452, 498, 515, 642 Greenwald, A. E., 519, 525, 646 Greenwood, H. H., 8, 27, 28, 30, 31, 35, 40, 41, 42, 48, 49, 64, 66 Greep, R. O., 189(51), 207(206), 224, 228 Gregory, J. E., 234, 867 Greig, J. R., 150, 156, 166 Greulich, W. W., 219(332), 231 Greville, G. D., 442, 447 Grey, C. E., 139, 164 1 Griffin, A. C., 290, 336, 302, 307, 317, 336,347, 348, 349, 360, 368, 369, 373, 376, 377, 378,391, 396, 474, 477, 498 Grob, D., 533, 642 Grollman, A., 518, 642 Gross, J., 214(289), 230 Gross, L., 135, 156, 158, 166, 526, 640, 642 Grubgeld, L. E., 527, 643 Gruenstein, M., 284, 337 Grunwald, E., 433, 448 Gruschow, J., 318, 336 Gruskin, B., 505, 526, 527, 642
Gudjonsson, S., 478, 498 Guerin, M., 201(137, 138), 226,245, 251, 255, 261, 262, 263, 269, Cuerin, P., 201(137, 138), 226, 251, 261, 269 Guest, M. M., 533, 642 Guggenheim, K., 474, 498 Gulland, J. M., 398, 410, 411, 447 Gumbreck, L. G., 189(48), 224 Curd, F. R. N., 510, 511, 512, 521, 640, 644 Gurin, S., 417, 448 Guthneck, B. T., 377, 396 Gutman, A. B., 504, 505, 506, 507, 510, 528, 529, 642, 646 Gutmann, H. R., 288, 336 Gye, W. E., 106, 123, 136, 147, 163, 166, 247, 256, 258, 267 Gyorgy, P., 348, 349, 391, 392
H Haagensen, C. D., 106,112,114,133,136, 138, 140, 142, 166, 207(207), 998 Haagen-Smit, A. J., 307, 334 Haaland, M., 117, 127, 166 Haddow, A., 50, 66, 65, 100, 157, 166, 243, 264, 268, 280, 336, 353, 354, 399, 399, 421, 436, 442, 444, 445, 448, 484, 485, 498 Hagopian, F., 120, 160 Hahn, A., 505, 522, 642 Hahn, L., 312, 336 Hahn, P. F., 275, 336 Hakanson, E. Y., 505, 534,64Z Hall, G. G., 52, 66 Hall, G. H., 527, 642 Halter, C. R., 346, 352, 353, 355, 358, 362, 377, 387, 392,396,474,'477, 499 Halvorson, H. O., 146, 166, 252, 266 Ham, A., 116, 141, 160 Ham, T. H., 524, 64.2 Hamburger, C., 208(219, n o ) , 228 Hamilton, J. B., 178(14), 923 Hammett, F. S., 158, 166 Hammett, L. P., 433, 44.8 Hamperl, H., 345, 346, 390 Hanby, W. E., 411, 412, 413, 448 Hane, S., 290, 336, 360, 368, 369, 396 Hanke, M. E., 524, 642
558
AUTHOR INDEX
Hankwitz, R. F., 525, 647 Harde, E., 213(268), 629 Harman, J. W., 345, 346, 394 Harris, J., 411, 412, 447 Harris, M., 526, 646 Harris, P. N., 347,348,392, 474,478,439 Harris, R. J. C., 238, 242, 249, 252, 254, 255, 257,259, 260, 866, 268 Harris, R. J., 349, 353, 354, 382, 392 Harris, T. N., 534, 646 Hart, R. W., 529, 640 Hartley, G. S., 411, 412, 413, 448 Hartwell, J. L., 4, 66, 341, 343, 352, 353, 379,384, 392 Hasirnoto, H., 372, 393 Haslewood, G. A. D., 380, 391 Hatschek, R., 530, 647 Hausmann, R., 530, 647 Havel, J., 245, 255, 269 Hawk, B. O., 517, 525,641 Hawkins, J. A., 256, 268 Heath, N. S., 433, 447 Heep, W., 341, 392 Heidelberger, C., 274, 276, 279, 280, 281, 283, 284, 285,280,287, 288, 289, 290, 291, 294,296, 300,302, 319,320, 333, 334, 336,336, 337, 364, 389, 392, 393, 396 Heidelberger, M., 513, 646 Heilman, F. R., 141, 161, 166, 207(200), 228
Heiman, J., 215(304), 210(311), 230, 231 Heinrich, M., 318, 336 Heller, C. G., 189(56), 624 Hellwig, C. A., 139, 166 Helmer, 0. M., 250, 259, 668, 269 Hendry, J. A., 436, 442, 4 9 Henle, G., 248, 871 Henle, W., 248, 250, 253, 266, 271 Henry, R. J., 517, 519, 523, 525, 64.9 Henshaw, P. S., 206(190), 627 Herbert, F. K., 529, 6-42? Herken, H., 530, 642 Herriott, R. M., 258, 666, 398, 408, 409,
w
Hertz, R., 174(10), 223 Hem, E. L., 538, 639 Heston, W. E., 105, 100, 107, 108, 109, 110, 111, 113, 118, 121, 124, 129, 130, 153, 155, 157, 158, 159,163, 166, 166,
168, 171, 200(127), 213(275), 215, (291), 217(275, 291), 218(323, 324), 626, 230, 231, 457, 475, 487, 499, 600, 601 Hevesy, G., 276, 312, 313, 314, 315, 320, 334, 336 Hewett, C. L., 359, 380, 381, 382, 384, 385,390,391 Hieger, I., 380, 391 Higgins, H., 377, 392 Hilfinger, M. F., 378, 396 Hilliard, J., 505, 533, 647 Hinsberg, K., 524, 535, 642 Hirshfeld, S., 534, 642 Hirst, J., 400, 448 Hisaw, F. L., 180(17), 223, 207(206), 228 Hitchcock, C. R., 302, 334, 336 Hoagland, C. L., 255, 268 Hoan, N., 293,336 Hoch, H., 377, 392, 520, 643 Hoch-Ligetti, C., 216(312), 231, 343, 358, 377,384,385,391,392,491,499,520, 643 Hodges, C. V., 529, 530, 643 Hofer, M. J., 377, 896 Hoffman, D. C., 259, 26'8 Hoffman, F. L., 489, 499 Hoffman, R. S., 244, 267 Hoffstadt, R. E., 260, 268 Hogberg, B., 530, 639, 641 Hogeboom, G. H., 249, 668, 270 Hognew, K. R., 507, 508, 510, 523, 646 Hoilomon, J. H., 92, 93, 100 Holmes, B. E., 258, 269, 315, 336 Holmgren, N., 527, 643 Homburger, F., 506, 513, 514, 530, 641, 643 Hooker, C. W., 194(74, 79), 195, 197(83), 264, 666, 203, 270 Hopwood, F. L., 398, 409, 410, 437, 446 Horne, H. W., 158, 166 Homing, E. S., 120, 162, 201, 204(184), 205,220(337,338, 339)) 226,227, 231 Horwitt, B. N., 377, 396 Hospelhorn, V. 0.) 505, 516, 518, 519, 520,643 Hoster, H. A., 139, 166 Hoster, M. S., 139, 166 Hotchkiss, R. D., 249, 268 Howe, P. E., 506, 643
559
AUTHOR INDEX
Hoyle, L., 163, 248, 868 Huddleson, I. F., 526, 646 Htickel, E., 21, 28, 66 Huff, J. W., 323, 337 Huggins, C., 219(333), 231, 504, 516,517, 518, 519, 520, 529, 530, 643 Hughes, E. D., 420, 448, 510, 517, 519, 520, 528, 640, 643 Hummel, K. P., 117, 133, 134, 143, 150, 151, 166 Humphrey, J., 644 Humphreys, E. M., 483, 498 Hungate, R. E., 141, 166, 171 Hunter, A., 489, 499 Hunter, S. W., 302, 336 Hurlbert, R. B., 302, 325, 336, 336 Huseby, R. A., 107, 108, 110, 111, 122, 125, 128, 129, 130, 131, 137, 138, 139, 143, 160, 162, 166, 200(125), 212, 213(255), 213(276), 214 (286, 287), 217(316), 218(276), 226, 229, 230, 231, 314, 326, 334, 466, 499 Hutchinson, M. C., 511, 646 Hutchison, 0. S., 331, 336 I Iki, H., 372, 393 Imagawa, D. T., 136, 145, 146, 162, 166 Inkley, J. J., 517, 525, 641 Introzai, P., 398 Israel, H. L., 537, 643 Ivy, A. C., 527, 643 Izar, G., 526, 639
J Jablonski, C. F., 382, 392 Jackson, J., 120, 148, 160 Jacobi, H. P., 365, 379, 390, 398, 468, 469, 482, 497, 499 Jacobs, J., 23, 24, 25, 33, 64, 66 Jacques, R., 16, 66 Jager, B. V., 507, 513, 523, 643 James, W. H., 471, 498 Janeway, C. A., 526, 640 Janota, M., 526, 639 Jean, M., 16, 20, 66 Jeener, R., 250, 268 Jefferies, M. E., 185(37), 186(37), 224 Jeffrey, G. A., 29, 65
Jenrette, W. V., 515, 642 Jensen, E. V., 505, 516, 517, 518, 519, 520, 643 Jensen, K. A,, 445, 448 Jobling, J. W., 235, 250, 256, 268, 270 Johnson, B. A., 235, 250, 270 Johnson, J. M., 482, 601, 530, 644 Johnson, M. J., 530, 640 Johnson, P., 136, 138, 139, 157, 169 Johnson, R. O., 457, 469, 473, 483, 500 Jones, E. E., 120, 166, 215(302), 230 Jones, F., 515, 639 Jones, H. B., 281, 284, 285, 301, 315, 336, SS6 Jones, R. N., 44, 66, 281, 336 Jordan, D. O., 398, 410, 411, 447 Jorgensen, H., 208(220), 228 Judd, T., 487, 499 Jungck, E. C., 189(56), 224
K Kabat, E. A., 147,166,260,263,668,507, 510,513,525, 528,642, 643,646, 647 Kadesch, R. G., 448 Kahler, H., 132, 136, 137, 141, 168, 166 Kahn, H., 505, 514, 524, 526, 642, 643 Kahnt, F. W., 510, 511, 512, 521, 640 Kamen, M. D., 75,100,157, 170,276,336 Kaminer, G., 530, 641, 543 Kaplan, H., 198(104), 226 Kaplan, H. S., 123, 167, 191(62, 62a), 192(62), 206(191, 193), 207(203), 984, 227, 228 Kaplan, M. H., 533, 643 Karnofsky, D. A., 508, 646 Kay, H. D., 505, 529, 643 Keighley, G., 307, 334 Kekwick, R. A., 528, 643 Keller, E. B., 307, 336 Kellcy, L. S., 315, 336 Kelley, V. C., 523, 534, 642, 643 Kelly, K. H., 505, 535, 537, 646 Kelly, M. G., 108, 171, 457, 601 Kelsch, J. J., 139, 164 Kendall, E. C., 207(200), 928 Kennaway, E. L., 359, 380,381, 382,384, 385, 390, 391, 490, 499 Kennaway, N. M., 358, 380, 381, 382, 384, 385, 390,3091
560
AUTHOR INDEX
Kensler, C. J., 341, 346, 347, 349, 352, 353, 355, 358, 362, 363, 364, 377,378, 386, 387, 388, 392, 393, 396, 474, 477, 478, 499 Kerwin, J. F., 413, 448 Keye, W. R., 617, 533,647 Keys, A., 507, 647 Khanolkar, V. R., 108, 166,214(285) Khanolkar, V. T., 111, 128, 166 Kidd, J. G., 91, 100, 247, 248, 867, 868, 270, 464, 600, 526, 641, 643,64.6 Kiefer, L., 517, 64.3 Kielley, M., 298, 336 Kienle, R. H., 515, 641 Kilpatrick, M., 429, 430, 434, 447 Kilpatrick, Mary, 429, 430, 432, 447 King, C. J., 452, 458, 462, @9 King, J. T., 108, 121, 166, 214(288), 830 King, J. W., 239, 867 King, T.T., 108, 162 Kingsley, G. R., 507, 643 Kinosita, R., 341, 343, 344, 345, 346,352, 353, 356, 360, 362, 380, 387, 392 Kiprianov, A., 434, 448 Kirby, A. H. M., 344, 355, 356, 379, 380, 381, 382, 392 Kiriluk, L. B., 534, 643 Kirk, I., 445, 448 Kirk, M. R., 283, 336 Kirkman, H., 221(357, 358), 232 Kirschbaum, A,, 121, 123, 124, 125, 126, 128, 131, 135, 155, 168, 164, 167, 188 (43), 191(61), 198(108, 109, 110), 199(110), 200(126), 204(172, 175), 205, 206(188), 218(322), 224, 226, 226, 227, 231, 263, 270, 469, 487,499 Kirtz, M. M., 121, 168, 212, 229 Kishi, S., 372, 391, 398, 394 Kit, S., 298, 306, 307, 336,336 Kits von Waveren, E., 244, 266 Klatt, T., 516, 518, 646 Klein, M., 187, 224 Kleiner, I. S., 505, 519, 525, 644 646 Klinck, G. H., 213(261, 263), 929 Kline, B. E., 341,346,348,349,350,362, 376, 377, 379,390, 392, 393,396, 452, 457, 468, 469, 470, 471, 473, 474, 478, 479, 483, 484, 499, 600 Klotz, I. M., 518, 643 Klumpar, J., 522, 640
