Drug Metabolism Concepts (ACS symposium series ; 44)

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.fw001

Drug Metabolism Concepts

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.fw001

Drug Metabolism Concepts Donald M . Jerina, EDITOR The National Institutes of Health

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.fw001

A symposium co-sponsored by the Division of Medicinal Chemistry and the Division of Analytical Chemistry at the 172nd Meeting of the American Chemical Society, San Francisco, Calif., August 31,

ACS

1976

SYMPOSIUM

SERIES

AMERICAN CHEMICAL SOCIETY WASHINGTON, D. C. 1977

44

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.fw001

Library of Congress CIP Data Drug metabolism concepts. (ACS symposium series; 44 ISSN 0097-6156) Includes bibliographical references and index. 1. Drug metabolism—Congresses. 2. Cytochrome P-450 —Congresses. 3. Benzpyrene—Metabolism—Congresses. 4. Carcinogenesis—Congresses. I. Jerina, Donald M., 1940- . II. American Chemical Society. Division of Analytical Chemistry. III. American Chemical Society. Division of Medicinal Chemistry. IV. Series: American Chemical Society. Symposium series; 44. RM301.D76 ISBN 0-8412-0370-9

615'.7 ACSMC8

77-2279 44 1-196

Copyright © 1977 American Chemical Society All Rights Reserved. No part of this book may be reproduced or transmitted in any form or by any means—graphic, electronic, including photocopying, recording, taping, or information storage and retrieval systems—without written permission from the American Chemical Society. PRINTED IN THE UNITED STATES OF AMERICA

ACS Symposium Series

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.fw001

Robert F. Gould, Editor

Advisory Board Donald G. Crosby Jeremiah P. Freeman E. Desmond Goddard Robert A. Hofstader John L. Margrave Nina I. McClelland John B. Pfeiffer Joseph V. Rodricks Alan C. Sartorelli Raymond B. Seymour Roy L. Whistler Aaron Wold

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.fw001

FOREWORD The ACS SYMPOSIUM S E R I E S was founded in 1974 to provide a medium for publishing symposia quickly in book form. The format of the S E R I E S parallels that of the continuing ADVANCES IN CHEMISTRY S E R I E S except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. As a further means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book. Papers published in the ACS SYMPOSIUM S E R I E S are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.

PREFACE he metabolism of drugs and other environmental chemicals has attracted the attention and concern of scientists whose specialties range from analytical and physical chemistry to toxicology, biology, and ecology. The efforts of enzymologists, biochemists, and pharmacologists have established that the basic components of microsomal drug metabolism consist of a membrane or lipid environment, reductases for electron transport, and a family of heme-containing terminal oxidases at which half of the oxygen molecule is reduced to water and the other half is incorporated into substrate. The mechanism by which dioxygen undergoes two-electron reduction to form water and an "active oxygen' species has fascinated inorganic and organic chemists alike. Rates and pathways of metabolism are critically important to medicinal chemistry, pharmacology, and clinical medicine. Chemically reactive intermediates are of concern to toxicologists and to biologists studying carcinogenicity and provide a special fascination for the chemist in terms of structure-activity relationships. Thus, the metabolism of drugs and other xenobiotics not only effects detoxication by conversion to polar, readily excretable compounds but, in many instances, forms reactive intermediates which are responsible for toxic or carcinogenic effects. The concept that chemical carcinogens induce neoplasia via conversion to reactive metabolites which cause chemical modification of critical cellular molecules has made the field of drug metabolism very relevant to the effects of drugs and environmental pollutants on man and his world.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.pr001

A

This volume is the outgrowth of a symposium titled "Recent A d vances in the Study of Drug Metabolism." For this symposium, an effort was made to assemble scientists who would represent a broad spectrum of research in drug metabolism. Hopefully, this interdisciplinary collection will prove of value. The National Institutes of Health Bethesda, Md. December 1976

DONALD M. JERINA

ix

1 Cytochrome P-450—Its Role in Oxygen Activation for Drug Metabolism R. W. ESTABROOK and J. WERRINGLOER

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

Department of Biochemistry, University of Texas Health Science Center, Dallas, TX 75235

At t h i s time many of our colleagues are directing their a t t e n t i o n (1,2) to e v a l u a t i n g the e x i s t e n c e of oxygen on the p l a n e t Mars - an effort d i c t a t e d by the d e s i r e to l e a r n whether any life form, as we know it, e x i s t s on that p l a n e t . What is it t h a t conf e r s on oxygen s p e c i a l p r o p e r t i e s t h a t make it so critical f o r the f u n c t i o n i n g of cellular metabolism. Much has been written on the "fitness of oxygen" (3) p o i n t i n g to the capability to metabolically reduce atmospheric oxygen to hydrogen peroxide or water by two or f o u r e l e c t r o n t r a n s f e r processes, r e s p e c t i v e l y . More important to the t o p i c of this symposium is the ability to enzymatically " a c t i v a t e " molecular oxygen, p e r m i t t i n g the i n c o r p o r a t i o n of one atom of oxygen i n t o an organic s u b s t r a t e molecule concomitant w i t h the r e d u c t i o n of the other atom of oxygen to water. The enzyme systems r e s p o n s i b l e f o r c a t a l y z i n g these types of r e a c t i o n s (Figure 1) are termed mixed-function oxidases, hydroxylases or oxygenases. In many i n s t a n c e s , the i n t r o d u c t i o n of a h y d r o x y l group to the hydrophobic s u b s t r a t e molecule provides a site f o r subsequent conjugation w i t h h y d r o p h i l i c compounds thereby i n c r e a s i n g the solubility of the product f o r its t r a n s p o r t and e x c r e t i o n from the organism. C e n t r a l to the f u n c t i o n i n g of many mixed-function o x i d a t i o n r e a c t i o n s i s a f a m i l y of hemoproteins g e n e r a l l y classified as cytochromes P-450. The present symposium is directeded to f u r t h e r our understanding of how t h i s unique hemoprotein p a r t i c i p a t e s in "oxygen activation" and " s u b s t r a t e h y d r o x y l a t i o n " . Studies w i t h the perfused r a t l i v e r by Thurman and Scholz (4j 5^) as w e l l as S i e s , et a l . (6,7) i n d i c a t e t h a t approximately 60 percent of c e l l u l a r r e s p i r a t i o n by t h i s organ i s s e n s i t i v e to i n h i b i t i o n by Antimycin A - a chemical w e l l recognized as a powerf u l i n h i b i t o r of the m i t o c h o n d r i a l r e s p i r a t o r y c h a i n . Of the r e maining 40 percent of c e l l u l a r r e s p i r a t i o n approximately h a l f i s s e n s i t i v e to the i n h i b i t o r sodium cyanide. A s i g n i f i c a n t p o r t i o n

Supported i n p a r t by a r e s e a r c h grant from the N a t i o n a l I n s t i t u t e s of Health (NIGMS - 16488).

1

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

2

DRUG M E T A B O L I S M CONCEPTS

of the antimycin A i n s e n s i t i v e r e s p i r a t i o n of the l i v e r c e l l i s presumed to occur by o x i d a t i v e enzymes a s s o c i a t e d w i t h the endoplasmic r e t i c u l u m , i . e . the microsomal f r a c t i o n . A c o n s i d e r a t i o n of microsomal e l e c t r o n t r a n s f e r r e a c t i o n s (Figure 2) r e v e a l s the f u n c t i o n i n g of a t l e a s t three f l a v o p r o t e i n s , two hemoproteins, and an i r o n c o n t a i n i n g p r o t e i n . The f l a v o p r o t e i n s f u n c t i o n as reduced p y r i d i n e n u c l e o t i d e dehydrogenases and they p a r t i c i p a t e i n the t r a n s f e r of reducing e q u i v a l e n t s to cytochrome 1>5 from NADH, v i a the f l a v o p r o t e i n ( f p i ) , NADH-cytochrome b$ reductase, or from NADPH by (fp2>, NADPH-cytochrome P-450 reductase ( 8 ) . Reduced cytochrome b$ may i n t e r a c t w i t h a r e c e n t l y i s o l a t e d i r o n cont a i n i n g p r o t e i n (9) i n the cyanide s e n s i t i v e d e s a t u r a t i o n of f a t t y acyl-Coenzyme A compounds (10,11). The reduced f l a v o p r o t e i n , f p 2 , can a l s o undergo o x i d a t i o n by f e r r i c cytochrome P-450 i n r e a c t i o n s to be described below, o r , by an as y e t p o o r l y understood r e a c t i o n , t h i s reduced f l a v o p r o t e i n has been p o s t u l a t e d (12,13) to r e a c t w i t h molecular oxygen g i v i n g r i s e to a superoxide anion f o r the i n i t i a t i o n of l i p i d p e r o x i d a t i o n or heme degradation. A t h i r d f l a v o p r o t e i n , f p 3 , p a r t i c i p a t e s i n the o x i d a t i o n of t e r t i a r y amines g i v i n g r i s e to N-oxides as described by Z i e g l e r et a l . (14,15). Of i n t e r e s t are recent r e s u l t s reported by Z i e g l e r et a l . (16) t h a t t h i s f l a v o p r o t e i n , f p 3 , may a l s o f u n c t i o n i n an oxygen dependent o x i d a t i o n of s u l f h y d r y l groups f o r the format i o n of d i s u l f i d e bonds. Cytochrome P-450 has many i n t e r e s t i n g p r o p e r t i e s that serve as a challenge to the biochemist concerned w i t h understanding the f u n c t i o n of t h i s hemoprotein as i t p a r t i c i p a t e s i n a broad spectrum of o x i d a t i v e r e a c t i o n s . I t s n a t u r a l environment i n most mammalian t i s s u e s i s the membrane s t r u c t u r e c a l l e d the endoplasmic r e t i c u l u m ; t h e r e f o r e , considerable i n t e r e s t i s d i r e c t e d to understanding the i n f l u e n c e imposed by a presumed r e s t r i c t e d m o b i l i t y of p r o t e i n molecules i n a m i l e u of l i p i d as t h i s pigment i n t e r a c t s w i t h oxygen, s u b s t r a t e molecules, and the f l a v o p r o t e i n e l e c t r o n donor. F u r t h e r , i t r e q u i r e s the s k i l l and perseverance of groups such as those l e d by Coon e t a l . (17-19) as w e l l as Lu and L e v i n (20-22) to i s o l a t e and p u r i f y v a r i o u s forms of t h i s hemoprotein f o r p h y s i c a l and chemical c h a r a c t e r i z a t i o n . Such s t u d i e s have revealed that the f a m i l y of cytochromes P-450 have molecular weights i n the range of 46,000 to 52,000 and d i s p l a y a great p r o p e n s i t y to aggregate because of t h e i r hydrophobic p r o p e r t i e s . F u r t h e r , cytochrome P-450 i s r e a d i l y i n d u c i b l e upon treatment of an animal w i t h v a r i o u s drugs or p o l y c y c l i c hydrocarbons. Although t h i s property i s p o o r l y understood i t appears to be r e s p o n s i b l e i n p a r t f o r the long observed phenomenon of drug t o l e r a n c e (23«24). As w i l l be discussed by Dr. Coon during t h i s symposium (25), cytochrome P-450 can e x i s t i n m u l t i p l e forms w i t h an ever i n c r e a s i n g r o s t e r of new p r o t e i n s i d e n t i f i e d as p u r i f i c a t i o n methodology becomes more s o p h i s t i c a t e d and w i d e l y used.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

1.

ESTABROOK A N D WERRINGLOER

Cytochrome

P-450 in Oxygen Activation

3

I n i t i a l i n t e r e s t i n microsomal mixed-function o x i d a t i o n r e a c t i o n s occured i n the 1950's; such s t u d i e s were focused on three g e n e r a l areas of b i o m e d i c a l importance. Brodie and h i s c o l l a b o r a t o r s (26,27) recognized the p o t e n t i a l r o l e of h y d r o x y l a t i o n r e a c t i o n s i n the o x i d a t i v e t r a n s f o r m a t i o n of many drugs - hence t h i s r e a c t i o n system was looked upon as a general mechanism f o r d e t o x i f i c a t i o n of f o r e i g n chemicals. In c o n t r a s t , the M i l l e r s and t h e i r colleagues (28,29) were concerned w i t h the o x i d a t i v e conversion of p r e c a r c i n o g e n i c chemicals, such as p o l y c y c l i c hydrocarbons, to carcinogens - r e a c t i o n s of great p o t e n t i a l harm to the maintenance of the v i a b i l i t y of the organism. A t h i r d group, the s t e r o i d e n d o c r i n o l o g i s t s , were a c t i v e l y studying (30, 31) the o x i d a t i v e processes r e s p o n s i b l e f o r the enzymatic conv e r s i o n of c h o l e s t e r o l to g l u c o c o r t i c o i d s and m i n e r a l o c o r t i c o i d s products of c r i t i c a l importance f o r the maintenance of homeos t a s i s of the organism. We now know t h a t the general f a m i l y of hemoproteins, cytochromes P-450, p l a y p i v i t o l r o l e s i n d i r e c t i n g the o p e r a t i o n of each of these, as w e l l as other s i m i l a r r e a c t i o n s . Thus t h i s c l a s s of hemoproteins has a d u a l i t y of f u n c t i o n (Figure 3) jL.e_., i t may serve as e i t h e r a panacea or a plague f o r the c e l l . Proposed C y c l i c F u n c t i o n of Cytochrome P-450. How do we c u r r e n t l y v i s u a l i z e the f u n c t i o n of t h i s unique hemoprotein, cytochrome P-450? One p o s t u l a t e d scheme (32) i l l u s t r a t i n g the c y c l i c p a t t e r n of r e d u c t i o n and oxygenation of cytochrome P-450 as i t i n t e r a c t s w i t h s u b s t r a t e molecules, e l e c t r o n donors, and oxygen i s shown i n F i g u r e 4. B r i e f l y , these r e a c t i o n s may be summarized as f o l l o w s : ^ A. The f e r r i c hemoprotein (Fe'") can i n t e r a c t w i t h a molec u l e of s u b s t r a t e (R) r e s u l t i n g i n a complex (Fe ^»R) analogous to an enzyme - s u b s t r a t e complex. This i n t e r a c t i o n can be d i r e c t l y measured s i n c e the s u b s t r a t e molecule appears to be c l o s e l y a s s o c i a t e d w i t h the heme moiety of cytochrome P-450 r e s u l t i n g i n a s p e c t r a l p e r t u r b a t i o n measurable by e i t h e r o p t i c a l absorbance spectrophotometry or e l e c t r o n paramagnetic resonance spectrometry (33-36). The o r i e n t a t i o n of the s u b s t r a t e molecule as i t s i t s i n p r o x i m i t y of the heme i r o n , the presumed b i n d i n g s i t e f o r oxygen, remains a conjecture although the r o l e of s t e e r i n g groups on the s u b s t r a t e molecule (37) or the i n f l u e n c e of the hydrophobic environment o f the heme 738) may be considered as p o s s i b l e d i r e c t ing f o r c e s . B. The s u b s t r a t e complex of f e r r i c - cytochrome P-450 (Fe 3*R) undergoes r e d u c t i o n to a f e r r o u s cytochrome P-450 s u b s t r a t e complex (Fe ^»R) by e l e c t r o n s o r i g i n a t i n g from NADPH and t r a n s f e r r e d by the f l a v o p r o t e i n (fp2)> NADPH-cytochrome P-450 r e ductase. The q u e s t i o n of a stimultaneous two e l e c t r o n t r a n s f e r to cytochrome P-450, as suggested by Coon et a l . (39,40), or the e x i s t e n c e of two d i s c r e t e one e l e c t r o n donating s t e p s , as demons t r a t e d f o r the p u r i f i e d b a c t e r i a l cytochrome P-450 by Tyson e t +

+

+

DRUG M E T A B O L I S M

4

CONCEPTS

oxygenase 2 e ~ + Substrate + 0

Product +

2 M

(hydrophobic)

F

a

H 0 2

(hydroxylated)

Figure 1. General representation of microsomal-catalyzed oxygenase or mixed-function oxidation reactions

Conjugation i t Excretion

F A T T Y ACID DESATURATION

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

,NADH fp

—•fpi

>b /

3

/

N A D P H — * ffPp p2a, ^ f

-

•* X ,

x

2

V...

•

5

x

P-450

*0

2

MIXED FUNCTION OXIDATION

\

LIPID LI PI PEROXIDATION

N-OXIDATION

Figure 2. Schematic of microsomal electron transport reactions

0 ,e~ — • 2

A.

Figure 3. The duality of cytochrome P-450-catalyzed reactions

A c t i v e Drug

Inactive

Drug

0 ,e 2

B.

Precarcinogen

•

Carcinogen

CO

I 02

Figure 4. Schematic of the proposed cyclic function of cytochrome P-450. The substrate molecule is designated R. The valence state of the heme iron of cytochrome P-450 is indicated.

CO

1.

ESTABROOK A N D WERRINGLOER

Cytochrome

P-450 in Oxygen Activation

a l . (41) as w e l l as Peterson (42), remains as a p o i n t o f uncert a i n t y . F o r the present d i s c u s s i o n the scheme presented i s based on the premise t h a t two separate e l e c t r o n donating r e a c t i o n s o c cur. C. Reduced cytochrome P-450 ( F e * R ) can r e a c t w i t h carbon monoxide t o form a d e r i v a t i v e which i s r e a d i l y i d e n t i f i a b l e s p e c t r o p h o t o m e t r i c a l l y by an absorbance band maximum a t about 450 nm - hence the o r i g i n (43) o f the name cytochrome P-450. A l t e r n a t i v e l y , reduced cytochrome P-450 can r e a c t w i t h oxygen t o form a complex termed (44) oxycytochrome P-450 (Fe 2»02*R). Knowledge o f the chemistry o f oxycytochrome P-450 should provide the needed c l u e t o evaluate the f i r s t step o f "oxygen a c t i v a t i o n " f o r h y d r o x y l a t i o n r e a c t i o n s ; t h e r e f o r e , much e f f o r t has been d i r e c t e d to understanding the parameters t h a t i n f l u e n c e the g e n e r a t i o n and subsequent u t i l i z a t i o n o f t h i s i n t e r m e d i a t e i n cytochrome P-450 c a t a l y z e d r e a c t i o n s . D. Oxycytochrome P-450 (Fe 2»02*R) can presumably d i s s o c i a t e to g i v e a superoxide a n i o n (02~) concomitant w i t h the r e g e n e r a t i o n of the f e r r i c hemoprotein. The r e s u l t a n t hydrogen p e r o x i d e formed by d i s m u t a t i o n o f the superoxide a n i o n may be a measure o f t h i s a b o r t i v e "uncoupling" (45,46) o f cytochrome P-450 f u n c t i o n , .i.e.., a r e a c t i o n which d i v e r t s the t e r n a r y complex o f oxygen, hemoprot e i n , and s u b s t r a t e from i t s r o l e i n oxygen a c t i v a t i o n and subsequent s u b s t r a t e h y d r o x y l a t i o n . A l t e r n a t i v e l y , the complex o f oxycytochrome P-450 may undergo f u r t h e r r e d u c t i o n t o form the equival e n t o f a p e r o x i d e a n i o n d e r i v a t i v e o f the s u b s t r a t e bound hemoprot e i n . Studies (47-50) w i t h the membrane bound cytochrome P-450 of l i v e r microsomes suggests t h a t a d o n a t i o n o f a proposed second e l e c t r o n occurs v i a cytochrome b$ (51). T h i s c o n c l u s i o n i s the b a s i s f o r e x p l a i n i n g the s y n e r g i s t i c T e f f e c t observed d u r i n g the concomitant o x i d a t i o n o f NADPH and NADH by t h i s system (52). E. The proposed p e r o x i d e a n i o n complex o f cytochrome P-450 may undergo p r o t o n a t i o n and d i s s o c i a t e as hydrogen peroxide o r i t may rearrange t o form an oxene d e r i v a t i v e (53) concomitant w i t h the r e l e a s e of water. The e x i s t e n c e o f oxygen as an oxenoid species i s c o n j e c t u r e although s t u d i e s w i t h o r g a n i c p e r o x i d e supported h y d r o x y l a t i o n r e a c t i o n s (54-58) mediated by cytochrome P-450 demonstrate the e x i s t e n c e o f an EPR species s i m i l a r t o Complex I of peroxidase as an i n t e r m e d i a t e i n the r e a c t i o n (59). C l e a r l y , more r e s e a r c h i s needed t o f i r m l y e s t a b l i s h the presence of t h i s form o f oxygen as " a c t i v e oxygen". F. L e a s t understood i s the mechanism o f d i s s o c i a t i o n o f the hydroxylated product and the r e s t o r a t i o n o f the low s p i n form of f e r r i c cytochrome P-450. I n some i n s t a n c e s (60) epoxide i n t e r mediates e x i s t , such as occurs i n those r e a c t i o n s i n v o l v i n g p o l y c y c l i c hydrocarbons (61). I n other c a s e s , product adducts r e s u l t (62-65) which impede the f u r t h e r f u n c t i o n o f cytochrome P-450 as i t p a r t i c i p a t e s i n s u b s t r a t e h y d r o x y l a t i o n r e a c t i o n s . +2

+

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5

+

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6

DRUG M E T A B O L I S M CONCEPTS

Hydrogen Peroxide Formation, The generation o f hydrogen peroxide during NADPH o x i d a t i o n by l i v e r microsomes has been r e cognized f o r a number o f years (66,67). L i t t l e a t t e n t i o n was p a i d to t h i s phenomenon - i n l a r g e p a r t because o f the a s s o c i a t i o n of c a t a l a s e w i t h the microsomal f r a c t i o n r e s u l t i n g i n the breakdown o f hydrogen peroxide as r a p i d l y as i t was formed. Recent s t u d i e s (54-58) on the p e r o x i d a t i c f u n c t i o n o f cytochrome P-450 as w e l l as t h e coupled r e a c t i o n o f hydrogen peroxide w i t h c a t a l a s e f o r the o x i d a t i o n o f a l c o h o l s (68-70) during NADPH o x i d a t i o n by l i v e r microsomes has s t i m u l a t e d a f u r t h e r c o n s i d e r a t i o n o f the r e a c t i o n s i n v o l v e d i n hydrogen peroxide formation. I n a d d i t i o n , an understanding o f the mechanism o f hydrogen peroxide generation may provide a needed c l u e f o r b e t t e r exami n i n g the p r o p e r t i e s o f " a c t i v a t e d oxygen" proposed t o be a necessary intermediate i n cytochrome P-450 c a t a l y z e d r e a c t i o n s (71). As i l l u s t r a t e d i n F i g u r e 5, the a d d i t i o n o f NADPH t o a suspension o f r a t l i v e r microsomes incubated i n the presence o f sodium a z i d e t o i n h i b i t a d v e n t i t i o u s c a t a l a s e , r e s u l t s i n a s t o i c h i o m e t r i c r e d u c t i o n o f oxygen. Approximately 50 percent o f the oxygen reduced can be accounted f o r as hydrogen peroxide generated during the r e a c t i o n * Repeated a d d i t i o n s of NADPH r e s u l t s i n t h e stepwise formation of equal i n c r e ments o f hydrogen peroxide i n d i c a t i n g the a b i l i t y t o a d d i t i v e l y form t h i s product. The f a i l u r e t o observe a s t o i c h i o m e t r i c amount o f hydrogen peroxide formed t o the amount o f oxygen reduced o r NADPH o x i d i z e d has been a t t r i b u t e d (72) t o the p r e sence o f "endogenous s u b s t r a t e s " a s s o c i a t e d w i t h the microsomal f r a c t i o n which can undergo mixed f u n c t i o n o x i d a t i o n r e a c t i o n s . The nature of the proposed "endogenous s u b s t r a t e s " remains t o be b e t t e r d e f i n e d . A number o f p o s s i b i l i t i e s e x i s t t o e x p l a i n the source o f hydrogen peroxide. As proposed i n the scheme presented i n F i g u r e 4 hydrogen peroxide may a r i s e e i t h e r from d i s s o c i a t i o n o f oxycytochrome P-450 o r from the two e l e c t r o n reduced form o f the t e r n a r y complex o f oxygen, s u b s t r a t e , and cytochrome P-450. A l t e r n a t i v e l y one must consider t h a t hydrogen peroxide format i o n may n o t i n v o l v e cytochrome P-450, such as by the autox i d a t i o n o f reduced f l a v o p r o t e i n s . I n order t o f i r m l y e s t a b l i s h the r o l e o f cytochrome P-450 i n hydrogen peroxide gene r a t i o n a s e r i e s o f experiments were c a r r i e d out t o examine the i n f l u e n c e o f i n h i b i t o r s o f cytochrome P-450 f u n c t i o n , such as carbon monoxide o r metyrapone, as w e l l as determine the i n f l u e n c e o f temperature, pH, and the e f f e c t o f v a r i o u s l e v e l s o f NADPH on the r e a c t i o n . I n each i n s t a n c e comparat i v e s t u d i e s were c a r r i e d out t o determine the changes observed i n the r a t e o f N-demethylation o f ethylmorphine - a s u b s t r a t e recognized t o undergo o x i d a t i v e t r a n s f o r m a t i o n c a t a l y z e d by cytochrome P-450.

1.

ESTABROOK A N D W E R R I N G L O E R

Cytochrome

P-450 in Oxygen Activation

7

n moles/min/mg

9.3

if ^no

u u n MM n U g

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

2

73liMNADPHQuid. 72 M Og utillztd M

min. 2 mg

Pb

liver m i c r o t o m e t / m l

Figure 5. The formation of hydrogen peroxide during NADPH oxidation. Liver microsomes from phenobarbital-treated rats were incubated at 2 mg of protein per ml in a reaction medium containing 50mM tris-chloride buffer, pH 7.5, 150mM KCl, 10 mM MgCl and ImM NaN . At times equal to 0 and 10 min, aliquots of NADPH were added to initiate the reaction. In the experiment shown in dashes, NADPH was added only at 0 time. Samples were removed at the points indicated and after addition to trichloroacetic acid the concentration of hydrogen peroxide formed was determined colorometrically with potassium thiocyanate and ferrous ammonium sulfate (S3). Oxygen use was measured in a comparable series of experiments polarographically, and NADPH oxidation was determined spectrophotometrically. 2>

s

y

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

8

DRUG M E T A B O L I S M

CONCEPTS

Incubation o f l i v e r microsomes i n a v e s s e l designed t o m a i n t a i n f i x e d r a t i o s o f carbon monoxide t o oxygen r e s u l t s i n an e q u i v a l e n t i n c r e a s e i n i n h i b i t i o n o f both hydrogen peroxide formation and ethylmorphine N-demethylation as the concentrat i o n o f carbon monoxide i n c r e a s e s r e l a t i v e t o oxygen as shown i n F i g u r e 6. The o b s e r v a t i o n t h a t hydrogen peroxide formation i s i n h i b i t e d by carbon monoxide i s strong presumptive evidence f o r the r o l e o f cytochrome P-450 i n t h i s r e a c t i o n (73). The f a c t t h a t t h e extent o f i n h i b i t i o n i s e f f e c t i v e l y i d e n t i c a l t o t h a t observed f o r the metabolism o f ethylmorphine r e i n f o r c e s t h i s c o n c l u s i o n . However, t o date no photochemical a c t i o n spectrum s t u d i e s (74,75) have been c a r r i e d out t o c o n f i r m the r o l e o f c y t o chrome P-450 i n the formation o f hydrogen peroxide a s has been done f o r other s u b s t r a t e s o x i d a t i v e l y metabolized by t h i s hemop r o t e i n . I t i s o f i n t e r e s t t o note t h a t t h e degree o f carbon monoxide i n h i b i t i o n i s independent o f the presence o f NADH where a s y n e r g i s t i c (47-50) e f f e c t on product formation d u r i n g t h e NADPH supported r e a c t i o n may occur (see below). Studies w i t h t h e i n h i b i t o r metyrapone ( 2 - m e t h y l - l , 2 - b i s ( 3 p y r i d y l ) - l - p r o p a n o n e ) are more e q u i v o c a l s i n c e hydrogen peroxide formation i s maximally i n h i b i t e d about 50 percent by t h i s compound whereas the metabolism o f ethylmorphine i s g r e a t e r than 90 percent i n h i b i t e d (Figure 7 ) . One p o s s i b l e e x p l a n a t i o n f o r t h i s d i f f e r e n c e r e l a t e s t o the o b s e r v a t i o n t h a t o n l y 50 percent o f t h e cytochrome P-450 a s s o c i a t e d w i t h l i v e r microsomes from p h e n o b a r b i t a l t r e a t e d r a t s r e a c t s w i t h metyrapone (76) t o form a s p e c t r a l l y i d e n t i f i a b l e complex. This would imply t h a t a l l forms o f c y t o chrome P-450 present i n l i v e r microsomes can p a r t i c i p a t e i n hydrogen peroxide formation whereas a unique metyrapone b i n d i n g form f u n c t i o n s i n ethylmorphine N-demethylation r e a c t i o n s . C l e a r l y more experiments w i l l be r e q u i r e d t o support t h i s hypothesis. An examination o f the i n f l u e n c e o f v a r y i n g suboptimal steady s t a t e l e v e l s o f NADPH shows (Figure 8) t h a t the apparent Km's f o r NADPH r e q u i r e d t o support the generation o f hydrogen peroxide and t h e N-demethylation o f ethylmorphine a r e i d e n t i c a l . L i k e w i s e s t u d i e s on the i n f l u e n c e o f temperature (Figure 9) on the r a t e s o f t h e two r e a c t i o n s shows an e q u i v a l e n t c a l c u l a t e d energy o f a c t i v a t i o n o f approximately 20 k i l o c a l o r i e s per degree. Thus a wide range o f d i f f e r e n t f a c t o r s have been examined t o e s t a b l i s h the s i m i l a r i t y o f r e a c t i o n s i n which c y t o chrome P-450 serves as the pigment i n t e r a c t i n g w i t h oxygen f o r the formation o f hydrogen peroxide. The q u e s t i o n remains unanswered, however, as t o the mechanism o f hydrogen peroxide gene r a t i o n , i..je. by d i s m u t a t i o n o f t h e superoxide anion d i s s o c i a t i n g from oxycytochrome P-450 o r by p r o t o n a t i o n of the proposed two e l e c t r o n reduced s t a t e e q u i v a l e n t t o the peroxide anion complex of cytochrome P-450.

ESTABROOK A N D W E R R I N G L O E R

Cytochrome

PDnn. IPT PRODUCT

IOC

HCHO

u n "2 2

• • • A

U

>

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

§

AnniTinMC ADDITIONS o o A

80

P-450 in Oxygen

EM EM EM

CONTROL , ACTIVITY - i , -i)

( n m o l e s

m i n

NADPH NADPH NADPH+ NADH

13.3 10.7 23.5

EM

NADPH NADPH NADPH+ NADH NADPH+ NADH

9.2 10.0 10.2 10.0

1

1

EM

Activation

m q

60-

o 8

4 0

O

20

i

1

1

1

1

1

>

RATIO

C

%

Figure 6. The inhibition by carbon monoxide of the generation of hydrogen peroxide and the N-demethylation of ethyl morphine. A series of experiments was carried out as described in Figure 5 in a reaction vessel designed to permit equilibration with various gas mixtures of carbon monoxide, oxygen, and nitrogen. The concentration of oxygen was maintained at 20% for all experiments. The reaction mixture was supplemented by adding 5mM sodium isocitrate, 0.5 units of isocitrate dehydrogenase per ml, 2mM of 5'AMP, and 5/AM rotenone. The reaction was initiated by adding 200fiM NADPH and 200[xM NADPH where indicated. Samples were removed every 30 sec for the initial 5 min of the reaction and analyzed for hydrogen peroxide (S3) or formaldehyde (84;.

10

DRUG M E T A B O L I S M

CONCEPTS

EM(HCHO)

1412.

10-

H

E

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

I

2°2

8-

Figure 7. The effect of metyrapone 6on the rate of hydrogen peroxide for-1 e mation as compared with the rate of 4'N-demethylation of ethylmorphine. Liver microsomes from phenobarbital< treated rats were incubated at 1 me of 2protein per ml in the reaction medium described in Figure 5 supplemented with an NADPH generating system (cf. Figure 6). Varying concentrations of metyrapone were added as indicated. The initial rates of product formation were determined as described in Figure 6.

0 .02

.1

.5 2

0 .02

METYRAPONE

.1

.5

2

(mM)

80-

Figure 8. The influence of varying g 60steady state concentrations of NADPH ^> on the rate of formation of hydrogen ^ peroxide and the N-demethylation of § ethylmorphine. 2 40Liver microsomes from phenobarbitaU &. treated rats were incubated in a reaction medium as described in Figure 5 supplemented with 5mM sodium isocitrate and 2 0 0.5 units of isocitrate dehydrogenase per ml. Where indicated, 5mM ethylmorphine was present. Varying concentrations of NADPH were added to initiate the reaction, and the rate of hydrogen peroxide or formaldehyde formed was determined as described in Figure 6.

• X

// if il §[

In m I HCHO o-«o

• ' ' • | 5

| 10 >)M

NADPH

H 0 2

I 15

2

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

1. E S T A B R O O K A N D W E R R I N G L O E R

Cytochrome

P-450 in Oxygen Activation

11

The I n f l u e n c e of Substrates o f Cytochrome P-450 on t h e Generation o f Hydrogen Peroxide. Presumably oxycytochrome P-450 serves a t a p i v i t o l p o i n t i n the c y c l i c f u n c t i o n o f cytochrome P-450 ( c f . F i g u r e 4 ) . T h i s t e r n a r y complex o f oxygen, s u b s t r a t e and cytochrome P-450 can undergo d i s s o c i a t i o n t o g i v e r i s e t o hydrogen peroxide r e g e n e r a t i n g t h e complex o f f e r r i c cytochrome P-450 w i t h s u b s t r a t e o r i t can proceed, by a mechanism as y e t unknown, t o a c t i v a t e oxygen f o r i n s e r t i o n i n t o the s u b s t r a t e r e s u l t i n g i n the formation o f a hydroxylated product and t h e low s p i n uncomplexed form o f f e r r i c cytochrome P-450. Therefore i t was o f i n t e r e s t t o c a r r y out experiments t o evaluate the i n f l u ence o f v a r i o u s s u b s t r a t e s o f cytochrome P-450, known t o undergo enzymatic h y d r o x y l a t i o n , and t o determine how such s u b s t r a t e s i n f l u e n c e t h e r a t e o f hydrogen peroxide formation. As i l l u s t r a ted i n F i g u r e 10, the presence o f ethylmorphine markedly a t tenuated t h e extent of hydrogen peroxide formed when a l i m i t i n g amount o f NADPH i s added t o i n i t i a t e the r e a c t i o n ( c f . F i g u r e 5 ) . In a d d i t i o n a s m a l l b u t r e p r o d u c i b l e i n h i b i t i o n o f t h e i n i t i a l r a t e o f hydrogen peroxide formation was observed. The decrease i n the extent o f hydrogen peroxide formation i n the presence o f ethylmorphine can be a t t r i b u t e d i n l a r g e p a r t t o t h e s t i m u l a t i o n of NADPH o x i d a t i o n observed i n t h e presence o f t h i s s u b s t r a t e , I.e. a n e a r l y a d d i t i v e e f f e c t o f NADPH o x i d a t i o n occurs when a Ndemethylation r e a c t i o n f u n c t i o n s concomitant w i t h hydrogen p e r oxide formation. Of i n t e r e s t i s the o b s e r v a t i o n t h a t a slow but p e r c e p t i b l e r a t e o f N-demethylation o f ethylmorphine cont i n u e s a f t e r the t o t a l o x i d a t i o n o f NADPH. T h i s slow r e a c t i o n ( o c c u r r i n g a f t e r 4 minutes i n t h e experiments shown i n F i g u r e 10) appears r e l a t e d t o a s t i m u l a t i o n i n the r a t e o f u t i l i z a t i o n of hydrogen peroxide i n the presence o f sodium a z i d e . As d i s cussed below, the a b i l i t y t o support the o x i d a t i v e metabolism o f a v a r i e t y o f s u b s t r a t e s by hydrogen peroxide, i n the absence o f reducing e q u i v a l e n t s generated from NADPH, d i r e c t l y demonstrates the p e r o x i d a t i c f u n c t i o n o f cytochrome P-450. A f u r t h e r s e r i e s o f experiments were c a r r i e d o u t t o examine the e f f e c t o f other s u b s t r a t e s on t h e r a t e o f formation o f hydrogen peroxide as summarized i n F i g u r e 11. I n t h i s case NADPH c o n c e n t r a t i o n was maintained by the a d d i t i o n o f sodium i s o c i t r a t e and i s o c i t r a t e dehydrogenase. I n agreement w i t h t h e experimental r e s u l t s d e s c r i b e d i n F i g u r e 10, the presence o f ethylmorphine r e s u l t e d i n approximately a 20 percent i n h i b i t i o n o f the r a t e o f hydrogen peroxide f o r m a t i o n . I n c o n t r a s t , s u b s t r a t e s o f c y t o chrome P-450 such as benzphetamine and h e x o b a r b i t a l caused a marked s t i m u l a t i o n i n the r a t e o f hydrogen peroxide f o r m a t i o n . U l l r i c h and D i e h l (45), Hildebrandt e t a l (46) and Werringloer e t a l (70) have d e s c r i b e d t h e a b i l i t y o f v a r i o u s compounds t o serve as "uncouplers" o f cytochrome P-450 f u n c t i o n , chemicals which s t i m u l a t e the r a t e o f NADPH o x i d a t i o n and oxygen u t i l i z a t i o n w i t h out a corresponding i n c r e a s e i n the r a t e o f s u b s t r a t e h y d r o x y l a t i o n . There appear t o be two c l a s s e s o f such "uncouplers"; those

12

DRUG M E T A B O L I S M

CONCEPTS

100 50 •X.

V o Z> Q O

or o_

V

HCHO

1

1

10 5

20 Kcal udegree" jimole" X

H 0 2

2

21 Kcal xdegree" jtmole"

1

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

1

Figure 9. The effect of varying temperatures on the rate of hydrogen peroxide formation and the rate of Ndemetnylation of ethylmorphine. A series of experiments similar to those described in Figure 6 were carried out at the temperatures indicated.

27°

37° 3.2

33

17°

3.4

3.5, 3.6 J-.IO 5

80—EM 60-

40-

Figure 10. The influence of ethylmorphine on the rate and extent of hydrogen peroxide formation in the presence of a limiting concentration of NADPH. A series of experiments similar to those described in Figure 5 were carried out in the presence or absence of 5mM ethylmorphine or ImM sodium azide. The amount of hydrogen peroxide or formaldehyde formed * were determined as described in Figure 6. An NADPH generating system was omitted during this series of experiments. The reactions were initiated by addition of 145uM NADPH.

20•

+EM

+ Az 80-Az 60-

40-

20-

10 TIME (minutes)

1.

ESTABROOK A N D W E R R I N G L O E R

Cytochrome

P-450 in Oxygen Activation

13

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

compounds t h a t r e s u l t i n an i n c r e a s e d r a t e o f f o r m a t i o n o f hydrogen peroxide where t h e i n c r e a s e d r a t e o f u t i l i z a t i o n o f oxygen i s accompanied by an e q u i v a l e n t i n c r e a s e i n the r a t e o f NADPH o x i d a t i o n (such as h e x o b a r b i t a l o r benzphetamine) and a second type o f "uncoupling" where non-metabolized s u b s t r a t e s such as p e r f l u o r i nated, a l i p h a t i c f l u o r o c a r b o n s (45) o r compounds l i k e halothane (Werringloer, J and Estabrook, R.W., unpublished r e s u l t s ) r e s u l t i n a f a i l u r e t o see any a d d i t i o n a l hydrogen peroxide formed upon s t i m u l a t i o n o f oxygen u t i l i z a t i o n , l*e., two moles o f NADPH a r e u t i l i z e d f o r each a d d i t i o n a l mole o f oxygen reduced o f f s e t t i n g the s t o i c h i o m e t r y o f 1 mole o f oxygen u t i l i z e d p e r mole o f NADPH o x i d i z e d . The d e t a i l s o f how "uncouplers" d i v e r t the f u n c t i o n o f cytochrome P-450 i n a presumed a b o r t i v e r e a c t i o n r e s u l t i n g i n t h e d i s s i p a t i o n o f the oxycytochrome P-450 t e r n a r y complex remains as a c h a l l e n g e f o r f u r t h e r experimental examination. E l e c t r o n paramagnetic resonance s t u d i e s . The above d i s c u s s i o n centers on the p o t e n t i a l r o l e o f a superoxide anion as an i n t e r m e d i a t e i n the formation o f hydrogen peroxide d u r i n g NADPH o x i d a t i o n by l i v e r microsomes. Since the superoxide anion i s a f r e e r a d i c a l i t should be d e t e c t a b l e by e l e c t r o n p a r a magnetic resonance spectroscopy (77). When l i v e r microsomes are incubated i n an oxygen s a t u r a t e d b u f f e r i n the presence of sodium a z i d e and a sample i s r a p i d l y f r o z e n i n l i q u i d n i t r o g e n soon a f t e r i n i t i a t i o n o f the r e a c t i o n by the a d d i t i o n of NADPH, examination by EPR spectroscopy r e v e a l s t h e f o r m a t i o n of a s i g n a l a t about g • 2.0 as shown i n F i g u r e 12. The gene r a t i o n o f t h i s new EPR s i g n a l , however, does n o t appear t o be r e l a t e d t o hydrogen peroxide f o r m a t i o n o r t h e g e n e r a t i o n o f a f r e e r a d i c a l o f the superoxide anion type. The same EPR s i g n a l a t g » 2.0 i s obtained i f NADH i s employed r a t h e r than NADPH and t h e s i g n a l i s u n a l t e r e d i f sodium a z i d e i s omitted from the medium when reduced p y r i d i n e n u c l e o t i d e i s added. F u r t h e r the s i g n a l remains when the f r o z e n sample of microsomes i s warmed from the temperature o f l i q u i d n i t r o g e n t o -10° and i t s power s a t u r a t i o n c h a r a c t e r i s t i c s resemble those d e s c r i b e d f o r a s i m i l a r f r e e r a d i c a l s i g n a l d e s c r i b e d by Iyanagi and Mason (78) which they a t t r i b u t e d t o a f l a v i n f r e e r a d i c a l generated during t h e f u n c t i o n o f the microsomal f l a v o p r o t e i n , NADPH-cytochrome c reductase. Thus no p o s i t i v e evidence f o r the presence o f the superoxide anion c o u l d be obtained by examining l i v e r microsomes by EPR spectroscopy. A number o f exp l a n a t i o n s c o u l d be o f f e r e d t o r a t i o n a l i z e t h i s n e g a t i v e r e s u l t , such as the presence of an a c t i v e superoxide dismutase a s s o c i a t e d w i t h the microsomal f r a c t i o n ; evenso, t h i s approach to d e f i n e the presence o f the superoxide anion does remain t o be f u r t h e r e x p l o r e d . NADH Synergism. A number o f years ago (47,48) i t was r e cognized t h a t NADH o x i d a t i o n by l i v e r microsomes concomitant

DRUG M E T A B O L I S M CONCEPTS

20-

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

E

16 Figure 11. The "uncoupling effect" of various sub-c strates on the rate of gen- E eration of hydrogen peroxide during NADPH 12oxidation by rat liver microsomes. A series of experiments were carried out using liver micro- 8somes from phenobarbitaU CM treated rats incubated in I a reaction mixture as described in Figure 7 containing an o 4NADPH generating system.£ c Where indicated 5mM ethylmorphine (EM), ImM benzphetamine (BPh), or 2mM hexobarbital (Hx) were added to the reaction mixture.

E M

B

P

h

Hx

ImM

-

AZIDE

NADPH

+ NADPH

2600

2800

3000

3200

3400 H

(Oersted)

Figure 12. Changes in the electron paramagnetic resonance signals of liver microsomes associated with the initiation of NADPH oxidation by liver microsomes. Liver microsomes from phenobarbital-treated rats were suspended at 10 mg protein per ml in an oxygenated reaction medium containing 50mM tris-chbride buffer, pH 7.5, 150mU KCl, lOmU MgCL, and ImM sodium azide. An aliquot was removed, placed in a calibrated EPR tube, and rapidly frozen in liquid nitrogen (upper curve). NADPH (final concentration, 200fiM) was then added to the suspension and an aliquot removed and frozen within 15 sec (lower curve). First derivative spectra were obtained with an E-4 Varian EPR with the samples maintained at the temperature of liquid nitrogen.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

1.

ESTABROOK AND WERRINGLOER

Cytochrome

P-450 in Oxygen Activation

15

w i t h t h e o x i d a t i o n o f l i m i t i n g c o n c e n t r a t i o n s o f NADPH r e s u l t e d i n a marked enhancement i n the o x i d a t i v e metabolism o f s u b s t r a t e s of cytochrome P-450 such as aminopyrine and ethylmorphine. As shown i n F i g u r e 13 the i n i t i a l r a t e o f N-demethylation o f e t h y l morphine by r a t l i v e r microsomes i s n e a r l y doubled when NADH i s added i n the presence o f NADPH. T h i s s t i m u l a t i o n o f a c t i v i t y i s r e f l e c t e d not o n l y i n the r a t e but a l s o t h e extent o f product formed. T h i s observed e f f e c t o f NADH g r e a t l y exceeds an a d d i t i v e e f f e c t o f the a c t i o n of the two forms o f reduced p y r i d i n e n u c l e o t i d e s s i n c e the N-demethylation of ethylmorphine i s r e l a t i v e l y slow i n the presence of NADH a l o n e . This type o f experiment t o gether w i t h measurements on changes i n the extent of steady s t a t e r e d u c t i o n o f cytochrome b$ (51) have served as one o f the foundat i o n s f o r the development of the c y c l i c scheme o f cytochrome P-450 f u n c t i o n as d e s c r i b e d i n F i g u r e 4. I t i s proposed t h a t NADH serves t o donate, v i a the f l a v o p r o t e i n reductase and c y t o chrome b^, the e l e c t r o n r e q u i r e d t o reduce oxycytochrome P-450 to an i n t e r m e d i a t e w i t h an o x i d a t i o n s t a t e e q u i v a l e n t t o a p e r oxide a n i o n form. Of i n t e r e s t was the q u e s t i o n whether a s i m i l a r NADH synergism would be observed d u r i n g t h e g e n e r a t i o n o f hydrogen p e r o x i d e a s s o c i a t e d w i t h the o x i d a t i o n o f NADPH. I n t h i s way i t may be p o s s i b l e t o g a i n some i n s i g h t i n t o which form o f the oxygen comp l e x o f cytochrome P-450 serves as t h e source o f hydrogen p e r oxide. As shown i n F i g u r e 14, NADH does not have a s i g n i f i c a n t syne r g i s t i c a f f e c t on the g e n e r a t i o n o f hydrogen p e r o x i d e d u r i n g NADPH o x i d a t i o n . A s m a l l i n c r e a s e i n the i n i t i a l r a t e o f hydrogen peroxide f o r m a t i o n i s observed i n the presence o f NADH and NADPH, r e l a t i v e t o the r a t e observed when NADPH alone i s used as the donor o f r e d u c i n g e q u i v a l e n t s , b u t t h i s i n c r e a s e i s a p p r o x i mately equal t o the r a t e observed when NADH i s used t o support the r e a c t i o n . The extent o f hydrogen peroxide f o r m a t i o n observed does i n c r e a s e measurably b u t t h i s may be a t t r i b u t e d i n l a r g e p a r t to a " s p a r i n g e f f e c t " of r e d u c i n g e q u i v a l e n t s from NADPH used t o support the mixed f u n c t i o n o x i d a t i o n o f "endogenous s u b s t r a t e s " as d e s c r i b e d i n an e a r l i e r s e c t i o n o f t h i s paper. On the b a s i s o f these r e s u l t s i t i s concluded t h a t the same type o f NADH synerg i s t i c e f f e c t c h a r a c t e r i s t i c o f h y d r o x y l a t i o n r e a c t i o n s mediated by cytochrome P-450 does not f u n c t i o n f o r the g e n e r a t i o n o f hydrogen p e r o x i d e . T h i s o b s e r v a t i o n may r e s u l t from an a l t e r a t i o n o f the r o l e f o r a needed second e l e c t r o n t o form the p e r o x i d e a n i o n form o f cytochrome P-450 o r from t h e dominant r o l e o f t h e d i s s o c i a t i o n o f oxycytochrome P-450 g i v i n g r i s e t o the superoxide a n i o n . The l a t t e r h y p o t h e s i s would p r e c l u d e the need f o r donation o f a second e l e c t r o n i n the f o r m a t i o n o f hydrogen p e r o x i d e . The p e r o x i d a t i c f u n c t i o n o f cytochrome P-450. Hrycay and O'Brien (54-56) have d e s c r i b e d experiments which demonstrate t h e

DRUG M E T A B O L I S M

CONCEPTS

72pM NAOPH + 96JIM NADH

A" 4-

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

4-

2

Figure 13.

4

6 8 TIME (minutes)

10

12

The synergistic effect of NADH on the NADPH-dependent N-demethylation of ethylmorphine. Liver microsomes from phenoharhital-treated rats were incubated at 1 mg of protein per ml in the presence of 5mM ethylmorphine. NADPH and NADH were added in the concentrations indicated to initiate the reaction. Samples were removed and the amount of formaldehyde formed determined by the Nash reagent (84). Sodium azide and an NADPH generating system were omitted from the reaction medium.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

1.

ESTABROOK A N D W E R R I N G L O E R

Cytochrome

P-450 in Oxygen Activation

17

TIME (minutes)

Figure 14. The effect of varying concentrations of NADH on the rate and extent of formation of hydrogen peroxide associated with the oxidation of a limiting concentration of NADPH by liver microsomes. A series of experiments similar to those described in Figure 5 were carried out in the presence of NADPH and NADH as indicated.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

18

DRUG M E T A B O L I S M

CONCEPTS

a b i l i t y o f cytochrome P-450 o f l i v e r microsomes t o serve as a peroxidase. About the same t i m e , Kadlubar e t a l (57) demonstrated t h e a b i l i t y o f o r g a n i c hydroperoxides t o support N-demethylat i o n r e a c t i o n s when l i v e r microsomes a r e added t o the r e a c t i o n mixture. R e c e n t l y c o n s i d e r a b l e i n t e r e s t has centered on the mechanism o f these peroxide c a t a l y z e d r e a c t i o n s as they i n v o l v e cytochrome P-450 s i n c e such r e a c t i o n s may p r o v i d e a d i f f e r e n t means of e v a l u a t i n g the presence and nature o f " a c t i v e oxygen". When l i v e r microsomes i n t e r a c t w i t h ethylmorphine and hydrogen p e r o x i d e , i n the presence o f sodium a z i d e t o i n h i b i t contamina t i n g c a t a l a s e , one observes (Figure 15) a s t o i c h i o m e t r i c u t i l i z a t i o n o f hydrogen p e r o x i d e concomitant w i t h the f o r m a t i o n o f formaldehyde - the product o f N-demethylation o f ethylmorphine (58). T h i s r e a c t i o n i s n o t dependent on the presence o f oxygen and i s not i n h i b i t e d by carbon monoxide. R e l a t i v e l y h i g h l e v e l s o f hydrogen p e r o x i d e a r e r e q u i r e d t o o b t a i n maximal r a t e s o f the Ndemethylation r e a c t i o n and an apparent Km o f approximately 20 mM f o r hydrogen p e r o x i d e has been determined (58). Under o p t i m a l cond i t i o n s r a t e s o f N-demethylation a r e observed i n the presence o f hydrogen peroxide which a r e g r e a t e r than 50 f o l d the r a t e o b t a i n e d when NADPH supports the o x i d a t i v e t r a n s f o r m a t i o n of ethylmorphine. Both o p t i c a l (Figure 16) and e l e c t r o n paramagnetic resonance (Figure 17) spectroscopy s t u d i e s (59) r e v e a l e d changes i n the o x i d a t i o n p r o p e r t i e s of microsomal cytochrome P-450 upon a d d i t i o n o f o r g a n i c hydroperoxides. T r a n s i e n t changes i n the o p t i c a l s p e c t r a were observed upon a d d i t i o n o f cumene hydroperoxide and these s p e c t r a l changes d i f f e r e d from those r e p o r t e d (44) f o r oxycytochrome P-450 observed d u r i n g the a e r o b i c steady s t a t e o x i d a t i o n o f NADPH by l i v e r microsomes. F u r t h e r , e l e c t r o n paramagnetic resonance s t u d i e s (Figure 17) r e v e a l e d the f o r m a t i o n of EPR s i g n a l s i n the a r e a o f g * 2.0 d i f f e r e n t from those d e s c r i b e d i n F i g u r e 12. Indeed, t h e unique nature o f the EPR s i g n a l s observed a t about g • 2.0 when cumene hydroperoxide r e a c t s w i t h microsomal cytochrome P-450 resemble t h e s i g n a l s observed when hydrogen peroxide r e a c t s w i t h metmyoglobin o r cytochrome c peroxidase (79, 80). From these r e s u l t s i t was concluded (59) t h a t cytochrome P-450 may o b t a i n h i g h e r v a l e n c e s t a t e s o f the heme i r o n i n a manner analogous t o t h a t proposed by Yamazaki e t a l (81) f o r p e r o x i dases. T h i s would suggest t h a t the e q u i v a l e n t o f aiT^oxene" form of oxygen might be formed d u r i n g t h e a c t i v a t i o n o f oxygen by cytochrome P-450 as i t f u n c t i o n s i n h y d r o x y l a t i o n r e a c t i o n s . I t i s o f i n t e r e s t t o note t h a t the EPR s i g n a l observed when p e r o x i des i n t e r a c t w i t h l i v e r microsomal cytochrome P-450 i s v e r y s i m i l a r t o t h a t r e p o r t e d by Vanneste e t a l (82) f o r the complex o f oxygen w i t h reduced cytochrome P-450 o b t a i n e d i n r a p i d f r e e z e quenching experiments. Concluding Remarks. The experimental r e s u l t s d e s c r i b e d i n the preceeding s e c t i o n s have been presented t o p r o v i d e some background as t o the present s t a t u s of our understanding o f " a c t i v e

1.

ESTABROOK A N D WERRINGLOER

R A ITI IUOH -C H -Q RM 2

2

3

too

!{

g g 4

.

A

2

2

u t i l i 2 e <

,j

*

nmoles x min" n

m

• B o *

fi

o

l

e

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

O O

x

1

$

d *

mg" O

* ^ < QC hZ LU O

P-450 in Oxygen Activation

°

1

H 02 utilized 2

« * (minus SUBSTRATE)

80! 60

65 t *>,

3.3

nmoles

J HCH0

o ? g .. liberated

It

^ £ ^

(plus

I A '

xmin '

40-

J*

s

20

i i ,

ft X " 4

8

PB microsomes/ml,

Figure 15.

5mM

ETHYLMORPHINE) H 0 * 1 utilized I 65 2

2mg

19

* i|

b e r a t e d

0.4 °*

,

• • 8

Cytochrome

12 I mM

2

16 20 TIME (minutes) Azide :

A o a

aerob ARGON CARBON MONOXIDE

The hydrogen peroxide-dependent N-demethylation of ethylmorphine as catalyzed by liver microsomes. Liver microsomes from phenobarbital-treated rats were diluted to 2 mg protein per ml in a reaction mixture similar to that described in Figure 5. Where indicated, 5mM ethylmorphine was added. The reaction was initiated by adding lOOuM. hydrogen peroxide. Special reaction vessels were used to permit equilibration with various gas mixtures and to permit sampling the reaction at the times indicated. The changes in the concentrations of hydrogen peroxide used or formaldehyde formed were determined colorometrically.

20

DRUG M E T A B O L I S M CONCEPTS

Z

1

41

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

-^%Sh

442-500nm -0.005

^^^^^^

I

\vj W V422

-0.06-

400

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^

-0.000

|j

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8 <

! 3mg/ml Robbit Mvcr 7 -0.015 i mkrotomts (9>iM P-450) 50>iM CumtntOOH

450

500

550

600

WAVELENGTH (not)

Figure 16.

Spectrophotometric measurement of changes occurring during the oxidation of cumene hydroperoxide by rat liver microsomes. Liver microsomes from phenobarbital-treated animals were diluted to a protein concentration of 3 mg per ml in a reaction mixture containing 50mM tris-chloride buffer, pH 7.5, 150mM KCl, and 5mM MgCl . After recording a baseline of equal light absorbance, 50fiM cumene hydroperoxide was added to the contents of the sample cuvette, and the change of absorbance with time was determined by repetitive scanning at 2 nm per sec. (Insert) The kinetics of formation and decay of the absorbance at 442 nm at different concentrations of cumene hydroperoxide. 2

ESTABROOK A N D W E R R I N G L O E R

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

1.

Cytochrome

P-450 in Oxygen Activation

21

Biochemical and Biophysical Research Communications

Figure 17. Changes in the EPR spectra associated with the interaction of cumene hydroperoxide and liver microsomes. Experiments were carried out using liver microsomes from phenobarbital-treated rabbits. (A) no additions; (B) after adding 1.5mM cumene hydroperoxide; (C) after adding 46mM cyclohexane followed by cumene hydroperoxide; (D) 50/AM metmyoglobin mixed with 1.5mM cumene hydroperoxide; (E) a baseline obtained in the absence of liver microsomes

HO

Oxonium ion

+.p..

H0 2

fOxene

> e

2 Oxygen w

—

H

+

H0

H 0

2

2

Perhydroxyl radical

H+

HO*

2

Hydrogen peroxide

pK *4.5

7*

pK *ll.8

0

0

+

H +H0 Superoxide ion

2

Hydroperoxide Hy ion

I +

H +0

2

Peroxide ion

Hydroxy I radical

^ H M+

2

0 Water

I PK 7 S

0

H++OH" Hydroxyl ion

Figure 18. The possible various states of oxygen during its stepwise reduction to water

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

22

DRUG M E T A B O L I S M

CONCEPTS

oxygen" formed d u r i n g cytochrome P-450 c a t a l y z e d r e a c t i o n s . Oxygen may e x i s t i n a number o f o x i d a t i o n s t a t e s as i l l u s t r a t e d i n F i g u r e 18. As yet i t i s not p o s s i b l e t o a s s i g n a s p e c i f i c f u n c t i o n i n h y d r o x y l a t i o n r e a c t i o n s f o r the superoxide a n i o n , peroxide a n i o n , oxene o r t h e i r protonated forms. The a b i l i t y to observe the formation of hydrogen peroxide during NADPH o x i d a t i o n by l i v e r microsomes and to a s s i g n a r o l e f o r cytochrome P-450 i n the generation of hydrogen peroxide lends credence to the scheme p r o posed i n F i g u r e 4. F u r t h e r the experimental observations obtained during the p e r o x i d a t i c f u n c t i o n of cytochrome P-450 a l l p o i n t to the c e n t r a l r o l e f o r a peroxide anion complex of t h i s pigment p l a y i n g a c e n t r a l and p i v i t o l r o l e i n the a c t i v a t i o n of oxygen. New approaches and more experiments w i l l be r e q u i r e d to b e t t e r e s t a b l i s h the v a l i d i t y of the c u r r e n t hypotheses on the proposed intermediates formed during cytochrome P-450 f u n c t i o n . Oxygen i s c e n t r a l to the maintalliances of the l i f e of higher organisms as we now understand i t . In a d d i t i o n to r e a c t i o n s i n the c e l l where oxygen i s reduced to water concomitant w i t h the c o n s e r v a t i o n of energy i n the form of ATP, as c a t a l y z e d by the m i t o c h o n d r i a l r e s p i r a t o r y c h a i n , oxygen p l a y s a key r o l e i n the s y n t h e s i s and degradation of a wide d i v e r s i t y of n a t u r a l compounds, such as s t e r o i d s , as w e l l as f o r e i g n chemical agents. For these l a t t e r r e a c t i o n s cytochrome P-450 p l a y s a c r i t i c a l r o l e s e r v i n g t o a c t i v a t e oxygen f o r i n t e r a c t i o n w i t h these organic s u b s t r a t e s . Undoubtably the f u t u r e w i l l provide many new s u r p r i s e s as we delve deeper to g a i n a f u l l e r understanding of t h i s important enzyme system.

Literature Cited 1.

2.

3.

4. 5. 6. 7.

N i e r , A.O., Hanson, W.B., S e i f f , A., McElroy, M.F., Spencer, N.W., Duckett, R.J., Knight, T.C.D., and Cook, W.S., Science (1976) 193, 786-788. K l e i n , H.P., Horowitz, N.H., L e v i n , G.V., Oyama, V . I . , Lederberg, J., R i c h , A., Hubbard, J.S., Hobby, G.L., S t r a a t , P.A., Berdahl, B.J., C a r l e , G.C., Brown, F.C., and Johnson, R.D., Science (1976) 194, 99-105. George, P., in "Oxidases and R e l a t e d Redox Systems", e d i t e d by King, T.E., Mason, H.S., and M o r r i s o n , M., V o l . 1, pgs. 3-32, John Wiley and Sons, Inc., New York, 1965. Thurman, R.G. and Scholz, R., Eur. J . Biochem. (1969) 10, 459-467. Thurman, R.G. and Scholz, R., Eur. J . Biochem. (1973) 38, 73-78. S i e s , H. and Brauser, B., Eur. J . Biochem. (1970) 15, 531540. Brauser, B., S i e s , H., and Bucher, Th., FEBS L e t t e r s (1969) 2, 170-176.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

1.

ESTABROOK A N D W E R R I N G L O E R

Cytochrome P-450 in Oxygen Activation

23

8. Masters, B.S.S., Baron, J., T a y l o r , W.E., Isaacson, E . I . , and L o S p a l l u t o , J., J. Biol. Chem. (1971) 246, 4143-4150. 9. S t r i t t m a t t e r , P., Spatz, L., Corcoran, D., Rogers, M.J., Setlow, B., and R e d l i n , R., Proc. Nat'l. Acad.Sci.(USA) (1974) 71, 4565-4569. 10. Holloway, P.W., Biochemistry (1971) 10, 1556-1560. 11. Oshino, N., Imai, Y., and Sato, R., J. Biochem. (Tokyo) (1971) 69, 155-168. 12. K e l l o g g , E.W. and Fridovich, I . , J. Biol. Chem. (1975) 250, 8812-8817. 13. Masters, B.S.S. and Schacter, B.A., Annals o f Clinical Research (1976), Vol. 8, s u p p l . 17, 18-27. 14. Z i e g l e r , D.M. and Mitchell C.H., A r c h i v e s Biochem. Biophys. (1972) 150, 116-125. 15. Kadlubar, F.F. and Ziegler, D.M., A r c h i v e s Biochem. Biophys. (1974) 162, 83-92. 16. Z i e g l e r , D.M., Hyslop, R.M., and Poulsen, L.L., HoppeS e y l e r ' s Z. Physiol. Chem. (1976) 357, 1067. 17. Coon, M.J. and Lu, A.Y.H., in "Microsomes and Drug O x i d a t i o n s " , e d i t e d by Gillette, J.R., Conney, A.H., Cosmides, G.J., Estabrook, R.W., F o u t s , J.R., and Mannering, G.J., pgs. 151166, Academic P r e s s , New York (1969). 18. van der Hoeven, T.A., and Coon, M.J., J. Biol. Chem. (1974) 249, 6302-6310. 19. Haugen, D.A., van der Hoeven, T.A., and Coon, M.J., J. Biol. Chem. (1975) 250, 3567-3570. 20. L u , A.Y.H. and L e v i n , W., Biochem. Biophys. Res. Comm. (1972) 46, 1334-1339. 21. L u , A.Y.H., L e v i n , W., and Kuntzman, R., Biochem. Biophys. Res. Comm. (1974) 60, 266-272. 22. Ryan, D., L u , A.Y.H., West, S., and L e v i n , W., J. Biol. Chem. 250, 2157-2163. 23. Remmer, H. and Merker, H.J., Annals o f the New York Acad. Sci. (1965) 123, 79-97. 24. Conney, A.H., Pharmacol. Rev. (1967) 19, 317-366. 25. Coon, M.J., V e r m i l i o n , J.L., Vatsis, K.P., French, J.S., Dean, W.L. and Haugen, D.A., T h i s volume. 26. B r o d i e , B.B., Science (1955) 121, 603. 27. Cooper, J.R. and B r o d i e , B.B., J. Pharmacol. exp. Ther. (1955) 114, 409-417. 28. Conney, A.H., Miller, E.C., and Miller, J.A., J. Biol. Chem. (1957) 228, 753-766. 29. Miller, E.C., Miller, J.A., Brown, R.R., and MacDonald, J.C., Cancer Res. (1958) 18, 469-477. 30. M u e l l e r , G.C. and Rumney, G., J. Amer. Chem. Soc. (1957) 79, 1004-1005. 31. Ryan, K.J. and Engel, L.L., J. Biol. Chem. (1957) 225, 103114. 32. Estabrook, R.W., Schenkman, J.B., Cammer, W., Remmer, H., Cooper, D.Y., Narasimhulu, S., and Rosenthal, O. in

24

33.

34. 35. 36.

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37. 38.

39. 40. 41. 42. 43. 44.

45. 46. 47. 48. 49. 50. 51. 52.

DRUG M E T A B O L I S M

CONCEPTS

"Biological and Chemical Aspects o f Oxygenases" e d i t e d by K. Bloch and O. H a y a i s h i , pgs. 153-170, Maruzen Co. Ltd., Tokyo (1966). Remmer, H., Schenkman, J. B., Estabrook, R.W., Sasame, H., Gillette, J., Narasimhulu, S., Cooper, D.Y., and R o s e n t h a l , 0., Molec. Pharmacol. (1966) 2, 187-190. Schenkman, J.B., Remmer, H., and Estabrook, R.W., Molec. Pharmacol. (1967) 3, 113-123. Cammer, W., Schenkman, J.B., and Estabrook, R.W., Biochem. Biophys. Res. Comm. (1968) 23, 264-268. Estabrook, R.W., Baron, J., Peterson, J. and Ishimura, Y. in "Biological Hydroxylation Mechanisms", e d i t e d by Boyd, G.S. and S m e l l i e , R.M.S., pgs. 159-186, Academic P r e s s , London (1972). Estabrook, R.W., M a r t i n e z - Z e d i l l o , G., Young S., Peterson, J.A., and McCarhty, J., J. S t e r o i d Biochem. (1975) 6, 419-425. Narasimhulu, S., in "Proceedings o f the T h i r d I n t e r n a t i o n a l Symposium on Microsomes and Drug O x i d a t i o n s " , e d i t e d by Ullrich, V., Roots, I., H i l d e b r a n d t , A.G., Estabrook, R.W., and Conney, A., Pergamon P r e s s , Oxford, in press (1977). B a l l o u , D.P., Veeger, C., van der Hoeven, T.A., and Coon, M.J., FEBS L e t t e r s (1974) 38, 337-340. Guengerich, F.P., Ballou, D.P., and Coon, M.J., J. Biol. Chem. (1975) 250, 7405-7414. Tyson, C.A., Lipscomb, J.D. and Gunsalus, I.C., J. Biol. Chem. (1972) 247, 5777-5784. P e t e r s o n , J.A., A r c h i v e s Biochem. Biophys. (1971) 144, 678693. Omura, T. and Sato, R., J. Biol. Chem. (1964) 239, 2370-2378. Estabrook, R.W., H i l d e b r a n d t , A.G., Baron, J., N e t t e r , K . J . , and Leibman, K., Biochem. Biophys. Res. Comm. (1971) 42, 132139. Ullrich, V. and Diehl, H., Eur. J. Biochem. (1971) 20, 509412. H i l d e b r a n d t , A.G., Speck, M., and Roots, I., Biochem. Biophys. Res. Commun. (1093) 54, 968-975. Cohen, B.S. and Estabrook, R.W., Arch. Biochem. Biophys. (1971) 143, 46-53. Cohen, B.S. and Estabrook, R.W., Arch. Biochem. Biophys. (1971) 143, 54-65. C o r r e i a , M.A. and Mannering, G., M o l . Pharmacol. (1973) 9, 455-469. C o r r e i a , M.A. and Mannering, G.J., M o l . Pharmacol. (1973) 9, 470-485. H i l d e b r a n d t , A. and Estabrook, R.W., A r c h i v e s Biochem. Biophys. (1971) 143, 66-79. Peterson, J.A., Ishimura, Y., Baron, J., and Estabrook, R.W., in "Oxidases and R e l a t e d Redox Systems" e d i t e d by K i n g , T.E., Mason, H.S., and M o r r i s o n , M., pgs. 565-581, U n i v e r s i t y Park P r e s s , B a l t i m o r e , Md. (1973).

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

1.