Knight, C. A., 249,251,253,254.261.268 Knight, R. A., 147, 167 Knorr, L., 434, 448 Knowlton, N. P., Jr., 70, 100 Knox, J. C., 478, 600 Knox, R., 260, 262, 267, $68 Kocher, R. A,, 482, 499 Kock, A. M., 438, 449 K(lg1, F., 304, 336 Kohman, T. P., 311, 336 Koller, P. C., 483, 484, 485, 498 Kon, G. A. R., 353, 354, 392, 399, 442, 443, 444, 448 Koneff, A. A., 203(165, 166), 206(199), 297, 228 Koomen, J., 248, 270 Kopac, M. J., 419, 4.48 Kopacaewski, W., 505, 528, 643 Kortweg, R., 105, 106, 107, 112, 118, 121, 122, 127, 129,166,166, 167, 212, 214 (283, 284), 829, 930,244, 266 Kraemer, Dorothy Ziegler, 71, 100 Krahl, M. E., 347,348, 398, 474, 499 Krakower, C., 207(207), 228 Krejci, L. E., 526, 643 Kremen, A. J., 302, 334, 336, 534, 643 Krimsky, I., 250, 268, 269 Krueckel, B. J., 328, 337 Krysa, H. F., 66, 100 Kiipfmuller, K., 343, 391 Kuhn, R., 357, 387, 393 Kung, S. K., 65, 73, 74, 92, 100 Kunkel, H. G., 524,643 Kupke, D. W., 317, 336 Kupske, 0. W., 376, 391 Kurzrok, R., 209(2!26), 2.28 Kuttelwascher, H., 526, 646 Kynette, A., 141, l7f L
Lacassagne, A., 38, 64, 105, 122, 123, 167, 174(4, 5), 204(173), 205, 210, 213 (267, 269), 215, 216(305, 306, 307, 308), 223, 227, 830, 259, 868, 328, 336 La Due, J. S., 517, 641 Lafay, B., 240,269 Lafaye, J., 364, 396 Laird, A. K., 376, 394
AUTHOR INDEX
561
Lamerton, L. F., 483, 484, 485, 498 Levinson, S. A., 527, 643 Landau, J. L., 526, 643 Lewis,J. H., 533, 6.44 Landsteiner, K., 261, 269 Lewis, M. R., 249, 250, 257, 268, 399, 436, Lane, C. E., 196(95), 226 448 Langemann, H., 378, 388, 392, 393 Li, C. H., 194(76,77), 198(105),203(166), Langer, E., 345, 393, 535, 644 206(105, 198, 199), 826, 227, 228 Langham, W. H., 331, 337 Li, M. H., 186, 187, 191, 192, 193(71), Lansing, A. I., 74, 75, 100 197(102a), 198(104, 105), 224 Lardy, H. A., 364, 394 Liang, Hsu-mu, 64, 73, 95, 100 Larizza, P., 522, 644 Lichtenstein, H. J., 430, 448 Larsen, C. D., 457, 475, 499 Lick, L., 191, 224 Laser, H., 243, 244, 267 Liebler, J. B., 529, 640 Lasnitzki, I., 136, 169 Lindegren, C., 157, 170 Lathrop, A. E. C., 105, 119, 120, 167 Lindegren, G., 157, 170 Lauffer, M. A., 253, 266 Linsteed, R. P., 401, 402, 403, 404, 405, Lavin, G. I., 255, 268, 288, 336, 376, 394 407, 446 Lavik, P. S., 468, 469, 470, 479, ,499 Lippincott, S. W., 519, 525, 526, 530, Law, L. W., 104, 106, 120, 132, 135, 145, 641, 647 166, 167, 205(189), 206(189, 195), Lipschtitz, A., 174(6), 185(34), 186(34), 227, 344, 379, 380, 393 208(6, 217), 223, 228 Lawrason, F. D., 123, 167, 469, 499 Lischer, C. E., 65, 73, 96, 100 Lawrence, J. H., 310, 311, 337 Little, C. C., 104, 105, 106, 109, 111, 112, Lawrence, J. S., 528, 639 113, 115, 117, 118, 120, 130, 131, 132, Lawson, W. E., 406, 448 133, 134, 143, 148, 150, 151, 162, 164, 166, 167,169, 171, 172, 198(111, 112, Leblond, C. P., 68, 73, 100 Lecocq, J., 38, 47, 64 113, 114, 116, 117), 199(118, 119, Leese, A., 478, 600 122), 226, 226 Lefavre, H., 123, 167 Little, M. S., 517, 519, 523, 525, 642 Le Fevre, R. G., 490, 600 Little, P. A,, 240, 268, 269, 486, 499 Lehman, I., 523, 642 Littlefield, W., 517, 643 Lehmann-Facius, H., 526, 644 Liu, C. H., 510, 511, 512, 521, 640 Lehninger, A. L., 299, 337, 505, 530, 646 Llombart, A., 243, 268 Leidler, H. V., 194(75), 226 Loeb, L., 105, 117, 119, 120, 121, 122, Leiner, G., 536, 644 167, 168, 174(7), 210, 212(251), 213 (270, 273, 274), 214(251, 278), 223, Leiter, J., 326, 337 Le Maistre, J. W., 433, 448 229, 230 Lennard-Jones, J. E., 21, 24, 52, 66 Loeser, A. A., 215(303), 230 Lennette, E. H., 261, 268 Loewenthal, H., 258, 268 Leonard, A., 517, 646 Loftfield, R. B., 297, 305, 307, 338, 378, 396 Le Page, G. A., 280, 300, 302, 319, 320, 336,336, 337, 378, 396 Long, M. L., 204(177, 178), 205, 227, 481, Leseur, A., 157, 170 497 Long, S., 530, 646 Leslie, I., 309, 310, 336 Levaditi, C., 259, 268 Longsworth, L. G., 508, 510, 521, 528, Lever, W. F., 510, 511, 512, 521,640, 644 536, 644 Levi, A. A., 288, 336 Longuet-Higgins, H. C., 23, 24, 25, 26, Levillah, W. D., 106, 126, 153, 155, 166, 27, 40, 46, 47, 64, 66 218(323), 231 Loomis, E. C., 533, 644 Levina, M., 535, 640 Lord, D. D., 400, 448 Levine, M., 244, 256, 268 Lorens, E., 124, 168
562
AUTHOR INDEX
Lorenz, E., 218(324), 231, 255, 266 Lorenz, L., 533, 534, 541 Lotthammer, R.,524, 544 Loveless, A., 398, 399, 437, 442, 445, 446,
w
Lowenhaupt, E., 343, 393 Lowenstein, B. E., 519, 525, 646 Lowy, P. H., 307, 334 Luck, J. M., 302, 307, 336, 373, 376, 377, 378, 991 Luce-Clausen, E. M., 64, 100 Ludford, R. J., 117, 168, 243, 268 Ludwig, H., 519, 64.2 Luetscher, J. A., 507, 508, 520, 644 Luria, S. E., 246, 268 Lusbaugh, C. C., 483, 485, 498 Lustig, B., 535, 536, 644,64.5
M Ma, Chung K., 74, 100 Mabee, D., 283, 284, 936 McBee, B. J., 139, 166 McCarthy, E. F., 507, 646 McCarty, K., 139, 164 McCarty, K. S., 158, 166 McCawley, E. L., 137, 162 McCay, C. M., 458, 499 McClellan, V., 507, 510, 528, 642 McCombie, H., 407, 444, ,447, 4.48 McCord, W. M., 232 MacDonald, J. C., 343, 361, 364, 393 McEleney, W. J., 106, 107,142,169,160, 217(317), 220, 231 McEndy, D. P., 204(185), 205(185), 206(186), 2.27 McEuen, C. S., 201(144), 202, 203(144, 163), 206(197), 209(197), 216, 228 McFarlane, A. S., 513, 644 MacFarlane, R. G., 531, 533, 644 MacInnes, D. A., 608, 521, 528, 536, 644 McInnes, G. F., 517, 640 McIntosh, J., 240, 252, 255, 258, 261, 266, 268, 269, 270 MacIntyre, A., 531, 639 McKennis, H., 405, ,449 MacKenzie, I., 464, 499,526, 644 Mackenzie, R. D., 235, $69 McKewon, T., 219(334), 231 Macklin,LlM., 158, 168
McLean, I. W., 253, 256, 270 MacLeod, C. M., 531, 640 McQuarrie, I., 523, 64.3 Madden, 8. C., 514, 515, 644 Magat, M., 45, 66 Magill, J. W., 364, 392 Magnus, H. von, 156, 168 Magnus, P. von, 156, 168, 248, 249, 267 Maguigan, W. H., 384, 396 Maher, I. E., 537, 643 Mahoney, E. B., 64, 100 Maisin, J., 123, 168 Majoor, C. L. H., 507, 510, 644 Malmgren, R. A., 146, 168 Malmros, H., 528, 644 Malpress, F. H., 212(248, 249), 229 Mandelstam, J., 490, 498 Mann, I., 136, 166, 168 Mann, L. S., 526, 644 Mann, W., 318, 336 Marangos, G., 506, 514, 532, 640 Marcelet, J., 530, 645 March, N. H., 52, 66 Marcus, S.,534, 641 Marder, S. N., 207(203), 228 Mark, D. D., 373, 378, 393 Marks, H. H., 489, 498 Marle, E. R., 433, 447 Marshak, A., 311, 336 Martin, M., 38, 47, 64, 66 Martin, N. H., 507, 644 Martin, R. H., 382, 390 Marvin, H. N., 533, 647 Marx, W., 257, 267, 534, 644 Masayama, T., 372, 373, 39.9 Matthews, V. S., 221(357), 232 Matthews, W. B., 398, 448 Mattson, S., 434, 449 Maun, M. E., 216, 230,456, 468, 498 Maver, M. E., 257, 266, 482, 601, 506, 514, 530, 644 Mawson, C. A., 256, 267 Max, P. F., 65, 92, 100 Maxwell, L. C., 481, 497 Mayer, K., 518, 523, 536, 644, 647 Mayer, N., 377, 996 Mayer, R. L., 529, 644 Maynard, L. A., 458, 499 Mayneord, W. V., 380, 391 Mayot, M., 16, 66
AUTHOR INDEX
Meek, E. C., 517, 525, 641 Mehl, J. W., 508, 510, 521, 523, 528, 534, 5361 6&16&7 6471 648 Meinken, M. A., 438, 449 Meisel, D., 141, 171, 196, 197(102), 226, 329, SS7 Meister, A., 378, 393, 515, 642 Meitus, A. C., 533, 647 Melckionna, R. H., 220(336), 231 Mellanby, E., 236, 269, 468, 470, 501 Mellors, R. C., 378, S9S Meloche, V. W., 516, 518, 645 Mendelsohn, W., 250, 268 Mershimer, W. L., 519, 525, 646 Merten, R., 524, 535, 642 Meyer, F., 505, 518, 519, 647 Meyer, K., 536, 64.4 Meyer, L. M., 487, 499 Meyer, R. K., 189(48, 49, 50, 52), 224 Meyer-Heck, P., 517, 518, 644 Michaelis, L., 257, 268 Mider, G. B., 64, 90, 100, 108, 123, 124, 168, 171, 206(194), 227, 457, 474, 601,504, 508, 509,514,524, 525,644 Milford, J. J., 237, 238, 269 Millard, A., 136, 16S, 169 Miller, C . S., 323, SS7 Miller, E. C., 67, 100, 288, 290, 336, 343, 344,346,347,348,349,350, 351,353, 354, 355,356,357, 358,359,360,362, 363,364,365,366,367,369, 370, 371, 372, 373, 376, 377, 379, 380, 385,387, 388, 390, 391,393, S94, S96, 477, 478, 498,4.99, Miller, E. E., 510, 520, 524, 525, 644, 647 Miller, E. W., 106, 114, 120, 168, 210, 220, 221 (349, 350, 351), 329, 231 Miller, G. L., 510, 520, 525, 644, 647 Miller, G. M., 505, 516, 517, 519, 64.3 Miller, H., 74, 100 Miller, J. A., 290, 304, 3S6, 341, 343, 344, 345, 346, 347, 348, 349, 350, 351, 353, 354,355, 356,357,358, 359, 360,362, 363, 364, 365, 366, 367, 369, 370, 371, 372,373,376,377, 379,380,385,387, 388, 389, 390, 391, S92, S93, 394, 396, 452, 468, 471, 474, 477, @9, 600 Miller, J. K., 260, 869 Miller, 0. J., 188, 190(55), 224
563