ESTABROOK A N D W E R R I N G L O E R

Cytochrome P-450 in Oxygen Activation

25

53. Ullrich, V. and Staudinger, Hj., in 'Microsomes and Drug O x i d a t i o n s " edited by Gillette, J.R., Conney, A.H., Cosmides, G.J., Estabrook, R.W., Fouts, J.R., and Mannering, G.J., pg. 199-217, Academic P r e s s , New York (1969). 54. Hrycay, E.G. and O'Brien, P.J., A r c h i v e s Biochem. Biophys. (1972) 153, 480-494. 55. Hrycay, E.G. and O'Brien, P.J., A r c h i v e s Biochem. Biophys. (1973), 157, 7-22. 56. Hrycay, E.G. and O'Brien, P.J., Archives Biochem. Biophys. (1974), 160, 230-245. 57. Kadlubar, F.F., Morton, K.C., and Ziegler, D.M., Biochem. Biophys. Res. Comm. (1973) 54, 1255-1261. 58. W e r r i n g l o e r , J. in "Proceedings o f the T h i r d I n t e r n a t i o n a l Symposium on Microsomes and Drug O x i d a t i o n s " , e d i t e d by Ullrich, V., Roots, I., H i l d e b r a n d t , A.G., Estabrook, R.W., and Conney, A., Pergamon P r e s s , Oxford, in press (1977). 59. Rahimtula, A.D., O'Brien, P.J., Hrycay, E.G., Peterson, J.A., and Estabrook, R.W., Biochem. Biophys. Res. Commun. (1974) 60, 695-702. 60. Thakker, D.R., Y a g i , H., L u , A.Y.H., L e v i n , W., Conney, A.H., and J e r i n a , D.M., Proc. N a t ' l . Acad. Sci. (USA), (1976) 73, 3381-3385. 61. H e i d e l b e r g e r , C., Ann. Rev. Biochemistry (1975) 44, 79-121. 62. P h i l p o t , R.M. and Hodgson, E., Mol. Pharm. (1972) 8, 204-214. 63. F r a n k l i n , M., X e n o b i o t i c a (1971) 1, 581-591. 64. Schenkman, J.B., Wilson, B.J., and Cinti, D.L., Biochem. Pharmacol. (1972) 21, 2373-2383. 65. W e r r i n g l o e r , J. and Estabrook, R.W., Life Sciences (1973) 13, 1319-1330. 66. Gillette, J.R., B r o d i e , B.B., and LaDu, B.N., J. Pharm. Exp. Therap. (1957) 119, 532-540. 67. Orme-Johnson, W.H. and Z i e g l e r , D.M., Biochem. Biophys. Res. Comm. (1965) 21, 78-85. 68. Oshino, N., Oshino, R., and Chance, B., Biochem. J. (1973) 131, 555-563. 69. I s s e l b a c h e r , K.J. and C a r t e r , E.A., Biochem. Biophys. Res. Comm. (1970) 39, 530-537. 70. W e r r i n g l o e r , J., Chacos, N., Estabrook, R.W., Roots, I., and H i l d e b r a n d t , A.G. in " A l c o h o l and Aldehyde M e t a b o l i z i n g Systems" e d i t e d by Thurman, R.G., W i l l i a m s o n , J.R., D r o t t , H. and Chance, B., Academic P r e s s , New York (1977) in p r e s s . 71. Estabrook, R.W. and W e r r i n g l o e r , J. in "Proceedings o f the T h i r d I n t e r n a t i o n a l Symposium on Microsomes and Drug O x i d a t i o n s " e d i t e d by Ullrich, V., Roots, I., H i l d e b r a n d t , A.G., Estabrook, R.W. and Conney, A., Pergamon P r e s s , Oxford, in press (1977). 72. W e r r i n g l o e r , J. and Estabrook, R.W., in p r e p a r a t i o n . 73. Estabrook, R.W., F r a n k l i n , M.R., and H i l d e b r a n d t , A.G., Annals New York Acad. Sci. (1970) 174, 218-232.

26 74. 75. 76. 77. 78. 79. 80.

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81.

82. 83. 84.

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CONCEPTS

Estabrook, R.W., Cooper, D.Y. and Rosenthal, O., Biochem. Zeit. (1963) 338, 741-755. Cooper, D.Y., L e v i n , S., Narasimhulu, S., Rosenthal, O. and Estabrook, R.W., Science (1965) 147, 400-402. W e r r i n g l o e r , J. and Estabrook, R.W., A r c h i v e s Biochem. Biophys. (1975) 167, 270-286. Knowles, P.F., Gibson, J.F., P i c k , F.M. and Bray, R.C. Biochem. J. (1969) 111, 53-58. I y a n a g i , T. and Mason, H.S., Biochemistry (1973) 12, 22972308. K i n g , N.K. and Winfield, M.P., J. Biol. Chem. (1963) 238, 1520-1528. Yonetani, T. and Schleyer, H., J. Biol. Chem. (1967) 242, 1974-1979. Yamazaki, I., Nakajima, R., M i y o s h i , K., Makino, R., and Tamura, M., in "Oxidases and R e l a t e d Redox Systems" e d i t e d by K i n g , T.E., Mason, H.S., and M o r r i s o n , M., Vol. 1, pg. 407-418, U n i v e r s i t y Park P r e s s , B a l t i m o r e , Md. (1973). Vanneste, M.Y., Vanneste, W.H., and Mason, H.S., Biochem. Biophys. A c t a (1972) 267, 268-274. H i l d e b r a n d t , A.G. and Roots, I., A r c h i v e s Biochem. Biophys. (1975) 171, 385-397. Nash, T., Biochem. J. (1953) 55, 416-421.

2 Synthetic Models for the Reaction Stages of Cytochrome P-450 JAMES P. COLLMAN and THOMAS N. SORRELL

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch002

Department of Chemistry, Stanford University, Stanford, CA 94305

Metalloenzymes often exhibit primary coordination spheres which have no counterparts among structurally-characterized, s y n t h e t i c complexes. The p h y s i c a l and c h e m i c a l p r o p e r t i e s o f t h e s e m e t a l c e n t e r s may a l s o be u n u s u a l and may not have a n a l o g i e s t o the corresponding p r o p e r t i e s of s y n t h e t i c coordination compounds. The relationship between protein s t r u c t u r e and t h e s e uncommon p h y s i c a l and c h e m i c a l properties is essential to understanding the f u n c t i o n s of metal p r o t e i n active sites. In this paper I will summarize t h e work o f our group in c h a r a c t e r i z i n g model complexes f o r t h e i r o n c e n t e r i n t h r e e o f the five r e c o g n i z e d r e a c t i o n s t a g e s o f the hemoprotein cytochrome P-450 f a m i l y (1, 2). T h i s work is f a r from complete so t h a t the p r e s e n t paper is a p r o g r e s s report. Many o t h e r l a b o r a t o r i e s have a l s o c o n t r i b u t e d t o m o d e l l i n g t h e s e P-450 s t a g e s , but t h e p r e s e n t a c c o u n t will not attempt t o r e v i e w the c o n t r i b u t i o n s o f o t h e r groups in any d e p t h . Consider a simplified reaction c y c l e f o r the soluble, bacterial camphor h y d r o x y l a s e , P-450 , from Pseudomonas p u t i d a (3). Using the h i g h l y purified soluble P-450 system, G u n s a l u s , et al. have been a b l e t o reassemble components o f t h e enzyme system i n vitro and t h u s t o o b s e r v e and t o c h a r a c t e r i z e f o u r s t a b l e i n t e r m e d i a t e s i n the P-450 c y c l e as w e l l as the i n a c t i v e f e r r o u s c a r b o n y l s t a g e (4). These s t u d i e s have r e s u l t e d i n the e l u c i d a t i o n o f t h e r e a c t i o n sequence shown i n F i g u r e 1. I t s h o u l d be emphasized t h a t t h e b a c t e r i a l P - 4 5 0 c y c l e shown i n F i g u r e 1 d i f f e r s i n some degree from t h a t o f t h e membrane bound m i c r o s o m a l c y c l e deduced by Coon (5^, 6). The e l e c t r o n t r a n s p o r t components w h i c h a r e d i f f e r e n t i n the P - 4 5 0 and P - 4 5 0 i systems a r e o m i t t e d h e r e as t h e s e a r e f a r beyond the p r e s e n t scope cam

cam

cam

c a m

c a m

m

27

28

DRUG M E T A B O L I S M CONCEPTS

of modelling* Because o f t h e i r p r i o r r e c o g n i t i o n , the stages i n t h e P - 4 5 0 c y c l e ( F i g u r e 1) s e r v e d a s t a r g e t s f o r t h e m o d e l l i n g s t u d i e s t o be d e s c r i b e d herein. F i g u r e 2 shows a s i m p l i f i e d v e r s i o n o f t h e r e a c t i o n c y c l e along with the d i s t i n c t i v e properties a s s o c i a t e d w i t h each stage which a r e necessary t o p r o v i d e f o r a s s e s s i n g t h e v a l u e o f any s y n t h e t i c model « It i s c l e a r that there are strong s i m i l a r i t i e s between r e c o g n i z e d r e a c t i o n s t a g e s o f t h e P " 4 5 0 and t h e P - 4 5 0 i r e a c t i o n c y c l e s . B e f o r e d i s c u s s i n g models f o r t h e s e s t a g e s o f cytochrome P-450, i t i s a p p r o p r i a t e t o r e v i e w t h e s t r u c t u r a l c h a r a c t e r i s t i c s o f low and h i g h s p i n i r o n porphyrins. These r e l a t i o n s h i p s which were f i r s t p r e d i c t e d by Hoard (7) a r e now w e l l e s t a b l i s h e d and have no e x c e p t i o n s among s t r u c t u r a l l y c h a r a c t e r i z e d synthetic porphyrins. The s i t u a t i o n i s summarized i n T a b l e I . F e r r o u s i r o n has s i x d e l e c t r o n s . I n t h e c a m

c a m

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch002

m

Table I Structural Characteristics of I r o n i n Hemoproteins Biological Example

Oxidation and S p i n S t a t e S=2

5

2.09

d

6

S=0

6

2.00

3 +

d

5

S=5/2

5

2.07

3 +

d

5

S=l/2

6

1.99

Mb0 ,Hb0 ,HbCO

Pe

2 +

P

Pe

3 +

^ ) Fe . . . Pe

2

P

450°2' B 450 ( A ,

2

P

s u

450

C O

s t r a t e

F

o

Fe-N A

6

2 +

5 Q

Expected

d

Pe

Mb, Hb, P ^

Coord. No.

e

Radius o f p o r p h y r i n c o r e

-2.01,

d i a m a g n e t i c (S=0) s t a t e , i r o n ( I I ) p o r p h y r i n s p o s s e s s two a x i a l l i g a n d s a f f o r d i n g o v e r a l l s i x - c o o r d i n a t i o n . Low s p i n i r o n ( I I ) has a c o v a l e n t r a d i u s w h i c h f i t s without s t r e s s i n t o the porphyrin core. Welle s t a b l i s h e d examples a r e d i a m a g n e t i c f e r r o u s c a r b o n y l d e r i v a t i v e s s i m i l a r t o s t a g e D i n F i g u r e 1. H i g h s p i n i r o n ( I I ) has f o u r u n p a i r e d e l e c t r o n s (S=2) and a c o v a l e n t r a d i u s t o o l a r g e t o be accommodated by t h e porphyrin core. Such h i g h s p i n f e r r o u s complexes have a s i n g l e a x i a l l i g a n d ( o v e r a l l 5 - c o o r d i n a t e ) and i r o n i s d i s p l a c e d out o f the plane o f the porphyrin r i n g a s , f o r example, i n deoxymyoglobin. The f e r r i c s t a t e

2.

C O L L M A N A N D SORRELL

Reaction

Stages of Cytochrome

P-450

29

with f i v e d electrons i s s t r u c t u r a l l y s i m i l a r to the f e r r o u s s t a t e . Low s p i n f e r r i c p o r p h y r i n s have one u n p a i r e d e l e c t r o n (S=%), two a x i a l l i g a n d s , and i n p l a n e i r o n . High s p i n f e r r i c p o r p h y r i n s have f i v e u n p a i r e d e l e c t r o n s , (S=5/2), a s i n g l e a x i a l l i g a n d , and i r o n o u t o f t h e p o r p h y r i n p l a n e . Low S p i n F e r r i c Stage A. C o n s i d e r t h e r e s t i n g s t a g e o f cytochrome P - 4 5 0 , s t a g e A, F i g u r e 2. The heme i r o n i s i n t h e l o w - s p i n f e r r i c s t a t e and must, t h e r e f o r e , have two a x i a l l i g a n d s . Mason was t h e f i r s t t o r e c o g n i z e t h a t t h e a x i a l l i g a t i o n i n P-450 i s u n c o n v e n t i o n a l and t o s u g g e s t s u l f u r c o o r d i n a t i o n (£,9.) • The e s r s p e c t r a o f t h i s low s p i n form have u n u s u a l rhombic g v a l u e s which a r e s i m i l a r t o t h e g v a l u e s a f f o r d e d by a d d i t i o n o f t h i o l s t o methemog l o b i n and m y o g l o b i n ( 1 0 r i i ) • On t h e b a s i s o f model complexes g e n e r a t e d i n situ by o u r group (12) and t h a t o f my c o l l e a g u e , Holm (13), i t seems c e r t a i n t h a t s t a g e A has one a x i a l t h i o l a t e l i g a n d and a n o t h e r u n s p e c i f i e d a x i a l ligand. The t h i o l a t e l i g a n d u n d o u b t e d l y r e s u l t s from the m e r c a p t i d e a n i o n o f c y s t e i n e . Solutions of t h i o l a t e complexes o f f e r r i c p o r p h y r i n s a r e i n t r i n s i c a l l y u n s t a b l e , s p o n t a n e o u s l y a f f o r d i n g d i s u l f i d e and f e r r o u s p o r p h y r i n s as shown i n e q . 1. The scope and

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch002

c a m

2RS-Pe

II3:

( P ) (B) + 2B

(P r e p r e s e n t s base)•

>

R-S-S-R + 2 B F e

1 1

(P) (B) (1)

any p o r p h y r i n a t o group and B any a x i a l

d e t a i l e d mechanism o f t h i s redox r e a c t i o n i s a t present unclear. The p r o t e i n and/or l i p i d membrane o f cytochrome P-450 must somehow s e r v e t o i n h i b i t t h i s redox p r o c e s s . S y n t h e t i c models o f f e r a c l u e as to the i n h i b i t i o n o f t h i s r e a c t i o n . We have been able t o prepare a s t a b l e , c r y s t a l l i n e f e r r i c t h i o l a t e complex, 1, as i l l u s t r a t e d i n e q . 2. The

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch002

30

DRUG M E T A B O L I S M

CONCEPTS

complex Fe(TPP)(SCgHc)(HSCgHs) i s v e r y u n u s u a l i n t h a t i t i s the only i s o l a t e d low-spin t h i o l a t e f e r r i c p o r p h y r i n and t h e o n l y i s o l a t e d f e r r i c p o r p h y r i n h a v i n g two d i f f e r e n t a x i a l l i g a n d s t o our knowledge. In s o l u t i o n t h i s t h i o l a t e complex i s u n s t a b l e and undergoes a redox d i s p r o p o r t i o n a t i o n r e a c t i o n s i m i l a r t o t h a t shown i n eq. 1. However i n t h e c r y s t a l l i n e s t a t e , the f e r r i c t h i o l a t e complex 1 i s q u i t e s t a b l e . The p h y s i c a l p r o p e r t i e s o f 1 a r e v e r y u n u s u a l and o f f e r a p o s s i b l e analogy to~the f a c i l e s p i n e q u i l i b r i u m e x h i b i t e d by cytochrome P-450 systems which pass from low t o h i g h s p i n f e r r i c (stage A t o B) upon substrate binding. Single c r y s t a l s of 1 are pred o m i n a t e l y h i g h s p i n (]i =5.4 BM) a t 25°; however, as the temperature i s l o w e r e d , a low s p i n form i s o b s e r v e d which e v e n t u a l l y becomes dominant. This u n u s u a l temperature-dependent e q u i l i b r i u m i s q u i t e r e v e r s i b l e and does n o t a f f e c t t h e m o s a i c i t y o f s i n g l e crystals. We have been m o n i t o r i n g t h i s change w i t h a b a t t e r y o f p h y s i c a l t e c h n i q u e s w h i c h i n c l u d e X-ray d i f f r a c t i o n , Mossbauer s p e c t r o s c o p y , e s r , and magnetic measurements (14). These s t u d i e s a r e s t i l l i n p r o g r e s s but some p o i n t s a r e becoming c l e a r . The e s r o f t h e dominant h i g h s p i n form has g v a l u e s (-8.6, -3.4) comparable w i t h t h o s e o f t h e s u b s t r a t e - b o n d e d P-450 (stage B ) . A t -196° t h e e s r shows a dominant low s p i n form whose g v a l u e s (2.40, 2.25, 1.97) a r e s i m i l a r t o t h o s e o f the r e s t i n g form o f P-450 (stage A ) . X-ray d i f f r a c t i o n a t -160° shows t h e low s p i n form t o r e p r e s e n t -*70% o f t h e m o l e c u l e s . This r e s u l t i s c o n s i s t e n t w i t h p r e l i m i n a r y Mossbauer s t u d i e s w h i c h i n d i c a t e t h a t a s p i n e q u i l i b r i u m i s o c c u r r i n g between two s p i n s t a t e s o f a s i n g l e s p e c i e s . Two v e r y d i f f e r e n t i r o n - s u l f u r d i s t a n c e s a r e a p p a r e n t from t h e X - r a y d a t a a t 25°, c o n s i s t e n t w i t h t h e assumption t h a t one s u l f u r i s n o t c o o r d i n a t e d i n the h i g h s p i n form. Under vacuum J. l o s e s b e n z e n e t h i o l , p r o d u c i n g the h i g h s p i n complex 2, w h i c h has a s i n g l e a x i a l t h i o l a t e l i g a n d (eq. 3 ) • S m a l l gaseous l i g a n d s s u c h as ammonia

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch002

2.

C O L L M A N A N D SORRELL

Reaction

Stages of Cytochrome

P-450

31

and methylamine w i l l p e n e t r a t e s o l i d samples o f 2 a f f o r d i n g l o w - s p i n complexes w i t h e s r g v a l u e s s i m i l a r t o t h o s e o f s t a g e A o f t h e P-450 c y c l e ( F i g u r e 3 ) . S o l u t i o n s o f 2 i n t h e p r e s e n c e o f a x i a l bases a r e u n s t a b l e w i t h r e s p e c t t o t h e redox r e a c t i o n (eq. 1 ) . However, when c o l d t o l u e n e s o l u t i o n s o f 2 a r e mixed w i t h v a r i o u s a x i a l bases and t h e n f r o z e n , t h e r e s u l t i n g g l a s s e s e x h i b i t e s r g v a l u e s t y p i c a l o f t h e low s p i n s t a g e A o f P-450. S i m i l a r s t u d i e s have been c a r r i e d o u t by Holm (13) who employed p r o t o p o r p h y r i n I X d i m e t h y l e s t e r , PPIXDME, r a t h e r t h a n t e t r a p h e n y l p o r p h y r i n , TPP. T a b l e I I e x h i b i t s r e p r e s e n t a t i v e g values f o r low-spin f e r r i c porphyrins having d i f f e r e n t combinations o f a x i a l l i g a t i o n along w i t h r e p r e s e n t a t i v e v a l u e s f o r cytochrome P-450 and cytochrome c . Comparison o f t h e s e v a l u e s c l e a r l y i n d i c a t e s t h a t one a x i a l t h i o l a t e l i g a n d i n combination w i t h v i r t u a l l y a l l o t h e r p o s s i b l e modes o f l i g a t i o n w i l l a f f o r d g v a l u e s s i m i l a r t o t h o s e found f o r t h e r e s t i n g s t a g e A o f P-450. However t h e g v a l u e s f o r o t h e r c o m b i n a t i o n s o f l i g a t i o n w i t h o u t an a x i a l m e r c a p t i d e do n o t c o r respond as c l o s e l y t o t h o s e o f P-450. On t h e b a s i s o f such i n s i t u s t u d i e s we c o n c l u d e t h a t one a x i a l l i g a n d i n s t a g e A o f P-450 i s v e r y p r o b a b l y t h e c y s t e i n e t h i o l a t e b u t t h e o t h e r a x i a l base cannot be d i s t i n g u i s h e d from t h e f o l l o w i n g p o s s i b i l i t i e s : oxygen (water, amide c a r b o n y l , s e r i n e h y d r o x y l ) , n i t r o g e n ( i m i d a z o l e o r l y s i n e amino), o r n e u t r a l s u l f u r (methionine t h i o e t h e r o r c y s t e i n e t h i o l ) . The r o l e o f s u l f u r as one l i g a n d i n s t a g e A i s a l s o c o n s i s t e n t w i t h P e i s a c h ' s r e c e n t s t u d i e s u s i n g e s r i n an e l e c t r i c f i e l d (15) and w i t h Holm's l i m i t e d s t u d y o f low temperature U V - v i s i b l e s p e c t r a o f model compounds generated i n s i t u (13). One experiment p r o v i d e s a p o s s i b l e e x p l a n a t i o n f o r t h e s t a b i l i z a t i o n o f t h e low s p i n f e r r i c - t h i o l a t e p o r p h y r i n by t h e p r o t e i n o r t h e l i p i d i n cytochrome P-450, s t a g e A. R e a c t i o n between c o l d t o l u e n e s o l u t i o n s o f t h e h i g h - s p i n complex 2 and p o l y s t y r e n e bonded i m i d a z o l e a f f o r d s a s o l i d l o w - s p i n p o l y m e r i c complex, F e T P P ( S C H ) ( N ^ N - p o l y s t y r e n e ) / 3, which i s s t a b l e f o r months a t 25°C i n t h e absence o f s o l v e n t (12). I t i s a p p a r e n t t h a t i m m o b i l i z a t i o n k i n e t i c a l l y s t a b i l i z e s such low s p i n t h i o l a t e complexes. In t h e same v e i n , a m e r c a p t o p r o p y l group c o v a l e n t l y a t t a c h e d t o s i l i c a g e l r e a c t s w i t h [FePj^O i n t h e p r e s e n c e o f a base B ( B = p y r i d i n e o r N-Melm) t o give s t a b l e species with epr g values i d e n t i c a l t o t h o s e o f t h e c o r r e s p o n d i n g complexes g e n e r a t e d i n s i t u 6

5

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch002

32

DRUG M E T A B O L I S M

® F e L O W SPIN

CONCEPTS

-(|)Fe HIGH SPIN

3 +

3+

ESR: g=2.45, 2.26,191

ESR:g=8,4,1.8

X max: 417,534,568 nm

Xmax:391,520,540,646r

©

Fe «(0 ) -

•©Fe

2+

2

MOSSBAUER A E INDEPENDENT OFT

2 +

A E INDEPENDENT OFT Q

Q

©Fe »(CO) 2+

Figure 2. Distinctive properties for the reaction stages of P-45Q wm

X max:=370,450,555 nm CHARACTERISTIC MCD SPECTRUM

i CH

3

Figure 3. Scheme for the preparation of models for the resting stage (A) of P-450

B = NH

3

CH NH 3

2

3

4

5

5

5

5

5

SC H

SC H

SC H

SC H

2.40

S, N

cytochrome C

3.06

2.40

2.25

2.25

2.26

1.24

1.93

1.91

1.95

( r e f . 13).

P=TPP; o t h e r p o r p h y r i n s g i v e s i m i l a r r e s u l t s

s-, ?

P-450 (TBM)

2.45

2.27

1.97

1.94

1.96

1.96

1.57

1.92

1.85

1.95

1.93

(b)

s", ?

p-450 (cam)

2.36

2.25

2.27

2.25

2.22

2.29

2.15

2.21

2.25

2.26

Most s p e c t r a were o b t a i n e d i n f r o z e n t o l u e n e g l a s s e s a t -196°

S~, S

THT

5

S~, S

6

HSC H

2.37

2.38 2.34

S~, 0

S~, N

camphor

2

2.90

S~, 0

N H

N, N

0~, N

2.43

2.61

N

0~,

3 THF

C H

N-Melm

N-Melm

N-Melm

2.37

2.39

^1

S~, N

S~, N

Donor Set

(a)

6

6

6

6

6

SC H

N-Melm

OCH

6

OC H N0

3

2

?

SC H N-Melm

N-Melm

5

SC H

6

B

X

Table I I EPR S p e c t r a f o r Low S p i n F e r r i c P o r p h y r i n Complexes, F e ( P ) ( X ) ( B ) * , b

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch002

34

DRUG M E T A B O L I S M CONCEPTS

a t low t e m p e r a t u r e ( F i g u r e 4 ) ( 1 6 ) . A t t e m p t s t o remove the base B under vacuum have been m a r g i n a l l y s u c c e s s f u l , l e a d i n g t o some d e c o m p o s i t i o n o f t h e p o r p h y r i n . F u r t h e r work aimed a t p r e p a r i n g t h e f i v e - c o o r d i n a t e a l k y l t h i o l a t e complexes i m m o b i l i z e d on s i l i c a i s s t i l l i n p r o g r e s s , as w e l l as p a r a l l e l s t u d i e s on t h i o methylpolystyrene, (p)— CH2SH (16). H i g h S p i n F e r r i c S t a g e B. Upon s u b s t r a t e b i n d i n g , the r e s t i n g l o w - s p i n form A o f P - 4 5 0 m i s c o n v e r t e d i n t o a h i g h - s p i n f e r r i c stage B (Figure 1). The u n u s u a l epr and e l e c t r o n i c s p e c t r a l c h a r a c t e r i s t i c s o f B have been r e p r o d u c e d i n model f e r r i c p o r p h y r i n s h a v i n g a s i n g l e a x i a l t h i o l a t e l i g a n d (12^,13,r7) . Examples o f such i s o l a t e d f e r r i c complexes a r e presented i n Table I I I . I t i s i n t e r e s t i n g to note t h a t Fe(OEP) and Fe(PPIXDME) form f i v e - c o o r d i n a t e t h i o l a t e complexes d i r e c t l y whereas w i t h F e ( T P P ) , t h e s i x c o o r d i n a t e s p e c i e s 1 i s o b t a i n e d which must be f u r t h e r t r e a t e d t o give"the d e s i r e d h i g h - s p i n adduct. The d i s s i m i l a r p h y s i c a l p r o p e r t i e s ( T a b l e I I I ) e x h i b i t e d by t h e s e complexes may r e f l e c t some c r y s t a l p a c k i n g f o r c e s which a r e a f u n c t i o n o f the mode o f preparation. Holm, e t a l . have c h a r a c t e r i z e d by X-ray d i f f r a c t i o n one such model complex, Fe(PPIXDME)(SC6H4NO2), 4 , and s t u d i e d t h i s compound w i t h a wide range o f s p e c t r o s c o p i c t e c h n i q u e s (13). He found t h a t d i s t i n c t i v e f e a t u r e s i n t h e u v - v i s i b l e , MCD, epr, and Mossbauer s p e c t r a o f 4 c l o s e l y p a r a l l e l t h e a n a l o g o u s s p e c t r a l f e a t u r e s o f cytochrome P - 4 5 0 s t a g e B whereas o t h e r a x i a l l i g a n d s gave l e s s s a t i s f a c t o r y correspondence. Our own more l i m i t e d s t u d i e s (12) show t h a t F e ( T P P ) ( S C H ) , 2 , has e p r £-values s i m i l a r t o t h o s e o f s t a g e B, a l t h o u g h our complex i s l e s s w e l l d e f i n e d due t o i t s r e l a t i v e instability. In f a c t , even a t low t e m p e r a t u r e , F e ( T P P ) ( S C H ) undergoes a slow d e c o m p o s i t i o n t o g i v e a s p e c i e s w i t h e p r £-values o f 6 . 0 and 2 . 0 . Thus, the a b s o r b a n c e s o f £ = 8 . 6 and £ = 3 . 4 appear as s h o u l d e r s on t h e £ = 6 peak and can o n l y be approximated; i n r e a l i t y , they a r e p r o b a b l y c l o s e r t o 8 and 4 . Another consequence o f t h e i n s t a b i l i t y o f 2 i s t h e appearance of a broad a b s o r p t i o n a t £ = 2 which obscures the h i g h f i e l d r e g i o n and t h e e x p e c t e d t h i r d r e s o n a n c e a t £ = 1 . 8 . T a b l e IV shows t h a t no o t h e r t y p e o f e x p e r i m e n t a l l y a c c e s s i b l e a x i a l l i g a t i o n w i l l produce s i m i l a r epr spectral features. O g o s h i has p r e p a r e d a model f o r s t a g e B i n which t h e a x i a l l i g a n d i s an a l k y l t h i o l a t e (1J7) (Table I I I )

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch002

c a

c a m

6

6

5

5

2.

C O L L M A N A N D SORRELL

Reaction

Stages of Cytochrome

P-450

35

Table I I I Models f o r S u b s t r a t e Bonded H i g h S p i n F e r r i c P-450 Ref.

9 Values

y (B.M.)

Complex

12

5.8

~8.6 , -3.4,

FePPIXDME ( S C ^ C l )

5.9

7.2, 4.8, 1.8

13

Fe(OEP)(SC H ) 5

5.9

7.2, 4.7, 1.9

13

Fe(OEP)(t-BuS)

5.9

6.4, 4.4, 2.0

17

P-450

5.2

8.0, 4.0, 1.8

3

6

5

g

•substrate

—

a

FeTPP(SC H )

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch002

cam (a)

See t e x t f o r e x p l a n a t i o n .

T a b l e IV EPR Data f o r H i g h S p i n F e r r i c Complexes

Porphyrin

Ref.

Donor

Complex

—

12

4.8

1.8

13

6.4

4.4

2.0

17

o"

6.6

5.3

1.9

16

FePPIXDME(OC H N0 )

o"

5.9

5.9

2.0

13

FePPIXDME(0 CCH )

0"

5.9

5.9

2.0

13

Met-Hb

N

6.0

6.0

2.0

18

P-450 (TBM)

s"

8.3

3.3

—

19

P-450 (cam)

s"

8.0

4.0

1.8

s"

-8.6

-3.4

FePPIXDME(SC H C1)

s"

7.2

FeOEP(S-t-Bu)

s"

FeTPP(OCH )

FeTPP(SC.H ) c

D

0

6

4

3

6

2

4

2

3

3

36

DRUG M E T A B O L I S M CONCEPTS

i n c o n t r a s t w i t h t h e a r y l t h i o l a t e s employed by Holm, e t a l * and our own group. Such a l k y l t h i o l a t e f e r r i c porphyrins are f a r l e s s s t a b l e than the a r y l analogues with regard to the s e l f - d e g r a d a t i v e redox r e a c t i o n s u c h as t h a t shown i n eq. 1 , which l e d us t o our a t t e m p t s t o i m m o b i l i z e such complexes on s i l i c a . The P 2 p a r a m e t e r s r e p o r t e d by O g o s h i a r e n o t as s i m i l a r t o t h o s e o f s t a g e B as t h o s e o f t h e a r y l t h i o l a t e complexes; however, O g o s h i s £ v a l u e s a r e somewhat s u s p e c t due t o t h e u n u s u a l s p l i t t i n g o f t h e low f i e l d resonance. I n v i e w o f t h i s d i s c r e p a n c y i t seems d e s i r a b l e t o examine a d d i t i o n a l models f o r s t a g e B using a l k y l t h i o l a t e s . e

r

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch002

v

Stage C. To d a t e , w e l l d e f i n e d f i v e - c o o r d i n a t e h i g h - s p i n f e r r o u s p o r p h y r i n s h a v i n g an a x i a l t h i o l a t e o r an a x i a l t h i o l l i g a n d have n o t been i s o l a t e d o r even c h a r a c t e r i z e d i n s i t u so t h a t comparison cannot be made w i t h t h e u n i q u e p h y s i c a l p r o p e r t i e s o f t h e i r o n c e n t e r i n cytochrome P - 4 5 0 stage C (Figure 2 ) . Recent Mossbauer s t u d i e s o f s t a g e C i n t h e p r e s e n c e o f s t r o n g a p p l i e d m a g n e t i c f i e l d s have shown d i s t i n c t i v e parameters w h i c h have so f a r n o t been r e p r o d u c e d by model s t u d i e s and a r e d i f f e r e n t from t h o s e p r o p e r t i e s o f deoxymyoglobin i n w h i c h i m i d a z o l e i s t h e a x i a l l i g a n d (2J)) . c a m

S t a g e D. The f e r r o u s c a r b o n y l form ( s t a g e D) o f cytochrome P - 4 5 0 i s c h a r a c t e r i z e d by an u n u s u a l S o r e t a t about 450 nm w h i c h i s r e d s h i f t e d from t h e S o r e t o f "normal" f e r r o u s c a r b o n y l hemes w h i c h a p p e a r s around 420 nm. The u n u s u a l 450 nm a b s o r p t i o n has been i n v a l u a b l e as an a n a l y t i c a l a i d f o r d e t e r m i n i n g t h e p r e s e n c e o f t h i s cytochrome. Small d i f f e r e n c e s i n the p o s i t i o n o f t h i s band (from 446 t o 453 nm) have been o b s e r v e d i n t h e s p e c t r a o f d i f f e r e n t members o f t h e cytochrome P - 4 5 0 f a m i l y . Hanson, e t a l . (21) have proposed a t h e o r e t i c a l e x p l a n a t i o n f o r t h i s unusual S o r e t on t h e b a s i s o f t h e o r e t i c a l c a l c u l a t i o n s and the experimental o b s e r v a t i o n of another o p t i c a l t r a n s i t i o n a t 363 nm h a v i n g t h e same p o l a r i z a t i o n as t h e 446 peak i n t h e b a c t e r i a l cytochrome P " 4 5 0 . These "hyperbands" can be e x p l a i n e d by an i n t e r a c t i o n between a p — e (TT*) ( p o r p h y r i n ) and t h e normal porphyrin a ( i r ) , a2 (ir) — e (ir*) S o r e t t r a n s i t i o n r e s u l t i n g i n t h e o b s e r v e d p a i r o f bands. Such t r a n s i t i o n s c o u l d be c a u s e d by b i n d i n g o f an a x i a l thiolate ligand. S t e r n and P e i s a c h f i r s t r e p o r t e d t h a t t h e unu s u a l 450 nm S o r e t c o u l d be g e n e r a t e d i n s i t u i n an c a m

g

l u

U

g

2.

C O L L M A N A N D SORRELL

Reaction

Stages of Cytochrome

37

P-450

Fe(PPIX) t h i o l / C O DMSO-EtOH s o l u t i o n under v e r y s t r o n g l y b a s i c c o n d i t i o n s (22) . I n t e r p r e t a t i o n o f t h e i r d a t a i s c o m p l i c a t e d by t h e l a r g e e x c e s s o f t h i o l and base r e q u i r e d , t h e p o t e n t i a l f o r c o o r d i n a t i o n o f the DMSO and e t h a n o l s o l v e n t , and t h e p r e s e n c e o f a n o t h e r "normal" S o r e t , as w e l l as t e m p o r a l c h a r a c t e r i s t i c s o f t h e spectrum. Under c o n d i t i o n s o f c o n t r o l l e d s t o i c h i o m e t r y and s o l v e n t environment we have been a b l e t o q u a n t i t a t i v e l y r e p r o d u c e t h e f u l l e l e c t r o n i c and magnetic c i r c u l a r d i c h r o i s m s p e c t r a o f s t a g e D i n t h e P-450 sequence. T h i s was a c c o m p l i s h e d by combining e q u i v a l e n t amounts o f t h e sodium crown-ether m e t h y l m e r c a p t i d e s a l t w i t h i r o n ( I I ) p o r p h y r i n s and CO i n anhydrous benzene as shown i n F i g u r e 5 (23). This f e r r o u s c a r b o n y l complex i s e x t r e m e l y s e n s i t i v e towards oxygen. The i n f r a r e d vCO band a t 1945 cm" is c l o s e t o t h e 1938 cm"l v a l u e r e p o r t e d by Caughey, e t a l . f o r t h e s u b s t r a t e bonded s t a g e D, P-450 m (24). However t h i s s i m i l a r i t y may n o t be m e a n i n g f u l i n t h a t s e v e r a l f a c t o r s such as l o c a l p o l a r i t y and t i l t i n g o f t h e CO group from t h e a x i s normal t o the p o r p h y r i n can influence vco values (25). The e l e c t r o n i c and MCD s p e c t r a (26) o f our mercaptide f e r r o u s carbonyl porphyrins generated i n s i t u are d i s p l a y e d along with those of h i g h l y p u r i f i e d P - 4 5 0 i i n F i g u r e s 6 and 7. The q u a n t i t a t i v e s i m i l a r i t y between the PPIXDEE d e r i v a t i v e and t h e n a t u r a l system i s s t r i k i n g . The s p e c t r a l band a t 370 nm, f i r s t d e s c r i b e d and e x p l a i n e d by Hanson, e t a l . (21) i s c l e a r l y e v i d e n t i n each. These s p e c t r a l comparisons p r o v i d e s t r o n g e v i d e n c e t h a t an a x i a l m e r c a p t i d e l i g a n d i s p r e s e n t i n s t a g e D o f cytochrome P-450. I t i s noteworthy t h a t no o t h e r l i g a n d (RO", PhO~, RC05, RSH, i m i d a z o l e , RCONH2, ROH, o r RNH2) i s c a p a b l e o f p r o d u c i n g t h i s u n u s u a l chromophore. We a l s o d i s c o v e r e d t h a t t h e 449 nm S o r e t c o u l d be s h i f t e d t o l o n g e r wavelengths by i n c r e a s i n g t h e p o l a r i t y o f the s o l v e n t . T h i s o b s e r v a t i o n provides a p o s s i b l e e x p l a n a t i o n f o r the v a r i a t i o n i n the Soret of stage D o b s e r v e d f o r d i f f e r e n t members o f t h e P-450 f a m i l y . Subsequently, Dolphin, e t a l reported s i m i l a r o p t i c a l s p e c t r a f o r models o f s t a g e D g e n e r a t e d i n s i t u (27). We a r e c u r r e n t l y a t t e m p t i n g t o p r e p a r e a c r y s t a l l i n e model f o r s t a g e D. V a r i o u s r e a g e n t s d e n a t u r e cytochrome P-450 y i e l d i n g a p r o t e i n whose f e r r o u s c a r b o n y l e x h i b i t s a S o r e t band a t 420 nm. We have r e p r o d u c e d t h e MCD spectrum o f t h i s s o - c a l l e d P-420 by combining F e ( I I ) P P I X D E E , CO, and e i t h e r N-methyl i m i d a z o l e o r an a l k y l mercaptan (26). These s p e c t r a a r e shown~Tn c a m

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch002

1

ca

m

M

38

DRUG M E T A B O L I S M

CONCEPTS

-3 Et OH (Si0 ) + (Et 0) - Si CH CH CH SH 2

x

3

2

2

2

(Fe PPIXDME) 0 2

(Si0 ) -SiCH CH CH SH 2

x

2

2

2

(Si0 ) - SiCH CH CH S-0e - B Figure 4.

x

2

2

2

:

B= PYRIDINE, g=2.34, 2.25,1.96 N-Melm, g=2.38, 2.26,1.95

Scheme for immobilization

of low spin ferric porphyrin thiolate complexes on silica gel [(SiO»)z]

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch002

2

E

P

R

B

=

V CH S-SCH 2

3

I) Na/NH

3

(Na )SCH +

3

2) CROWN ETHER IN BENZENE

Fe (PPIXDEE) CO INC H 31

6

A max = 375,449,555nm Figure 5. Scheme for the prepa- »co = 1945 cm" ration of a model for the carbonyl (P i/Co i938cm ) stage (D) of P-450

6

1

H

450

s

(g)

e :

\H

3

3

2. C O L L M A N A N D S O R R E L L

Reaction

Stages of Cytochrome

P-450

39

F i g u r e 8. T h i s r e s u l t i n d i c a t e s that the nature o f the a x i a l l i g a n d i n P-420 cannot be a s s i g n e d a t p r e s e n t and r a i s e s t h e p o s s i b i l i t y t h a t P-420 c o u l d r e p r e s e n t a v a r i e t y o f d i f f e r e n t modes o f a x i a l l i g a t i o n . However t h e s e r e s u l t s demonstrate t h a t s t a g e D o f P-450 does n o t have an a x i a l t h i o l l i g a n d . Oxygenated Stage E. The n a t u r e o f t h e o t h e r a x i a l l i g a n d i n t h e cytochrome P - 4 5 0 oxygen complex (stage E F i g u r e 1) i s u n c e r t a i n . On t h e b a s i s o f t h e above mentioned m o d e l l i n g s t u d i e s o f s t a g e s A, B, and D i t would be r e a s o n a b l e t o assume t h a t t h e a x i a l l i g a n d t r a n s t o d i o x y g e n i s m e r c a p t i d e . However, D o l p h i n has r e c e n t l y d e s c r i b e d low temperature s p e c t r a o f t h e c o m b i n a t i o n o f r e d u c e d i r o n p o r p h y r i n s , oxygen, and e x c e s s m e r c a p t i d e i n d i m e t h y l a c e t a m i d e c o n t a i n i n g 5% water (enough t o a f f o r d e q u i v a l e n t c o n c e n t r a t i o n s o f t h i o l ) (23). These s p e c t r a e x h i b i t a S o r e t a t 476 nm, whereas oxygenated P " 4 5 0 ( s t a g e E) i s r e p o r t e d t o have a normal S o r e t a t 418 nm. Dolphin r e p o r t s a 1:1 O2 t o Fe s t o i c h i o m e t r y from gas a b s o r p t i o n measurements on t h e d i l u t e ( 1 0 ~ * M ) , c o l d (-45°) s o l u t i o n s . The assignment o f t h i s spectrum t o t h a t o f an oxygen complex i s c r i t i c a l l y dependent on t h i s s t o i c h i o m e t r y i n view o f t h e f a c i l e o x i d a t i o n o f i r o n ( I I ) and m e r c a p t i d e and t h e redox r e a c t i o n o f t h e f e r r i c t h i o l a t e c o m b i n a t i o n shown i n eq. 1. D o l p h i n ' s r e s u l t s i n d i c a t e t h a t an a x i a l base d i f f e r e n t from m e r c a p t i d e must be p r e s e n t i n t h e oxygenated s t a g e E o f t h e P-450 c y c l e . The oxygenated s t a g e E o f P-450 e x h i b i t s a MSssbauer spectrum d i f f e r e n t from t h a t o f oxyhemoglobin (3). An e s p e c i a l l y d i s t i n c t i v e f e a t u r e i n t h e Mossbauer spectrum o f s t a g e E i s t h e l a c k o f tempera t u r e dependence o f t h e q u a d r u p o l e s p l i t t i n g p a r a meter which i s q u i t e temperature dependent i n oxyhemoglobin. We have i s o l a t e d c r y s t a l l i n e d i o x y g e n i r o n p o r p h y r i n complexes h a v i n g t h r e e d i f f e r e n t modes o f axial ligation: i m i d a z o l e n i t r o g e n (29), t e t r a h y d r o f u r a n oxygen (30), and t e t r a h y d r o t h i o p h e n e s u l f u r (31). The Mossbauer spectrum o f each o f t h e s e oxygenated complexes d i f f e r s from t h e spectrum o f P-450 s t a g e E — e s p e c i a l l y w i t h r e s p e c t t o the temperature dependence o f t h e q u a d r u p o l e s p l i t t i n g . However, t h e a x i a l l i g a n d may have l i t t l e e f f e c t on t h i s s p l i t t i n g parameter (32) i n which c a s e no good probe w i l l be a v a i l a b l e t o d e t e r m i n e t h e n a t u r e o f t h e a x i a l base i n s t a g e E. The t h i o e t h e r d i o x y g e n " p i c k e t f e n c e " p o r p h y r i n c a m

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch002

f

c a m

DRUG M E T A B O L I S M

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch002

40

*7

Figure 8. MCD spectra for P'420 carbonyl (—), FePPlXDEE + N-Melm + CO in benzene ( ), and FePPlXDEE + C H SH + CO in benzene (—)

CONCEPTS

15H

LM

S

7

350

400

450

500

550

WAVELENGTH (nm)

600

650

700

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch002

2.

C O L L M A N A N D SOBRELL

Reaction

Stages of Cytochrome

P-450

41

i s r e l e v a n t t o t h e P-450 models because t h i o e t h e r i s e x p e c t e d t o have a l i g a n d f i e l d c h a r a c t e r s i m i l a r t o t h a t o f t h i o l (33). The r e s u l t s o f an X-ray d i f f r a c t i o n study o f t h i s complex a r e i l l u s t r a t e d i n F i g u r e 9 (31). The s t r u c t u r a l f e a t u r e s o f t h i s t e t r a h y d r o t h i o p h e n e complex a r e s i m i l a r t o t h o s e o f our e a r l i e r N-methylimidazole " p i c k e t fence" dioxygen complex e x c e p t t h a t t h e t e r m i n a l oxygen atom does n o t appear t o be d i s o r d e r e d i n t h e former and i s f o u r way d i s o r d e r e d i n t h e l a t t e r . The poor q u a l i t y o f o u r c r y s t a l s and t h e r e l a t i v e l y s m a l l number o f r e f l e c t i o n s do n o t p e r m i t a s u f f i c i e n t l y a c c u r a t e d e t e r m i n a t i o n o f i n t e r e s t i n g bond d i s t a n c e s t o make r e a l i s t i c comparisons. We have a l s o p r e p a r e d an oxygen complex from a new t y p e o f p o r p h y r i n h a v i n g t h r e e " p i c k e t s " and a b u i l t - i n a x i a l t h i o e t h e r base. The s y n t h e t i c scheme i s shown i n F i g u r e 10 (3_1). O x y g e n a t i o n o f c o l d t o l u e n e s o l u t i o n s o f t h i s complex l e a d s t o slow d e c o m p o s i t i o n b u t t h e p r e s e n c e o f t h e oxygen complex has been demonstrated by Mossbauer s t u d i e s on f r o z e n solutions. However, we have n o t y e t i s o l a t e d and c h a r a c t e r i z e d a s t a b l e oxygen complex from t h i s system, a l t h o u g h a c r y s t a l l i n e c a r b o n y l adduct has been o b t a i n e d . T h i s system s h o u l d l e n d i t s e l f t o t h e s y n t h e s i s and f u l l c h a r a c t e r i z a t i o n o f model f e r r o u s complexes h a v i n g a x i a l t h i o l and t h i o l a t e l i g a n d s (3£) . E x p e r i m e n t s d i r e c t e d towards t h i s end a r e i n p r o g r e s s . An i m i d a z o l e d e r i v a t i v e o f t h i s " t h r e e p i c k e t " system undergoes r e v e r s i b l e o x y g e n a t i o n and we have r e c e n t l y i s o l a t e d an oxygen complex which i s i n t h e process of being f u l l y c h a r a c t e r i z e d (35). Summary Model complexes have been g e n e r a t e d which a c c u r a t e l y r e f l e c t s p e c t r o s c o p i c features of stages A, B, and D o f cytochrome P - 4 5 0 , a l t h o u g h a c r y s t a l l i n e model f o r D has n o t y e t been i s o l a t e d . The a x i a l l i g a n d common t o s t a g e s A, B. and D i s mercaptide. However t h e p r e s e n c e o f an a x i a l merc a p t i d e l i g a n d i n s t a g e s C and E i s u n c e r t a i n a t present. The r e a s o n b e h i n d N a t u r e ' s c h o i c e o f t h i s u n u s u a l l i g a t i o n i s u n c l e a r . One p o s s i b i l i t y has t o do w i t h s t a b i l i z i n g t h e a c t i v e P-450 o x i d a n t w h i c h must be two o x i d a t i o n l e v e l s above t h e f e r r i c s t a g e . In t h i s s t a g e perhaps one e l e c t r o n i s removed from i r o n a f f o r d i n g FeIV=0 and t h e o t h e r from m e r c a p t i d e g e n e r a t i n g a t h i o l r a d i c a l as suggested by Marchon (36). I t i s c l e a r t h a t more work must be done i n c a m

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch002

42

DRUG M E T A B O L I S M CONCEPTS

Figure 9. Crystal structure of Fe(TpivPP) (THT)O g

(CH3)3CC0CI

I

R

V"}

heat NH, CH 3 S(CH 2 ) 5 -COCI

Figure 10. Scheme for preparing "picket fence" porphyrins having an appended axial thioether ligand. X=NH and R=(CH ),CCONH-. t

t

» 1

I

I

NN <

A

.CO N 1CM 2 ) 5

A CH 3

' i

NH /

.co (CH 2 ) $

2. C O L L M A N A N D S O R R E L L

Reaction

Stages of Cytochrome

P-450

43

s y n t h e s i z i n g , i s o l a t i n g , and f u l l y c h a r a c t e r i z i n g model p o r p h y r i n complexes b e f o r e we c a n f u l l y unders t a n d t h e p r i m a r y c o o r d i n a t i o n s p h e r e s employed by cytochrome P-450 i n i t s c a t a l y t i c c y c l e . Abbreviations P-450: P-450 : P-450 : P-450 : c a m

l m

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch002

T B M

TPP: PPIXDEE: PPIXDME: PPIX: OEP: N-Melm: THF: THT: (N^SCH-:

DMSO: MCD: esr (or e p r ) :

cytochrome P-450 cytochrome P-450 camphor h y d r o x y l a s e cytochrome P-450 from l i v e r microsomes cytochrome P-450 from t u l i p b u l b microsomes meso-tetraphenylporphyrin dianion p r o t o p o r p h y r i n IX d i e t h y l e s t e r d i a n i o n p r o t o p o r p h y r i n IX d i m e t h y l e s t e r d i a n i o n p r o t o p o r p h y r i n IX d i a n i o n octaethylporphyrin dianion N-methylimidazole tetrahydrofuran tetrahydrothiophene sodium m e t h y l mercaptide-crown e t h e r complex dimethylsulfoxide magnetic c i r c u l a r d i c h r o i s m e l e c t r o n s p i n (paramagnetic) r e s o n a n c e

Acknowledgments We w i s h t o acknowledge t h e s u p p o r t o f t h e N a t i o n a l S c i e n c e F o u n d a t i o n , g r a n t number CHE-75-17018, and t h e N a t i o n a l I n s t i t u t e s o f H e a l t h , g r a n t number GM17880. Literature Cited (1) (2) (3)

(4)

(5)

Ullrich, V., Angew. Chem. I n t . E d . E n g l . (1972) 11, 701. Tomaszewski, J. E., Jerina, D. M., and D a l y , J. W., Annu. Rep. Med. Chem. (1974) 9, 290. G u n s a l u s , I . C., Meeks, J. R., Lipscomb, J. D., Debrunner, P., and Munck, E . , " M o l e c u l a r Mechanisms o f Oxygen Activation", O. H a y a i s h i , Ed., Academic P r e s s , New York, N. Y., 1973, C h a p t e r 14 and refs. t h e r e i n . G u n s a l u s , I . C., Tyson, C. A., Tsai, R., and Lipscomb, J. D., Chem. Biol. I n t e r a c t i o n s (1971) 4, 75. v a n d e r Hoeven, T. A. and Coon, M. J., J. Biol. Chem. (1974) 249, 6302.

44

(6) (7) (8)

(9) (10) (11) (12)

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(13)

(14) (15) (16) (17) (18)

(19) (20)

(21)

(22) (23) (24)

(25)

(26)

DRUG M E T A B O L I S M CONCEPTS

van d e r Hoeven, T. A. and Coon, M. J., Biochem. B i o p h y s . Res. Comm. (1974) 60, 569. Hoard, J. L., S c i e n c e (1971) 174, 1295. Mason, H. S., N o r t h , J. C., and V a n n e s t e , M., F e d . P r o c . F e d . Am. Soc. Exp. Biol. (1965) 24, 1172. Murikami, K. and Mason, H. S., J. Biol. Chem. (1967) 242, 1102. Roder, A. and B a y e r , E., E u r . J. Biochem. (1969) 11, 89. P e i s a c h , J., Blumberg, W. E . and A d l e r , A., Ann. N. Y. Acad. Sci. (1073) 206, 310. C o l l m a n , J. P., Sorrell, T. N., and Hoffman, B. M., J. Amer. Chem. Soc. (1975) 97, 913. Tang, S. C., Koch, S., P a p a e f t h y m i o u , G. C., F o u e r , S., F r a n k e l , R. B., I b e r s , J. A., and Holm, R. H., J. Amer. Chem. Soc. (1976) 98, 2414. S t r o u s e , C. E., Wickman, H. H., C o l l m a n , J. P., and Sorrell, T. N., u n p u b l i s h e d results. P e i s a c h , J. and Mims, W. B., P r o c . N a t l . Acad. Sci. U.S.A. (1973) 70, 2979. C o l l m a n , J. P. and Sorrell, T. N., u n p u b l i s h e d results. O g o s h i , H., Sugimoto, H. and Y o s h i d a , Z., T e t r a h e d r o n L e t t . (1975) 2289. P e i s a c h , J., Blumberg, W. E., W i t t e n b e r g , B. A., and Kampa, L., P r o c . N a t l . Acad. Sci. U.S.A. (1969) 63, 934. R i c h , P. R., Cammack, R., B e n d a l l , D. S., E u r . J. Biochem. (1975) 59, 281. Champion, P. M., Lipscomb, J. D., Munck, E., Debrunner, P., and G u n s a l u s , I . C., Biochem. (1975) 14, 4151. Hanson, L. K. E a t o n , W. A., Sligar, S. G., G u n s a l u s , I . C., Gouterman, M. and C o n n e l l , C. R., J . Amer. Chem. Soc. (1976) 98, 0000. S t e r n , J. D. and P e i s a c h , J., J. Biol. Chem. (1974) 249, 7495. C o l l m a n , J. P. and Sorrell, T. N., J. Amer. Chem. Soc. (1975) 97, 4133. V o l p e , J. A., Griffin, B., Peterson, J. A., and Caughey, W. S., u n p u b l i s h e d results cited in: Barlow, C. H., O h l s s o n , P . - I . , and P a u l o , K.-G., Biochem. (1976) 15, 2225. C o l l m a n , J. P., Brauman, J. I., Suslick, K. S., and H a l b e r t , T. R., P r o c . N a t l . Acad. Sci., U.S. (1976) s u b m i t t e d for publication. C o l l m a n , J. P., Sorrell, T. N., Dawson, J. H., Trudell, J. R., Bunnenberg, E . , and Djerassi, C., P r o c . N a t l . Acad., U.S. (1975) 73, 6.

2. C O L L M A N A N D S O R R E L L

(27) (28) (29)

(30) (31)

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(32)

(33) (34) (35) (36)

Reaction

Stages of Cytochrome

P-450

45

Chang, C. K. and D o l p h i n , D., J. Amer. Chem. Soc. (1975) 97, 5948. Chang, C. K. and D o l p h i n , D., J. Amer. Chem. Soc. (1976) 98, 1607. C o l l m a n , J. P., Gagne, R. R., Reed, C. A., H a l b e r t , T. R., Lang, G., and Robinson, W. T., J. Amer. Chem. S o c . (1975) 97, 1427. C o l l m a n , J. P., Gagne, R. R., and Reed, C. A., J. Amer. Chem. Soc. (1974) 96, 2629. C o l l m a n , J. P., Sorrell, T. N., Lang, G., S p a r t a l i a n , K., Jameson, G. B., R o d l e y , G. A., and Robinson, W. T., u n p u b l i s h e d results. S p a r t a l i a n , K., Lang, G., C o l l m a n , J. P., Gagne, R. R., and Reed, C. A., J. Chem. Phys. (1975) 63, 5375. Kuehn, C. G. and Taube, H., J. Amer. Chem. Soc. (1975) 98, 689. Sorrell, T. N. Groh, S., and C o l l m a n , J. P., unpublished data. H a l b e r t , T. R. and C o l l m a n , J. P., unpublished data. Marchon, J.-C., p r i v a t e communication.

3 Biochemical Studies on Drug Metabolism: Isolation of Multiple Forms of Liver Microsomal Cytochrome P-450 M. J. COON, J. L . VERMILION, K. P. VATSIS, J. S. FRENCH,† W. L . D E A N , ° †and D. A. HAUGEN‡

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

Department of Biological Chemistry, Medical School, The University of Michigan, Ann Arbor, MI 48109

One of the most interesting properties of the drug-metabolizing enzyme system of l i v e r microsomal membranes i s i t s remarkably broad substrate s p e c i f i c i t y . Not only drugs and anesthetics, but a variety of other foreign compounds and a number of naturally occurring substances also undergo chemical transformation by this system. Cytochrome P-450, the carbon monoxide-binding pigment of microsomes (1-3), was shown i n the pioneering studies of Estabrook and his associates (4-6) to function i n the hydroxylation of steroids and the oxidative demethylation and hydroxylation of drugs. Omura and Sato (7) made the important finding that the pigment was a cytochrome of the b type and showed that i t yielded an altered hemeprotein (cytochrome P-420) when solubilized with phospholipase or deoxycholate. However, hydroxylation activity was lost during the conversion to cytochrome P-420. In 1968 our laboratory reported the resolution of the enzyme system into i t s components, i n cluding a solubilized form of cytochrome P-450 which retained the a b i l i t y to hydroxylate various substrates (8,9). The present paper w i l l review further studies on the purification and reconstitution of this enzyme system and provide evidence for the occurrence of multiple forms of cytochrome P-450. The existence of a number of distinct forms helps to explain the v e r s a t i l i t y of this biological catalyst. In addition, evidence supporting the exis-

*This research was supported by Grant PCM76-14947 from the National Science Foundation and Grant AM-10339 from the United States Public Health Service. †Predoctoral Trainee of the United States Public Health Service, Grant GM-00187. °Present address, Department of Biochemistry, Duke University, Durham, North Carolina. ‡Postdoctoral Fellow of the United States Public Health Service. Present address, Argonne National Laboratory, Argonne, I l l i n o i s .

46

3.

COON E T A L .

Liver

Microsomal

Cytochrome

47

P-450

tence of two forms of NADPH-cytochrome P-450 reductase differing in apparent minimal molecular weight w i l l be described. The types of reactions generally attributed to P-450LM are shown i n Table I. In addition to hydroxylation (mixed function 1

TABLE I a

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

Reactions Attributed to Liver Microsomal Cytochrome P-450 Aromatic hydroxylation Aliphatic hydroxylation N-Dealkylation S-Dealkylation O-Dealkylation Deamination Desulfuration ^rom

N-Oxidation Sulfoxidation Dehalogenation Azoreduction Nitroreduction Peroxidation Epoxidation

a review by G i l l e t t e (10).

oxidation) reactions, including dealkylations resulting from the hydroxylation of alkyl groups at the carbon atom adjacent to a nitrogen, sulfur, or oxygen atom, the l i s t includes other reactions involving molecular oxygen, such as epoxidation, as well as chemical changes as diverse as dehalogenation and the reduction of n i tro and azo groups. A p a r t i a l l i s t of compounds which serve as substrates for P-450LM i s given i n Table I I . Included are physiol o g i c a l l y important substances as well as drugs and a variety of other foreign compounds. Some of these* are taken from an earlier review (10) and no attempt w i l l be made to document this l i s t i n view of the vast literature on the subject. I t i s impossible to estimate accurately the number of substrates attacked by cytochrome P-450 i n view of the variety of reactions catalyzed and the fact that newly manufactured xenobiotics of a l l sorts would be expected to undergo chemical transformation when incubated with this enzyme system. The alteration of drugs by the hepatic endoplasmic reticulum to form products which are inactive or, i n some cases, have quantitatively or even qualitatively different a c t i v i t i e s from the parent compounds, i s obviously of great interest to pharmacologists and toxicologists as well as to biochemists.

The abbreviations used are: P - 4 5 0 L M , l i v e r microsomal cytochrome P B , phenobarbital; B N F , G-naphthoflavone; d i l a u r o y l - G P C , dilauroylglyceryl-3-phosphorylcholine; S D S , sodium dodecyl sulfate; and P M S F , phenylmethane sulfonyl fluoride.

P-450;

American Chemical Society Library 1155 16th St., N.W. Washington, O.C. 20036

48

DRUG M E T A B O L I S M

CONCEPTS

TABLE II Examples of Substrates for Liver Microsomal Cytochrome P-450

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

Class

Examples

Drugs

Some compounds i n each category: narcotics, phenothiazine tranquilizers, anticoagulants, barbiturate sedatives, antidepressants, antihistamines, CNS stimulants, analgesicantipyretics, sulfonamides, etc.

Anesthetics

Methoxyflurane, fluroxene, etc.

Physiologically occurring compounds

Patty acids, prostaglandins, steroids (steroidal hormones, cholesterol, and b i l e acids)

Petroleum products

Aliphatic, a l i c y c l i c , and aromatic hydrocarbons

Insecticides

Dieldrin, parathion, etc.

Carcinogens

Polycyclic aromatic hydrocarbons, acetylaminofluorene, etc.

Resolution and Reconstitution of the Hydroxylation System of Liver Microsomal Membranes The mixed function oxidase system of l i v e r microsomal membranes was solubilized and resolved i n this laboratory into three fractions: Ax, containing a solubilized form of cytochrome P-450; A 2 , containing a solubilized form of NADPH-cytochrome P-450 reductase; and B, containing a heat-stable component (8,9). A l l three fractions were found to be necessary for the hydroxylation of fatty acids, drugs, and various other compounds i n the presence of NADPH and molecular oxygen (8,9,11-13). The solubilized cytochrome P-450 retained the properties of the membrane-bound form. For example, i t exhibited an electron paramagnetic resonance spectrum typical of a low spin f e r r i c hemeprotein, a carbon monoxide difference spectrum (after chemical or enzymatic reduction) with a maximum at about 450 nm, and typical type I and type II difference spectra with various substrates. Because of the glycerol and other protective agents used during detergent solubilization the cytochrome P-450 preparations contained only low levels of cytochrome P-420. The detergent-solubilized reductase retained the a b i l i t y of the membrane-bound form to transfer electrons from NADPH to cytochrome P-450 as well as to a r t i f i c i a l acceptors such as cytochrome c, and could not be replaced i n the reconstituted hydroxylation system

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

3.

COON E T AL.

Liver Microsomal

Cytochrome

P-450

49

by the steapsin-solubilized NADPH-cytochrome £ reductase described by Williams and Kamin (14) and kindly furnished by Dr. Kamin. The heat-stable microsomal factor was shown to have the solub i l i t y characteristics of a l i p i d and was subsequently identified as phosphatidylcholine (15). On the other hand, the microsomal phosphatidylethanolamine fraction was inactive. Dilauroyl-GPC and dioleoyl-GPC were at least as effective as the isolated microsomal phosphatidylcholine fraction, whereas dipalmitoyl-GPC and lysolauroyl-GPC were relatively poor. Both the phospholipid (15,16) and a substrate (17) were required for the rapid phase of reduction of cytochrome P-450 by NADPH i n the presence of reductase. To obtain f u l l a c t i v i t y i n the reconstituted enzyme system, the cytochrome P-450, reductase, and sonicated l i p i d must f i r s t be mixed i n concentrated form; the buffer and substrate are then added, and the mixture i s incubated for several minutes before the f i n a l addition of NADPH to i n i t i a t e the reaction. Small amounts of a detergent, such as deoxycholate, are sometimes also added to ensure maximal activity (18). The dilauroyl compound i s used routinely as the phospholipid i n hydroxylation assays because i t i s the most effective and because, unlike the microsomal phosphatidylcholine fraction containing unsaturated fatty acids, i t does not readily undergo chemical or enzymatic peroxidation. It should be noted that the hydroxylation a c t i v i t i e s toward most substrates i n the reconstituted enzyme system, expressed as turnover numbers (that i s , mol of substrate hydroxylated per mol of P-450m per min), are at least as great as i n microsomal suspensions. In some instances the a c t i v i t i e s are considerably higher upon reconstitution, since P-450LM can easily be made the rate-limiting component by the addition of saturating levels of reductase and phospholipid, whereas the cytochrome may not necessarily be the rate-limiting component in intact microsomes. The isolated P-450LM and NADPH-cytochrome P-450 reductase individually form small aggregates, and a mixture of the two proteins and the phospholipid behaves similarly. Reconstitution i n either the presence or absence of substrate does not cause the formation of very large aggregates or membrane-like structures, as shown by sedimentation studies, gel exclusion chromatography, and electron microscopy (19). We have reported evidence indicating that a mixture of the purified cytochrome P-450 and reductase exists as a complex with an apparent molecular weight of about 500,000 i n the presence of phospholipid and substrate (20). By reconstitution of the microsomal system we are referring to the recovery of hydroxylation a c t i v i t y when the individual components are combined i n the proper fashion, not to reassembly of the o r i ginal membrane. The latter task w i l l require greater knowledge of the various other protein, l i p i d , and carbohydrate components of microsomal membranes.

50

DRUG M E T A B O L I S M CONCEPTS

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

Purification and Characterization of Multiple Forms of Liver Microsomal Cytochrome P-450 Experimental Approaches and Proposed Nomenclature. The challenging question of whether the many catalytic a c t i v i t i e s attributed to P-450LM reside i n one or more forms of this cytochrome has been the subject of much investigation. As indicated i n several earlier reviews (10,21-23), the various patterns of a c t i v i t i e s observed i n l i v e r microsomes of animals treated with different i n ducing agents suggest that numerous forms of the cytochrome might be involved. On the other hand, kinetic data obtained with microsomal suspensions (24-26) or with the reconstituted system (13) indicated that a number of substrates act as mutually competitive inhibitors, thereby showing that they may be acted on by a single enzyme. Spectral evidence was reported suggesting the occurrence of two forms of cytochrome P-450 i n l i v e r microsomes, induced either by PB or polycyclic aromatic hydrocarbons (27-31), and cyanide titrations of microsomes indicated the possible occurrence of three forms with different binding constants (32). A study of the genetic regulation of aryl hydrocarbon hydroxylation also supported the existence of a separate cytochrome responsible for this a c t i v i ty (33), and i t was further shown- that substrate s p e c i f i c i t y resides i n the different cytochrome fractions as tested i n reconstitued systems (34-36). Recently, some of the forms of P-450LM have been obtained i n a highly purified state, thereby permitting the unequivocal conclusion that these are d i s t i n c t proteins with different catalytic a c t i v i t i e s . Our laboratory has reported the isolation of the PBinducible and BNF-inducible forms of rabbit P-450LM i n an electrophoretically homogeneous state (18,37,38) as well as the separation and characterization of other forms with different physical and catalytic properties (38). More recently, as w i l l be described below, we have characterized i n detail P-450LM isolated from PB- and BNF-induced microsomes, respectively (39), and shown that these proteins also exhibit homogeneity by the Ouchterlony immunochemical technique (20,40). Highly purified P-450LM has also been obtained recently from induced animals by two other laboratories. The isolation of the cytochrome from 3-methylcholanthrenetreated rats and rabbits and from PB-treated rats has been reported by investigators at Hoffmann-La Roche (41,42). Immunochemical studies by the same group (43) showed that P-450LM from the carcinogen-treated rats was probably homogeneous but that the preparation from PB-treated rats contained at least three hemeproteins. The cytochrome has also been isolated i n electrophoretically homogeneous form from PB- and 3-methylcholanthrene-treated rabbits by investigators i n Japan (44,45). The latter preparation was reported to contain bound carcinogen, thereby accounting for the observed optical spectrum. a n d

2

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

3.

COON E T A L .

Liver

Microsomal

Cytochrome

51

P-450

The selection of a suitable nomenclature for the multiple forms of P-450LM i s d i f f i c u l t because of our present inadequate knowledge of their properties and functions. Naming these cytochromes by the spectral characteristics of the reduced CO complex (e.g., P-450 and P-448), as frequently seen in the literature, has the serious drawback that several forms have almost identical spectra (38). Furthermore, i n no instance do the CO complexes of the cytochromes from rabbit l i v e r absorb exactly at 450 nm. A nomenclature based on the inducer used (e.g., P-450PB* P-450BNF* etc.) appears inadequate at the present time, since several forms of the hemeprotein are not yet known to be inducible. In addition, certain forms which are inducible (as by BNF) are also present i n cont r o l animals at significant levels. Although i t would be logical to name the cytochromes by function, that i s , by the substrates attacked, unfortunately the evidence so far available indicates that each of the proteins has a broad spectrum of a c t i v i t i e s , though with some quantitative differences (38). A further d i f f i culty i s that, with the a v a i l a b i l i t y of new""3rugs and other foreign compounds, the picture w i l l gradually change. Until a nomenclature based on the functions of these versatile catalysts becomes possible, i t seems advisable to use the electrophoretic method which has proved useful and reproducible (38,39). The proteins are submitted to discontinuous polyacrylamide gel electrophoresis i n the presence of SDS by the procedure of Laemmli (46), which i n our experience gives the best resolution of the electrophoretic methods presently available. Beginning with the major microsomal band of greatest electrophoretic mobility, the bands are numbered according to decreasing mobility and increasing molecular weight (38), as shown i n Figure 1. I t can be seen that P-450LM clearly corresponds to the protein band induced by PB but absent from normal and BNF-induced microsomes, and P-450i M corresponds to the protein band induced by BNF and also present i n significant amounts i n normal and PB-induced microsomes. In addition, four other forms have been p a r t i a l l y purified, and tentatively identified by their specific electrophoretic bands, as w i l l be described below. To i l l u s t r a t e the usefulness of the electrophoretic method of nomenclature, the purified cytochrome from 3-methylcholanthreneinduced rabbits, kindly furnished by Dr. A. Y. H. Lu and his associates, and the purified cytochrome from 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rabbits (47), kindly furnished by Dr. E. F. Johnson and Dr. U. Muller-Eberhard, are electrophoretically indistinguishable from our BNF-induced P-450LMi » Accordingly, i t can assumed that our three laboratories are working with the same protein and should thus avoid unnecessary confusion by the use of separate names based on the inducer used or the substrates tested by a particular research group. Clearly, i t i s advisable that forms of cytochrome P-450 isolated from l i v e r microsomes of rabbits treated in various ways, as well as from other organelles, tissues, and species, be exchanged by the various investigators for electrophoretic characterization. 2

i

f

tf

52

DRUG M E T A B O L I S M CONCEPTS

Purification of P-450LM2

^

e

steps used i n the puri-

fication of P-450ui2 from PB-induced rabbit l i v e r microsomes ( 1 8 , 37, 39) are summarized i n Table III. The microsomes are extracted with pyrophosphate to remove hemoglobin and certain other contaminating proteins. This step i s followed by solubilization of the membranes with cholate i n the presence of glycerol and other protective agents and fractionation with polyethylene glycol, which precipitates the various forms of P-450LM and leaves NADPH-cytochrome P-450 reductase i n the supernatant fraction. Column chromatography of the cytochrome fraction on DEAE-cellulose i n the presence of Renex 690, a nonionic detergent, provides further extensive purification. Finally, hydroxylapatite column chromatography gives electrophoretically homogeneous P-450ui2 ^ yields as high as 9.5% from the starting mixture of various forms of P-450LM* The y i e l d would be considerably higher (perhaps approaching twofold) i f calculated on the basis of the starting P-450LM2' concentration of this form of the cytochrome i n microsomes i s not

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

n

b u t

t n e

TABLE III Purification of P-450LM2

f

r

o

m

PB-Induced Rabbit Liver Microsomes

P-450LM

Preparation

Pyrophosphate-treated

content

13

Yield

nmol per mg protein

%

2.6-4.0

100

5.4-7.1

40

9.0-15.2

24

microsomes

3

Cholate-solubilized preparation; polyethylene glycol precipitate (8

to

10%)

DEAE-cellulose column eluate (0.1% Renex) Hydroxylapatite-silica gel column eluate ( 0 . 1 % Renex) Calcium phosphate gel eluate a

d

13.0-19.7°

9.5

13.2-20.1

The detailed procedure i s published elsewhere ( 3 9 ) .