Miller, W. L., Jr., 63, 100, 343, 350, S94, S96 Miller, Z. B., 418, 447 Millington, R. A., 295, 337 Mills, C. A., 108, 164, 168, 171 Milne, J., 507, 510, 646 Miner, D. L., 346, 347, 348, 394, 474, 477, 478, 499 Mitchell, E. B., 525, 642 Mitchell, J. H., Jr., 320, 331, 332, S34, 536, 336, 3S7 Mittelman, D., 510, 511, 512, 521, 640 Mixer, H., 191(61), 205(188), 206(188), 224, 227 Miyaji, T., 366, 996 Moen, J. K., 244, 269 Moeschlin, S., 525, 646 Mo5tt, W. E., 13, 16, 25, 66 Moloney, J. B., 255, 257, 266 Monod, J., 247, 269 Moon, H. D., 194(76, 77), 198(105), 203 (166), 206(105, 198, 199), 225, 227, 228
Moore, A. M., 409, 448 Moore, D. H., 136, 138, 140, 166, 507, 510, 528, 534, 642, 646 Moore, G. E., 302, 336 Moore, L. V., 538, 659 Moore, R. A., 220(336), 231 Moore, S., 402, 405, 407, 408, 448, 4.49 Moosey, M. M., 142, 144, 145, 166 Moreschi, C., 481, 499 Morgan, W. C., 135, 160 Mori, K., 348, 379, 394 Morrione, 378, 394 Morris, H. P., 108, 162, 163, 291, 336, 3S7, 452, 476, 477, 482, 486, 4.99 Morris, R., 507, 64.4 Morrow, A. G., 221(355), 232 Morse, A. H., 208(221), 228 Morton, D. E., 207(204), 228 Morton, J. J., 64, 90, 100, 123, 168, 206 (194), 227, 504, 508, 509, 514, 644 Moser, H., 445, 446 Moskop, M., 210, 213(270, 273, 274), 229, 2.90 Mottram, J. C., 64, 67, 99, 100, 243, 269, 464, 499 Moulder, P. V., 219(333), 231 Moulton, F. R., 174(8), 211(8), 22.3
564
AUTHOR INDEX
Mouton, R. F., 510, 511, 512, 521,640 Moyer, A. W., 252,269 Mucke, K.,524,642 Mtihlbock, O.,108, 120, 121, 133, 135, 150, 167, 168, 181(21), 182(23, 24), 218(21), 223 Mueller, G. C., 347, 358, 362, 363, 365, 366,370, 387, 393,394,477, 499 Mueller, J. H., 258, 269 Mtiller, 0.H., 518, 523, 646 Muether, R. O.,517, 525, 641 Mulinos, M. G.,465,499 Mulligan, R. M.,211(246), 929 Mulliken, R.S., 17,21, 22, 24, 27, 33, 66 Munro, L. A., 528,646 Murphy, J. B., 204(180), 205, 206(186), 207(201, 202), 927, 228, 235, 236, 237,243,250,260,261,967,269,270 Murray, J. A., 104, 117, 160,168 Murray, W.S., 105, 106, 109, 110, 111, 112,120,122,143,148,162, 167,168. 169,212, 229 Muxart, R.,293,336 Mylroie, A.,445,44.9
Noble, R. L., 216, 227 Noda, L.,376, 377, 391 Norberg, E.,302,336, 515, 646 Northrup, J. M.,398, 409,448 Norris, T.A., 141, 171 Novak, F. V., 522, 640 Novak, J., 535, 536,646 Novikoff, A. B.,378,394 Nye, W.,290, 336 Nye, W.N., 376,377,391 0
Oberling, C., 201(137, 138), 226, 245,251, 255, 261, 262, 263, 969 Ogston, A. G.,399, 401, 402, 403, 404, 405,408,415, 439, 449 Ok46, A.,406, 449 Olcott, H. A., 441,449 Olcott, H.S., 367,391 Oleson, J. J., 240, 868, 269, 486,@9 Olson, R.E.,294, 296, 336, 378, 394 Omachi, A.,249, 969 Oncley, J. L., 510, 511,512,513, 517,519, 528,640,646 N Opie, E.L.,344, 345, 348, 349, 376, 304, 468, 478,600 Nagao, N., 352, 356, 357, 380, 394 Orgel, L. E.,23, 66 Nakahara, W.,255, 271, 372, 391, 399, Orr, J. W.,70, 100, 123, 124, 125, 126, 394,515, 646 162, 163, 169, 218(325, 327), 221 Nassau, E.,535, 536,644 (349),231, 343,344, 345, 346, 394 Nathanson, I. T., 215(301), 230 Ostergard, R. P.,349,392 Nebbia, G.,8, 16, 27, 33, 34,66 Oswald, W.,526, 646 Needham, D.M., 399,408, 441,442,447, Ott, M. L.,534,641 Ottesen, J., 312, 313,336 4& Nelson, W. O., 189(56), 202, 209(224, Overholser, M. D.,207(205), 928 226), 215(152), 216, 2B4, 226,228 Nero, W. J., 135,162 P Neukom, H.,409, 449 Neurath, H.,253, 269,520, 646 Pack, G . T., 174(9), 211(9), 219(9),223, Newton, B. L., 207(211, 212), 208(211, 490, 600,514,515,639 212), 211(211), 213(264), 228, 229, Paesi, F. J. A., 189(54), 924 533,640 Page, O.,506, 513, 514,643 Newton, M. A., 323,332, 337 Palade, G. E.,531, 646 Niasi, S.,535, 538, 646 Paletta, F. X., 65, 71, 72, 73, 92, 95,96, Nickerson, M., 507, 513, 523,643 99, 100 Nielson, P. E.,66,95,99 Pan, S. C., 208(214), 210, 211(232, 234), Nibson, I., 535, 646 928,929 Ninni, M., 398 Pardee, A. B.,294, 296, 336 Nitsche, G . A., 513, 646 Parfentjev, I. A., 525, 646
565
AUTHOR INDEX
Parker, F., 259, 268 Parker, R. C., 244, 269 Parkes, H. S., 190, 224 Parkinson, M., 139, 164 Passey, R. D., 135, 136, 138, 139, 150, 157, 163, 169, 478, 600 Pading, L., 6, 7, 8, 10, 13, 15, 23, 29, 66 Payne, A. H., 316, 336 Payne, L. D., 347, 348, 391 Peacock, P. R., 246, 269, 355, 356, 381, 382, 392 Pearce, L., 520, 646 Pearsall, H. R., 508, 528, 529, 641, 646 Pearson, B., 378, 394 Pearsons, J., 120, 167 Peckham, B. M., 185, 186(37), 224 Pedersen, K. O., 513, 646 Penn, H. S., 505, 527, 642, 646 Penney, G. W., 20, 25, 66 Pennington, D., 141, 171 Pentimalli, F., 235, 236, 269 Perkins, M. S., 283, 336 Perlmutter, M., 534, 646 Perloff, W. H., 209(226), 228 Perlzweig, W. A., 474, 600, 527, 528, 646 Perry, I. H., 123, 269, 210, 229 Petermann, M. L., 507, 508,510,523,646 Peters, R. A., 409, 442, 449 Peyron, A., 240, 269 Pfaff, M. L., 505, 535, 536, 537, 646 Pfeiffer, C. A., 178(12, 13), 180(19), 188, 194(74, 79, 83), 195, 197(102a, 83), 204(87), 207(208), 208(222, 223, 12), 221(348, 354), 223, 224, 828 Pfeiffer, P. H., 643 Philips, F. S., 398, 408, 436, 445, 446, 447, 449 Pickels, E. G., 245, 255, 266 Pierson, H., 209(227), 228 Pikovski, M., 239, 262, 269 Pillemer, L., 511, 646 Pirani, c. L., 536, 646 Pirie, A., 250, 258, 259, 269, 409, 449 Pirie, N. W., 253, 269 Plant, G. W. E., 364, 394 Player, M. A., 505, 516,618,519,520,643 Plescia, A. M., 290, 336,364, 393 Podloucky, F. H., 530, 640 Poling, E. C., 348, 3991
Pollack, A. D., 191, 192(65), 224 Pollack, M. A., 377, 394, 395 Pollak, 0. J., 517, 646 Pollard, A., 252, 255, 256, 257, 258, 259, 260, 869 Pomerantz, L., 465, 499 Ponder, E., 517, 646 Popjak, G., 507, 646 Pople, J. A., 52, 65, 66 Porter, K. R., 140, 169, 245, 255, 266 Potter, V. R., 78, 100, 157, 169, 249, 270, 294, 296, 299, 302, 325, 333, 336, 336, 377, 378, 387, 390, 394 Poumeau-Delille, G., 240, 269 Powell, E. O., 411, 412, 413, 448 Prato, M., 8, 33, 34, 66 Preer, J. T., 157, 169, 170 Prelog, V., 416, 4 4 Pressman, D., 275, 336 Preston, J. M., 437, 449 Price, C. C., 400, 449 Price, D. E., 344, 394 Price, J. M., 345, 346, 370, 371, 372, 373, 376, 377, 390, 392, 394, 396 Prickett, C. O., 263, 266 Prhzmetal, M., 523, 646 Pullinger, B. D., 70, 71, 89, 100, 121, 128, 169 Pullinger, B. O., 219(328), 231 Pullman, A., 3, 6, 7, 13, 14, 15, 16, 18, 28, 30, 31, 32, 33, 34, 35, 36, 37, 41, 42, 43, 44, 47, 64, 66, 66, 279, 336 Pullman, B., 7, 16, 33, 42, 43, 44, 66, 279, 336 Purdy, W. J., 147,166, 256,258,262, 266, 267, 268 Purr, A., 517, 646 Pybus, F. C., 106,114,120,168,210,220, 221(349, 350, 351), 229, 231
Q Quadbeck, G., 357, 393 Quimby, E. H., 275, 336 Quinlin, P. M., 438, 443
R Rachele, J. R., 409, 449 Racker, E., 250, 268, 269
566
AUTHOR INDEX
Ragnotti, R., 236, 269 Ranadive, K. J., 111, 128,166, 214(285), 830 Randall, H. T.,106, 112, 114, 133, 136, 138, 140, 142, 166 Rapaport, I. A., 399, 445,&9 Rapaport, S. I., 633,647 Rask-Nielsen, R., 123, 164,206(196),228 Rathery, F.,535,640 Ray, F. E.,293,336 Raynaud, A., 216(307,308), 830 Reding, R., 522, 646 Reed, R., 136, 138, 139, 167,169 Reid, E.E.,406,448 Reid, J. C.,274,276,279, 301, 328, 334, S36 Reiner, L.,482,498 Rciner, M., 507,646 Reinhard, M.C.,110,171 Rekers, P. E.,514, 6S9 Reller, H.C.,68,69,70, 95,99, 100 Revell, S.,398,399,448 Rhees, M. C., 526,647 Rhoads, C.P.,288, 336, 342, 346, 347, 352,353,355,358,359,360, 362,363, 377,384,386,387,398,396, 399,436, 447,474, 477,478,499,514,639 Richardson, H. L., 346,346,351, 394 Richert, D.A., 378, 396 Richmond, V.,505, 536,536, 537, 646 Richter, M. N., 139,164 Riddle, O.,180(18),223 Riegel, C.,537,646 Rieke, C.A., 17,27,66 Rieke, H.S., 280, 291,336 Riley, V. T., 141,162,240, 241,242, 251, 259, 266, 269,270 Rimington, C.,523, 535, 536, 646,646 Ria, H., 373, 393 Ritchey, M. G.,76, 100, 101 Rittenberg, D.,278,301, 338,337 Rivers, T.M., 255,868 Rizzone, G. P.,643 Roberts, E., 76,77, 78, 81, 82, 96, 100 Roberta, J. J., 443,444,&8 Robertson, W.van B., 487, 600 Robinson, A. M., 518, 64.6 Robinson, D.,534, 646 Robinson, W.D.,534,641
Robson, J. M., 195, 197(86), 226 Roche, J., 530, 646 Roe, E. M. F.,65,100, 353, 354,392 Roll, P. M.,319,S34,966 Rolnick, H.A., 139,166 Rose, F. L.,436,442,443,445, 449 Rosenbohm, A.,644 Rosenfeld, L.,524, 646 Rosenthal, O., 297, SS6 Rosenthal, T.B.,74,75, 100 Roskelley, R. C., 377, 396 Ross, M.H.,526, 64.8 Ross, S.D.,433,447 Ross, W.C.J., 399, 400, 405, 409, 413, 414,419,421,422,425,426,427,428, 429,430,431,432,433,436,437,442, 444,445,446,447,448, 449 Rossiter, L.J., 490,498 Rothe-Meyer, A.,263,270, 971 Rothen, A.,242, 246, 252,257, 259, 266 Rothman, S.,66,76, 100, 101 Rothwell, J. T.,505, 517, 525, 641 Roue, P.,91,100,156,169,235,237,248, 260,268,870,464,481,498,499,600 Ruangsiri, C.,72 Rudali, G.,38,47,64 Rudall, K.M., 65,100 Rumsfeld, H.W.,Jr., 63,100, 343, 396 Rundles, R. W.,528,646 Rupp, J. J., 204(177, 1781, 205, 827 Rusch, H.P.,108,162,280,304, 311, 334, 336,341,343,346,347,348,349,350, 351,362,376,377,379, 390,393,394, 3g6,452,457,459,466,468,469,470, 471,473,474,477,478,479,482,483, 497,498, 499,600,516, 518, 646 Rush, B. F.,188(44), 224 Russ, S.,259, 270 Russel, B. R. G., 117,169 Russel, M.,517, 646 Rutenberg, A. M.,330,397 Ryder, A., 533,644 Rydon, H.N.,411,412,413, .bbs Rygaard, J., 206(192), 227 S