The values given show the P - 4 5 0 L M content, based on spectral assay of the reduced CO complex, i n the various fractions i n a series of experiments. c • Whereas several forms of the cytochrome are present i n previous steps, after hydroxylapatite column chromatography the P - 4 5 0 L M 2 preparation i s electrophoretically homogeneous. d

T h i s step removes excess detergent.

3.

Liver Microsomal

COON ET A L .

Cytochrome

53

P-450

known accurately. The heme i s lost by dissociation to a varying extent, and the preparations are electrophoretically homogeneous after the hydroxylapatite chromatography step but vary i n the r e l a tive amount of holoenzyme present. Calcium phosphate gel adsorption i s usually carried out to remove excess detergent; following this step, the best preparations had a content of 20.1 nmol of holoenzyme per mg of protein (39). The procedure followed for the purification of P-450u4 from BNF-induced microsomes uses methods similar to those already described for P-450LM » A S shown i n Table IV, electrophoretically homogeneous P - 4 5 0 L M I * i s obtained i n yields as high as 9.8% from the starting mixture of various forms of P-450^; i f calculated on the basis of the starting LM^ i n microsomes, the y i e l d would be greater. The best preparations obtained following the calcium phosphate gel adsorption step had 16.8 nmol of P-450LM per mg of protein, and the best preparations of P-450LMI from PB-induced microsomes had 17.2 nmol per mg of protein. lf

2

lf

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

+

Properties of Purified P-450LM from PB-Induced Microsomes and 2

LM^ from BNF-induced Microsomes. P-450LM

f 2

r

o

m

The subunit molecular weights of P-450LMI+

PB-treated animals and

from BNF-induced ani-

TABLE IV Purification of P-450LM from BNF-induced k

Rabbit Liver Microsomes Preparation

a

P-450LM content

b

Yield

nmol per mg protein

%

Pyrophosphate-treated microsomes

2.3-2.4

100

Polyethylene glycol precipitate

3.2-6.3

61

DEAE-cellulose column eluate

6.1-9.2

16

Hydroxylapatite column chromatography

9.5-14.4°

(Calcium phosphate gel eluate)d a

9.8 c

(11.1-16.8)

The detailed procedures are published elsewhere (39). """"""""

The values given show the P-450TJI content i n a series of experiments. Q

Whereas several forms of the cytochrome are present i n previous steps, after hydroxylapatite column chromatography the P-450i Mi preparation i s electrophoretically homogeneous. T h i s step removes excess detergent. J

d

f

54

DRUG M E T A B O L I S M CONCEPTS

mals were estimated to be 50,000 and 54,000, respectively, by SDSpolyacrylamide gel electrophoresis with standardization by proteins of known molecular weight (38). Values based on amino acid analysis (kindly carried out by Dr. Karl Dus, Department of Biochemistry, St. Louis University) and allowing for the heme and carbohydrate content are 48,700 for P-450LM and 55,300 for I M ^ . These proteins tend to aggregate, and therefore exhibit molecular weights higher than the subunit values under non-denaturing conditions. For example, the apparent molecular weight of solubilized but relat i v e l y crude preparations of P-450LM was estimated by sedimentation velocity measurements, sucrose density gradient centrifugation, and gel exclusion chromatography to be about 350,000 (19), and that of purified P-450LM about 270,000 (20). These are i n f a i r l y good agreement considering the experimental error i n the measurements. Additional analytical data on these purified proteins are presented i n Table V. Heme, determined as the reduced pyridine hemo2

t o

b e

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

2

TABLE V Properties of Purified Cytochromes

Component analyzed

P-450LM2

P-450LMi

f

20

17

Heme (mol/mol protein)

1

1

Carbohydrate (residues/mol protein)

3

3

424

482

Cytochrome P-450 (nmol/mg protein)

Amino acids (residues/mol protein)

0.3

Phospholipid (mol/mol protein) Cytochrome b$, NADPH-cytochrome P-450 reductase, or NADH-cytochrome b$ reductase

none detected

0.5

none detected

chrome (48), i s present i n an amount not exceeding one mol per mol of protein subunit. Both proteins contain three carbohydrate residues (hexose or hexosamine) and, as expected from the molecular weight differences, P-450LM^ * s about 60 more amino acid residues per polypeptide chain than does P-450IM « Following extensive d i alysis of the proteins against d i s t i l l e d water to remove phosphate buffer, phospholipids were extracted and estimated by determination of the phosphorus content. The results indicated that during purification the phospholipid content i s reduced to very low levels. As shown i n the table, several other microsomal enzymes h

2

3.

COON E T A L .

Liver Microsomal

Cytochrome

55

P-450

could not be detected i n these highly purified P-450^ preparations. The Renex 690 concentration i n the purified cytochromes was determined by a method developed for nonionic detergents containing polyethoxy groups (49,50); the cobalt ammonothiocyanate complex of the detergent was extracted into chloroform, and the concentration was determined from the absorbance at 322 nm. Earl i e r studies showed that the detergent concentration i n preparations of P-450LM2 p a r t i a l l y purified by DEAE-cellulose chromatography was found to be about 0.4 mg per mg of protein (18), whereas the concentration i n the highly purified proteins i s much less (about 0.05 mg per mg of protein) (39). Evidence for Additional Forms of P-450LM» In addition to P-450LM and LM^, which have now been isolated and well characterized, as already described, l i v e r microsomes contain hemeproteins other than cytochrome b»5. These were f i r s t detected by staining for heme after the preparations were treated with SDS at 5° i n the absence of mercaptoethanol (according to the method of Welton and Aust (51)) and submitted to polyacrylamide gel electrophoresis (38). During the purification of P-450LM and LM^, the various fractions were examined for cytochrome P-450 by spectral analysis of the CO complexes and also submitted to gel electrophoresis after treatment with mercaptoethanol and SDS. It soon became evident that forms of P-450u4 other than LM and LM^ were being isolated i n other fractions. We have already reported the isolation of a fraction containing P-450LM (a mixture of LMj and LM7) and the further separation of these two forms (38) . Such preparations, as well as others which have been designated P-450ui and 1^3^, a l l exhibit spectra as the reduced CO complexes typical of cytochrome P-450. P-450LM LM , M , and LM have not yet been highly purified or thoroughly characterized, however, and u n t i l that has been accomplished the p o s s i b i l i t y should be considered that these might be electrophoretic artifacts or somehow derived from the two well characterized forms. On the other hand, these four forms do not increase i n amount when rabbits are induced by the administration of PB or BNF, as might have been expected i f they were derived from P-450^^ and LM^. Because of the lack of known inducers, we presently have no way of increasing the amounts of these four forms i n order to f a c i l i t a t e their isolation from microsomes. Figure 2 shows a l l six forms of P-450LM i n the 45,000 to 60,000 molecular weight range as seen upon SDS-polyacrylamide gel electrophoresis of the purified samples. For reference, the pattern of polypeptides from PB-induced microsomes i s also shown. The bands corresponding to P-450LM and 1^3^ are barely distinguishable i n the microsomes, but are more clearly seen to be distinct proteins when the purified samples are compared. As we have already i n d i cated, only P-450LM and LM^ give single electrophoretic bands; M l , M 3 , M315, and M 7 have been extensively purified but s t i l l contain additional protein components. Accordingly, their designation i n this manner by our electrophoretic method of nomenclature should be considered tentative.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

2

2

2

lf7

3a

lf

3a

3b

3a

2

a

?

DRUG M E T A B O L I S M

Figure 1. Slab polyacrylamide gel electrophoresis of P-450 fractions in a discontinuous buffer system. The preparations were treated with mercaptoethanol and SDS at 100° and submitted to electrophoresis according to Laemmli (46) with a 7.5% separating gel. Migration was from top to bottom. The gel was faced in 65:25:10 water - isopropanol - acetic add, stained with 0.05% Coomassie Blue in the same solvent, and destained with 80:10:10 water-isoprooanolacetic acid. The following amounts of protein were analyzed: PB-induced, normal (control), and BNF-induced microsomes, 6fig each; highly purified P-450 (from PBinduced microsomes) and P450 (from BNF-induced microsomes), 1 fig each.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

LM

LMi

LM4

Figure 2. Slab gel electrophoresis of P-450 fractions. The conditions were as in Figure 1, and the various preparations were as follows with the amounts of protein indicated: PB-induced microsomes (6 fig), LM (1.5 fig), LM (0.5 v%), LMsa (2.0 fig), LM (1.5 fig), LM (1.0 f*g), and LM (1.5 pg). LM

t

t

sb

k

7

CONCEPTS

3.

COON E T A L .

Liver Microsomal

Cytochrome

P-450

57

Immunochemical Studies on Different Forms of Cytochrome P-450. We have also used immunological techniques to compare the properties of various forms of cytochrome P-450. Such studies were begun i n collaboration with Dr. Karl M. Dus i n order to determine possible similarities between P-450 nu the camphor-hydroxylating cytochrome of Pseudomonas putida (52^54), which has been obtained i n crystalline form (55), and phenobarbital-inducible rabbit l i v e r microsomal cytochrome P-450, the f i r s t such mammalian cytochrome to be obtained i n a highly purified state (37). These two cytochromes d i f fer markedly i n substrate specificity, solubility, and the requirement of the bacterial cytochrome for an iron-sulfur protein and of P-450LM for a phospholipid for hydroxylation a c t i v i t y . Despite these differences, the two highly purified proteins showed immunological cross reaction by radioimmune assay (56,57). Similar techniques have now been used to compare the various forms of P-450LM (40). Rabbits and goats were inoculated with purified rabbit P-450LM or LMit, and i t was found that rabbits produced antibodies against P-450LM only, whereas goats produced antibodies against both cytochromes. P-450LM i s present i n only trace amounts i n uninduced rabbit l i v e r and apparently, therefore, acts as a foreign protein i n e l i c i t i n g an immune response i n the same species. A comparison of the immunochemical properties of some of the forms of P - 4 5 0 m by Ouchterlony double diffusion analysis i s shown in Figure 3. A strong precipitin line was observed i n the reaction of anti-P-450LM antibodies with PB-induced microsomes or with P-450LM * but not with P-450i M , P-450LMJ , or control or BNF-induced microsomes. Careful examination of the Ouchterlony plate showed that the p r e c i p i t i n band with P-450LM did not extend into the well containing control microsomes, thereby indicating that trace amounts of P-450LM are present i n uninduced rabbits. In other experiments, competitive binding studies with radiolabeled cytochromes confirmed that rabbit anti-LM does not cross-react with P-450T M . On the other hand, slight but significant crossreactions were detected by this technique between goat anti-LM and P-450i M , and between goat anti-LM^ and P - 4 5 0 L M . T O summarize, such immunochemical studies show that P-450LM and LM^ have significant structural differences. This conclusion i s i n accord with other information already presented. In view of the immunochemical differences observed between P-450LM and the other rabb i t hepatic cytochromes, P-450LMJ, TM^ and I M 7 , i t i s indeed surprising that the drug-inducible P-450LM resembles bacterial P-450 nu as judged both by immunological methods and by amino acid analysis of the heme peptides of these two proteins (56,57). ca

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

2

2

2

2

2

2

1

tf

7

2

2

2

J

lf

2

J

2

lf

2

2

t

2

ca

Comparison of P-450TM Preparations from Three Types of J

lf

Rabbit Liver Microsomes. Since, as already indicated, P-450LM i s present i n only trace amounts i n l i v e r microsomes of uninduced animals, one need not be concerned that the cytochrome isolated from PB-treated animals might be a mixture of somewhat different endo2

58

DRUG M E T A B O L I S M CONCEPTS

genous and induced forms. On the other hand, this p o s s i b i l i t y exi s t s with P-450LMi / since this protein i s present i n about half as great an amount i n control animals as i n those induced with BNF (cf. Figure 1). To determine whether the endogenous and induced forms of this cytochrome might be electrophoretically similar, but different i n other respects, we have recently purified P-4501^ to homogeneity from BNF-induced, PB-induced, and uninduced animals (39). No differences were seen among these three cytochrome preparations i n amino acid content, identity of the C-terminal amino acids, or spectral properties. Indeed, the absolute spectra of the oxidized forms, reduced forms, and CO complexes are i n distinguishable both i n location of the absorption maxima and i n the magnitude of the extinction coefficients. In addition, electrophoretically homogeneous P-450LMi from the three kinds of microsomes was examined by the Ouchterlony technique, using antisera to the BNF-induced preparation of the cytochrome, and the three preparations exhibited complete identity. The p o s s i b i l i t y remains, nevertheless, that the various preparations of P-450LMi d i f f e r only s l i g h t l y , perhaps i n a few amino acid residues, and are so similar that they are not distinguished by the techniques so far employed. f

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

f

f

Substrate Specificity of Forms of P-450LM» The data i n Table VI summarize some of our present information about the catalytic a c t i v i t i e s of the purified forms of P-450LM toward various subTABLE VI a

Substrate Specificity of Different Forms of Cytochrome P-450

Position of hydroxyl group in product

Substrate

p-Nitrophenetole p-Nitroanisole Benzphetamine Ethylmorphine Aniline Biphenyl M

Testosterone it it Benzo(a)pyrene a

Activity of LM 2.0

2.5 9.6

2 4 63 7a 16a

2

10.5 5.7 56.8 6.1 1.0 0.7 5.4 0.02 0.02 0.43 0.04

P-450LM

LMi+ 5.5 3.4 4.9 3.0 0.4 0.3 0.4 0.02 0.04 0.02 (Trace)

preparation LM "*1,7 7

2.0 3.3 7.7 3.0 0.6 0.4 0.6 0.32 0.02 0.07 0.5

1.0 1.8 13.6

Some of these data are taken from an earlier publication (38). The P - 4 5 0 L M I was isolated from l i v e r microsomes of BNF-induced rabbits. +

3.

COON E T A L .

Liver Microsomal

Cytochrome

59

P-450

strates. The picture which emerges i s that some forms have higher turnover numbers with certain substrates (for example, P-450LM with benzphetamine, the 4 position of biphenyl, and the 16a position of testosterone), but that a l l forms seem to have at least slight a c t i v i t y with a l l of the substrates. We have also examined the P-450i M isolated from microsomes of rabbits treated i n v a r i ous ways for a c t i v i t y toward three different substrates, as shown in Table VII. The results indicate that, within experimental error, the preparations of P-450LMIJ. from uninduced, PB-induced, and BNF-induced microsomes have the same a c t i v i t y toward the substrates tested. Such results are in accord with other properties of these three preparations, as already discussed, and provide no indication for more than one form of P-450LM . 2

i

lf

I+

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

TABLE VII Catalytic A c t i v i t i e s of P-450LMU from Various Sources

Source of P-450i M J

Substrate

p-Nitrophenetole p-Nitroanisole Benzphetamine

Uninduced animals 3.7 2.7 5.0

PB-induced animals 4.2 2.2 3.9

lf

BNF-induced animals 5.3 3.4 4.9

Purification and Characterization of Liver Microsomal NADPH-cytochrome P-450 Reductase The enzyme originally called NADPH-cytochrome £ reductase was i n i t i a l l y isolated by Horecker (58) from pig l i v e r acetone powder and was purified from lipase-solubilized l i v e r microsomes by Williams and Kamin (14) and from trypsin-solubilized microsomes by P h i l l i p s and Langdon (59). The biological role of this flavoprotein was uncertain, since cytochrome £ i s a normal constituent of mitochondria rather than microsomes. However, i t was demonstrated by Ernster and Orrenius (60) that cytochrome P-450, drug-metabolizing a c t i v i t i e s , and NADPH-cytochrome £ reductase a c t i v i t y of microsomes were a l l increased following the administration of drugs to animals. In addition, immunochemical studies established that antibodies prepared against the purified cytochrome £ reductase were effective inhibitors of microsomal NADPH-linked drug hydroxylations (61). It i s now clear from studies with the reconstituted system, u t i l i z i n g the detergent-solubilized reductase (11,62,63), that cytochrome P-450 i s the natural electron acceptor for this flavoprotein. In contrast to the detergent-solubilized flavoprotein, re-

60

DRUG M E T A B O L I S M CONCEPTS

ductase preparations purified after solubilization with a protease such as bromelain (64) or with a lipase (65) believed to contain a protease (66) do not retain significant a c t i v i t y toward cytochrome P-450 (67). Proteolytic solubilization apparently removes part of the polypeptide chain (68,69), possibly a hydrophobic portion which aids i n binding the enzyme to the membrane. As shown by gel f i l tration studies, the intact (detergent-solubilized) reductase i s capable of binding to purified P-450LM2' proteasetreated reductase apparently i s not (20). We have obtained the detergent-solubilized NADPH-cytochrome P-450 reductase from rat l i v e r microsomes i n a highly purified form and shown that i t contains both FMN and FAD (62,63). Iyanagi and Mason (70) were the f i r s t to report the presence of both f l a v i n coenzymes i n purified trypsin-solubilized and p a r t i a l l y purified detergent-solubilized reductase preparations. Recently, Dignam and Strobel (71) have obtained an apparently homogeneous reductase preparation from rat l i v e r and have made use of NADP-Sepharose aff i n i t y column chromatography to isolate the enzyme i n high y i e l d (72). Also, Yasukochi and Masters (73,74) have used 2 ,5'-ADPSepharose a f f i n i t y column chromatography to isolate the apparently homogeneous enzyme i n very good yield.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

w

h

e

r

e

a

s

t h e

,

Specificity of Reductase toward Cytochrome P-450 from Various Sources. I t appears that neither the reductase nor the phospholipid confers substrate s p e c i f i c i t y on the microsomal hydroxylation system, but that such s p e c i f i c i t y resides i n the various forms of the cytochrome, as already discussed. The rat l i v e r reductase fraction (tested e a r l i e r with less purified preparations than presently available) and the synthetically prepared dilauroyl-GPC function effectively with various cytochrome P-450 preparations. Thus, cytochrome P-450 from rat (13), rabbit (12), mouse (36), and human l i v e r (75), and from a yeast (Candida tropicalis) (76,77) as well as the carcinogen-inducible cytochrome from mouse (36) and rat l i v e r (34) a l l couple effectively with the rat l i v e r microsomal reductase in the presence of a phospholipid. Purified Reductase from Rabbit Liver Microsomes. The detergent-solubilized reductase from PB-induced rabbit l i v e r microsomes has been purified i n this laboratory as shown i n Table VIII. After extraction with pyrophosphate, the microsomes are solubilized with cholate, and this preparation i s then treated with polyethylene glycol to precipitate the various forms of P-450^ (18). Following the removal of polyethylene glycol, the supernatant fraction i s submitted to DEAE-cellulose ion exchange column chromatography. The p a r t i a l l y purified reductase i s subsequently passed over a c o l umn of 2',5'-ADP-Agarose [Agarose-bound (6-aminohexyl)-adenosine 2',5*-diphosphate, obtained from P-L Biochemicals] according to the method used by Yasukochi and Masters (73,74) for the rat and pig l i v e r reductases. The resulting preparation catalyzed the reduction of 60 umol of cytochrome £ per min per mg of protein i n an assay mixture containing 0.3 M potassium phosphate buffer, pH 7.7, at

3.

Liver Microsomal

COON E T A L .

Cytochrome

61

P-450

TABLE V I I I P u r i f i c a t i o n o f NADPH-cytochrome P-450 Reductase from Rabbit L i v e r

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

Preparation

Microsomes

Specific a c t i v i t y

Yield

ymol cyt. £ reduced per min per mg protein

%

Pyrophosphate-extracted microsomes

0.3-0.6

100

Polyethylene glycol (12%) supernatant fraction

0.6-1.0

60

DEAE-cellulose column chromatography (0.4% Renex)

6.0-12.0

42

1

1

2 ,5 -ADP-Agarose column chromatography (0.1% Renex)

59.8

26

30°. In addition, Figure 4A (sample a) shows that the purified enzyme appears to be homogeneous by SDS-polyacrylamide gel electrophoresis performed according to the method of Laemmli (46). The minimal molecular weight was estimated to be 74,000 by calibrated gel electrophoresis. When chromatography on ADP-Agarose was replaced by chromatography on DEAE-cellulose i n the presence of 0.1% deoxycholate, the p a r t i a l l y purified enzyme was further fractionated into two d i f ferent reductase preparations having specific a c t i v i t i e s of 44 and 36 ymol cytochrome £ reduced per min per mg of protein, respectively. The electrophoretic properties of the former preparation were identical to those of the purified enzyme described above. In contrast, the latter preparation contained as i t s major constituent a polypeptide with a minimal molecular weight of 68,000 (Fig. 4A, sample b). Both of these enzyme preparations contain FMN and FAD, and both catalyze the reduction of P-450LM ' I^t*, and LMi,7, as determined by NADPH oxidation i n a reconstituted system with benzphetamine as the substrate. In a l l cases the 74,000 molecular weight form was significantly more active than the 68,000 molecular weight form. When either of the reductase preparations was treated with trypsin and then submitted to SDS-polyacrylamide gel electrophoresis, a single major band was seen i n the 68,000molecular weight region, as shown i n Figure 4A (sample d). The combined results from the above experiments demonstrate that two distinct forms of NADPH-cytochrome P-450 reductase, differing by 6,000 i n apparent molecular weight, may be isolated from rabbit l i v e r microsomes. 2

62

DRUG M E T A B O L I S M

CONCEPTS

The Structural Basis of Membrane Function

Figure 3. Ouchterlony double diffusion analysis showing reaction of rabbit antibody against P-450 with microsomes or purified cytochrome preparations. The agar contained 0.5% sodium deoxychelate. The center weU contained anti-P-450 y-globulin (1.5 mg of protein) and the other wells the following: (1) PB-induced rabbit Uver microsomes (0.3 nmol of P-450 ); (2) P-450 (0.3nmol); (3) P-450 (0.9 nmol of a mixture of about equal amounts of P-450 and ); (4) P-450 (0.9 nmol); (5) BNF-induced microsomes (0.9 nmol of P-450LM)i (6) control microsomes (0.9 nmol of P-450 ). The plates were incubated at 5° for 24 hr, placed in 1% NaCl containing 0.1% deoxycholate for 24 hr, and then stained with Coomassie Blue (20). LM2

LM2

LM

LM2

LMlt1

LMl

LMl

LM4

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

LM

Figure 4.

Slab polyacrylamide gel electrophoresis of rabbit liver microsomal NADPHcytochrome P-450 reductase. The amount of protein for each sample is indicated in parentheses. (A) purified reductase a, 74,000 molecular weight form of reductase (0.5 fig); b, 68,000 molecular weight form o reductase (0.5 fig); c, mixture of samples a and b; d, mixture of samples a and b after treatment with trypsin. (B) purified reductase (e same as c above) ana pyrophosphate-treate microsomes (f through j, 24 pg each) isolated 3, 6, 9, 13, or 27 hr after a homogenate was prepared from PB-induced rabbit liver. The homogenate was kept at 4° during this time.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

3.

COON E T AL.

Liver Microsomal

Cytochrome

P-450

63

A d d i t i o n a l s t u d i e s were c a r r i e d out i n order t o determine i f the h i g h e r molecular weight form was converted t o the lower molecul a r weight form during the p r e p a r a t i o n o f the microsomal f r a c t i o n . Figure 4B (samples £ through j_) shows the r e s u l t s o f SDS-polyacrylamide g e l e l e c t r o p h o r e s i s o f pyrophosphate-extracted microsomes i s o l a t e d 3,6,9,13, or 27 hr f o l l o w i n g the p r e p a r a t i o n o f a homogenate from l i v e r s o f PB-induced r a b b i t s . During t h i s time p e r i o d , there was o n l y a small decrease i n the i n t e n s i t y o f the band c o r responding t o the 74,000 molecular weight reductase and c e r t a i n l y no i n c r e a s e i n the i n t e n s i t y of the band corresponding t o the 68,000 molecular weight reductase. In f a c t , the 68,000 molecular weight form o f the reductase appears to be more l a b i l e , and d i s appears with a h a l f - l i f e o f 21 hours a t 4 ° . T h i s experiment prov i d e s no evidence f o r a d i r e c t conversion o f the l a r g e r form o f the reductase to the s m a l l e r form during prolonged storage o f the l i v e r homogenate a t 4 ° . P r e l i m i n a r y experiments with the protease i n h i b i t o r PMSF (78) a l s o suggest t h a t the 68,000 molecular weight form i s not a p r o t e o l y t i c a r t i f a c t produced during p u r i f i c a t i o n o f the reductases. In a b r i e f r e p o r t , Satake e t a l . (69) s t a t e d t h a t the molecular weight o f the r a b b i t l i v e r microsomal reductase changed from 91,000 to 84,000 upon treatment with t r y p s i n . The cause f o r the d i s p a r i t y between t h e i r r e p o r t e d molecular weights and those we have determined i s not known. P u r i f i e d Reductase from Rat L i v e r Microsomes. Evidence has a l s o been obtained f o r the e x i s t e n c e o f two forms' o f the reductase i n PB-induced r a t l i v e r microsomes. We reported p r e v i o u s l y a p r o cedure f o r the p u r i f i c a t i o n o f the d e t e r g e n t - s o l u b i l i z e d reductase which leads t o the i s o l a t i o n o f enzyme p u r i f i e d over 100-fold with r e s p e c t t o the cytochrome £ reductase a c t i v i t y o f microsomes (62). Table IX summarizes an a l t e r n a t e procedure developed i n t h i s labor a t o r y which u t i l i z e s R e n e x - s o l u b i l i z e d microsomes (71) and a l s o y i e l d s a c t i v e cytochrome P-450 reductase, but with a s l i g h t l y higher f l a v i n content and s p e c i f i c a c t i v i t y (79). Enzyme p u r i f i e d by e i t h e r o f these procedures e x h i b i t s a s i n g l e major band when submitted t o SDS-polyacrylamide g e l e l e c t r o p h o r e s i s . However, as shown i n F i g . 5 (samples b,£, and d ) , the two p r e p a r a t i o n s d i f f e r i n e l e c t r o p h o r e t i c m o b i l i t y . Enzyme obtained as d e s c r i b e d p r e v i o u s l y (62) has a r e l a t i v e m o b i l i t y corresponding t o a minimal molec u l a r weight o f 78,000, whereas t h a t o f the enzyme obtained by the procedure o u t l i n e d i n Table IX corresponds t o 76,000. F i g u r e 5 (sample a) a l s o shows t h a t microsomes c o n t a i n two bands w i t h mobili t i e s i d e n t i c a l t o those o f the two p r e p a r a t i o n s o f p u r i f i e d enzyme d e s c r i b e d above. Although PB-induced microsomes were used as s t a r t i n g m a t e r i a l i n both p u r i f i c a t i o n procedures, the method o f drug a d m i n i s t r a t i o n was d i f f e r e n t . In the e a r l i e r work (62), both PB and h y d r o c o r t i sone were administered v i a i n t r a p e r i t o n e a l i n j e c t i o n but i n the more recent .studies (79) animals r e c e i v e d PB only i n the d r i n k i n g water. To determine T f the i s o l a t i o n o f d i f f e r e n t forms o f the r e -

64

DRUG M E T A B O L I S M CONCEPTS

TABLE IX Purification of NADPH-cytochrome P-450 Reductase from Rat Liver Microsomes

Preparation

Specific a c t i v i t y

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

umol cyt. £ reduced per min per mg p r o t e i n

Yield % a

Microsomes

0.4

100

F i r s t DEAE-cellulose column eluate (0.5% Renex); calcium phosphate gel eluate

8.6

60

Second DEAE-cellulose column eluate (0.1% deoxycholate); calcium phosphate gel eluate

38.6

37

Third DEAE-cellulose column eluate (0.1% Renex, 0.1% deoxycholate); calcium phosphate gel eluate

61.4

b

21

Cytochrome £ reductase a c t i v i t y was determined at 30° i n 0.3 M potassium phosphate, pH 7.7 (59). ^he f i n a l preparation catalyzed the oxidation of 2.4 ymol of NADPH per min per mg of protein at 30° with rabbit l i v e r P-450LM; as the electron acceptor and benzphetamine as the substrate. ductase was a result of the use of different induction procedures, l i v e r microsomes were prepared from animals receiving no treatment, or PB pretreatment by either of the two methods described above. These preparations were submitted to SDS-polyacrylamide gel electrophoresis and, as shown i n Fig. 6 (samples a through £ ) , two bands of approximately equal intensity were observed i n the 76,000 to 78,000 molecular weight region (indicated by the arrow), regardless of the manner i n which the animals were treated* These results do not suggest a preferential induction of either band by PB pretreatment, nor does i t appear that hydrocortisone alters the relative proportion of these two bands i n microsomes. To examine the p o s s i b i l i t y that the lower molecular weight form was produced during isolation of the microsomes by proteolytic degradation of the larger polypeptide, homogenization and subcellular fractionation were carried out i n the presence of the protease inhibitor PMSF. As shown i n Fig. 6 (samples £ and d), no s i g n i f i cant change i n the electrophoretic pattern of microsomes resulted from the use of this inhibitor. The p o s s i b i l i t y s t i l l remains

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

3.

COON ET

AL.

Liver Microsomal

Cytochrome

P-4S0

Figure 5. SDS-polyacrylamide gel electrophoresis of PBinduced rat liver microsomes, purified NADPH-cytochrome P-450 reductase, and trypsintreated reductase as follows. Sample (a) microsomes, 8 fig of protein; (b) reductase obtained by the procedure summarized in Table IX, 0.7 fig of protein; (c) reductase obtained by the procedure described earlier (62), 1.0 fig of protein; (d) mixture of (a) and (b); (e) mixture of both forms as in (a) following trypsintreatment, 0.7 fig of protein. Slab eel electrophoresis was performed by a slight modification of the method of Laemmli (46).

Figure 6. SDS-polyacrylamide gel electrophoresis of microsomes prepared as follows. (a) From untreated rats, 6 and 8 fig of protein; (b) from rats pretreated with PB and hydrocortisone, 6 and 8 ag of protein; (c) from rats pretreated with PB only in the drinking water, 6 and 8 fig of protein; (d) from rats pretreated as described in (c) but prepared in the presence of the protease inhibitor PMSF, 6 and 8 fig of protein. In the latter case, the homogenizing buffer was supplemented with 0.4mM PMSF, and all other buffers used in the preparation of microsomes contained O.lmM PMSF.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

66

DRUG M E T A B O L I S M CONCEPTS

t h a t a protease i n s e n s i t i v e t o PMSF may be r e s p o n s i b l e f o r the exi s t e n c e o f the 76,000 molecular weight form. However, as shown i n F i g . 5 (sample e), treatment o f e i t h e r form of the p u r i f i e d enzyme with t r y p s i n y i e l d s a s i n g l e l a r g e p o l y p e p t i d e with an apparent minimal molecular weight o f 69,000 which i s s i g n i f i c a n t l y l e s s than t h a t o f the s m a l l e r form o f the d e t e r g e n t - s o l u b i l i z e d reductase. These s t u d i e s are the f i r s t t o p r o v i d e evidence f o r the e x i s tence o f more than one form o f cytochrome P-450 reductase i n r a t o r r a b b i t l i v e r microsomes. Therefore, the p o s s i b i l i t y should be cons i d e r e d t h a t there may be d i s c r e t e pathways o f e l e c t r o n t r a n s f e r i n v o l v i n g the s e l e c t i v e i n t e r a c t i o n o f a p a r t i c u l a r form o f the r e ductase with a c e r t a i n form o f P - 4 5 0 L M . More e x t e n s i v e s t u d i e s on the s p e c i f i c i t y o f the two forms o f the reductase f o r P-450LM are undoubtedly r e q u i r e d . M u l t i p l e forms o f P-450LM w i t h d i f f e r ent c a t a l y t i c a c t i v i t i e s may e x p l a i n the broad s u b s t r a t e s p e c i f i c i t y o f the microsomal mixed f u n c t i o n oxidase; on the other hand, m u l t i p l e forms o f the reductase may p l a y an important r o l e i n det e r m i n i n g the o v e r a l l r a t e s o f metabolism o f v a r i o u s s u b s t r a t e s , p a r t i c u l a r l y under c o n d i t i o n s i n which the reductase becomes the l i m i t i n g component i n the membrane-bound system. Mechanistic Studies with P u r i f i e d Enzymes As a l r e a d y i n d i c a t e d , the p u r i f i e d and r e c o n s t i t u t e d system has been very u s e f u l i n the study o f m u l t i p l e forms o f cytochrome P-450LM and i n the e l u c i d a t i o n o f the r o l e o f the p h o s p h o l i p i d . I t has a l s o proved v a l u a b l e i n s t u d i e s on the mechanism o f the hyd r o x y l a t i o n r e a c t i o n . Only b r i e f mention o f such experiments w i l l be made, s i n c e they have r e c e n t l y been reviewed elsewhere (80-83), and are not the primary s u b j e c t o f the present paper. In a r e c e n t study we have shown t h a t h i g h l y p u r i f i e d P - 4 5 0 L M ' f r e e o f other known e l e c t r o n c a r r i e r s , c a t a l y z e s the hydroperoxide-dependent hyd r o x y l a t i o n o f a v a r i e t y o f s u b s t r a t e s i n the absence o f NADPH, NADPH-cytochrome P-450 reductase, and molecular oxygen (84). The f e r r o u s form o f the cytochrome i s a p p a r e n t l y not i n v o l v e d i n such r e a c t i o n s , as i n d i c a t e d by the l a c k o f i n h i b i t i o n by carbon monoxide. Hydrogen peroxide i s formed d u r i n g s u b s t r a t e h y d r o x y l a t i o n i n the u s u a l complete r e c o n s t i t u t e d enzyme system, and must be taken i n t o account i n determining the s t o i c h i o m e t r y o f O 2 and NADPH u t i l i z a t i o n r e l a t i v e t o product f o r m a t i o n . Whether peroxide u t i l i z a t i o n and formation represent the r e v e r s e o f a common pathway i s not y e t c l e a r . Evidence has been presented elsewhere t h a t P-450LM2 i s a two-electron acceptor and a l s o donates two e l e c t r o n s t o molec u l a r oxygen o r t o a r t i f i c i a l e l e c t r o n acceptors (85,86). As shown by stopped flow methods, the reduced p r o t e i n r e a c t s with molecular oxygen t o form two d i s t i n c t o x y f e r r o complexes (87); Complex I i s 2

2

Nordblom, G. D.,

and Coon, M. J . , unpublished data.