Sack, T., 326,337 Sakami, W.,364, 396 Salmon, W.D.,388, 391,478,498
AUTHOR INDEX
Salter, W. T., 377, 696 Salzberg, D. A., 290, 866, 360, 368, 369, S96 Sampath, A., 240, 368 Samuels, L. T., 131, 169, 218(319), 2Sl Sanders, E., 526, 646 Sandin, R. B., 354, 388, ,9995 Sandorfy, C., 16, 47, 66 Sannie, C., 201(138), 226 Saphir, O., 526, 659 Sapp, R. W., 344,347,355,357,358,360, 366, 367, 369, 370, 379, 380, 385, 387, s9s Sarett, H. P., 474, 600 Saaaki, T., 341, 379, 596 Sauberlich, H. E., 377, S96 Savignac, R. J., 505, 519, 646 Saxton, J. A., Jr., 201(134), 202(153, 1541, 207(134), 226, 456, 457, 482, 498, 600 Sayers, G., 466, 600 Scatchard, G., 513, 518, 646, 646 Schaible, P. J., 526, 646 Schenken, J. R., 220, 221(347), 2S1, $32 Schenken, L. R., 121, 122, 168 Schiff, A., 95, 99 Schiller, W., 382, 596 Schmid, K., 510, 511, 512, 521, 523, 536, 640, 6/14, 546 Schmidt, G. W., 235, 236, 267 Schmidt, H. W., 523, 646 Schmidt, I. G., 208(218), 228 Schmidt, M. B., 341, 396 Schmidt, O., 4, 5, 6, 49, 51, 66 Schmitt, L. H., 533, 647 Schneider, W. C., 78, 100, 249, 270, 296, 336, 367, 373, S96 Schoenbach, E. B., 505, 508, 516, 517, 520, 523, 528, 642,646, 647 Schoenewaldt, E. F., 329, 3S6, 357 Schoenheimer, R., 276, 336 Schoental, R., 280, 334, 410,447,491,498 Schultz, E. L., 511, 521, 644 Schuster, M. C., 139, 164 Schwarta, S., 214(289), 2SO Schwartz, T. B., 507, 513, 643 Schweigert, B. S., 377, 396 Scott, G. M., 259, 270 Scudder, J., 521, 536, 646 Seegers, W. H., 533, 643
567
Segaloff, A., 201(147, 148), 202, 216, 220 (335), 221(147), 226, 2Sl Seibert, F. B., 504, 505, 508, 521, 535, 536, 537, 646 Seibert, M. V., 504, 505, 508, 521, 535, 536, 537, 646 Selbie, F. R., 252, 255, 258, 261, 969, 370 Seligman, A. M., 326, 327, 330, 337 Sells, M. T., 257, 266 Selye, H., 201(144), 202, 203(144, 163), 2261 227 Sevringhaus, E. L., 185, 201(131), 22S, 226
Seymour, R. B., 433,448 Shacter, B., 534, 646 Shapiro, D. M., 487, 600 Shapiro, J. R., 200(126), 226 Sharp, D. G., 251,253,254,255,256,257, 258, 263, 266, 260, 370,271, 520,646 Sharpless, G. R., 252, 269 Shaw-MacKenzie, J. A., 505, 526, 646 Shay, H., 284, 3S7 Shear, M. J., 326, SSY, 341, 379, 380, 396, 480,600 Shedlovsky, T., 508, 521, 528, 536, 644, 646 Shemin, D., 250, 254, 270, 301, SS7 Sherman, J., 13, 16, 66 Shetlar, C. L., 505, 535, 536, 537, 648 Shetlar, M. R., 505, 535, 536, 537, 646 Shimkin, M. B., 105, 106, 107, 108, 110, 119, 120, 121, 122, 123, 128, 132, 134, 135, 136, 142, 148, 150,160,162, 169, 170, 182(22), 195, 204(87, 183),205, W S , 226, 227, 284, 3S4, 534, 646 Shinowara, G. Y., 524, 646 Shope, R. E., 247, 270 Shrigley, E. W., 236, 239, 267, 270 Shubik, P., 93, 100, 464, 497 Sibley, J. A., 505, 530, 646 Siedentopf, H. A., 106, 132, 136, 171 Siegel, I., 364, 396 Siekevita, P., 307, 337, 364, 396 Sievers, O., 524, 646 Silberberg, M., 122, 170, 183(26, 27), 194 (75), 204(181, 182), 205, 212, 215 (299), 223, 226, 227, 230 Silberberg, R., 122, 170, 183(26, 27), 194 (75), 204(181, 182), 205, 212, 215 (299), 223, 226,227, 2SO
568
AUTHOR INDEX
Silverstone, H., 108, 170, 171, 360, 396, 457,459,462,463,464,465,468,469, 471,472,473,474,480,482,483,484, 600 Silvertsen, I., 108, 171 Simkin, B.,523, 646 Simpson, L.,323, 332,337 Simpson, M. E., 185, 194(76, 77), 198 (105), 203(165, 166), 206(105, 198, 199), 223,626,227, 228 Simpson, W.L., 67,73, 74,96,99,100 Sittenfield, M.J., 235,250, 970 Sivertsen, I., 457,601 Sizer, I. W.,505,519,646 Skarzynski, B.,530,641 Skegga, H.R.,323,337 Skipper, H.E.,320, 331, 332, 334, 336, 336,337 Sklar, A. L., 14,66 Skorodumov, V. A.,436,447 Slaughter, D.P.,490,498 Sloviter, H.A.,327, 337 Slye, M.,104, 170 Smadel, J. E.,255, 268 Small, G.,433,447 Smiljanic, A. N.,66,76, 100, 101 Smith, B. W.,505,533,647 Smith, C.A. H., 458,499 Smith, E.L.,507,513,521,536,643,646 Smith, F., 110, 122, 129, 130, 162,166 Smith, F.W.,120,128,129,130,170,185 (33), 188(33), 198(104, 115), 199, 283,226,286 Smith, G. M., 123, 127, 164, 171, 201 (136), 210, 214(280), 218(326), 220 (340),926,229, 231, 358, 380, 396 Smith, K. A., 410,419,445,447 Smith, L.,434, 449 Smith, W.,66,95,99, 156, 170 Smith, W.B.,524, 646 Smith, W.E., 248,270 Smyth, I. M., 505,510,521,522,523,536, 648 Snell, G. D.,104, 170 Snider, H.,141, 166 Sobel, H.,186, 193(68, 70), 194(69), 8.34 Sobotka, H.,506, 514,646 Sonne, J. C.,318,334 Sonneborn, T.M.,157, 170 Sorof, S.,371,396
Spaey, J., 522,642 Spangler, J. M.,347, 396, 478,498 Speakman, J. B., 435,44.9 Speer, F.D., 525,640 Sperling, G.,458, 499 Spiegel, A., 198(106), B26 Spiegelman, S., 157, 170 Spirtes, M.A.,299, 337 Spitz, S.,384, 396 Sprinson, D.B.,319, 323, 336 Sproul, E.E., 250, 254, 256,268, 270 Stacey, M.,536, 646 Stadie, W.C.,519, 534, 646 Stahmann, M.A.,398,414,418,437,448, 449 Stanley, W.M.,136, 138, 140, 166,253, 270 Stare, F. J., 294,336 State, D.,535, 538,646 Staube, A. M., 536,646 Steele, J. M.,297, 307, 338,378, 396 Stein, W.H.,398, 400,401, 402,405,407, 408, 415,416,417,418,437,448,449 Steiner, P. E.,31,66 Stephan, V., 416, 449 Stephenson, M. L., 297, 305, 307, 338, 378,396 Stern, K., 135, 162, 452, 476, 480, 486, 600, 506,514, 526, 531, 532, 646 Stern, K.G., 250,252,255, 263,270,507,
646
Stettner, M.M., 519, 525, 646 Stevens, C. D.,405, 409, 411, 438, 441, 445, 447,449 Stevens, S., 256,268,270 Stevenson, E.S.,359,360, 363,396 Stickland, L. H.,136, 140, 163 Stier, A. R.,351, 394 Stock, C.C.,399, 436,445,446,447, 449 Stoerck, H.C.,486,600 Stoeaz, P.A., 300,337 Storey, W.F.,68, 100 Stout, A. P.,196, 197(102), 926 Stowell, E.C., 526, 647 Stowell, R.E.,63, 06,72,90,95,99, 100, 101, 376,396 Strait, L.A., 137, 161 Straw, A. A., 526,639 Strong, A.,123, 167 Strong, F. M.,377, 891
569
AUTHOR INDEX
Strong, L. C., 91, 101,104,115,118,123, Taylor, A. R., 251, 253, 254, 255, 256, 257, 258,269,270, 271 125,126,127, 135,148,164,167,170, 171, 196(98), 201(136), 204(175), Taylor, D.R.,141, 171 205, 210, 211(233), 213(265), 214 Taylor, H.C.,188(45), 224 (279,280), 218(321, 326), 220(340), Taylor, H.C.,Jr., 127,171 226,226,227,228,$29,231, 358,380, Taylor, H.F. W., 398,410,411,447 Taylor, H.L.,507, 646 396 Taylor, J., 377,394 Strong, L. E., 510, 517, 519, 528, 640 Taylor, J. C.,214(277), 230 Stulberg, C. R., 252, 266 Sturm, E.,204(180), 205, 207(201, 202) Tempereau, C.E., 533,647 227,228,235,250,259, 261, 269,270 Tenenbaum, E.,244, 267 Teresi, J. D.,302,307, 336,378, 391 SubbaRow, Y.,240, 268,486, 499 Sugiura, K.,237,241,250,258,270,319, Tesluk, H.,514, 6.44 323,334,341,342,343,346,347,350,Teutschlaender, O.,255, 271 352,353,355,357,358, 362,364,377, Thacker, J., 141, 171,471,498 378,382,387,392,393, 396, 436,443, Thiersch, J. B.,436,445,446, Thoma, G.E., 517,525, 641 474,477,478,481, 498,499,600 Sullivan, B. H.,517,6.43 Thomas, F.,121, 167,214(283), 230 Suntzeff, V., 62,63,66, 74,75,76,77,81, Thomas, M.A., 257, 266 83, 84, 92, 93, 94, 95, 96, 99, 1 , Thompson, C. R.,196(99, loo), 226 101,121,122,168,210,213(270, 273, Thompson, F.I,.,524,640 Thompson, H.C.,Jr., 70,99 274), 229, 230 Surgenor, D.M., 510, 511, 512, 521,640 Thompson, H.P.,140, 169 Sutton, L. E.,23, 66 Thompson, J. W.,482, 483, 601, 530, Svartholm, N. V.,6, 9, 16,66 644 Sveusson, H.,507, 510, 535, 536, 640, Thompson, R. C., 141, 166,377,394 Thornton, H.,526,647 643 Swain, C. G., 401,433,447 Timmis, G.M.,65, 100,399, 437,4.49 Sweeney, L.,526,643 Tinozsi, E. P.,524,647 Sweet, B.,326,337 Tiselius, A., 507,510,611,525, 535, 536, Swift, E.F., 471,498 640,647 Swift, R. W., 471, 498 Tishkoff, G. H.,82, 96,100 Sylven, B.,534, 642 Toennies, G.,504, 647 Symeonidis, A.,216(310), 230 Tolbert, B. M.,274, 276, 279, 328, 334, 337 Syverton, J. T., 145, 146, 166, 248,270 Tomarelli, R., 349,392 T Toosy, M.H.,66,97, 101 Totter, J. R.,319, 337 Tagnon, H.J., 531,646 Tracy, M.M.,312, 319,334 Taki, I., 366,396 Traub, E.,152, 171 Tannenbaum, A., 108,170, 171,452,457, Trebing, J., 532, 6.40 458,459,460,461,462,463, 464,465, Treffers, H.P., 513, 647 468,469,470,471,472,473,480,481,Trentin, J. J., 108, 121, 171, 181(20), 182(20), 183(25), 214(288a), 218(20), 482,483,484,488,600 Tapley, D. F., 516,643 223, 230 Tamer, H.,369, 396 Tripi, H.B., 260, 268 Tatum, E.L.,76, 100, 101 Troiser, J., 271 Tauber, H.,533, 647 Tropp, C., 518,647 Taylor, A,, 117, 141, 166, 171, 377, 394, Tung, T. C.,378, 396 396, 479,600 Turba, F.,511, 647
a?
570
AUTHOR INDEX
Turner, C. W., 108, 171, 212(247), 214, (288a), 2.99, 230 Turner, F. C., 326, 337 Turner, R. A,, 290, 334, 364, 390 Tuttle, L. W., 310, 311, 337 Twigg, G. H., 430, 431, 448, 44.9. Twombly, G. H., 123, 141, 171, 174(9), 188(45), 196, 197(102), 211(9), 223, 224, 926, 329, 336,337 Tyner, E. P., 302, 320, 937 Tytler, W. H., 235, 270 Tyzzer, E. E., 115, 167
W
Wagner, J. C., 252, d71 Wakefield, L. D., 400, 449 Wakelin, R. W., 409, 4.49 Walaszek, E., 333, 334 Waldron, D. M., 535, 647 Waldschmidt-Leitz, E., 517, 518, 530,647 Waldvogel, M. J., 533, 647 Walker, T. T., 259, 268 Wallace, E. W., 108, 171 Wallace, H., 108, 171 Walple, A. L., 436, 442, 449 Waltma D, C. A., 127, 171 U Wdtman, I#&"., 214(277), 230 Waitor, A. . R., 524, 647 Uhl, E., 263, 271 Waihrg, O., 293, 337, 505, 530, 647 Ullyot, G. E., 413, 448 Ward, R., 490, 601 Ungar, G., 533, 647 Ware, A. G., 533, 642 Unna, K., 347,390 Warner, S. G., 110, 111, 143, 169, 171 Uphoff, D., 218(324), 231 Warner, W., 104, 160 Uroma, E., 510, 511, 512, 521, 640, 644 Warren, F. L., 358, 384, 391 Warren, S., 505, 517, 525, 641 V Waser, P., 378, 396 N., 452, 601, 531, 647 Waterman, Van Artsdalen, E. R., 411,412,447 Watson, A. F., 468, 470, 601 van der Scheer, J., 252, 266 Watson, C. J., 132, 135, 16.9 Van Dyke, J. H., 70, 99 Waymouth, C., 244, $71, 373, 391 Van Eck, G., 192(64), 294 Wayne, A., 530, 641 van Gulik, P. J., 106, 107, 112, 121, 127, Weaver, J. C., 328, 336,337 166, 167, 214(284), 230 Weber, G. M., 344, 360, 366, 367, 369, van Thoai, N., 530, 646 370,371,372,373,376,377,393,394 van Wagenen, G., 194(78, 80), 208(221), Webster, M. B., 537, 643 219(331), 226, 228,231 Weed, L. L., 325, 337 van Wagtendonk, W. J., 157, 170 Wegelin, C., 489, 601 van Winkle, Q., 139, 166 Weigert, F., 49, 64, 67, 99, 100 Vasquea-Lopez, E., 203(164), 221(164), Weimer, H. E., 510,621, 536, 646, 647 227 Weinhouse, S., 284, 295, 298, 299, 387 Vassel, B., 515, 641 Weinstein, L., 209(230), 928 Vimtrup, B., 478, 498 Weisbrod, F. G., 537, 647 Viollier, G., 377, 378, 388, 396 Weisburger, E. K.,291, 336, 337 Visscher, M. B., 106, 108, 110, 128, 129, Weisburger, J. H., 291, 336, 337 132,136,160,162,166,167,171, 214 Weiss, E., 526, 647 (287, 288), 230, 457, 458, 462, 466, Weiss, 8.M., 281, 284, 286, 288,336, 389, 497, 499, 601 392 Voegtlin, C.,482, 483, 499,601 Weissman, N., 505, 508, 516, 517, 520, Volkin, E., 319, 337 528, 646, 647 Voorhees, V., 259, 266 Weitkamp, A. W., 75, 76, 101 Vroelant, C., 16, 20, 47, 66, 66 Welker, W. H., 526,644 Vyeki, E., 333,334 Wells, B. B., 633,647
57 1
AUTHOR INDEX Wells, E. B., 248, 270 Weltman, O., 528, 647 Werner, C. E., 295, 299,337 Werner, H. W., 196(99, loo), 226 Werner, I., 537, 647 West, P. M., 341, 377, 396, 505, 517, 533, 634, 641, 642, 647 West, R., 505, 535, 647 Westerfeld, W. W., 378, 396 Westergaard, M., 445, 448 Westman, A., 196(96), 226 Westphal, V., 505, 518, 519, 647 Wicks, L. F., 63, 75, 76, 96, 100,101 Wiener, M., 248, 871 Wiest, W. G., 285,287, 288, 289,55, 389, 396 Wiggins, L. F., 431, 435, 449 Wilheim, R., 452, 476, 480, 486, 600,506, 514, 526, 531, 532, 646 Williams, A. H., 444, 448 Williams-Aahman, H. G., 299, 337 Williams, R. J., 377, 394, 396, 479, 600 Williams, W. L., 123, 124, 125, 126, 128, 131, 167, i r i , i98(108), 204(176), 205, 208(215), 218(321, 322), 226, 227, 228, 231 Willie, R . A., 126, 171 Wilson, D. W., 318, 325, 336, 337 Wilson, J. G., 178(14), 223 Wilson, R. H., 354, 396 Winder, W. R., 70, 100 Winfield, K., 252, 269 Winnick, T., 301, 302, 306, 336, 337 Winstein, S., 433, 448 Winzler, R. J., 476, 486, 498, 505, 510, 513, 517, 519,521, 522,523, 525, 532, 534, 536, 642, 644, 646, 647, 648 Witschi, E., 185(32), 223 Wharton, D. R. A,, 525, 647 Wharton, M. L., 525, 647 Wheland, G. W., 6, 13, 15, 23, 24, 28, 66 Whipple, G. H., 514, 64.6 White, C. C., 257, 266 White, F. R., 108, 171, 348, 353,355, 356, 362,387, 396,457, 462, 474, 475, 483, 601 White, J., 108, 124, 171, 343, 345, 346, 348, 353, 355, 356, 362, 379, 387, 3.90, 391, 396, 457, 462, 474, 475,601, 515, 642
White, L., Jr., 331, 332, 334, 337 White, M. R., 316, 336 Whitney, R., 534, 641 Woglom, W. H., 208(213), 228,341, 377, 396 Wolbach, S. B., 65, 101 Wolf, C. G. L., 532, 648 Wolf, G., 287, 337 Wolfe, J. M., 201(1.32, 139, 140, 141, 142), 202, 208(216), 213(140, 261, 262, 263), 226, 828, 229 Wolff, E., 505, 522, 648 Wolfson, W. Q., 507, 640 WOU, E., 240, 2ri Wollman, E., 247, 269 Wolstenholme, J. T . , 193(72), 194(72), ,924 Wood, D. J. C., 431, 44.9 Wood, J. L., 288, 336, 409, 411, 441, 447,
449
Wood, M. T., 257, 266 Woodard, H. Q.; 378, 396, 529, 640 Woodhouse, D. L., 506, 514, 532, 535, 647, 648 Woods, A., 377, 394 Woods, M. W., 157, 163, 171, 172 Woodward, F. N., 444, 448 Woolley, G. W., 106, 115, 120, 130, 131, 132, 164, 172, 198(111, 112, 113, 114, 116, 117), 199(118, 119, 122), 200 (1231, 201(130), 226, 226 Wormall, A., 398, 405, 407, 409, 410, 428, 437, 442, 4461 447 Woywood, E., 185(34), 186(34), 223 Wright, A. W., 201(132, 139, 140, 142), 202, 213(140, 262, 263), 227 Wright, L. D., 323, 337 Wright, W. M., 532, 648 Wyckoff, R. W. G., 258, 266, 271 Wyman, R. S., 120, 122, 134, 170, 204 ( l a ) , 205, 227
Y Yankwich, P. E., 274,276,279, 334 Yaoi, H., 255, 271 Yates, F., 461, 498 Yokoyamrt, T., 372, 373, 393 Yoshida, T., 341, 379, 380, 396, 396 Young, G., 346, 371, 378, 390 Young, L. E., 533, 6-40
572
AUTHOR INDEX
Young, N. F., 346, 377, 392, 474, 477, 499, 507, 510, 514, 643, 646 Young, W. C., 178(14), 2?23 Yvan, P., 47, 66
z
Zamecnik, P. C., 297, 301, 305, 307, 309, 387, 888,378, 396
Zerahn, K., 315, 334 Ziegler, D. M., 71, 101 Zilversmit, D. B., 279, 313, 838 Zirkle, C. L., 413, 448 Zondek, B., 185, 201(143), 202, 223, 226, H7 Zuckerman, S., 219(334), 131 Zweifach, B. W., 93, 99
Subject Index A AAT, see under o-Aminoazotoluene AB, see under 4Aminoazobenzene Abwehrferment, 532 Acetate, labeled, metabolism, 294-296 2-Acetylaminofluorene, carcinogenic activity, 291 of 3-methyldiaminoazobenzene and, 351 mammary cancer in mice and, 124 metabolism, 291 Adrenals, effect on lymphomagenic action of estrogens, 207 of X-rays, 207 intermitotic time in, 70 ovary and, 188, 190 tumors, experimental, 198-200 caloric intake and, 458 gonadotropins and, 199 hormonal effects of, 199 strain differences in, 198, 199 types of, 198 Adrenocorticotropic hormone, caloric restriction and formation of, 466 gonadal hormones and, 199 tumorigenic activity of, 200 Adrenocorticotropin, see under Adrenocorticotropic hormone Alanine, incorporation into proteins of normal and tumor tissue, 305, 306,308 enzymes and, 307 reaction with aliphatic nitrogen mustards, 415,416 Albumin, egg, reaction of epoxides with crystalline, 434-435 plasma, effect of cancer on, 504, 509, 514,524
Albumin A, of plasma, cancer and, 524 Aldolase, serum, neoplastic disease and, 530 Alginic acids, reactions of epoxides with, 435 4-Alkyl-l-phenylpiperazines, formation, 416 Alkylating agents, see also under names of individual compounds cytotoxic, biological activity, 439ff. structure and, 442, 443, 444, 445, 446 chemistry, 397-449 reaction with nucleophilic groups in biological systems, 439ff. radiomimetic, 444 Amines, reaction of mustard gas with, 406 Amino acids, see also under names of individual compounds, carcinogenic aminoazo dyes and, 348, 377 epidermal, 82 effect of methylcholanthrene on, 81 of squamous cell carcinoma on, 82 incorporation into proteins, 309 labeled, incorporation into tumor proteins, 301-309 in vitro, 304-309 in vivo, 301-304, 307 reaction of alkyl-2-chloroethylamines with, 416,417 of mustards with, 407-408 D-Amino acids, in tumors, 304 4Aminoazobenzene, structure, 342 Aminoaao dyes, carcinogenic, see also under names of special dyes, 339-390 activity, 353, 354,355 amino acids and, 348 chemical structure and, 352-359,370
673
574
SUBJECT INDEX
dietary effects on, 346-351 mechanism, 371, 383-390 speaiea differences, 376 vitamins, 346-348 effecton chemical composition of liver, 371-378 liver tumors induced by, chemical composition of, 371-378 protein synthesis in, 378 o-Aminoazotoluene, carcinogenic activity, of, and its derivatives, 341, 379-380 sex differences in, 379 structure, 342 2-Aminofluorene, carcinogenic activity, 293 Amino groups, nucleophilic character, 427, 434 Ammonia, preformed, in epidermis, 78 Androgens, effect on carcinogenic action of estrogens, 215-216, 217, 219 on lymphagenic action of x-rays, 206 Aniline, carcinogenic activity, 353 Anthracene, carcinogenic activity, 37 K-region of, 37 Anticarcinogenesis, 2 Apyrase, epidermal, activity, effect of skin cancer on, 77 Arginase, epidermal, 78 effect of carcinogens on, 79, 81 of skin cancer on, 77, 81 role in urea formation in mammals, 78 Ar yl-2-chloroalk ylamines, reaction with anions, 425-427 Ascorbic acid, deficiency, effect on guinea pig sarcoma, 487 Atoms, meso, 5, 6, 30 %Azaguamine, tumor-inhibition by, 323, 487 uptake of labeled by nucleic acids, 323 Azo dyes, carcinogenic, see also under Amimoazo dyes and under names of individual dyes activity, diet and, 491 labeled, 290-293
2,2’-Azonaphthalene, carcinogenic activity of, and derivntives, 380-381 possible mechanism, 385 Azulene, ionic structures, 16
B Bases, reaction of I ,2-epoxides with, 434 of mustards with, 406-408, 416-418, 427-429 Bence-Jones protein, in multiple myeloma, 528 1 ,a-Benzacridine, K-region of, and its derivatives, 37, 39 carcinogenic activity and, 37, 39, 41 3,PBenzacridine, K-region of, and of derivatives, 4, 37 carcinogenic activity and, 37, 41 1,2-Benzanthracene, carcinogenic activity of, and its derivatives, 37, 42 K-region and, 37, 42, 43, 44 structure and, 40 K-region of, 3, 4, 5, 6, 32, 37 meso atoms of, 6, 30 molecular diagram, 30, 35 Benzene, I bond orders in, 15 molecular diagram, 17, 26 electron patterns, IOff. 3,4Benephenanthrene, carcinogenic activity of, and its derivatives, 31 K-region and, 36, 37, 43 K-region of, 3, 32 Benzpyrene, labeled, carcinogenic activity, 284 rate of elimination, 284-286 metabolism, 288 3,4-Benspyrene, carcinogenic activity, 67 caloric intake and, 463 labeled, 280 metabolism, 281, 282 Biological systems, nucleophilic groups in, 439-440 reaction of cytotoxic alkylatingagents with, 439ff.