3.

COON E T A L .

Liver Microsomal

Cytochrome

67

P-450

formed r a p i d l y and then decays t o form Complex I I which resembles the s t e a d y - s t a t e intermediate observed by Estabrook el: a l . ( 8 8 ) with microsomal suspensions. Such s t u d i e s w i t h the p u r i f i e d c y tochrome p r o v i d e v a l u a b l e i n f o r m a t i o n necessary f o r understanding the d e t a i l e d mechanism by which one o f the atoms o f molecular oxygen i s reduced t o water by the uptake o f two e l e c t r o n s , while the other, presumably as an a c t i v a t e d s p e c i e s (most l i k e l y oxene bound to the heme i r o n atom), i s i n s e r t e d i n t o the s u b s t r a t e .

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

ABSTRACT The occurrence o f m u l t i p l e forms o f r a b b i t liver microsomal cytochrome P - 4 5 0 ( P - 4 5 0 L M ) has been e s t a b l i s h e d by the i s o l a t i o n of two forms, p h e n o b a r b i t a l - i n d u c i b l e P-450LM2 and ß-napthoflavone­ - i n d u c i b l e P-450LM4, in an e l e c t r o p h o r e t i c a l l y homogeneous s t a t e and by the p a r t i a l p u r i f i c a t i o n o f s e v e r a l o t h e r forms. The e x i s t e n c e of m u l t i p l e forms o f the cytochrome may help t o account f o r the a b i l i t y o f h e p a t i c microsomes t o hydroxylate a v a r i e t y o f drugs and other f o r e i g n compounds as w e l l as n a t u r a l l y o c c u r r i n g s u b s t r a t e s such as s t e r o i d s and f a t t y a c i d s . P - 4 5 0 L M 2 a n d L M 4 d i f f e r i n t h e i r chemical, immunological, and c a t a l y t i c p r o p e r t i e s , and are c l e a r l y d i f f e r e n t p r o t e i n s . NADPH-cytochrome P-450 reductase has a l s o been i s o l a t e d in an e l e c t r o p h o r e t i c a l l y homogeneous s t a t e and shown to c o n t a i n both FMN and FAD. Two forms o f the reductase, differing i n apparent minimal molecular weight, have been i s o l a t e d from both r a t and r a b b i t liver microsomes. A l l o f these forms o f the reductase r e t a i n the a b i l i t y t o t r a n s f e r e l e c t r o n s from NADPH t o P-450LM, as judged by NADPH o x i d a t i o n in a r e c o n s t i t u t e d enzyme system with benzphetamine as s u b s t r a t e . Literature Cited 1.

Ryan, K. J., and Engel, L. L., J. Biol. Chem. (1957) 225,

2. 3. 4. 5.

Klingenberg, M., Arch. Biochem. Biophys. (1958) 75, 376-386. G a r f i n k e l , D., Arch. Biochem. Biophys. (1958) 77, 493-509. Estabrook, R. W., Cooper, D. Y., and Rosenthal, O., Biochem. Z. (1963) 338, 741-755. Cooper, D. Y., L e v i n , S., Narasimhulu, S., Rosenthal, O.,

6.

Omura, T., Sato, R., Cooper, D. Y., Rosenthal, O., and

103-114.

and

Estabrook, R. W.,

Estabrook, R. W.,

7. 8.

Science

Fed. Proc.

(1965) (1965)

147, 24,

400-402. 1181-1189.

Omura, T., and Sato, R., J. Biol. Chem. (1964) 239, 2370-2378. Lu, A. Y. H., and Coon, M. J., J. Biol. Chem. (1968) 243, 1331-1332. 9. Coon, M. J . , and Lu, A. Y. H., in "Microsomes and Drug O x i dations" (Gillette, J. R., et al., eds.) pp. 151-161, Academic Press, New York, 1969. 1 0 . Gillette, J. R., Adv. Pharmacol. (1966) 4, 219-261.

68 11. 12. 13. 14. 15. 16.

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17. 18. 19. 20.

21. 22. 23. 24. 25. 26 27. 28. 29. 30. 31. 32. 33. 34.

DRUG M E T A B O L I S M CONCEPTS

Lu, A. Y. H., Junk, K. W., and Coon, M. J . , J . B i o l . Chem. (1969) 244, 3714-3721. Lu, A. Y. H., Strobel, H. W., and Coon, M. J . , Biochem. Biophys. Res. Commun. (1969) 36, 545-551. Lu, A. Y. H., Strobel, H. W., and Coon, M. J . , Mol. Pharmacol. (1970) 6, 213-220. Williams, C. H., Jr., and Kamin, H., J . B i o l . Chem. (1962) 237, 587-595. Strobel, H. W., Lu, A. Y. H., Heidema, J . , and Coon, M. J . , J. B i o l . Chem. (1970) 245, 4851-4854. Coon, M. J . , Autor, A. P., Boyer, R. F., and Strobel, H. W., in "Oxidases and Related Redox Systems". (Proc. 2nd Int. Symp. (King, T. E., Mason, H.S., and Morrison, M., eds.) pp. 529-553, University Park Press, Baltimore, 1973. Guengerich, F. P., and Coon, M. J . , Fed. Proc. (1975) 34, 622. van der Hoeven, T. A., and Coon, M. J . , J . B i o l . Chem. (1974) 249, 6302-6310. Autor, A. P., Kaschnitz, R. M., Heidema, J . K., and Coon, M. J., Mol. Pharmacol. (1973) 9, 93-104. Coon, M. J., Haugen, D. A., Guengerich, F. P., Vermilion, J . L., and Dean, W. L., i n "The Structural Basis of Membrane Function" (Hatefi, Y., and Djavadi-Ohaniance, L., eds.) pp. 409-427, Academic Press, New York, 1976. Conney, A. H., Pharmacol. Rev. (1967) 19, 317-366. Gelboin, H. V., Adv. Cancer Res. (1967) 10, 1-81. Gillette, J. R., Fed. Eur. Biochem. Soc. Symp. (1969) 16, 109-124. Rubin, A., Tephly, T. R., and Mannering, G. J., Biochem. Pharmacol. (1964) 13, 1007-1016. Tephly, T. R., and Mannering, G. J., Mol. Pharmacol. (1968) 4, 10-14. Wada, F., Shimakawa, H., Takasugi, M., Kotake,T.,and Sakamoto, Y., J. Biochem. (Tokyo) (1968) 64, 109-113. Sladek, N. E., and Mannering, G. J., Biochem. Biophys. Res. Commun. (1966) 24, 668-674. Alvares, A. P., Schilling, G., Levin, W., and Kuntzman, R., Biochem. Biophys. Res. Commun. (1967) 29, 521-526. Hildebrandt, A., Remmer, H., and Estabrook, R. W., Biochem. Biophys. Res. Commun. (1968) 30, 607-612. Nebert, D. W., and Gelboin, H. V., J . Biol. Chem. (1968) 243, 6250-6261. Jefcoate, C. R. E., and Gaylor, J. L., Biochemistry (1969) 8, 3464-3472. Comai, K., and Gaylor, J. L., J. Biol. Chem. (1973) 248, 49474955. Gielen, J . E., Goujon, F. M., and Nebert, D. W., J . B i o l . Chem. (1972) 247, 1125-1137. Lu, A. Y. H., Kuntzman, R., West, S., Jacobson, M., and Conney, A. H., J . B i o l . Chem. (1972) 247, 1727-1734.

3. COON ET AL.

35. 36. 37. 38. 39.

Liver Microsomal

Cytochrome

Lu, A. Y. H., and Levin, W., Biochem. Biophys. Res. Commun. (1972) 46, 1334-1339. Nebert, D. W., Heidema, J . K., Strobel, H. W., and Coon, M. J., J . Biol. Chem. (1973) 248, 7631-7636. van der Hoeven, T. A., Haugen, D. A., and Coon, M. J . , Biochem. Biophys. Res. Commun. (1974) 60, 569-575. Haugen, D. A., van der Hoeven, T. A., and Coon, M. J . , J . B i o l . Chem. (1975) 250, 3567-3570. Haugen, D. A., and Coon, M. J . , J . B i o l . Chem. (1976) i n press.

40.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

P-450

Dean, W. L., Doctoral Thesis, The University of Michigan, 1976. 41. Ryan, D., Lu, A. Y. H., Kawalek, J . , West, S. B., and Levin, W., Biochem, Biophys. Res. Commun. (1975) 64, 1134-1141. 42. Kawalek, J . C., Levin, W., Ryan, D., Thomas, P. E., and Lu, A. Y. H., Mol. Pharmacol. (1975) 11, 874-878. 43. Thomas, P. E., Lu, A. Y. H., Ryan, D., West, S. B., Kawalek, J., and Levin, W., J . B i o l . Chem. (1975) 251, 1385-1391. 44. Imai, Y., and Sato, R., Biochem. Biophys. Res. Commun. (1974) 60, 8-14. 45. Hashimoto, C., and Imai, Y., Biochem. Biophys. Res. Commun. (1976) 68, 821-827. 46. Laemmli, U. K., Nature (1970) 227, 680-685. 47. Johnson, E. F., and Muller-Eberhard, U., Abstracts, Meeting of the American Chemical Society, San Francisco, 1976. 48. Antonini, E., and Brunori, M., i n "Hemoglobins and Myoglobins in Their Reactions with Ligands" (Neuberger, A., and Tatum, L., eds.) North Holland Publishing Co., London, 1971. 49. Garewal, H. S., Anal. Biochem. (1973) 54, 319-324. 50. Goldstein, S., and Blecher, M., Anal. Biochem. (1975) 64, 130-135. 51. Welton, A. F., and Aust, S. D., Biochem. Biophys. Res. Commun. (1974) 56, 898-906. 52. Hedegaard, J . , and Gunsalus, I. C., J . B i o l . Chem. (1965) 240, 4038-4043. 53. Katagiri, M., Ganguli, B. N., and Gunsalus, I. C., J . B i o l . Chem. (1968) 243, 3543-3546. 54. Peterson, J . A., Arch. Biochem. Biophys. (1971) 144, 678-693. 55. Yu, C-A., Gunsalus, I. C., Katagiri, M., Suhara, K., and Takemori, S., J . B i o l . Chem. (1974) 249, 94-101. 56. Dus, K., L i t c h f i e l d , W. J . , and Miguel, A. G., van der Hoeven, T. A., Haugen, D. A., Dean, W. L., and Coon, M. J . , Biochem. Biophys. Res. Commun. (1974) 60, 15-21. 57. Dus, K., L i t c h f i e l d , W. J., Miguel, A. G., van der Hoeven, T. A., Haugen, D. A., Dean, W. L., and Coon, M. J . , i n "Cytochromes P-450 and b : Structure, Function, and Interaction" (Cooper, D. Y., Rosenthal, O., Snyder, R., and Witmer, C., eds.) Adv. Exp. Med. B i o l . 58, pp. 47-53, Plenum Press, New York, 1975. 5

70 58. 59. 60. 61. 62.

DRUG METABOLISM CONCEPTS

Horecker, B. L., J. Biol. Chem. (1950) 183, 593-605. P h i l l i p s , A. H., and Langdon, R. G., J. Biol. Chem. (1962) 237, 2652-2660. E r n s t e r , L., and O r r e n i u s , S., Fed. Proc. (1965) 24, 1190-1199. Masters, B. S. S., Baron, J., T a y l o r , W. E., Isaacson, E. L., and L o S p a l l u t o , J., J. Biol. Chem. (1971) 246, 4143-4150. V e r m i l i o n , J. L., and Coon, M. J., Biochem. Biophys. Res. Commun. (1974) 60, 1315-1322.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch003

63.

V e r m i l i o n , J. L., and Coon, M. J., in " F l a v i n s and F l a v o p r o t e i n s " (Singer, T. P., ed.) pp. 674-678, E l s e v i e r , Amsterdam, 1976. 64. Aust, S. D., Roerig, D. L., and Pederson, T. C., Biochem. Biophys. Res. Commun. (1972) 47, 1133-1137. 65. Masters, B. S. S., W i l l i a m s , C. H., Jr., and Kamin, H., Methods Enzymol. (1967) 10, 565-573. 66. Buege, J. A., and Aust, S. D., Biochim. Biophys. Acta (1972) 286, 433-436. 67. Coon, M. J., S t r o b e l , H. W., and Boyer, R. F., Drug Metab. Disp. (1973) 1, 92-97. 68. Welton, A. F., Pederson, T. C., Buege, J. A., and Aust, S. D., Biochem. Biophys. Res. Commun. (1973) 54, 161-167. 69. Satake, H., Imai, Y., and Sato, R., A b s t r a c t s , Ann. Meeting Jap. Biochem. Soc., 1972. 70. Iyanagi, T., and Mason, H. S., Biochemistry (1973) 12, 22972308. 71. Dignam, J. D., and S t r o b e l , H. W., Biochem. Biophys. Res. Commun. (1975) 63, 845-852. 72. Dignam, J. D., and S t r o b e l , H. W., Biochemistry (1976) in press. 73. Yasukochi, Y., and Masters, B. S. S., Fed. Proc. (1976) 35, 1654. 74. Yasukochi, Y., and Masters, B. S. S., J. Biol. Chem. (1976) in press. 75. K a s c h n i t z , R. M., and Coon, M. J., Biochem. Pharmacol. (1975) 24, 295-297. 76. Lebeault, J.-M., Lode, E. T., and Coon, M. J., Biochem. B i o phys. Res. Commun. (1971) 42, 413-419. 77. Duppel, W., Lebeault, J.-M., and Coon, M. J., Eur. J. Biochem. (1973) 36, 583-592. 78. Fahrney, D. E., and Gold, A. M., J. Am. Chem. Soc. (1963) 85, 997-1000. 79. V e r m i l i o n , J. L., D o c t o r a l T h e s i s , The U n i v e r s i t y o f Michigan, 1976. 80. Coon, M. J., van d e r Hoeven, T. A., Haugen, D. A., Guengerich, F. P., V e r m i l i o n , J. L., and B a l l o u , D. P., in "Cytochromes P-450 and b : S t r u c t u r e , Function, and I n t e r a c t i o n " (Cooper, D. Y., Rosenthal, O., Snyder, R., and Witmer, C., eds.) Adv. Exp. Med. Biol. 58, pp. 25-46, Plenum Press, New York, 1975. 5

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3. COON ET AL.

Liver Microsomal Cytochrome P-450

71

81. Coon, M. J., B a l l o u , D. P., Guengerich, F. P., Nordblom, G. D., and White, R. E. in "Iron and Copper P r o t e i n s " (Yasunobu, K. T., Mower, H. F., and H a y a i s h i , O., eds.) Adv. Exp. Med. Biol. 74, pp. 270-280, Plenum Press, New York, 1976. 82. Coon, M. J., B a l l o u , D. P., Haugen, D. A., K r e z o s k i , S. O., Nordblom, G. D., and White, R. E., T h i r d I n t e r n a t i o n a l Symposium on Microsomes and Drug Oxidations, B e r l i n , Pergamon Press, Oxford, in p r e s s , 1976. 83. Coon, M. J . , White, R. E., Nordblom, G. D., and B a l l o u , D. P., Scientific Conference on Cytochrome P-450: S t r u c t u r a l Aspects, Primošten, Yugoslavia, in p r e s s , 1976. 84. Nordblom, G. D., White, R. E., and Coon, M. J., Arch. Biochem. Biophys. (1976) 175, 524-533. 85. B a l l o u , D. P., Veeger, C., van d e r Hoeven, T. A., and Coon, M. J., FEBS L e t t . (1974) 38, 337-340. 86. Guengerich, F. P., B a l l o u , D. P., and Coon, M. J., J. Biol. Chem. (1975) 250, 7405-7414. 87. Guengerich, F. P., B a l l o u , D. P., and Coon, M. J., Biochem. Biophys. Res. Commun. (1976) 70, 951-956. 88. Estabrook, R. W., Hildebrandt, A. G., Baron, J., N e t t e r , K. J . , and Leibman, K., Biochem. Biophys. Res. Commun. (1971) 42, 132-139.

4 Resolution of Multiple Forms of Rabbit Liver Cytochrome P-450 ERIC F. JOHNSON and URSULA MULLER-EBERHARD

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch004

Scripps Clinic and Research Foundation, Department of Biochemistry, La Jolla, CA 92037

Recognition of the existence of multiple forms of cytochrome P-450 is an important step in understanding the complexities of drug and carcinogen metabolism. The characterization of individual forms of cytochrome P-450 is expected to contribute to the knowledge of their function in various metabolic pathways. Investigations undertaken in our laboratory have been directed toward resolving and characterizing forms of cytochrome P-450 from liver microsomes of rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD. Our efforts have resulted in the partial purification of three distinct forms of cytochrome P-450. These forms possess properties which are unlike those of phenobarbital-inducible cytochrome P-450. The forms of cytochrome P-450 induced by TCDD, 3-methyl-cholanthrene, and related inducers are often referred to as cytochrome P-448. These names are derived from differences in the characteristic absorption maximum of the reduced carbonyl cytochrome complex. In addition, the spectra of the cytochromes contrast in the presence of other ligands (1). The induction of cytochrome P-448 is oTten linked to increases of several monooxygenase a c t i v i t i e s . This was extensively examined with inbred mouse strains where a genetic association was found between the induction of a number of monooxygenase a c t i v i t i e s and mouse liver cytochrome P-448 (2). A prototype activity in this respect is aryl hydrocarbon hydroxylase (benzo(a)pyrene 3-hydroxylase). However, this enzyme is not induced by TCDD or related inducers nor by phénobarbital in liver microsomes of adult rabbits (3-6). Likewise, very few correlate monooxygenase a c t i v i t i e s are increased. However, acetanilide hydroxylase (6), 2-acetylaminofluorene N-hydroxylase (6), and 7-ethoxyresorufin" O-deethylase (7) are increased concomitantly with the induction of rabbiT liver cytochrome P-448 by TCDD or 3-methylcholanthrene. The presence of multiple forms of cytochrome P-450 in rabbit liver microsomes following TCDD treatment might be related to these observations. The form comprising a major portion of

72

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4.

JOHNSON AND MULLER-EBERHARD

Rabbit

Liver

Cytochrome

P-450

73

cytochrome P-450 d i s p l a y s a spectrum s i m i l a r t o that observed w i t h microsomal p r e p a r a t i o n s f o l l o w i n g TCDD treatment. T h i s cytochrome f u n c t i o n s i n the c a t a l y s i s o f TCDD-inducible monooxygenase r e a c t i o n s . A d d i t i o n a l forms o f cytochrome P-450 are a c t i v e i n those monooxygenase r e a c t i o n s which are not a s s o c i a t e d w i t h TCDD- o r p h e n o b a r b i t a l - i n d u c t ton and are d i s t i n g u i s h a b l e from the p h e n o b a r b i t a l - i n d u c i b l e cytochrome P-450. We have employed column chromatography t o separate i n d i v i d u a l forms o f cytochrome P-450. P r i o r t o chromatography, l i v e r microsomes from TCDD-treated r a b b i t s were s o l u b i l i z e d w i t h sodium c h o l a t e and f r a c t i o n a t e d by p o l y e t h y l e n e g l y c o l p r e c i p i t a t i o n as d e s c r i b e d by van der Hoeven and Coon (8). T h i s method was developed f o r the i s o l a t i o n o f the p h e n o b a r b i t a l - i n d u c i b l e form o f cytochrome P-450. The cytochromes from TCDD-induced microsomes p r e c i p i t a t e over a broad range o f p o l y e t h y l e n e g l y c o l concentrations. Therefore, f r a c t i o n s containing substantial p o r t i o n s o f the p a r t i a l l y p u r i f i e d cytochromes were pooled before chromatography HydroxylapaTite was adopted as the i n i t i a l s e p a r a t i o n medium. Two p r i n c i p a l f r a c t i o n s o f cytochrome P-450 a r e resolved u s i n g a step-wise c o n c e n t r a t i o n g r a d i e n t o f potassium phosphate b u f f e r , pH 7*4, c o n t a i n i n g g l y c e r o l (20%), EDTA (0.1 mM), and Nonidet P-40 ( O . U ) , a n o n i o n i c detergent. The two p r e p a r a t i o n s a r e r e f e r r e d t o as cytochrome P-450ab and P-450c (JJ). Cytochrome P-450ab represents ea. s i x percent (6%) o f t h e microsomal cytochrome and c o n t a i n s 5.8 nmoles o f cytochrome P-450 per mg o f p r o t e i n . The cytochrome P-450c p r e p a r a t i o n c o n t a i n s oa. eighteen percent (18$) o f the o r i g i n a l cytochrome P-450 pool and 10.0 nmoles per mg p r o t e i n . One major p e p t i d e i s detected in the cytochrome P-450c f r a c t i o n when analyzed by polyacrylamide gel e l e c t r o p h o r e s i s in the presence o f sodium dodecyl s u l f a t e . Whereas, s e v e r a l major peptides are observed i n cytochrome P-450ab

(3).

Immunological d i f f e r e n c e s between the cytochromes were i n v e s t i g a t e d w i t h antiserum produced a g a i n s t the major peptide i n the cytochrome P-450c p r e p a r a t i o n . The major peptide was separated from contaminants by polyacrylamide g e l e l e c t r o p h o r e s i s in the presence o f sodium d o d e c y l s u l f a t e . A segment o f the g e l which contained the peptide was used t o educe antibody formation i n a goat. The antiserum was examined by double d i f f u s i o n In agarose, and a s i n g l e p r e c i p i t i n l i n e Is seen when i t r e a c t s w i t h p u r i f i e d cytochrome P-450c o r w i t h sodium c h o l a t e s o l ubt 11 zed, l i v e r microsomes from untreated r a b b i t s o r r a b b i t s t r e a t e d w i t h TCDD o r p h e n o b a r b i t a l . The antiserum t o cytochrome P-450c does not c r o s s - r e a c t w i t h cytochrome P-450ab (Si). In a d d i t i o n , the antibody was t e s t e d f o r i t s a b i T l t y t o i n h i b i t microsomal monooxygenase a c t i v i t i e s . For use In these experiments, an immunoglobulin f r a c t i o n was i s o l a t e d from the goat serum by DEAE-cellulose chromatography (10). Experiments recorded i n Figure 1 d e p i c t the i n h i b i t i o n o f microsomal

74

DRUG M E T A B O L I S M

CONCEPTS

acetan H i d e h y d r o x y l a t i o n achieved by a d d i t i o n o f i n c r e a s i n g amounts o f anti-cytochrome P-450c immunoglobulin. A maximum i n h i b i t i o n o f ca. 80% i s observed. Immunoglobulin p u r i f i e d i n the same manner from nonimmune goat serum served as a n e g a t i v e c o n t r o l . N e i t h e r antibody i n t e r f e r e d w i t h the e x t r a c t i o n o r q u a n t i t a t i o n o f the r e a c t i o n products. Thus, antibody t o the major f r a c t i o n o f cytochrome P-450 (form c) i n h i b i t s an a c t i v i t y which i s induced by TCDD. Comparable amounts o f antibody do not i n h i b i t four monooxygenase a c t i v i t i e s which are not a s s o c i a t e d w i t h TCDD-induct Ion. These a c t i v i t i e s are a r y l hydrocarbon hydroxylase (benzo(a)pyrene h y d r o x y l a s e ) , amlnopyrlne N-demethyl a s e , 7-ethoxycoumarin O-deethylase, and coumarin hydroxylase

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch004

(2)Two s p e c t r a l p r o p e r t i e s o f cytochrome P-450c agree w i t h t h e concept t h a t t h i s form i s the TCDD-inducible cytochrome P-448. The Soret maximum o f the CO complex o f the reduced cytochrome Is 447 nm, s l i g h t l y lower than the v a l u e o f 449 nm observed f o r cytochrome P-450ab. In the presence o f jv-octylamine the o x i d i z e d cytochromes e x h i b i t d i f f e r e n t s p e c t r a , as shown i n F i g u r e 2. The spectrum o f cytochrome P-450c i s s i m i l a r t o the n-octylamine d i f f e r e n c e s p e c t r a observed f o r l i v e r microsomes f o l l o w i n g i n d u c t i o n by TCDD o r r e l a t e d inducers. The spectrum o f c y t o chrome P-450ab resembles t h a t o f uninduced microsomes (9). By t h r e e c r i t e r i a , cytochrome P-450ab and P-450c appear t o be d i f f e r e n t forms o f cytochrome P-450. They can be chromatog r a p h i c a l l y r e s o l v e d , a r e immunologically d i s c r e t e , and a r e s p e c t r a l l y d i s t i n g u i s h a b l e . Moreover, the s p e c i f i c i n h i b i t i o n o f a c e t a n i l l d e h y d r o x y l a t i o n by antI-cytochrome P-450c suggests that these cytochromes c a t a l y z e d i f f e r e n t monooxygenase r e a c t i o n s However, p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s experiments i n d i c a t e d cytochrome P-450ab might be composed o f more than one form o f the cytochrome. To f u r t h e r r e s o l v e t h i s p r e p a r a t i o n , DEAE-cellulose was employed. Two f r a c t i o n s were resolved and are r e f e r r e d t o as cytochrome P-450a and P-450b (9). Anti-cytochrome P-450c shows no immunological c r o s s - r e a c t i v i t 7 w i t h e i t h e r o f these forms. DEAE-cellulose i s a l s o used t o p u r i f y cytochrome P-450c t o near homogeneity (9). The i s o l a t i o n scheme i s o u t l i n e d i n F i g u r e 3. For comparison, cytochrome P-450 was p u r i f i e d from phenobarbital t r e a t e d r a b b i t s . The m a j o r i t y o f the p h e n o b a r b i t a l - i n d u c i b l e cytochrome P-450 was i n the f r a c t i o n corresponding t o cytochrome P-450a. Anti-cytochrome P-450c d i d not c r o s s - r e a c t w i t h t h i s cytochrome e i t h e r . These c o n d i t i o n s may not be optimal f o r p u r i f i c a t i o n o f t h i s form, but i t was obtained w i t h a content o f 11.0 nmoles per mg p r o t e i n and a y i e l d o f eighteen percent (18%). I t Is presumably the cytochrome P-450 LhL described by van der Hoeven et al (11). However, a d i r e c t comparison has not yet been made, and we w i l l denote t h i s form as cytochrome P-450d. The four cytochrome p r e p a r a t i o n s are summarized In Table I.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch004

JOHNSON AND MULLER-EBERHARD

Rabbit Liver

Cytochrome

Cytochrome P450 Cytochrome P450

P-450

AB

c

T 0.02 O.D. i I—N~

350nm\

400nm/

Figure 2.

1 450nm

500nm

n-Octylamine difference spectra

76

DRUG M E T A B O L I S M CONCEPTS

Table I Summary o f Cytochrome P-450 P u r i f i c a t i o n * Cytochrome P-450

Content

2

M o l e c u l a r Weight

Vleld(S)

a

9.7

48,000

b

12.0

60,000

2

c

17.7

54,500

5

d

11.0

51,000

16

3

2

51,000

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch004

1

V a l u e s a r e presented f o r t y p i c a l 2

preparations

nmoles o f cytochrome P - 4 5 0 per mg p r o t e i n based on t o t a l microsomal cytochrome P - 4 5 0 The f o u r forms o f cytochrome P-450 appear t o c o n s i s t o f peptides o f d i f f e r e n t molecular weight. These have been d e t e r mined by polyacrylamide g e l e l e c t r o p h o r e s i s i n the presence o f sodium dodecyl s u l f a t e . R e s u l t s o f these experiments a r e shown in F i g u r e 4 and a r e presented i n Table 1. With the e x c e p t i o n o f cytochrome P - 4 5 0 c , the cytochromes seem t o be a s s o c i a t e d w i t h a s i n g l e major e l e c t r o p h o r e t i c band. Cytochrome P - 4 5 0 a comprises two p r i n c i p l e p e p t i d e s ; one o f these corresponds t o the phenob a r b i t a l - i n d u c i b l e form. The f o u r cytochromes c a t a l y z e d i f f e r e n t monooxygenase r e a c t i o n s when r e c o n s t i t u t e d w i t h NADPH cytochrome P-450 reduct a s e . For these experiments, h i g h l y p u r i f i e d reductase Is r o u t i n e l y i s o l a t e d from r a b b i t l i v e r microsomes. We use a s l i g h t m o d i f i c a t i o n o f t h e a f f i n i t y chromatographic procedure d e s c r i b e d by Yasukochi and Masters (12). These reductase p r e p a r a t i o n s c a t a l y z e the r e d u c t i o n o f "55-55 nmoles o f cytochrome c/mln/mg p r o t e i n and are obtained w i t h 38fc t o 55% o v e r a l l y i e l d s . In a d d i t i o n , the p h o s p h o l i p i d d l l a u r o y l - L - a - l e c t t h l n i s included i n the assay mixture. The a c t i v i t y o f t h e i n d i v i d u a l cytochrome P - 4 5 0 p r e p a r a t i o n s w i t h a v a r i e t y o f s u b s t r a t e s i s shown i n Table I I . When r e c o n s t i t u t e d , the f o u r cytochromes show c l e a r d i f f e r e n c e s i n t h e i r metabolism o f t h e s u b s t r a t e s we have t e s t e d thus f a r . Cytochrome P-450d r a p i d l y N-demethylates benzphetamine which Is i n accord w i t h our suggestion t h a t t h i s i s the phenob a r b i t a l - i n d u c i b l e form described by van der Hoeven et al (11)* In a d d i t i o n , cytochrome P - 4 5 0 c a c t i v e l y hydroxylates a c e t a n i l i d e . T h i s was expected as t h i s form o f t h e cytochrome and t h i s

4.

JOHNSON AND MULLER-EBERHARD

Rabbit Liver Cytochrome

P-450

77

Microsomes

Sodium c h o l a t e

solubilization

Polyethylene glycol

fractionation

H y d r o x y l a p a t i t e column chromatography

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch004

Cytochrome P-450ab

Cytochrome P-*»50c

DEAEcellulose column chromatography

Cytochroie P-450a (P-450d) Figure 3.