SUBJECT INDEX
Biotin, azo dye-induced liver tumors and, 478 Bladder, tumors, fl-naphthalimine and, 175 Blood, adsorption of b u s No. 1 sarcoma agent on elements of, 236 Body weight, tumorigenesis and, 464 Boneb), neoplastic response to sex hormones, 22 1 tumors, alkaline serum phosphatase and, 530 Bradosol (fl-phenoxyethyl-dimethyldodecyl ammonium bromide), 529 Butadiene, molecular diagram, 26, 34 1,1Butanediol, dimethanesulfonyl ester, 437 Butter yellow, liver tumors produced by, 175 Butyl 2-chloroethylsulfide, reaction with tobacco mosaic virus, 441 C
Calcium, in normal and precancerous hyperplastic epidermis, 93 Cancer, see also under Tumors breast, see under Cancer, mammary effect on plasma globulins, 520, 524, 525-528 on serum protein-bound carbohydrate, 535-538 epidermal, 58 gastric cancer and, 58-61 gastric, epidermal cancer and, 58-61 hypoalbuminemia in, 514 mammary, in laboratory animals, 211, 212 hormonal imbalance and, 215 size of glands and, 215 in man, milk agent and, 158 in mice, 2-acetylaminofluorene and, 124 age and, 154 classification, 104 differentiation, 108 effect of pituitary on, 120
575
environmental factors and, 108 genetic factors and, 106, 108, 109ff., 119 hereditary factors and, 104, 105, 129 histology of, 125, 154 hormonal factors and, 106, 106, 108, 119, 129, 149 methylcholanthrene induced, 123 milk agent and, 103-172 effect of genetic factors on transmission of, 113 estrogens and, 121ff., 126 spontaneous, 118 in man, body weight and incidence of, 488489 dietary deficiencies and, 489-491 nutritional state and, 487-497 nitrogen mustards and chemotherapy of, 397 nutrition and, 451-497 plasma proteins and, 503-548 polarographic filtrate test for, 522-523 serodiagnosis of, 504, 514, 516-529, 530, 532 methods, 516-520, 525, 526-527, 530, 531, 532 skin, activity of epidermal enzymes and, 77, 78, 81 epidermal choline content and, 76 inositol content and, 76 squamous cell, experimental, 69, 90 chemical composition, 86, 87 effect on epidermal amino acids, 82 nitrogen metabolism, 80, 81 reducible substance in, 83 virus origin, 240 Carbohydrates, protein-bound of serum, distribution, 535 effect of cancer on, 535-538 nature of, 535-536 origin, 536-537 requirement for mitosis, 466 Carbonium ion, 412, 425, 426 affinity of iodide ion for, 427 formation, 411 reactions, 411, 415, 420, 422
576
SUBJECT INDEX
Carcinogenesis, see also under Tumorigenesis chemical, 280 mechanisms of, 384 effect on chemical composition of tissues, 87 electronic configuration and, 1-54 epidermal, 57-101 choline and, 86 hair follicle cycle and, 64-65 heredity and, 66 human, 66, 93-97 as compared with that in mice, 94-97
sunlight and, 94 latent period in, 89-90 in mice, 62-69 application of carcinogen, 63-64, 66 estrogen and, 65 factors influencing, 62-63, 64-65 nature of, 91, 97 stages in, 66-69, 91-93 mitotic activity of tissue and, 466 nutrition and, 495 radioisotopes in the study of, 273334
stages in, 66-09, 91-93, 464 vitamins and, 476-480 Carcinogens, see also under Hydrocarbons, carcinogenic and under names of individual compounds activity, 67 diet and, 461-462, 469-470, 485 factors influencing, 92 mechanism, 317 molecular size and, 2 structure and, 351 effect on chemical composition of epidermis, 72, 74-78 on cytoplasmic ribonucleic acid, 72 on dermis, 74 on epidermal alkaline phosphatase, 72
cells, 71, 72, 87-89, 90, 91, 92, 97 enzymes, 76-78 lipids, 76-76 minerals, 74-76 on hair follicles, 73 on sebaceous glands, 73
milk agent and, 123 responses to, 175 specificity, 175 transport through skin, 67, 68 Carcinolysis, 530-531 Carcinoma, see under Canwr Casein, tumorigenesis and dietary, 348, 472473
Catalase, cancer and activity of, in liver, 515 Catecholase, inhibition by serum, 534 Cell(s), divisibn, effect of ethyleneimine and its derivatives on, 436 epidermal, carcinogenic activity and, 92-93
number of, 70-71 spinous, effect of carcinogens on, 90, 91, 92, 97
tissue fluid environment of, 92 malignant, 244 conversion of normal into, 247 latent, 91 Rous No. 1 sarcoma agent and, 243-250
metabolism, effect of b u s No. 1 sarcoma agent on, 234, 246-250 virus-infected, metabolites of, 24 white, dendritic of human skin, pigment formation and, 66 Chick embryo agent, stability, 258 Chick embryo components, 253 chemical composition, 254 Chloroalkylamines, effect of structure on hydrolysis of, 424 2-Chloroethylamine (s), 411-429 biological activity of, and related compounds, 445 reaction with hexamethylenetetramine compounds, 418 in water, 411 N-2-Chloroethyl-N-3-chloropropylaniline, biological activity, 444 2-Chloroethyl sulfides, mutagenic action of, and related compounds, 445
SUBJECT INDNX
reaction mechanism in aqueous media, 4376. Choline, epidermal carcinogenesis and, 86 liver tumors and, 388, 478-479 Chromosomes, effect of mustard compounds on vege tal, 445 Chrysene, carcinogenic activity, 37 K-region of, 32, 37 Chymotrypsin inhibitor, of serum, neoplastic disease and, 533 Cocarcinogenesis, quantum-mechanical base of, 2 Colchicine, effect on incorporation of Pa* into DNA of tumors, 314 inhibition of Shope virus by, 240 labeled, metabolism in normal and tumor-bearing animals, 335 Configuration, carcinogenesis and electronic, 1-54 Cysteine, reaction of di-2-chloroethyl sulfide with, 405-406 Cystine, reaction with carcinogenic hydrocarbons, 288 Cytochrome oxidase, activity of epidermal, in cancer of skin and, 77 in precancerous hyperplastic epidermis, 77 Cytochromes, in tumors, 294 Cytoplaam, effect of carcinogenic hydrocarbons on, 289
D DAB, see under 4Dimethylaminoazobenzene DNA, see under Desoxyribose nucleic acid DN, see under Diphosphopyridine nucleotide Dehydrogenases, in transplantable tumors, 299 Dermis, effect of carcinogens on, 74
577
Desoxypentosenucleic acid, in liver following ingestion of carcinogenic aminoazo dyes, 372,373,374, 375-376 in viruses, 255 Desoxypyridoxine, 8-azaguanine and, 487 Desoxyribose nucleic acid, see also under Thymonucleic acid, 309 biosynthesis, 320 incorporation of Pa9 into, 312 colchicine and, 312,314,315, 316 radiation and, 31G315 tumors and, 312, 314, 315, 316 reaction of carcinogenic hydrocarbons with, 289 thymonucleic acid and, 312 1,2-5,6-Dianhydro-3, &acetone-mannitol, effect on wool fiber, 435 Dibenzanthracene, labeled, carcinogenic activity, 284, 288 metabolites, 283-284 rate of elimination, 284-286 lymphomagenic action, 206 1,2-Dibenzanthracene, K-region of, 286 metabolites, 286-288 1,2,5,6-Dibenzanthracene,33 K-region of, 3, 32 labeled, 280 metabolism, 281, 282, 283 1,2,7,8-Dibeneanthracenel33 K-region of, 32 1,2,3,4-Dibenaphenanthrenel carcinogenicity, 31 Di-2-chloroethylarylamines1 hydrolysis, 420ff. structure and rate of, 422, 423 tumor growth inhibitors, 421 NN-Di-2-chloroethyl-p-anisidine, cross linking in wool produced by, 429 Di-2-chloroethyl sulfide, 397, 399 reaction with amines, 406-407 with amino acids, 407 with anions, 402-406 with cysteine, 405-406 with proteins, 408 in water, 400, 401, 402 1,2,3,4Diepoxy-2-methylbutanebiological activity, 444
578
SUBJECT INDEX
Diet, liver cancer in humans and, 351 Diethyl-&iodoethylamine, labeled, metabolism in tumor-bearing mice, 330 Diethylstilbestrol, labeled, metabolism, 329 Di-2-halogenoalkylamines1 hydrolysis, structure and, 424 Di-2-hydroxyethylarylamines1 derivatives, tumor growth inhibition by, 437 4-Dimethylaminoarobeneene, administration, histological changes following, 343 pancreas and spleen tumors following, 344-346 carcinogenic activity, 341, 342-351 of derivatives, 343, 357-358 sex and species differences in, 343 of metabolites, 358-369 riboflavin and, 477, 478 thyroid and, 350-351 effect on chemical composition of normal and tumorous liver, 371378 on liver choline oxidase, 388 fluoro derivatives, carcinogenic activity, 385-386 labeled, metabolism, 290 metabolism by rats, 369-371 N-demethylation, 363-366 formation of protein-bound dyes, 366-371 hydroxylation of aniline ring, 363 pathway of, 361 reduction of azo linkage, 360, 362363 structure, 342 2,eDimet hoxy-6-et hyleneimino-l,3,6triarine, biological activity, 444 tumor growth inhibition by, 444 2',3-Dimethy1-~aminoaeobenrene1 see under o-Aminoarotoluene 9,I0-Dimethy1anthracenel3 3,l0-Dimethyl-6,6-benzacridinel 293 Dimethylbenzanthracene, auxinocarchogens, 46, 47 carcinogenophore, 46, 47
9,10-Dimethy1-1,2-dibenranthracene136 carcinogenic activity, 37 K-region of, 37 Dimethyl-2-chloroethylamine, reaction in water, 414 N-(2,4)-Dinitrophenyl-ethyleneimine tumor growth-inhibiting activity, 445 Diphenylamine reaction, 538 Diphosphopyridine nucleotide, effect on oxidative tumor metabolism, 299 Divinyl sulfone, radiomimetic effects, 437 Drugs, action, quantum-mechanical base of, 2 Dyes, see also under AZOdyes, Aminoazo dyes and under names of individual compounds protein-bound, 388, 389 formation of, 366-371 tumors induced by, 340ff. classification, 344-346 metastases, 345 origin, 345
E Electrons, methods of representing, in conjugated compounds, see under Valence bond method and under Molecular-orbital method II Electrons, 6-7, 8, 9, 20,21ff., 52ff. action of, 6 aromatic character of molecules and, 9-13 K-region and, 6, 9 nature of, 20 in pyridine, 19 Svartholm's model of, 6, 7 u Electrons, 8, 9, 52 Enryme inhibitors, in plasma, cancer and, 532-534 specificity of, 633 Enzymes, see also under names of individual enzymes effect of carcinogens on epidermal, 76-78 of mustards on sulfhydryl groups of, 441
SUBJECT INDEX
579
of tumors on, 515 with proteins, 434-435 of virus infection on cellular, 249-250 in water, 429-432 inactivation of Rous No. 1 sarcoma kinetics of, 430, 431 agent by, 258 mechanism of, 430-431, 438 in liver, effect of carcinogenic aminoazo Estradiol benzoate, effect on carcinodyes on, 377-378 genic activity, 92 oxidative, incorporation of labeled Estrogens, see also under names of indialanine into protein and, 307 vidual compounds inhibition by serum of cancer pacervical cancer in mice and, 176 tients, 534 effect on lymphomagenic action of location of, 298, 307 X-rays, 206 in tumors, 299, 306 on thymus, 204 plasma, neoplastic disease and, 529-532 on uterine cervix, 207 Epidermis, extrinsic, mammary tumorigenesis in calcium in normal and cancerous, 86, rodents and, 214ff., 218 87 inactivation by hepatic tissue, 185, 188 cancer, 58 incidence of liver tumors in mice and, gastric cancer and, 58-61 221 cells of, lymphoid tumors in rodents following growth, 70ff ., 58-59 treatment with, 204-205, 206 intermitotic time in, 70 adrenals and, 207 mitotic time in, 70 see differences in, 206 nuclei of, 71-72 strain differences in, 204 carcinogens and, 71, 72 mammary cancer in mice and, 121 number of, 70-71 milk agent and, 121, 123, 126 carcinogens and, 87-89 mammary response in mice to, 214ff., volume of, 71 218 chemical composition, 85-87, 97 pituitary tumors in rodents following effect of carcinogens on, 74-78 treatment with, 201, 202, 203 effect of carcinogens on nitrogen testicular response in mice to, 194, 195 metabolism, 78-82 strain differences in, 194, 196-197 experimental carcinogenesis in, 57-101 tumorigenic activity, 177, 207, 211, histochemistry of, 72-73 214ff. injuries to, 59, 60 androgens and, 215-216, 2 17, 219 carcinogenetic effect of, 59 progesterone and, 202, 204, 216-217 precancerous hyperplastic, 69 uterine response in rodents to, 208,209, biotin content, 76 210 calcium content, 93 vaginal response in mice to, 181-182 characteristics of, 89-90 strain differences in, 182, 183 cytochrome oxidase activity, 77 Estrone, mineral content of, 73 mammary response in mice to, 182 thymonucleic acid content, 72 strain differences in, 182-183 reducible substance in, 82, 83 Ethyl-di-2-chloroethylamine, replacement, 68 reactions with amino acids, 416, 417, urea content of, 78 418 1,2-Epoxides, with anions, 415, 416 cytotoxic action, 433, 436 with peptides, 416 reactions with anions, 432-434 Ethyleneimine, 436 with bases, 434 biological activity, 444, 445 with nucleic acid, 435-436 effect on cell division, 436
580
SUBJECT INDEX
on growth of animal tumors, 436 reaction in aqueous media, 438
F 4’-F-DAB, see under 4’-Fluoro-Pdimethylaminoazobenzene FSH, see under Follicle-stimulating hormone Fat, dietary, effect on tumorigenic activity of DAB and its derivatives, 469 growth of tumors and, 487 tumorigenesis and, 467-472 Fibrinogen, cancer and, 524-525 liver and synthesis of, 524 Fibroblasts, lymphoblasts and, 244 Roux No. 1 sarcoma agent and, 243, 244 transformation into macrophages, 244
4’-Fluoro-4dimethylaminoazobenzene, carcinogenic action, 343 effect on liver, 345 Folic acid, antagonists, effect on mouse leukemia, 48 Rous No. 1 sarcoma and, 240-241, 486 Follicle-stimulating hormone, ovarian tumors and, 189 testicular tumors and, 196, 197 Food dyes, commercial, carcinogenic activity, 380ff. Formate, labeled, incorporation into nucleic acid purines, 322, 323 Fowl leucoses, transmissible, 262-264 Rous No. 1 sarcoma and, 264 Fowl leucosis agent, isolation, 263 stability, 258 Fowl tumors, chemically-induced, 261-262 immunological properties, 262 Rous No. 1 sarcoma and, 261 Fuchs’ test, for cancer, 531-532 Fujinami tumor agent, enzymatic inactivation, 259
G
Gelatinases, bacterial, inhibitor of, in serum, 534 Glands, mammary, see under Mammary glands sebaceous, 73 effect of carcinogens on, 73 entry of carcinogens through, 68 a-2 Globulin, serum, effect of wasting diseases on, 536 a-Globulins, cancer and, 504, 508, 509,520-521 multiple myeloma and, 527-528 &Globulins, neoplastic disease and, 524, 527-528 y-Globulins, cancer and, 525-528 Glucose, labeled, metabolism, 295, 296, 297 D-Glutamic acid, labeled, metabolism in normal and tumorbearing animals, 304 Glycidol, biological activity, 445 Glycine, labeled, incorporation into proteins, 302-303,304,305,306 reaction of mustards with, 407 Glycol sulfonyl esters, reactions in aqueous media, 437 Glycolysis, in tumors, 294, 318 Gonadotropins, secretion of, age and, 188 ovary and, 185, 187, 188, 189 tumorigenic activity, 177, 194 Growth hormone, nucleic acids and, 326 tumorigenic action, 194, 203 Guanazolo, 323
H HN2, see under Methyldi-2-chloroethylamine HNs, see under Tri-2-chloroethylamine Hair follicles, cycle, 64-65 epidermal carcinogenesis and, 65 effect of carcinogens on, 73
58 1
SWJECT INDEX
Hexamethylenetetramine, reaction with chloroethylamines, 418 Hormonal imbalances, 178 cancer and, 178 contributing factors, 177 experimental, 178ff. lactation and, 212 reversibility of, 178, 179 types of, 178-180 ‘IHormonal influence, inherited,” mammary cancer in mice and, 129 Hormones, see also under names of individual hormones activity, quantum-mechanical base of, 2 caloric intake and production of, 465, 466 carcinogenic, 174, 176, 211, 222, 304 nature of, 176, 177 neoplastic response to, 175-176, 179ff. duration of stimulus and, 178 sex differences in, 183, 184 species differences in, 180 time factor and, 179 endogenous, hyperplasia induced by, 179 experimental tumorigenesia and, 173232 gonadal, adrenocorticotropin and, 199 gonadotropic, see under Gonadotropins growth, Bee under Growth hormone mammary, response to, 211, 212, 219 species differences in, 212 metabolites, carcinogenic activity, 176 sex, neoplastic response of bones to, 221 tumors of secondary male sex organs and, 219-220 Hyaluronidase, inhibitors of, in serum, 534 Hydrocarbons, carcinogenic, see also under names of individual compounds activity, 37 dietary cystine and, 288 fat and, 469 K-region and, 6, 30, 31-32, 33-40, 43-50, 52 of metabolites, 288 possible mechanisms, 50-5‘4
structure and, 3, 17, 36, 41, 46 cervical cancer in mice following application of, 211 effect on formation of protein-bound dyes in rat liver, 369, 370 on tumorigenic activity of 3‘methyldiaminoazobenzene, 351 interaction with tissue components, 288-289 K-region of, 3-8, 30-50, 279, 280 mammary cancer and, 123, 218 metabolism, 279-290 oxidation in vivo and in vitro, 49 synthesis of labeled, 280 Hyperconjugation, 17-18, 27-28 Hyperplasia, adrenal in mice, milk agent and, 130 endogenous hormones and, 179 Hypoalbuminemia, in cancer patients, 508, 514, 515
I Influenza virus, 251 composition, 248 host and, 263, 254 immunologicalproperties, 248,249,261 infective component, 253-254 metabolites, 248 Iodine deficiency, incidence of thyroid cancer and, 490 Irradiation, carcinogenic effects of, 275 by compounds localized in tumors, 327-328 inactivation of Rous No. 1 sarcoma agent by, 259 Isotopes, see also under names of individual compounds radioactive, methodology, 276-279 production and measurement, 276 in the study of carcinogenesia, 273334 of immunology of cancer, 276
K K-region, of carcinogenic hydrocarbons, bond orders in, 31-32 carcinogenic action and, 3-8,36,37,38, 39, 53, 279, 280
582
SUBJECT INDEX
charge at, 33-40 definition, 279 electrical properties, 6, 30-46 electronic distribution in and near, 8 free valences, 33 indexes, electrical, 30-31 molecular-orbital, 20-21 “Kappa ” factor, of paramecia, comparison with milk agent, 157 Ketosteroids, fecal excretion by mice with mammary tumor virus, 218 Kidneys, effect of sex on, of rodents, 221 Krebs cycle, conversion of carbohydrate to protein and, 307 localization of enzymes, 298 in tumors, 294-298, 307
L LH, see under Luteinieing hormone Lactation, hormonal imbalances and, 212 B-Lactoglobulin, reaction of epoxides with, 435 Lead-212 (thorium B), in the study of plant metabolism, 276 Leukemia, caloric intake and, 492 of mice, folic acid antagonists and, 487 milk agent and, 135 Lipids, effect of carcinogens on epidermal, 7576 Liver, cancer, see also under Liver, tumors, 490-491 diet and, in man, 351 etiologic factors, 490 liver cirrhosis and, 490 chemical composition, 85-87 effect of carcinogenic dyes on, 371378 effect of neoplastic disease on, 515 mitochondria in, 515 production of carbohydrate-rich plasma proteins by, 536 role in hypoalbuminemia of cancer patients, 515
tumors, see also under Liver, cancer butter yellow and, 175 caloric intake and experimental, 458, 492 choline and, 388, 478-479 dietary fat and, 468, 492 proteins and incidence of, 473-474, 475, 476, 477, 492 induced by aminoazo dyes, 340-343, 379-380 chemical composition of, 371-378 histology Of, 344-346 species differences in, 344 vitamin levels and, 477-478 in mice, hormones and, 221 sex differences in, 220-221 urethan and, 175 vitamins €3 and spontaneous, 480, 492 Luteinizing hormone, testicular tumors and, 196 Lymphoblasts, fibroblasts and, 244 Lymphoid tumors, experimental, 204-207 adrenals and, 207 following treatment with dibenzanthracene, 206 with estrogens, 204-205, 206 with methylcholanthrene, 206 sex differences in, 206 transplantability of, 204
M MAB, see under CMonomethylaminoasobeneene 3’-Me-DAB, see under 3’-Methyl-4dimethylaminoazobenzene Macrophages, fibroblasts and, 244 Malnutrition, tumorigenesis and, 491,496 Mammary glands, cancer, see also under Mammary glands, tumors caloric restriction and, 485, 492 dietary fat and, 468, 469, 492 proteins and, 472, 475, 476 hormones and, 212, 219 in mice, effect of labeled Nile blue 2B on, 327 vitamins B and incidence of, 477, 479-480, 486, 492
SUBJECT INDEX
hormonal influence on, 212 milk agent and structure of, 127 response to estrogens, inanition and, 214 tumors, experimental, 211-219 contributing factors, 218 ovarian function and, 214 role of hormones in, 211, 212, 217218 strain differences in, 213, 214, 217, 218 Metabolic pool, 276 Methionine, labeled, incorporation into prdteins of tumor-bearing animals, 302 reaction with di-2-chloroethyl sulfide, 408 5-Methylacridine, carcinogenic activity, 37 K-region of, 37 Methylbis (2-chloroethylamine), labeled, metabolism in normal and leukemic mice, 33 Methylcholanthrene, carcinogenic action, 63, 67, 69, 97, 175 effect on epidermal amino acids, 81 cells, 92, 93 epidermal hyperplasia and, 70, 71 labeled, 280 carcinogenic activity, 284 excretion into mother’s milk, 284 rate of elimination, 284-285 leukemia in mice induced by, 474 dietary proteins and, 474, 475 lymphomagenic action, 206 milk agent and, in mammary cancer in mice, 123 20-Methylcholanthrene, effect on carcinogenic activity of aminoazo dyes, 351, 370 labeled, distribution of radioactivity in tumors induced by, 283 2-Methyldiaminoazobenzene, carcinogenic activity, 356 4’-Methyldiaminoazobenzene, carcinogenic activity, 356 Methyldi-2-chloroethylamine, 392 reaction with amino acids, 416,417,418 with anions, 415, 416 with peptides, 416, 417
583
with proteins, 418 in water, 412413, 414 3’-Methyl-4-dimethylaminoazobenzene, carcinogenic action, 343 2-acetylaminofluorene and, 351 carcinogenic hydrocarbons and, 351 20-methylcholanthrene and, 351, 370 metabolism of labeled, 290 by rats, 360 7-Methylpteroylglutamic acid, synergism with desoxypyridoxine, 487 Milk agent, 152 adrenal hyperplasia and, 130 antigenic properties, 144 behavior in vivo, 140 chemical properties, 135 comparison with “kappa” factor of paramecia, 157 disappearance of, 142 distribution in body of mice, 107, 132, 133 effect of genetic factors on susceptibility to, 110, 114, 119 on transmiesion of, 113 on leukemia in mice, 135 hormonal factors and, 119, 152 inherited susceptibility to mammary cancer and, 129 introduction into body of mice, 107 isolation, 136 mammary cancer in mice and, 103-172 carcinogen-induced, 123ff ., 127 estrogen induced, 121ff., 126 in hybrid mice and, 148 in man and, 158 transplanted, 115 sarcomatous transformation in, 117 multiplication, 143 nature of, 147, 154, 156 neutralization, 144 occurrence, 132, 149, 150 physical properties, 135 purification, 140 serological behavior, 144 structure of mammary gland and, 115 transmission in mice, 107, 113, 140, 150 Minerals, carcinogenic action, 480 effect of carcinogens on epidermal, 74-75
584
BUBJECT INDEX
Mitochondria, in normal and tumorous liver, 298 Molecular-orbital method, 20-30 hyperconjugation, 27-28 LCAO representation, 21-24 magnitudes derived from, 24-26 molecular orbitals, 20-21 polarizabilities, 26-27 resulta, 25-26 tests of theory 28-30 Molecules, aromatic, r electrons and bond structure of, 9-13 carcinogenic potency, prediction of, 7 resonance energy and, 43 stability and, 42 electronic distribution in, effect of methyl groups on, 17-18, 27-28 structure, 6 “molecular diagrams,” 16 &Monomethylaminoazobenzene, carcinogenic activity, 356, 354 structure, 342 Mucoproteins, carbohydrate-containing of serum, 536 in normal and pathological plasma, 522,523 Muscle, chemical cornposition of epidermis and, 85-87 Mustards, see also under names of individual compounds antileukemic action, 329 biological effects, 398,410 relation between chemical activity and, 398-399 carcinogenic activity, 329 cross linking activity of, 442443 effect on sulfhydryl groups of enzymes,
with proteins, 398, 418,429 in water, 399402,411-415,437-439 sulfur, 399-411 Myeloma, multiple, serum globulins in, 527-528 therapeutic effect of stilbamidine in, 328
N
Naphthacene, carcinogenic activity, 37 K-region of, 32,37 Naphthhlene, T bond orders in, 15 carcinogenic activity, 37 ionic structures, 16 K-region, 37 molecular diagram, 17,26 valence bond structures, 12 &Naphthalimine, bladder tumors produced by, 175 2-Naphthyldi-2’-chloroethylamine, chemotherapy of cancer and, 398 Neoplastic disease, see also under Cancer and Tumors immunological aspects of, 526 plasma enzymes and, 529-532 protein stability in, 528-529 Nile blue 2B,labeled, 327 effect on mouse tumors, 327 properties, 327 Nitrogen, nucleophilic character, 412 Nitrogen metabolism, effect of carcinogens on epidermal, 78-82 Nitrogen mustards, see under Mustards, nitrogen Nucleic acids, see also under names of 441 individual compounds labeled, metabolism, 330-331 biochemistry, 310 nitrogen, aliphatic, 411-419 biosynthesis, 323 aromatic, 427-429 composition, 309 chemotherapy of cancer and, 397,398 cytoplasmic, DAB and, 344 radiomimetic, 398 growth and, 326 reactions with alanine, 415-416 incorporation of purines and pyrimiwith anions, 402-406,415-416 dines into, 308, 319,320,321 with bases, 406-408, 416-418, 427X-rays and, 320 429 metabolism, 310,312 with nucleic acids, 410,419 precursors, 314
SUBJECT lNDPlX
radioisotopes in the study of, 309-326 reactions with electrophilic groups in uico, 440, 441 of l12-epoxideswith, 435-436 of mustards with, 410411, 419 turnover rates, 312-314 uptake of Palby, 3106. Nutrition, cancer and, 451497 0
Orotic acid, labeled, 324-325 incorporation into nucleic acid pyrimidine nucleotides, 324 metabolism, 325 Ovary, adrenals and, 188, 189 effect on gonadotropin secretion, 185, 187 pituitary and, 188, 189 tumors, experimental, 184-194 age and, 187 gonadotropins and, 187-188, 189, 190, 194 histogenesis, 192-193 species differences in, 193 hormonal etiology, 184-187, 192 hormone production by, 194 induced by intrasplenic ovarian grafts, 185ff. by X-ray irradiation, 190-193 species differences in, 194 transplantability, 193
P PNA, see under Ribosenucleic acid Pancreas, tumors, following administration of, DAB, 343 Pectic acids, reactions of epoxides with, 435 Pentaphene, K-region of, 33 D-Peptidases, serum, neoplastic disease and, 530 Peptides, reaction of alkyl-2-chloroethylaminea with, 416, 417 Pharyngeal cancer, incidence in artic region, 490
585
Phenanthrene, carcinogenicderivatives, structure of, 3 double bond, see under Phenanthrene, K-region K-region of, 3, 4, 5, 6, 37 molecular diagram, 34 tumorigenic activity, 37 Pheochromocytomas, experimental, 198 Phosphatase, acid, metastatic prostatlic cancer and serum, 529 alkaline, effect of carcinogens on, 72 hyperplastic bone disease and, 529530 Phosphorus, radioactive, uptake by nucleic acids, 312 by nucleoproteins of normal and tumor-hearing mice, 310-312, 318 carcinogenic azo dyes and, 317 glucose and, 318 Phosphorylation, oxidative, in tumors, 299, 300 Pituitary, effect on mammary cancer in mice, 120 hormones, experimental testiculrtr tumors and, 194, 196, 197 ovary and, 188, 189 sex difference in function of, 178 in size of, 201 tumors, experimental, 200-204 following estrogen treatment, 201, 202, 203 nature of, 200, 202 possible etiology, 203 strain and species differences in, 201, 202, 203 thyroid and, 202-203 Plasma, electrophoretic components, 509 cancer and, 504-505, 509 enzymes, neoplastic disease and, 529532, 532-534 proteins, cancer and, 503-548 stability in neoplastic disease, 528529 Plasmin, 531 neoplastic disease and, 531 Polarographic filtrate test, for cancer, 522-523
586
SUBJECT INDEX
Polypeptides, in normal and pathological plasma, 522 Progesterone, carcinogenic action of estrogens and, 216-217
.