DEAEeellulose column chromatography

Cytochrome P-450b

Cytochrome P - 4 5 0 c

Schematic of the resolution of multiple forms of cytochrome P-450

Figure 4. Polyacrylamide gel electrophoresis of four forms of cytochrome P-450 (8 fig) in the presence of sodium dodecylsulfate

78

DRUG M E T A B O L I S M

CONCEPTS

TABLE II RECONSTITUTED ENZYME ACTIVITIES Cytochrome P-450a

P-450b

P-450c

P-450d

12

4.0

2.0

51

0.42

4.1

0.03

0.03

7 ethoxyresorufIn

0.04

0.4

0.4

0.003

Acetanl1Ide

1.2

1.3

6.1

1.1

Substrate Benzphetamlne Benzo(a)pyrene

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch004

_

A c t i v i t i e s a r e expressed as moles o f product formed per mole o f cytochrome P-450. NADPH cytochrome P-450 reductase was present In excess t o ensure t h a t cytochrome P-450 was t h e r a t e l i m i t i n g component. Reactions were performed In a t o t a l volume of 1 ml c o n t a i n i n g 0.05 M Hepes b u f f e r , pH 7.4 and d i l a u r o y l - L a - l e c l t h l n (30 ug). M g C l (15 umoles) i s present when benzphetamlne and benzo(a)pyrene a r e the s u b s t r a t e s t e s t e d . Products of each s u b s t r a t e were q u a n t i t a t e d as described by the f o l l o w i n g authors: benzphetamine as HC0, T. Nash (1953) Biochem. J . 416-421; benzo(a)pyrene as 3*hydroxybenzo(a)pyrene, D. Nebert and H. Gelboin Tl968) J . B i o l . Chem. 24^, 3242-6249; 7-ethoxyr e s o r u f l n as r e s o r u f i n , M. Burke and R. Mayer (1974) Drug Metab. Dispos. 2, 245-253; a c e t a n i l i d e as hydroxyacetani1ide, K. K r i s c h and H, Staudinger (1961), Biochem. Z. 334, 312-327. Reactions were i n i t i a t e d by a d d i t i o n o f NADPH ( o T T y m o l e s ) . 2

a c t i v i t y a r e both induced by TCDD, and the microsomal enzyme i s i n h i b i t e d by anti-cytochrome P-450c. Benzo(a)pyrene i s metabolized r a p i d l y by cytochrome P-450b. The turnoveF value observed w i t h t h i s cytocrome (9) i s comparable t o that reported f o r a h i g h l y p u r i f i e d p r e p a r a t i o n o f r a t l i v e r cytochrome P-448 (J£). Both the p h e n o b a r b i t a l - and the TCDDi n d u c l b l e forms (d and c) a r e much l e s s a c t i v e w i t h t h i s s u b s t r a t e as assayed by formation o f f l u o r e s c e n t phenols. Thus, r a b b i t l i v e r does appear t o c o n t a i n an a c t i v e a r y l hydrocarbon hydroxylase ( $ ) . The r e s u l t s we have presented a r e c o n s i s t e n t w i t h t h e presence o f m u l t i p l e forms o f cytochrome P-450 i n l i v e r microsomes o f r a b b i t s t r e a t e d w i t h TCDD. The major form, cytochrome P-450c has been p u r i f i e d t o near homogeneity and has s p e c t r a l p r o p e r t i e s a s c r i b e d t o cytochrome P-448. However, I t does not a c t i v e l y hydroxylate benzo(a)pyrene when r e c o n s t i t u t e d w i t h

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch004

4. JOHNSON A N D MULLER-EBERHARD

Rabbit

Liver

Cytochrome

P-450

79

NADPH-cytochrome P-450 reductase. T h i s form o f the cytochrome i s Immunologically d i s t i n g u i s h a b l e from the o t h e r forms we have described. A r y l hydrocarbon hydroxylase (benzo(a)pyrene hydroxylase) was r e c o n s t i t u t e d w i t h cytochrome P-450b."" T h i s second form o f the cytochrome has been e x t e n s i v e l y p u r i f i e d and appears t o be composed o f a p e p t i d e o f higher molecular weight than e i t h e r cytochrome P-450c o r cytochrome P-450d. The t h i r d form, cytochrome P-450a, may be a mixture o f s e v e r a l cytochromes. Although i t i s i s o l a t e d i n a manner s i m i l a r t o the i s o l a t i o n o f the p h e n o b a r b i t a l - i n d u c i b l e cytochrome, form d, i t metabolizes benzphetamine a t a slower r a t e . None o f t h e s u b s t r a t e s t e s t e d thus f a r i s r a p i d l y metabolized by t h i s c y t o chrome. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s experiments show t h a t t h i s p r e p a r a t i o n c o n s i s t s o f s e v e r a l peptides w i t h d i f f e r e n t m o b i l i t i e s . Two prominent bands are observed. One band comigrates w i t h cytochrome P-450d, and the presence o f t h i s c y t o chrome i n the p r e p a r a t i o n may account f o r the benzphetamine Ndemethylase a c t i v i t y observed. The o t h e r major band has a f a s t e r m o b i l i t y than those o f the o t h e r t h r e e cytochrome preparations. Several i n v e s t i g a t o r s have d e s c r i b e d m u l t i p l e forms o f cytochrome P-450. These i n c l u d e two h i g h l y p u r i f i e d forms i s o l a t e d from r a t l i v e r microsomes (12) and s e v e r a l forms i s o l a t e d from mouse l i v e r microsomes"Tl4). Several forms have a l s o beenresolved from uninduced (15) and phenobarbital-induced (16) r a b b i t l i v e r microsomes. The number o f d i s c r e t e forms o f cytochrome P-450 present In r a b b i t l i v e r microsomes i s u n c e r t a i n , and the correspondence between p r e p a r a t i o n s i s o l a t e d i n v a r i o u s l a b o r a t o r i e s i s not yet c l e a r . In order t o begin a c l a r i f i c a t i o n o f t h i s s i t u a t i o n , a comparison was made between cytochrome P-450 LM^ i s o l a t e d from G-naphthaflavone induced r a b b i t 1iver microsomes (16), cytochrome P-448 i s o l a t e d from 3-methylcholanthrene induced r a b b i t l i v e r microsomes 0 7 ) > cytochrome P-450c. TCDD, B-naphthaf l a v o n e , and 3-methylcholanthrene are r e l a t e d inducers o f c y t o chrome P-450. These t h r e e cytochrome P-450 p r e p a r a t i o n s react s t r o n g l y w i t h antibody prepared a g a i n s t cytochrome P-450 LM^ o r P-450c. They a l s o e x h i b i t i d e n t i c a l m o b i l i t i e s i n p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s experiments. These experiments were performed i n Dr. M.J. Coon's and our l a b o r a t o r i e s . The work we have d e s c r i b e d i n d i c a t e s that benzphetamine N-demethylase, benzo(a)pyrene 3-hydroxylase, and a c e t a n i l i d e hydroxylase are each a s s o c i a t e d w i t h d i s t i n c t forms o f cytochrome P-450. The demonstration o f s u b s t r a t e s p e c i f i c i t i e s f o r m u l t i p l e forms o f cytochrome P-450 i s an important aspect o f t h i s i n v e s t i g a t i o n and i n d i c a t e s each form may f u n c t i o n i n d i f f e r e n t metab o l i c pathways. In a d d i t i o n , i t may be p o s s i b l e t o use these o r o t h e r assays t o detect i n d i v i d u a l cytochromes i n t i s s u e preparations. a

n

d

o

u

r

80

DRUG M E T A B O L I S M C O N C E P T S

This research was supported by National Institutes of Health Grants HD-04445 and CA-17735 (Dr. U. Muller-Eberhard) and by a California Division, American Cancer Society Junior Fellowship Number J-301 (Dr. E . F . Johnson). We would like to thank Dr. Betty Sue Siler Masters for supplying a preprint of her work, and Dr. Russell Prough for providing samples of 7-ethoxyresorufIn and resoruftn. We would also like to express our appreciation to K. Cox, G. Schwab, and M. Zounes for their help in this investi­ gation. Literature Cited: 1.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch004

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

G i l l e t t e , J . R . , Davis, D . C . , and Sasame, H . A . , Ann. Rev. Pharm. (1972) 12, 57-84. Nebert, D.W., Robinson, J . R . , Niwa, Α . , Kumaki, Κ., and Poland, A . P . , J. C e l l . Physiol. (1975) 85, 393-414. Alvares, A . P . , S c h i l l i n g , G . , and Levin, W., J. Pharm. Exp. Therap. (1970) 175, 4-11. Nebert, D.W., Robinson, J . R . , and Kon, H., J. B i o l . Chem. (1973) 248, 7637-7647. Kawalek, J . C . and Lu, A . Y . H . , Mol. Pharmacol. (1975) 11, 201-210. Atlas, S . A . , Thorgeirsson, S . S . , Boobis, A . R . , Kumaki, Κ., and Nebert, D.W., Blochem. Pharmacol. (1975) 24, 2111-2116. Prough, R., personal communication. van der Hoeven, T.A. and Coon, M . J . , J. B i o l . Chem. (1974) 249, 6302-6310. Johnson, E . F . and Muller-Eberhard, U., in preparation. Weir, D.M., "Handbook of Experimental Immunology", F.A. Davis C o . , Philadelphia (1967). van der Hoeven, T . A . , Haugen, D . A . , and Coon, M . J . , Blochem. Biophys. Res. Commun. (1974) 60, 569-575. Yasukochi, Y. and Masters, B . S . S . , J. B i o l . Chem. (1976) 251, 5337-5344. Ryan, D . , Lu, A . Y . H . , Kawalek, J., West, S . B . , and Levin, W., Biochem. Biophys. Res. Commun. (1975) 64, 1134-1141. Huang, M . I . , West, S . B . , and Lu, A . Y . H . , J. B i o l . Chem. (1976) 251, 4659-4665. Philpot, R.M. and Arinc, E., Mol. Pharmacol.(1976) 12, 483-493. Haugen, D . A . , van der Hoeven, T . A . , and Coon, M . J . , J. B i o l . Chem. (1975) 250, 3567-3570. Kawalek, J.C., Levin, W., Ryan, D . , Thomas, P . E . , and Lu, A . Y . H . , Mol. Pharmacol. (1975) 11, 874-878.

5 Enantiomeric Selectivity and Perturbation of Product Ratios as Methods for Studying the Multiplicity of Microsomal Enzymes WILLIAM F. TRAGER

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch005

Department of Pharmaceutical Sciences, School of Pharmacy, University of Washington, Seattle, WA 98195 I t i s n o w well established that t h e process o f t h e biotransformation o f ingested exogenous substances m a y form reactive electrophilic intermediates which in turn may be responsible for various kinds of potentially lethal toxicities including carcinogenesis. Covalent binding of such intermediates to nucleophilic sites in critical catalytic and structural proteins or nucleic acids is being recognized as one of the major molecular mechanisms for the manifestation of such toxicities. As a consequence, intensified investigations have been initiated t o delineate a n d categorize the multiple forms of the enzymes present in the endoplasmic reticulum of mammalian tissues since these enzymes are largely responsible for such reactions. Intimate knowledge of the specific, catalytic and structural spectrum of the system would at the very least alert society to the real scope of the problem and would undoubtedly suggest methods by which potential toxic hazards could be recognized and therefore be either circumvented or corrected. The suggestion that more than one enzyme system was responsible f o r t h e oxidations o f drugs w a s m a d e almost t w o decades ago (1,2) and was based on the differential induction of drug metabolism by phenobarbital and polycyclic aromatic hydrocarbons. Subsequent studies directed towards the elucidation of t h e multiplicity o f t h e microsomal cytochrome P - 4 5 0 e n z y m e s have segregated along two main lines of investigation, direct and indirect. Direct studies a r e t h e m o s t recent a n d a r e largely biochemical in nature. Investigators employing such studies have focused on the isolation, purification, characterization and reconstitution of both the normal enzymes and various inducible forms from several different species. Levin and Lu et al.(3-11) utilizing chromatographic e l e c t r o p h o r e t i c and immunologic techniques have succeeded i n s e p a r a t i n g and p u r i f y i n g t h e m i c r o somal cytochrome P-450 s from both r a t and r a b b i t l i v e r a f t e r i n d u c t i o n w i t h e i t h e r phénobarbital (PB) o r 3-methylcholanthrene (3-MC). S i m i l a r l y Coon e t a l . (12-15) have i s o l a t e d and charact e r i z e d m u l t i p l e forms o f cytochrome P-450 as haveAust e£aj.. (16,17). 1

81

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch005

82

DRUG M E T A B O L I S M CONCEPTS

I n d i r e c t s t u d i e s are much more numerous and have a r r i v e d a t the c o n c l u s i o n of the m u l t i p l e nature of the cytochrome P-450's present i n crude microsomal p r e p a r a t i o n s by i n f e r e n c e . G e n e r a l l y d i f f e r e n c e s produced i n the c a t a l y t i c p r o f i l e s of the system produced by some p e r t u r b a t i o n are i n t e r p r e t e d as evidence f o r m u l t i p l i c i t y . T y p i c a l l y changes produced by e i t h e r i n d u c i n g agents (1,2,19-27) o r i n h i b i t o r s (28,29) are s t u d i e d and o f t e n c o n c l u s i o n s regarding m u l t i p l i c i t y are b u r i e d w i t h i n the manus c r i p t s i n c e the o r i g i n a l i n t e n t of the study was focused on some other q u e s t i o n (27,30,31). Two recent examples from the l i t e r a t u r e i l l u s t r a t e the b a s i s of the i n d i r e c t approach. Selander, J e r i n a and Daly (32) s t u d i e d changes i n the r a t i o s of the £, m and jg-chlorophenol m e t a b o l i t e s o f chlorobenzene produced by the hemoprotein-monoxygenase system a t v a r i o u s stages o f r e s o l u t i o n . I n a d d i t i o n they s t u d i e d the d i f f e r e n t i a l e f f e c t s produced by p e r t u r b a t i o n s such as the i n d u c i n g agents PB and 3-MC, the i n h i b i t o r s , carbon monoxide, metyrapone, g l u t a t h i o n e , SKF-525a and 7,8-benzoflavone and changes i n the dependence of product r a t e on s u b s t r a t e c o n c e n t r a t i o n . These d i f f e r e n t i a l e f f e c t s allowed the authors to conclude t h a t a t l e a s t three cytochrome P-450 s d i f f e r i n g i n r e g i o s e l e c t i v i t y and o p e r a t i n g by two d i s t i n c t mechanisms were i n v o l v e d i n the h y d r o x y l a t i o n o f chlorobenzene. Burke and Bridges (33) i n a c o n c e p t u a l l y analogous approach s t u d i e d changes i n the r a t i o s of the 2- and 4- hydroxybiphenyl m e t a b o l i t e s of b i p h e n y l produced by a range of p e r t u r b a t i o n s . These authors concluded t h a t a t l e a s t two independent enzyme systems must be r e s p o n s i b l e i n order to account f o r the data. I t i s somewhat s u r p r i s i n g t h a t i n the wealth of d i f f e r e n t p e r t u r b a t i o n s such as i n d u c e r s , i n h i b i t o r s , age, sex, s p e c i e s , aging o f microsomes, d i f f e r e n t p r e p a r a t i o n s , e t c . , t h a t have been used as t o o l s to probe the m u l t i p l i c i t y of the microsomal system, stereochemical f a c t o r s have been e s s e n t i a l l y ignored. The r e mainder o f t h i s chapter w i l l focus on work done i n our l a b o r a t o r i e s (Sprague-Dawley r a t s ) and the New York S t a t e Department of H e a l t h (Wistar r a t s ) on the use o f stereochemical f a c t o r s f o r such s t u d i e s and the i n t r o d u c t i o n and i n i t i a l development o f a systematic framework f o r the i n t e r p r e t a t i o n of such data. The o r a l a n t i c o a g u l a n t w a r f a r i n , jL, e x i s t s i n two e n a n t i o meric forms. In both man (34,35) and the r a t (36-38) the 5- enantiomer i s f i v e to s i x times as potent as the R-enantiomer. Moreover, i t i s known t h a t the drug i s s t e r e o s e l e c t i v e l y metabolized both q u a n t i t a t i v e l y and q u a l i t a t i v e l y i n man (39) and t h a t the metabolic p a t t e r n s are q u a n t i t a t i v e l y a f f e c t e d by p r i o r a d m i n i s t r a t i o n of other drugs (40,41). I n the r a t the metabolic p a t t e r n s both i n v i v o (42) and i n v i t r o (43) had been r e p o r t e d but the metabolic p a t t e r n s o f the i n d i v i d u a l enantiomers were r e p o r t e d only r e c e n t l y (44,45). Since the drug i s metabolized to f i v e i s o m e r i c hydroxylated products i t i s w e l l s u i t e d f o r probing the microsomal system from the viewpoint o f 1

5.

TRAGER

Enantiomeric

Selectivity and Perturbation

of Product Ratios

83

stereochemical f a c t o r s i n order t o e x p l o r e both t h e phenomenon of drug i n t e r a c t i o n s and m u l t i p l i c i t y . The Michaelis-Menten k i n e t i c parameters f o r the formation o f the i n d i v i d u a l hydroxylated m e t a b o l i t e s from each o f the enantiomers a r e g i v e n i n Table I and F i g u r e 1. I n the a n a l y s i s t o f o l l o w the r e s u l t s obtained from each o f the enantiomers w i l l be d i s c u s s e d s e p a r a t e l y and then i n combination. Table I . Comparative K i n e t i c s o f the O x i d a t i o n o f R and j5 W a r f a r i n by Normal Microsomes From Rat L i v e r (Sprague Dawley) Apparent V (nmolesAng p r o t e i n , 10 min i n c u bations*) d*f (R) 22 0.754±0.049 22 1.65310.113 22 0.44810.052 0.50010.042 19 19 1.69310.527 m a x

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch005

Warfarin Metabolite

Apparent Km(mM)* (R)

6-hydroxy 7-hydroxy 8-hydroxy 4'-hydroxy b e n z y l i c hydroxy

0.096±0.028 0.046±0.024 0.093±0.051 0.109±0.038 0.803±0.400

(S)

(S) 6-hydroxy 7-hydroxy 8-hydroxy 4'-hydroxy b e n z y l i c hydroxy

0.032±0.011 0.050±0.010 0.19810.048 0.067±0.021 0.22110.157

0.64410.023 0.45510.013 0.20010.015 0.65210.036 0.23610.060

23 23 19 23 19

*Data were d e r i v e d from weighted l e a s t - s q u a r e s l i n e a r r e g r e s s i o n o f [ S ] / v s . [ S ] . The data i s expressed as the means 1 standard e r r o r s w i t h degrees o f freedom ( d * f ) as shown. v

H y d r o x y l a t i o n o f R-Warfarin The apparent Km f o r the 6-, 7- and 8-hydroxylation o f R w a r f a r i n a r e s t a t i s t i c a l l y i n d i s t i n g u i s h a b l e a t t h e 95% confidence l e v e l . Since a s i n g l e s u b s t r a t e i s being transformed i n a chemic a l l y and s p a t i a l l y d i s c r e t e p a r t o f t h e molecule (coumarin r i n g ) i n t o three s t r u c t u r a l l y d i s t i n c t products, i t i s reasonable t o assume based on t h i s evidence alone t h a t the products a r e formed a t a s i n g l e enzymatic s i t e and t h a t product formation i s r a t e l i m i t i n g . S i m i l a r l y the Km f o r 4 - h y d r o x y l a t i o n i s i n d i s t i n g u i s h a b l e from those o f coumarin r i n g h y d r o x y l a t i o n . Hence t h i s l i n e o f evidence i s c o n s i s t e n t w i t h a s i n g l e hemoprotein being r e s p o n s i b l e f o r a l l the aromatic hydroxylated products obtained from R-warfarin and the observed d i f f e r e n c e s i n reflect d i f f e r e n c e s i n t h e v a r i o u s a c t i v a t i o n energies f o r product format i o n i r r e s p e c t i v e o f mechanism. Although t h e Km f o r b e n z y l i c h y d r o x y l a t i o n , Table I , appears to be l a r g e r than t h e o t h e r s , the i m p r e c i s i o n o f i t s determination 1

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch005

DRUG M E T A B O L I S M

S-(->-ISOMER

CONCEPTS

R-(+)-ISOMER 7 OH?

Figure 1. The theoretical velocity for the formation of each of the hydroxylated products was calculated from the experimentally determined Km and V values and plotted as a function of log [S], Such plots approach V asymptotically and have inflection point /"SJ — Km. W<M?

maa

5.

TRAGER

Enantiomeric

Selectivity and

Perturbation

of Product Ratios

85

(due to the l a c k of a s y n t h e t i c standard) does not a l l o w i t to be d i s t i n g u i s h e d s t a t i s t i c a l l y . However, other evidence suggests t h a t the Km f o r b e n z y l i c h y d r o x y l a t i o n i s l a r g e r than the Km f o r any o f the aromatic h y d r o x y l a t i o n s . For example, a s m a l l e r q u a n t i t y of the b e n z y l i c hydroxy m e t a b o l i t e was obtained i n a s i n g l e c o n c e n t r a t i o n study r e l a t i v e to the q u a n t i t i e s of the aromatic h y d r o x y l a t i o n producte (44) but the V for benzylic h y d r o x y l a t i o n i s g r e a t e r than the Vmax f o r e i t h e r 4'- or 8-hydroxylation. This can o n l y be t r u e i f the Km f o r b e n z y l i c h y d r o x y l a t i o n i s indeed l a r g e r than the Km f o r e i t h e r 4 - o r 8-hydroxylation. The f a c t t h a t the Km f o r 6- or 7-hydroxylations are no l a r g e r than the Km f o r 4 - or 8 - h y d r o x y l a t i o n i m p l i e s t h a t the Km f o r b e n z y l i c h y d r o x y l a t i o n must be l a r g e r than the Km f o r any of the aromatic h y d r o x y l a t i o n processes. This r e s u l t i s perhaps not too s u r p r i s i n g s i n c e b e n z y l i c h y d r o x y l a t i o n r e s u l t s from a fundamentally d i f f e r e n t chemical r e a c t i o n which i n v o l v e s o x i d a t i o n a t a s t e r i c a l l y hindered a l i p h a t i c s i t e . m a x

f

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch005

1

H y d r o x y l a t i o n of j[ W a r f a r i n U n l i k e the r e s u l t s obtained f o r R-warfarin the Km f o r 8-hydroxylation from ^ - w a r f a r i n i s s i g n i f i c a n t l y l a r g e r than the Km f o r e i t h e r 6- or 7-hydroxylation. Thus, i t i s c l e a r t h a t two k i n e t i c a l l y d i s t i n c t enzymatic processes must be i n v o l v e d d e s p i t e the p r o x i m i t y of the s i t e s o f metabolic a t t a c k . I f product formation i s r a t e l i m i t i n g the c o n t r i b u t i o n of the r a t e constant f o r product formation to Km should be n e g l i g i b l e ; hence, a l l the Km should be the same w h i l e the Vmax n&y he d i f f e r e n t . I f product formation i s not r a t e l i m i t i n g but a common intermediate i s i n v o l v e d , two cases are p o s s i b l e ; the r a t e of formation i s comparable to the r a t e of d i s s o c i a t i o n of the common intermediate back to s u b s t r a t e or the r a t e of formation of the common i n t e r mediate i t s e l f may be r a t e l i m i t i n g . In the f i r s t case the product which i s formed w i t h the g r e a t e s t must a l s o have the g r e a t e s t Km w h i l e i n the second case the Km f o r a l l the products must be the same. Since 8-hydroxylation occurs w i t h the l a r g e s t Km coupled to the s m a l l e s t V ax> c o n d i t i o n s are f u l f i l l e d . Hence, there can.be no common intermediate i n the formation of 8-hydroxywarfarin and e i t h e r 6- or 7-hydroxy m e t a b o l i t e s . As was the case f o r R-warfarin, the Km f o r 4 - and b e n z y l i c h y d r o x y l a t i o n alone y i e l d l i t t l e new i n f o r m a t i o n . Although the evidence i s l e s s convincing i n t h i s case, the data suggests t h a t Km f o r b e n z y l i c h y d r o x y l a t i o n i s l a r g e r than the r e s t . n

o

n

e

o f

t l i e

a

t

o

v

e

m

f

Comparative H y d r o x y l a t i o n of the Enantiomers A h i g h degree of s t e r e o s e l e c t i v i t y as evidenced by the d i f f e r i n g V x values f o r each of the enantiomeric p a i r of products i s d i s p l a y e d . The i n d i s t i n g u i s h a b i l i t y of the Km m a

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch005

86

DRUG M E T A B O L I S M CONCEPTS

f o r 6- and 7 - h y d r o x y l a t i o n o f a given enantiomer suggests on f i r s t a n a l y s i s that a common enzymatic s i t e i s r e s p o n s i b l e f o r t h e generation o f these two products. Since arene oxides are w e l l e s t a b l i s h e d intermediates i n aromatic h y d r o x y l a t i o n processes by microsomal mixed f u n c t i o n oxidases (45), the p o s s i b l e intermediacy o f a 6,7-epoxide i n t h e i r formation i s an a t t r a c t i v e i n i t i a l P o s t u l a t e . However s e v e r a l l i n e s o f evidence argue a g a i n s t t h i s p o s s i b i l i t y . When the Km f o r the two 6-hydroxylation products are compared they are found t o j u s t d i f f e r a t the 95% confidence l e v e l . This i s n o t t r u e f o r 7-hydroxylation. I f 6- and 7-hydroxylation occur v i a the intermediacy o f an epoxide, a s t e r e o s e l e c t i v e d i f f e r e n c e i n Km f o r t h e 6-hydroxy m e t a b o l i t e s should be r e f l e c t e d i n a corresponding i d e n t i c a l d i f f e r e n c e i n the Km f o r the 7-hydroxy m e t a b o l i t e s . This does n o t appear t o be t h e case i f the Km d i f f e r e n c e s a r e r e a l . Studies o f the a r o m a t i z a t i o n s o f arene oxides have shown t h a t the r a t e - l i m i t i n g step o f t h i s process i n v o l v e s the spontaneous opening o f t h e arene oxide. The d i r e c t i o n i n which the opening occurs depends on the s t a b i l i t y o f the c a r b o c a t i o n o i d t r a n s i t i o n s t a t e (47-54). In the case o f the p o s t u l a t e d arene oxide t h i s would imply t h a t r i n g opening should occur t o y i e l d p r i m a r i l y t h e 6-hydroxylated product because o f the ortho and para d i r e c t i n g e f f e c t o f t h e coumarin l a c t o n e oxygen. However, s i n c e twice as much 7- as 6-hydroxy product i s obtained from R-warfarin i t i s reasonable to conclude t h a t an epoxide i s probably not i n v o l v e d i n t h e formation o f the 6- and 7-hydroxy m e t a b o l i t e s from t h i s isomer. S i m i l a r e l e c t r o n i c arguments permit one t o conclude t h a t a 7,8-epoxide cannot be an i n t e r m e d i a t e i n the 7-hydroxylation of R-warfarin w h i l e the non involvement o f a common i n t e r m e d i a t e i n the formation o f t h e 7- and 8-hydroxy m e t a b o l i t e s o f ^-warf a r i n i s based on the k i n e t i c arguments presented e a r l i e r . Therefore, i f 7,8-epoxidation occurs i n the metabolism o f e i t h e r isomer i t must y i e l d 8-hydroxywarfarin almost e x c l u s i v e l y . U n l i k e coumarin h y d r o x y l a t i o n , 4 ' - h y d r o x y l a t i o n i s s t e r e o s p e c i f i c f o r the j>-enantiomer but these r e s u l t s a r e c o n s i s t e n t w i t h e i t h e r a s i n g l e o r multi-enzyme system. The apparently l a r g e r Km f o r b e n z y l i c h y d r o x y l a t i o n o f both isomers suggests t h a t i f product formation i s r a t e l i m i t i n g , a l i p h a t i c h y d r o x y l a t i o n i s probably the r e s u l t o f a d i s t i n c t microsomal enzyme. Thus, the data i s c o n s i s t e n t w i t h involvement o f a t l e a s t three k i n e t i c a l l y d i s t i n c t enzymes o r enzymatic s i t e s . One enzyme would be r e s p o n s i b l e p r i m a r i l y f o r the o x i d a t i o n o f jS-warfarin to 8-hydroxywarfarin. A second could be r e s p o n s i b l e f o r a l l t h e remaining p h e n o l i c products and a t h i r d f o r the formation o f b e n z y l i c hydroxywarfarin. To f u r t h e r probe t h e system the s t u d i e s were repeated a t a s i n g l e c o n c e n t r a t i o n a f t e r pretreatment o f the animals w i t h e i t h e r PB o r 3-MC (55). The r e s u l t s o f t h i s study are shown i n Table I I (Sprague-Dawley), Table I I I (Wistar) and F i g u r e 2. The data can most r e a d i l y be analyzed i n terms o f the f o l l o w i n g

5.

TRAGER

Enantiomeric

Table I I .

Selectivity

of Product Ratios

87

I n v i v o Comparative O x i d a t i o n o f R- and Warfarin by Normal, PB and 3-MC Induced Hepatic Microsomes from Sprague-Dawley Rats.

Warfarin Metabolites

Product Normal (R)

6-hydroxy 7-hydroxy 8-hydroxy 4 -hydroxy b e n z y l i c hydroxy Total f

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch005

and Perturbation

6-hydroxy 7-hydroxy 8-hydroxy 4'-hydroxy h e n z y l i c hydroxy Total

(nmoles/mg protein/10 min*) PB

3-MC (R)

0.69±0.05 1.92±0.13 0.4310.02 0.3810.02 0.2610.03 3.68

1.3510.04 4.3610.26 0.9210.04 0.8910.03 0.4010.01 7.92

2.7810.17 0.9410.13 4.9110.09 0.1610.02 0.0910.01 8.88

(1)

(1)

(S)

0.6410.02 0.4310.01 0.1310.01 0.6410.04 0.1110.01 1.95

1.2310.07 1.7010.05 0.4210.02 1.1210.12 0.9410.04 5.41

1.7010.06 0.4010.02 0.5410.03 0.2710.04 0.0910.01 3.00

*The data i s expressed as the means 1 standard d e v i a t i o n s and represents three analyses. A w a r f a r i n c o n c e n t r a t i o n o f 0.26 mM was employed i n these s t u d i e s .

6-OH R

S

7-OH R S

8-OH R S

4'and Benzylic OH R S

Normal microsomes

o c <0

3 - Methylcholanthrene induced microsomes H W i s t a r rats

|

Sprague - Dawley rats

Figure 2. Quantitative microsomal metabolism of the enantiomers of warfarin by normal-, PB-, and 3-MC-induced animals

DRUG METABOLISM CONCEPTS

88 Table I I I .

I n v i v o Comparative O x i d a t i o n o f R- and JS-Warfarin by Normal, PB and 3-MC Induced Hepatic Microsomes from W l s t a r Rats*

Warfarin Metabolites

Product (nmoles/mg p r o t e i n / 2 0 min*) Normal

6-hydroxy 7-hydroxy 8-hydroxy 4'-hydroxy p l u s b e n z y l i c hydroxy"*" Total

1. 36±0.12 3. 3110.18 0. 5910.12 1. 5410.02 6.80

PB (R) 3.4110.61 13.4111.09 1.9510.28 3.9010.95 22.67

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch005

(i) 6-hydroxy 7-hydroxy 8-hydroxy 4'-hydroxy p l u s b e n z y l i c hydroxy Total

1. 30+0.37 1. 0010.12 0. 1010.10 2. 0110.25 4.31

1.8310.07 2.8010.56 0.9710.37 1.4610.42 7.06

3-MC <*> 8.1610.23 1.6510.41 9.3011.46 0.1010.10 19.11 (!) 3.2010.36 0.9210.15 0.6410.09 1.2810.48 6.04

*The data a r e expressed as the means 1 standard d e v i a t i o n s and represent three analyses. À w a r f a r i n c o n c e n t r a t i o n o f 0.60 mM was employed i n these s t u d i e s . +4'-Hydroxy and b e n z y l i c hydroxy were not s e p a r a t e l y q u a n t i t a t e d . r e g i o s p e c i f i c h y d r o x y l a t i o n pathways: 6- and 8 - h y d r o x y l a t i o n , 7-hydroxylation and 4 - and b e n z y l i c h y d r o x y l a t i o n . f

6- and 8-Hydroxylation I t has been suggested t h a t i n d u c t i o n by 3-MC merely a l t e r s the r e l a t i v e p r o p o r t i o n s o f m u l t i p l e forms o f cytochrome P-450 (17,56-58). I f the r e s u l t s from normal r a t s a r e considered as a c o n t r o l and a r e s u b t r a c t e d from the 3-MC r e s u l t s , the r e s i d u a l q u a n t i t i e s must be due t o the 3-MC enzyme(s). When the data a r e viewed i n t h i s way i t i s c l e a r t h a t the 3-MC i n d u c i b l e enzyme(s) a r e s t e r e o s e l e c t i v e f o r the R-enantiomer and r e g l o se l e c t i v e f o r 8-hydroxylation o f the R-enantiomer and 6-hydroxybt i o n o f the j3-enantiomer. I f the r e s u l t s f o r the R and £ enantiomers a r e summed the apparent r e g i o s e l e c t i v i t y i s g r e a t l y reduced thus emphasizing the u t i l i t y o f enantiomers as probes o f microsomal systems. I f the PB i n d u c t i o n data a r e considered i n the same manner, the PB induced enzyme(s) a r e found t o be both l e s s s t e r e o s e l e c t i v e and l e s s r e g i o s p e c i f i c . The s t e r e o s e l e c t i v i t y and r e g i o s p e c i f i c i t y observed f o r normal enzymes a r e i n t e r m e d i a t e t o t h a t observed f o r 3-MC and

5.

Enantiomeric

TRAGER

Selectivity

and

Perturbation

89

of Product Ratios

PB induced microsomes. These data, on f i r s t a n a l y s i s , a r e t h e r e f o r e c o n s i s t e n t w i t h the p o s t u l a t e that normal microsomes are comprised o f a m i x t u r e o f 3-MC and PB i n d u c i b l e systems. However, the c o n t r i b u t i o n of the 3-MC i n d u c i b l e enzyme(s) must be minimal s i n c e the s t e r e o s e l e c t i v i t y o f the normal enzymes f o r 8-hydroxylat i o n are c l o s e to t h a t d i s p l a y e d by the PB induced system and d i f f e r e n t from the 3-MC system, F i g u r e 3. Moreover, the r e g i o s e l e c t i v i t y o f the 3-MC system f o r 8-hydroxylation o f the R isomer i s i n c o n s i s t e n t w i t h the p r e f e r e n t i a l formation o f the 6- hydroxy m e t a b o l i t e i n the normal system. Thus, the r e s u l t s are c o n s i s t e n t w i t h the 6- and 8-hydroxylase a c t i v i t y o f normal microsomes being comprised p r i m a r i l y o f PB i n d u c i b l e enzyme(s). The p o s s i b i l i t y o f a minor c o n t r i b u t i o n from 3-MC and/or non i n d u c i b l e enzyme(s) cannot be excluded. Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch005

7- H y d r o x y l a t i o n The enzymatic processes r e s p o n s i b l e f o r the formation o f 7- hydroxywarfarin can be d i s t i n g u i s h e d from coumarin 6- and 8- hydroxylase(s) on the observed d i f f e r e n c e s i n s t e r e o s e l e c t i v i t y produced i n both s t r a i n s of r a t s by i n d u c t i o n w i t h 3-MC o r PB

I Control, Q |

R(+)

3 - Methylcholanthrono

ioo%

Spragu« Dawloy

S H

P h é n o b a r b i t a l , or [ ]

un

0 %

100%

ι

6 OH

7

OH

8 OH

R(+)

100%—r-

j

j

Ss(-) (-)

ioo%—L 100%

J_

_L

Figure 3.

4'

OH

Benzylic O H

_ - r

-L

J

Stereoselectivity of the microsomal metabolism of the enantiomers of warfarin by normal-, PB-, and 3-MC-induced animals

90

DRUG M E T A B O L I S M C O N C E P T S

(Figure 3)· T h i s behavior i s i n marked c o n t r a s t to the s i m i l a r i t y i n s t e r e o s e l e c t i v i t y between the s t r a i n s f o r 6- and 8h y d r o x y l a t i o n . These r e s u l t s c l e a r l y suggest t h a t 7-hydroxylat i o n i s d i s s o c i a t e d from 6-,8-hydroxylase a c t i v i t y and represents a d i s t i n c t enzymatic process l e n d i n g weight to our p r i o r c o n c l u s i o n s based on k i n e t i c evidence. The a l t e r a t i o n s i n s t e r e o s e l e c t i v i t y f o l l o w i n g i n d u c t i o n suggest t h a t normal microsomes c o n t a i n a 7-hydroxylase which i s not i n d u c i b l e and which i s d i s t i n c t from the PB i n d u c i b l e 7-hydroxylase w h i l e changes i n r e g i o s e l e c t i v i t y of h y d r o x y l a t i o n of the coumarin r i n g caused by i n d u c t i o n p a r a l l e l s those reported f o r s i m p l e r systems (32»33).

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch005

4'-Hydroxylation The enzymatic a c t i v i t y f o r t h i s process p a r a l l e l s t h a t of the PB i n d u c i b l e 6- and 8-hydroxylase system but the reduced s t e r e o s e l e c t i v i t y suggests t h a t non-PB i n d u c i b l e and non-3-MC i n d u c i b l e 4'-hydroxylase a c t i v i t y i s present i n normal microsomes. Benzylic hydroxylation The p r e f e r e n t i a l f o r m a t i o n o f R - b e n z y l i c h y d r o x y w a r f a r i n a t h i g h s u b s t r a t e c o n c e n t r a t i o n s employing microsomes obtained from e i t h e r r a t s t r a i n was r e p o r t e d r e c e n t l y (59). I n Sprague-Dawley r a t s 3-MC i n d u c t i o n f a i l e d to i n c r e a s e the s y n t h e s i s o f t h i s m e t a b o l i t e from e i t h e r enantiomer whereas PB i n d u c t i o n causes a g r e a t e r i n c r e a s e i n h y d r o x y l a t i o n a t t h i s p o s i t i o n than a t any o f the o t h e r aromatic p o s i t i o n s except 7. Moreover, PB i n d u c t i o n reverses the observed s t e r e o s e l e c t i v i t y , F i g u r e 3, and a s i m i l a r o b s e r v a t i o n has been reported f o r another a l i p h a t i c h y d r o x y l a t i o n (27). T h i s f i n d i n g demonstrates t h a t normal microsomes c o n t a i n a n o n - i n d u c i b l e b e n z y l i c hydroxylase which i s s t e r e o s e l e c t i v e f o r the R-enantiomer and which i s k i n e t i c a l l y d i f f e r ent from normal coumarin hydroxylase. D i f f e r e n c e s i n the s t e r e o c h e m i c a l preferences o b t a i n e d from the microsomal enzymes prepared from normal, PB o r 3-MC p r e t r e a t e d animals permit s e v e r a l c l a s s e s o f hemoproteins to be d i s t i n g u i s h e d . The s i m p l i s t but by no means unique h y p o t h e s i s c o n s i s t e n t w i t h our f i n d i n g s i s t h a t l i v e r microsomes from normal animals c o n t a i n a t l e a s t f o u r hemoprotein monoxygenases, enzymes A-D, d i f f e r i n g i n t h e i r s t e r e o s e l e c t i v i t y and r e g i o s p e c i f i c i t y toward the enantiomers o f w a r f a r i n These a r e c l a s s i f i e d as f o l l o w s : 1) Enzyme A, which i s present i n normal animals but i s not i n d u c i b l e by PB o r 3-MC and i s s t e r e o s e l e c t i v e and r e g i o s e l e c t i v e f o r 7- and b e n z y l i c h y d r o x y l a t i o n o f R - w a r f a r i n and 4' -hydroxylat i o n o f S-warfarin.