effect on lymphomagenic action of estrogens, 204 of X-rays, 204 tumorigenia action of estrogens and, 202
Prostate, cancer, hormones and, 220 serum acid phosphataae and metastatic, 629 Proteins, dietary resistance to toxic agents and, 474
tumor growth and, 482485 tumorigenesia and, 472-476 effect on tumorigenic action of DAB, 348
incorporation of amino acids into, 301309
in liver, effect of carcinogenic aminoazo dyes on, 373, 374, 375, 376, 377, 378
plasma, cancer and, 503-548 determination of, 506-513 stability in neoplastic disease, 528529
reaction with electrophilic agents in vivo, 440, 441 of 1,2-epoxides with, 434-435 with mustards, 398, 408-410, 418, 429
synthesis, circulation and rate of, 307
Roua No. 1 sarcoma agent and, 247 Provirusea, 264 Pseudohypophysectomy, 465 Purines, incorporation of labeled formate into, 318, 323
into nucleic acids, 318, 319, 320 grrene, K-region of, 32 Pyridine, molecular diagram, 26 stmctures, 19
Pyridoxine, azo dye-induced liver tumors and, 478 deficiency, effect on mouse lymphosarcoma, 486 Pyrimidines, incorporation into nucleic acids, 318 of nucleic acids, 323-326 Pyruvate, labeled, metabolism in tumors, 300
Q Quinoline, molecular diagram, 26
R RNA, see under Ribosenucleic acid Reducible substance, in liver, 85 in muscle, 85 in normal and cancerous epidermis, 82, 83, 84
ultraviolet characteristics, 83, 84 Rennin inhibitor, in serum, neoplastic disease and, 533 Resonance method, see under Valence bond method, Riboflavin, azo dye liver tumors and, 477478,492 deficiency, effect on mouse lymphosarcoma, 486 inhibitory effect on tumorigenic action of aminoazo dyes, 346-347, 369 mechanism of, 362, 370 mammary cancer in mice and, 477 Ribonucleic acid, cytoplasmic, effect of carcinogens on, 72
Ribosenucleic acids, 309 Roue No. 1sarcoma agent, adaptation, 237 adsorption on blood elements, 236 age and, 242 antigen of, 249 assay of, 241-242 cell metabolism and, 234, 245-250 distribution, 236-237 effect of host environment on antigenic structure, 239
587
BUBJECT INDEX
on type of lesions produced by, 238, 239 on protein-synthesizing systems, 247 folio acid and, 240-241 fowl leucoses agents and, 263-264 hemorrhagic lesions produced by, 237, 238 infectiveness of, 242, 246 inhibition in vivo, 240 isolation, 250-255 lipid content, 255, 256 localization of, 235, 240, 244 origin, 264-265 preparations, purity, 234,253 properties, 233-265 immunological, 260-261 physico-chemical, 250ff. relationship to host, 235-242 size of, 251, 255-256 stability, 257-260 purification and, 258 survival time, 237 variations, 238-240 vitamin Ble and, 240, 241 b u s sarcoma, cells of, 243-245 electron microscopy of, 245 chemically induced fowl tumora and, 261, enzymes in, 249 folk acid deficiency and, 486 histogenesis of, 243-245 S
Sarooma, caloric intake and, 492 dietary protein and incidence of experimental, 473,476 effect of iodinated polysaccharide of Serratia matcesnes on mouse, 326 Scarlet red, carcinogenic action, 341 structure, 342 Serodiagnosis, of cancer, 504, 514, 516529, 530, 532 methods, 516-520, 525, 526-527, 530, 531,532 Serratia marcesnes, iodinated polysaccharide of, 326-327
effect on mouse sarcomas, 326 properties, 326 Serum, protein-bound carbohydrate of, cancer and, 535-538 sulfhydryl content, 505, 519 neoplastic disease and, 534 Sex organs, secondary tumors of male, hormones and, 219-220 Skin, effect of carcinogenic hydrocarbons on metabolism of, 289 transport. of carcinogens through, 67, 68 tumors, experimental, caloric intake and, 492 dietary fat and 468, 469,471, 492 protein and, 476 vitamins B and, 479,492 Spleen tumors, following administration of DAB, 343 Stilbamidine, labeled, 328 nucleic acids and, 328 Stomach cancer, epidermal cancer and, 58-61 mortality, 58 vitamin A deficiency and, 478 Succinate, labeled, metabolism, 296-297 Sulfur, mustards, see under Mustards, sulfur radioactive, uptake by nucleoproteins, 315,316 Sunlight, carcinogenic action, 94 Switzerland, incidence of thyroid cancer in, 489,490
T Testis, atrophy in mice, following estrogen treatment, 194 strain differences in, 194, 196-197 tumors, experimental, 194-197 androgenic effects, 197 histology of, 197 hormonal etiology, 194, 195, 196 induced by estrogen treatment, 195
588
SWJE1CT INDEX
metastases, 197 pituitary gonadotropins and, 194, 196, 197 transplantability, 197 Texas, incidence of skin cancer in, 59 Thiamine, effect on mammary carcinoma in mice, 486 Thiazans, synthesis of aryl-substituted, 427 Thymonucleic acid, desoxyribosenucleic acid and, 312 in precancerous hyperplastic epidermis, 72 reaction of di-2-chloroethyl sulfide with, 410 of epoxides with, 435, 436 Thymus, effect of estrogens on, 204 experimental lymphoid tumors of, 204 Thyroid, cancer, 489-490 incidence in Switzerland, 489, 490 iodine deficiency and, 490 effect on tumorigenic action of 3'-MeDAB, 350-351 experimental pituitary tumors and, 202-203 Thyrotropic hormone, carcinogenicity, 177 Tissue, cancerous, acidity of, 438 connective, Rous virus and tumors of, 175 hepatic, inactivation of estrogens by, 185, 188 interaction of carcinogenic hydrocarbons with components of, 288-289 mitotic activity, caloric restriction and, 466 cancer and, 466 Tobacco mosaic virus, reaction with butyl2-chloroethyl sulfide, 411 Toluene, charge distribution in, 28 molecular structures, 17, 18 Z(p-Toluenesulfonyl) aminofluorene, labeled, 293
Tracers, radioactive, see under Isotopes, and under names of individual elements Tri-Zchloroethylamine, 392 reactions with amino acids, 416, 417, 418 with anions, 415, 416 with peptides, 416 2,4,6-Triethyleneimino-l,3,5-triazine, 436 cross linking action, 437 Triphenylbromoethylene, labeled, metabolism, 328-329 Triphenylene, carcinogenic activity, 37 K-region of, 37 Triphenylethylene, tumorigenic activity of, and its derivatives, 195 Tritium, 328 Trypan blue, tumorigenic activity, 381 Trypsin inhibitor, in plasma, 532-533 neoplastic disease and, 533 Tryptophan-acid reaction, 537-538 Tumorigenesis, see also under Carcinogenesis, 455480 caloric intake and, 492 effect of dietary fats on, 467472 minerals on, 480 proteins on, 472476 experimental, 174ff. hormonal aspects of, 173-232 ovarian, 184-194 inhibitory effect of caloric restriction, 456-462, 492 factors influencing, 458-465 mechanism, 462-466 malnutrition and, 491 mustard gases and, 410 Tumors, see also under Cancer adrenal, experimental, 198-200 types of, 198 D-amino acids in, 304 effect on enzyme systems, 515 on Pal uptake by DNA, 312, 314, 315, 316, 317 glycolysis in, 318 growth of, 454, 484, 481487 caloric intake and, 481-482 dietary fat and, 482 protein and, 482-485
589
BUBJECT INDEX
effect of ethyleneimine and its derivatives on, 436 inhibition by di-2-chloroethylanilines, 421 mechanism, 303, 304 stages, 481 vitamins and, 485-487 induced by dyes, 340ff. classification, 344, 345 metastases, 345 origin, 345 by vitamin deficiency, 478 inhibition by &azaguanine, 487 Krebs cycle in, 294298, 307 liver, induced by azo dyes, 340, 341, 342, 343 lymphoid, experimental, 204-207 mammary, experimental, 211-219 metabolism, oxidative, 293-300 radioisotopes in the study of, 273334 ovarian, experimental, 184-194 pituitary, experimental, 200-204 proteins of, incorporation of labeled amino acids into, 301-309 in &TO, 304309 i n uiuo, 301-304 testicular, experimental, 194-197 hormonal etiology, 195ff. transplantable, dehydrogenases in, 299 uterine, experimental, 207-21 1 Tyrosinase, inhibition by serum of cancer patients, 534 Tyrosine, labeled, incorporation into proteins of tumorbearing animals, 301-302
U Urea, formation in mammals, arginase and, 78 Urethan, activity, antileukemic, 331 carcinogenic, 333 mechanism of, 332 labeled, metabolism in tumor-bearing mice, 331-332 lung tumors produced by, 175
Uric acid, biosynthesis, 318 Uterus, tumors, experimental, following estrogen treatment, 208209 types of, 208, 209
v Valence bond method, 8-20 aza replacement, 18-19 complicating features, 13-15 derived magnitudes, 15-17 methyl substitution, 17-18 Valence bond orders, 15, 26, 29 Penney’s, 18-20 in K-region, 31-32 Valence bonds, structures of, 9-13 numbers of, 13 Virus(es), see also under ROUENo. 1 sarcoma agent animal, immunological properties, 261 avian tumor, effect on cell metabolism, 235 effect on cellular enzymes, 250 equine encephalomyelitis (Eastern strain) chemical composition, 254, 255 Rous No. 1 sarcoma virus and, 25 stability, 257, 258 fowl-tumor, antigenic relationship between, 262 host and chemical composition, 253 mammary tumor, 176, 182, 215, 218, 219 rabbit papilloma (Shope), 247-248,251 chemical composition, 254 immunological properties, 261 inhibition by colchicine, 240 purity of preparations, 253 ROUE,production of connective tissue tumors by, 175 Vitamin deficiency, tumors induced by, 478 Vitamins, effect on carcinogenic activity, 76,346348, 373, 374, 375, 377, 476-480 on tumor growth, 485-487
590
SUBJBCT INDEX
Vitamins B, see also under name8 of individual vitamins tumorigenesis and, 479, 492
W
X X-rays, effect on Pa* uptake by DNA, 315 lymphomagenic action, adrenals and, 207 progesterone and, 207 mutagenic activity, 97 ovarian tumors in mice produced by, 190-193
Water, reaction of l,%epoxides in, 429-432 of mustards in, 39-15, 419425 Wool, z effect of 1,2-5,6dianhydro-3,4-acetonemannitol on, 435 Zymohexase, see under Aldolase