5.

TRAGER

Enantiomeric

Selectivity

and Perturbation

of Product Ratios

91

2) Enzyme B, which i s a l s o present i n normal animals, i s i n d u c i b l e o n l y by PB and i s r e g i o s e l e c t i v e f o r 6-, 8- and some 4 -hydroxylation. 3) Enzyme C, which i s present t o a l i m i t e d extent i n normal animals, i s i n d u c i b l e by PB o n l y , and i s r e g i o s e l e c t i v e f o r 7and b e n z y l i c h y d r o x y l a t i o n and more s t e r e o s e l e c t i v e f o r ^-warf a r i n than Enzyme A. 4) Enzyme D, which i s present i n normal animals t o a l i m i t e d extent, i s i n d u c i b l e o n l y by 3-MC, and i s s t e r e o s e l e c t i v e and r e g i o s e l e c t i v e f o r 6- and 8 - h y d r o x y l a t i o n o f R-warfarin. f

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch005

P e r t u r b a t i o n o f Product R a t i o s As s t a t e d e a r l i e r the i n d i r e c t method f o r studying m u l t i p l i c i t y i s not w e l l defined and g e n e r a l l y i n v o l v e s changes i n the system induced by v a r i o u s treatments o r p e r t u r b a t i o n s . To our knowledge n e i t h e r the assumptions nor the r u l e s f o r t h e i r a p p l i c a t i o n have been e x p l i c i t l y s t a t e d by authors r e p o r t i n g such s t u d i e s . Since a systematic framework f o r the i n t e r p r e t a t i o n o f changes i n enzymatic p r o f i l e s induced by p e r t u r b a t i o n s t o t h e system would a i d g r e a t l y i n both the i n t e r p r e t a t i o n and t h e design o f experiments, we a r e c u r r e n t l y t r y i n g t o develop such a framework u t i l i z i n g the model o f â s i n g l e s u b s t r a t e and m u l t i products (60) · I n developing the framework we assume t h a t product formation i s i r r e v e r s i b l e and t h a t the steady s t a t e k i n e t i c s o f Briggs and Haldane (61) a r e a p p l i c a b l e . For a s i n g l e s u b s t r a t e , s i n g l e enzymatic s i t e , m u l t i p l e product system, the f o l l o w i n g two cases a r e p o s s i b l e . Case 1. The s u b s t r a t e combines w i t h an enzyme t o form a s i n g l e enzyme-substrate complex which d i s s o c i a t e s t o m u l t i p l e products. That i s ;

Ε + S

D e r i v i n g the e x p r e s s i o n f o r E«S by the method of King and Altman (62) as described by Segel (63) and rearranging t o the form o f the M i c h a e l i s Menten equation y i e l d s ( E ) (S) o

Ε·S «

k

DRUG M E T A B O L I S M C O N C E P T S

k - "Hc-+k-+k, Therefore, a s i n g l e Km • — r — c h a r a c t e r i z e s the e n t i r e l system and i s independent o f e i t h e r enzyme o r s u b s t r a t e concen­ t r a t i o n . Since the i n d i v i d u a l v e l o c i t i e s f o r each o f the p r o ­ ducts a r e dPx/dt - k2(E*S), dP2/dt - k3(E*S) and dP3/dt - k4(E*S) the r a t i o of any two w i l l be constant and independent o f e i t h e r the c o n c e n t r a t i o n o f Ε o r S. K

Case 2. The s u b s t r a t e combines w i t h an enzyme t o form e n e r g e t i c a l l y d i s t i n c t E*S complexes each o f which d i s s o c i a t e s to a d i f f e r e n t product. That i s ; l k, Ε + S E*S — Ρ 1 - l k

k

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch005

k

2

k

, 5 E'S' - i

k

P

2

6 E-S" - 2 - P

3

-2

k

—

k

3

Once again d e r i v i n g the expressions f o r E»S, E«S' and E*S" by the method o f K i n g and Altman and r e a r r a n g i n g i n the form o f the Michaelis-Menten equation y i e l d s t h r e e equations: k

< .•2 5> ( k _ + k ) R k

l

E-S

+k

3

k E'S

(k

2

s

+

Km

f

ts]

6

(k k ) 3 +

+k

•l 4>

«

ts]

6

R

k

(k_ •l 4>

( k _ + k ) [ E ] [S]

+k

3

s

+

Km

2

E*S"

5

0

R

+

Km

S

Where R • k^ ( k _ + k ) ( k _ + k ) + k ( k _ + k ) 2

5

3

6

2

1

(k_ +k >

4

3

6

•AjOc^-H^) ( k _ + k ) 2

k

and Km

k

+ k

< _l"*4> < - 2 5

)

( k

-3

+ k

6

)

5

5.

TRAGER

Enantiomeric

Selectivity

and Perturbation

of Product Ratios

93

The Km again c h a r a c t e r i z e s the e n t i r e system. Since the v e l o c i t i e s f o r product formation are dPi/dt« k^(E*S),dP2/dt « k5(E*S ) and d P / d t » k (E*S") and s i n c e the r a t i o o f (dPx/dt)/dP2dt) f

3

(

k

6

+

k

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch005

Vl -2 5 k k ^ — t h e

r a t i o o f any two products i s a constant which i s

independent o f e i t h e r enzyme o r s u b s t r a t e c o n c e n t r a t i o n . I t should be noted t h a t i n d e r i v i n g the r a t e laws f o r Case 1 and Case 2 the o n l y assumptions t h a t were made were t h a t steady s t a t e k i n e t i c s were a p p l i c a b l e and t h a t product formation was i r r e v e r s i b l e . I t was not necessary t o assume t h a t product formation i s rate l i m i t i n g . Consider a microsomal p r e p a r a t i o n which a c t s on a s i n g l e s u b s t r a t e t o y i e l d m u l t i p l e products. Information r e g a r d i n g t h e enzymatic m u l t i p l i c i t y o f the p r e p a r a t i o n can now be gained by p e r t u r b i n g the system. Fundamentally two types o f p e r t u r b a t i o n s are p o s s i b l e ; 1) those t h a t a f f e c t enzyme o r s u b s t r a t e concent r a t i o n without a l t e r i n g the a c t i v e s i t e o r i n d i v i d u a l r a t e constants and 2) those t h a t can a l t e r the a c t i v e s i t e o r i n d i v i d u a l r a t e constants. Type-1 p e r t u r b e r s i n c l u d e such f a c t o r s as i n h i b i t o r s , i n ducers and s u b s t r a t e c o n c e n t r a t i o n s t u d i e s . F o r such p e r t u r b a t i o n s any s t a t i s t i c a l l y v a l i d d i f f e r e n c e i n the measured Km s o r any change i n product r a t i o s f o r e i t h e r Case 1 o r Case 2 systems i n d i c a t e s the e x i s t e n c e o f a t l e a s t two independent enzymatic s i t e s . Conversely l a c k o f changes i n Km o r product r a t i o s a r e not c o n c l u s i v e evidence f o r the presence o f a s i n g l e enzymatic s i t e but become i n c r e a s i n g l y c o n v i n c i n g as the number o f p e r t u r bations studied i s increased. Type-2 p e r t u r b e r s i n c l u d e such f a c t o r s as pH (by a f f e c t i n g i o n i z a b l e groups a t the a c t i v e s i t e ) i o n i c s t r e n g t h (change s o l v a t i o n o f the a c t i v e s i t e ) temperature ( s h i f t the steady s t a t e constants) and a l l o s t e r i c i n t e r a c t i o n s (by i n d u c i n g conformational changes a t the a c t i v e s i t e ) . For such p e r t u r b a t i o n s a l l combin a t i o n s o f changes i n Km and product r a t i o s are p o s s i b l e . That i s , three r e s u l t s are p o s s i b l e : 1) Km and product r a t i o s do n o t change. This r e s u l t i n d i c a t e s that the p e r t u r b a t i o n d i d n o t a f f e c t e i t h e r the r a t e constants o r the a c t i v e s i t e ; 2) Km changes but product r a t i o s do not (e.g., a temperature e f f e c t ) o r product r a t i o s change and Km does n o t (e.g., an a l l o s t e r i c i n t e r a c t i o n ) . These r e s u l t s i n d i c a t e a s i n g l e enzyme but are n o t c o n c l u s i v e (to have the s i t u a t i o n where two independent enzymes gave e x a c t l y the same product r a t i o s but had d i f f e r e n t Km* s o r the converse would be h i g h l y f o r t u i t o u s ) ; 3) Both Km and product r a t i o s change. This r e s u l t y i e l d s no i n f o r m a t i o n regarding multiplicity. Based on the above a n a l y s i s and assuming the model i s a p p l i c a b l e , i t would appear t h a t i n f o r m a t i o n r e g a r d i n g t h e m u l t i p l i c i t y o f microsomal cytochrome P-450's can most r e a d i l y 1

DRUG M E T A B O L I S M

94

CONCEPTS

be obtained by p e r t u r b i n g the system by f a c t o r s which o n l y a f f e c t enzyme o r s u b s t r a t e c o n c e n t r a t i o n s . To t e s t the method c o n s i d e r the w a r f a r i n data presented e a r l i e r i n Table's I and I I . R-Warfarin

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch005

The s t a t i s t i c a l i n d i s t i n g u i s h a b i l i t y o f t h e Km data f o r Itw a r f a r i n suggests t h a t a l l the h y d r o x y l a t e d products c o u l d be formed by a s i n g l e enzyme. I f one accepts the arguments advanced f o r b e n z y l i c hydroxywarfarin having a d i f f e r e n t Km then a t l e a s t two independent c a t a l y t i c s i t e s o r enzymes a r e r e s p o n s i b l e . The product r a t i o s f o r each o f the h y d r o x y l a t e d products from PB induced versus normal animals as determined from Table I I are shown below: 6-0H|^ - = 1.96 ± .15 Normal

. = 2.27 ± .20 Normal

benzylic-OHg^-1.5*

.18

The aromatic h y d r o x y l a t i o n processes a r e s t a t i s t i c a l l y i n d i s t i n g u i s h a b l e w h i l e a t t h e 90-95% confidence l e v e l b e n z y l i c h y d r o x y l a t i o n i s d i f f e r e n t i a l l y induced. ^-Warfarin The PB/Normal product r a t i o s f o r ^ - w a r f a r i n a r e shown below:

6

"

0 H

Srmal

β

X

8

"

0 H

^rmal " '

3

9 2

2 3

* °±

'

1 2

7

2 9

benzylic-OH ^

4

0 H

"

^

0

s m

a

l

Η

3

Srmal " ' fifrmal

m

Χ

·

7

9 5

5

* "

1 5

* °'

2 2

8.54 ± 0.86

In c o n t r a s t t o R - w a r f a r i n t h r e e product groupings a r e immediately apparent. These a r e 6- and 4 ' - h y d r o x y l a t i o n , 7- and 8 - h y d r o x y l a t i o n and b e n z y l i c h y d r o x y l a t i o n . Based on the d i f f e r e n c e i n Km a f u r t h e r d i s t i n c t i o n between 7- and 8- h y d r o x y l a t i o n can be made. Thus, ^ - w a r f a r i n i s h y d r o x y l a t e d by a t l e a s t f o u r d i s t i n c t enzymatic processes.

5.

TRAGER

Enantiomeric

Selectivity

and Perturbation

of Product Ratios

95

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch005

R + S-Warfarin Thus f a r the a n a l y s i s has considered a s i n g l e s u b s t r a t e o n l y . To compare two d i f f e r e n t s u b s t r a t e s one must e s t a b l i s h t h a t they are i n t e r a c t i n g w i t h the same enzymatic systems. Since b i o l o g i c a l systems are c h i r a l , enantiomers must be considered as d i f f e r e n t s u b s t r a t e s . I n the a n a l y s i s t o f o l l o w we assume t h a t the R and j> enantiomers are i n t e r a c t i n g w i t h the same group o f enzymes. Experiments are i n progress t o c l a r i f y t h i s assumption by u t i l i z i n g one enantiomer as a c o m p e t i t i v e i n h i b i t o r f o r the other. Assume f o r example, t h a t a s i n g l e enzyme generates-both 6-(R) and 6-(£)-hydroxywarfarin then the r a t i o o f β-ΟΗ^ should be constant upon p e r t u r b a t i o n o f enzyme o r s u b s t r a t e concentra­ t i o n . Therefore R R 6-0H-4- Normal « 6-0tt-=- PB

which i m p l i e s t h a t R-6-0H ^

r

a

a

l

- S-6-0H

T h i s r e l a t i o n s h i p holds f o r the comparison o f any two products and t h e r e f o r e a l l the necessary c a l c u l a t i o n s have a l r e a d y been done. Groups o f s t a t i s t i c a l l y n o n - d i s t i n g u i s h a b l e products obtained from the product r a t i o s c a l c u l a t e d above are shown below. Group 1 R-6-0H = 1.961.15 R-7-0H » 2.271.20 R-8-0H = 2.131.14 R-4'-0H * 2.341.15 S-6-0H » 1.921.12 S-4'-0H = 1.7510.22

Group 2 R-benzylic-OH 1.51.18 Group 3 ^-benzylic-OH 8.541.86

Group 4 S-7-0H = 3.951.15 Group 5 (Based on Kn) S-8-0H = 3.231.29

Thus the a n a l y s i s indicates that the combination of normal and PB induced microsomes c o n t a i n a minimum o f f i v e d i s t i n c t enzymatic processes. F u r t h e r d i f f e r e n t i a t i o n and determination o f the i n d i v i d u a l c a t a l y t i c r e g i o and s t e r e o s p e c i f i c i t i e s r e q u i r e s the study o f a g r e a t e r number o f p e r t u r b a t i o n s and u l t i m a t e l y the study o f the i s o l a t e d hemoproteins themselves. Such s t u d i e s are c u r r e n t l y i n progress. For example a s i m i l a r a n a l y s i s o f the 3-MC data i n d i c a t e s t h a t t h i s agent induces the formation o f a p p a r e n t l y abnormal isozymes which c a t a l y z e 6- and 8-hydroxyla­ t i o n . The isozymes r e s p o n s i b l e f o r the remaining h y d r o x y l a t i o n r e a c t i o n s are not induced. The a p p l i c a t i o n o f the product r a t i o technique t o the i n t e r p r e t a t i o n o f microsomal data appears t o be reasonably s u c c e s s f u l and i f v a l i d , g r e a t l y s i m p l i f i e s such i n t e r p r e t a t i o n s . The a p p l i c a b i l i t y o f t h i s method t o cases where M i c h a e l i s Menton

96

DRUG M E T A B O L I S M C O N C E P T S

k i n e t i c s a r e n o t f o l l o w e d such as: product i n h i b i t i o n , s u b s t r a t e i n h i b i t i o n , non h y p e r b o l i c k i n e t i c s i n g e n e r a l ; t o d i f f e r e n t mechanisms f o r P-450 o x i d a t i o n ; t o the i n f l u e n c e o f epoxide hydrase e t c . , a r e being s t u d i e d . Acknowledgements The author wishes t o express h i s g r a t i t u d e t o h i s c o l l a b o r a ­ t o r s and c o l l e a g u e s a t the New York S t a t e Department o f H e a l t h ; Dr.'s M i c h a e l Fasco, John Fenton, I I I and Mr. F r e d e r i c k Baker, h i s former students, D r . s Lance P o h l , W i l l i a m P o r t e r and Sidney Nelson and h i s present student, Mr. R i c h a r d Branchflower f o r t h e i r c o n t r i b u t i o n s t o the work d e s c r i b e d i n t h i s chapter. We a l s o a p p r e c i a t e the p e r m i s s i o n granted by Biochemical Pharmacology, t o reproduce the F i g u r e s and Tables. Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch005

1

Literature Cited (1) Conney, Α. Η., Gillette, J. R., Inscoe, J. Κ., Trams, E. R. and Posner, H. S., Science (1959), 130, 1478. (2) Conney, Α. Η., Pharmac. Rev. (1967), 19, 317. (3) L u , Α. Υ. Η., and L e v i n , W., Biochem. Biophys. Res. Commun. (1972), 46, 1334. (4) L e v i n , W., L u , Α. Υ. Η., Ryan, D., West, S., Kuntzman, R. and Conney, A. H., Arch. Biochem. Biophys. (1972), 153, 533. (5) L u , Α. Y. H., West, S. Β., Ryan, D. and L e v i n , W., Drug Metab. Dispos. (1973), 1, 29. (6) L e v i n , W., Ryan, D., West, S., and Lu, Α. Υ. H., J. Biol. Chem. (1974), 249, 1747. (7) Kawalek, J. C., L e v i n , W., Ryan, D., Thomas, P. E. and L u , A. Y. H., Mol. Pharmacol. (1975), 153, 533. (8) Ryan, D., Lu, Α. Υ. Η., West, S., and Levin, W., J. Biol. Chem. (1975), 250, 2157. (9) Ryan, D., Lu, Α. Y. H., Kawalek, J., West, S. B., and L e v i n , W., Biochem. Biophys. Res. Commun. (1975), 64, 1134. (10) Kawalek, J. C., L e v i n , W., Ryan, D. and L u , Α. Υ. Η., Drug Metab. Dispos. (1976), 4, 190. (11) Thomas, P. E., Lu, Α. Υ. Η., Ryan, D., West, S. B., Kawalek, J. and Levin, W., J. Biol. Chem. (1976), 251, 1385. (12) Van der Hoeven, T. A. and Coon, M. J., J. Biol. Chem. (1974), 249, 6302. (13) Van der Hoeven, Τ. Α., Haugen, D. A. and Coon, M. J., Biochem. Biophys. Res. Commun. (1974), 60, 569. (14) Haugen, D. Α., Van d e r Hoeven, T. A. and Coon, M. J., J . Biol. Chem. (1975), 250, 3567. (15) Haugen, D. A. and Coon, M. J., Pharmacol. (1975), 17, 186. (16) Haugen, D. Α., Coon, M. J. and Nebert, D. W., J. Biol. Chem. (1976), 251, 1817. (17) Welton, A. F. and Aust, S. D., Biochem. Biophys. Res. Commun. (1974), 56, 898.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch005

5. TRAGER

Enantiomeric

Selectivity

and Perturbation

of Product Ratios

97

(18) Welton, A. F., O'Neal, F. O., Chaney, L. C. and Aust, S. D., J. Biol. Chem. (1975), 250, 5631. (19) L e v i n , W., A l v a r e s , Α., Jacobson, M. and Kuntzman, R., Biochem. Pharmacol. (1969), 18, 883. (20) Pederson, T. C. and Aust, S. D., Biochem. Pharmacol. (1969), 18, 883. (21) Gloumann, Η., Chem. Biol. Interactions (1970), 2, 369. (22) Aust, S. D., and Stevens, J. B., Biochem. Pharmacol. (1971), 20, 1061. (23) Conney, A. H., L u , Α. Υ. Η., L e v i n , W., Somogyi, Α., West, S., Jacobson, Μ., Ryan, D. and Kuntzman, R., Drug Metab. Dispos. (1973), 1, 199. (24) Stonard, M. D., Biochem. Pharmacol. (1975), 24, 1959. (25) Hook, G. E. R., Orton, T. C., Moore, J. A. and Lucifer, G. W., Biochem. Pharmacol. (1975), 24, 335. (26) U l b r i c h , V., Wever, P., and Woolenberg, P., Biochem. Biophys. Res. Commun. (1975), 64, 808. (27) May, H. E., Bose, R. and Reed, D. J., Biochem, (1975), 14, 4723. (28) W e r r i n g l o e r , J. and Estabrook, R. W., Arch. Biochem. Biophys. (1975), 167, 270. (29) Wiebel, F. J. and G e l b o i n , H. V., Biochem. Pharmacol. (1975) 24, 1511. (30) Townsend, M. G., Odam, Ε. M. and Page, J. M. J., Biochem. Pharmacol. (1975), 24, 729. (31) Juchau, M. R., Namkung, M. J., B e r r y , D. L. and Zachariah, P. K., Drug Metab. Dispos. (1975), 3, 494. (32) Selander, H. G., Jerina, D. M. and Daly, J. W., A r c h i v . Biochem. Biophys. (1975), 168, 309. (33) Burke, M. D. and B r i d g e s , J. W., X e n o b i o t i c a (1975),5.,357. (34) Hewick, D. and McEwen, J., J. Pharm. Pharmacol. (1973), 25, 458. (35) O ' R e i l l y , R. Α., Clin. Pharmacol. Therap. (1974), 16, 348. (36) E b l e , Ν., West, Β. and L i n k , Κ., Biochem. Pharmacol. (1966), 15, 1003. (37) Breckenridge, A. and L'E Orme, M., Life Sciences (1974), 11 (Part II), 337. (38) Hewick, D., J. Pharm. Pharmacol. (1972), 24, 661. (39) Chan, Κ. Κ., Lewis, R. J. and Trager, W. F., J. Med. Chem. (1972), 15, 1265. (40) Lewis, R. J., Trager, W. F., Chan, Κ. Κ., Breckenridge, Α., L'E Orme, M., Rowland, R. and Scharry, W., J. Clin. Invest. (1974), 53, 1607. (41) O ' R e i l l y , R. Α., New E n g l . J. Med. (1976), 295, 354. (42) Barker, W. M., Hermodson, M. A. and L i n k , K. P., J . Pharmacol. Exp. Therap. (1970), 171, 307. (43) Ikeda, Μ., Ullrich, V. and Staudinger, Η., Biochem. Pharmacol. (1968), 17, 1663. (44) P o h l , L. R., Nelson, S. D., P o r t e r , W. R., Trager, W. F., Fasco, M. J., Baker, F. D. and Fenton, J. W., Biochem.

DRUG M E T A B O L I S M C O N C E P T S

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch005

98

Pharmacol. (1976), 25, 2153. (45) P o h l , L. R., B a l e s , R. and Trager, W. F., Res. Commun. Chem. Path. Pharmacol., in press. (46) Jerina, D. M. and Daly, J. W., Science (1974), 185, 573. (47) Daly, J. W., Jerina, D. H. and Witkop, Β., E x p e r i e n t i a (1972), 28, 1129. (48) Kasperek, G. J., and Bruce, T. C., J. Amer. Chem. Soc. (1972), 94, 198. (49) Kasperek, G. J., Bruice, T. C., Y a g i , H. and Jerina, D. Μ., J . Chem. Soc. D., Chem. Commun. (1972), 784. (50) Y a g i , Η., Jerina, D. Μ., Kasperek, G. J. and B r u i c e , T. C., Proc. N a t ' l . Acad. Sci., U.S.A. (1972), 69, 1985. (51) Kasperek, G. J., B r u i c e , T. C., Y a g i , H., Kaubisch, N. and Jerina, D. M., J. Amer. Chem. Soc. (1972), 94, 7876. (52) J e r i n a , D. Η., Kaubisch, N. and Daly, J. W., Proc. N a t ' l . Acad. Sci., U.S.A. (1971), 68, 2545. (53) Kaubisch, Ν., Daly, J. and Jerina, D. Μ., Biochem. (1972), 11, 3080. (54) Richardson, J. D., B r u i c e , T. C., Warasziewiez, S. M. and B e r c h t o l d , G. Α., J. Org. Chem. (1974), 39, 2088. (55) P o h l , L. R., P o r t e r , W. R., Trager, W. F., Fasco, M. J. and Fenton, J. W., Biochem. Pharmacol., in press. (56) Comai, K. and Gaylor, J. L., J. Biol. Chem. (1973), 248, 2947. (57) A l v a r e z , A. P. and Siekevitz, P., Biochem. Biophys. Res. Commun. (1973), 54, 923. (58) Muna, W. J., "Cytochrome P-450 and Cytochrome b L e v e l s in Rat L i v e r Microsomes During Prolonged A d m i n i s t r a t i o n o f Pheno­ barbital: Changes in the Topographical R e l a t i o n s h i p s o f Cytochrome b ," Ph.D. T h e s i s , U n i v e r s i t y o f Washington (1974). (59) P o h l , L. R., Nelson, S. D., Garland, W. A. and Trager, W. F., Biomed. Mass Spect. (1975), 2, 23. (60) P o r t e r , W. R., Branchflower, R. V. and Trager, W. F., Biochem. Pharmacol., in p r e s s . (61) B r i g g s , G. E. and Haldane, J. B. S., Biochem. J. (1925), 19, 388. (62) K i n g , E. L. and Altman, C., J. Phys. Chem. (1956), 60, 1375. (63) Segel, I. Η., "Enzyme Kinetics," John Wiley and Sons, I n c . , New York (1975), 506-515. 5

5

6 Role of Purified Cytochrome P-448 and Epoxide Hydrase in the Activation and Detoxification of

Benzo[α]pyrene

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

W. LEVIN, A. W. WOOD, A. Y. H. LU, D. RYAN, S. WEST, and A. H. CONNEY Department of Biochemistry and Drug Metabolism, Hoffman-La Roche Inc., Nutley, NJ 07110 D. R. THAKKER, H. YAGI, and D. M. JERINA National Institute of Arthritis, Metabolism and Digestive Disesases, National Institutes of Health, Bethesda, MD 20014

The liver microsomal monoxygenase system is a membranebound, multicomponent electron transport system which is responsible for the oxidative metabolism of a variety of endogenous and exogenous substrates such as steroids, fatty acids, drugs, insecticides and chemical carcinogens (1). Of the three components involved in microsomal drug metabolism (cytochrome P-450, NADPH-cytochrome c reductase and phosphatidylcholine), cytochrome P-450 is undoubtedly the most important because of its vital role in oxygen activation, substrate binding and in determining the overall substrate specificity of the enzyme system (2,3). The rate at which various compounds are metabolized by this enzyme system varies widely and depends on the species, strain, age, tissue and pretreatment of the animal (1). Over the last decade, numerous studies have suggested that different forms of cytochrome P-450 exist in liver microsomes, and, more recently, the purification and reconstitution of the monoxygenase system have established the existance of multiple forms of cytochrome P-450 having different substrate specificities. The various purified forms of cytochrome P-450 differ from one another not only in their substrate specificity but also in their spectral and immunological properties as well as in their minimum molecular weights as determined by SDS-gel electrophoresis (4-9). The importance of this rather versatile enzyme system has become increasingly apparent during the last 10 years. Today we live in a society that has become increasingly aware of the potential dangers of environmental pollutants which include chemical carcinogens. It has been estimated that 60-80% of a l l human cancers are caused by environmental factors (10-12), and many chemicals in our environment are metabolically activated to ultimate carcinogens by the microsomal monoxygenase system (13). One 99

100

DRUG M E T A B O L I S M C O N C E P T S

of these environmental p o l l u t a n t s , the p o l y c y c l i c aromatic hydro­ carbon benzolajpyrene (BP), may b e o n e o f t h e m o s t p r e v a l e n t c h e m i c a l c a r c i n o g e n s t o w h i c h man i s e x p o s e d (14).

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

1

S i n c e p o l y c y c l i c h y d r o c a r b o n s s u c h a s BP a r e c h e m i c a l l y i n e r t , t h e i r c a r c i n o g e n i c i t y i s thought to r e s u l t from metabolic a c t i v a t i o n by t h e m i c r o s o m a l monoxygenase system t o a c h e m i c a l l y reactive iηtermediate(s)(13,15-17). The o x i d a t i v e m e t a b o l i s m o f BP p r o c e e d s i n i t i a l l y t h r o u g h t h e f o r m a t i o n o f r e a c t i v e a r e n e oxides which spontaneously isomerize to phenols, are hydrated t o d i h y d r o d i o l s by microsomal epoxide hydrase or are c o n j u g a t e d with glutathione v i a the soluble glutathione S-transferases (ljj>« 16,18). In o r d e r t o e l u c i d a t e t h e r o l e o f m e t a b o l i s m i n t h e m u t a g e n i c i t y and c a r c i n o g e n i c i t y o f BP, a b a s i c understanding o f t h e p r o p e r t i e s and mechanism o f a c t i o n o f t h e enzymes involved i n t h e a c t i v a t i o n a n d i n a c t i v a t i o n o f BP i s e s s e n t i a l . Thus, t h e p u r i f i c a t i o n and r e c o n s t i t u t i o n o f t h e monoxygenase s y s t e m (19) i n t h e p r e s e n c e o r absence o f p u r i f i e d e p o x i d e hydrase (20) has e n a b l e d us t o s t u d y t h e r o l e o f t h e s e enzymes i n t h e m e t a ­ b o l i s m o f BP a n d B P d e r i v a t i v e s a n d t o m a n i p u l a t e t h e s o u r c e o f t h e p u r i f i e d c y t o c h r o m e P-450 as w e l l as t h e l e v e l o f e p o x i d e hydrase to generate mutagenic metabolites of t h i s p o l y c y c l i c aromatic hydrocarbon. F i n a l l y , the synthesis of approximately t h i r t y BP d e r i v a t i v e s a n d m e t a b o l i t e s ( 2 1 ) h a s p e r m i t t e d u s t o u t i l i z e t h e s e compounds as s u b s t r a t e s f o r t h e p u r i f i e d monoxy­ g e n a s e s y s t e m i n an e f f o r t t o i d e n t i f y t h e b i o a c t i v a t e d m e t a ­ b o l i t e s o f BP. M e t a b o l i s m o f B e n z o l a j p y r e n e and B e n z o l a j p y r e n e A r e n e O x i d e s b £ t h e P u r i f i e ? Wônoxygenase System and E p o x i d e H y d r a s e . Tïïë p u r i f i e d , r e c o n s t i t u t e d monoxygenase system w i t h and w i t h o u t a d d i t i o n o f p u r i f i e d e p o x i d e hydrase has been u t i l i z e d t o s t u d y the metabolism o f L CJ-benzolajpyrene (Table I). Although high pressure l i q u i d chromatography i s h i g h l y e f f i c i e n t f o r the separ a t i o n and q u a n t i t a t i o n o f d i h y d r o d i o l s , p h e n o l s and q u i n o n e s formed from BP, a l l p o t e n t i a l m e t a b o l i t e s w i t h i n each group have not been i d e n t i f i e d i n t h e s e s t u d i e s ( 2 2 J . Thus, d i o l fractions 1 , 2 a n d 3 c o r r e s p o n d t o BP 9 , 1 0 - , 4 , 5 - a n d 7,8-dihydrodiols, q u i n o n e f r a c t i o n 1 c o r r e s p o n d s t o BP 1 , 6 - , 3 , 6 - and 4,5-quinones, q u i n o n e f r a c t i o n 2 c o r r e s p o n d s t o BP 1 1 , 1 2 - a n d 6 , 1 2 - q u i n o n e s a n d BP 4 , 5 - o x i d e , p h e n o l f r a c t i o n 1 c o r r e s p o n d s t o 2 - , 6 - , 8and 9-HOBP and p h e n o l f r a c t i o n 2 c o r r e s p o n d s t o t h e o t h e r 8 i s o m e r i c p h e n o l s o f BP ( 1 - , 3 - , 4 - , 5 - , 7 - , 1 0 - , 1 1 - a n d 1 2 - H O B P ) . In t h e a b s e n c e o f e p o x i d e h y d r a s e , t h e r e c o n s t i t u t e d c y t o c h r o m e P - 4 4 8 c o n t a i n i n g m o n o x y g e n a s e s y s t e m m e t a b o l i z e s BP t o p h e n o l s and q u i n o n e s ( T a b l e I ) . Upon a d d i t i o n o f p u r i f i e d e p o x i d e h y d r a s e , t h e r a t e o f t o t a l BP m e t a b o l i s m i s u n c h a n g e d , b u t s i g n i f i c a n t amounts o f d i h y d r o d i o l s a r e p r o d u c e d a t t h e expense o f phenols. F o r m a t i o n o f d i o l f r a c t i o n s 2 and 3 (BP 4 , 5 - and 7 , 8 d i h y d r o d i o l s , r e s p e c t i v e l y ) r e a c h e d a maximum l e v e l w i t h t h e a d d i t i o n of 5 u n i t s of epoxide hydrase whereas f u r t h e r a d d i t i o n

6.

LEVIN E T A L .

Activation

and Detoxification

of Benzolajpyrene

101

o f t h e enzyme r e s u l t e d i n a c o n t i n u e d i n c r e a s e i n d i o l f r a c t i o n 1 (BP 9 , 1 0 - d i h y d r o d i o l ) , p r o b a b l y as a r e s u l t o f t h e marked i n s t a b i l i t y o f BP 9 , 1 0 - o x i d e ( t , < 2 m i n u t e s a t 3 7 ° i n 1 0 0 mM p o t a s s i u m p h o s p h a t e b u f f e r ) c o m p a r e d t o BP 4 , 5 - a n d 7 , 8 - o x i d e s . T h u s , h i g h e r amounts o f e p o x i d e h y d r a s e w o u l d be r e q u i r e d t o

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

/

2

Table

I

Metabolism o f B e n z o l a j p y r e n e b y a P u r i f i e d Cytochrome P-448 Dependent Monoxygenase System and Epoxide Hydrase

Epoxide Hydrase

Diol 1

(units)

None

2

3

(nmol

product

1

Quinone 2

formed/nmol

0.06

0.05

0.07

1.54

5

0.59

0.41

0.57

1.66

15

0.79

0.47

0.61

50

1.19

0.48

0.56

0.52

1

Phenol 2

Total

hemeprotein/min)

0.87

1.31

4.42

-

0.63

1.00

4.86

1.62

-

0.35

0.90

4.74

1.39

-

0.13

0.74

4.49

I n c u b a t i o n m i x t u r e s c o n t a i n e d 0 . 2 nmol c y t o c h r o m e P - 4 4 8 , 120 u n i t s o f NADPH c y t o c h r o m e c r e d u c t a s e , 0 . 1 mg o f l i p i d , 0 . 5 p m o l o f NADPH, 3 y m o l o f M g C l , 100 y m o l o f p o t a s s i u m p h o s p h a t e b u f f e r (pH 6.a) a n d 95 nmol o f ( ^ C J - B P i n a f i n a l v o l u m e o f 1 m l . One u n i t o f e p o x i d e h y d r a s e p r o d u c e s 1 nmol o f s t y r e n e g l y c o l p e r m i n from styrene oxide. M e t a b o l i t e s o f BP w e r e a n a l y z e d b y h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y as d e s c r i b e d b y H o l d e r e t jrt. ( 2 2 ) . 2

102

DRUG M E T A B O L I S M C O N C E P T S

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

c o m p l e t e l y c o n v e r t BP 9 , 1 0 - o x i d e t o t h e c o r r e s p o n d i n g dihydrodiol. A g o o d s t o i c h i o m e t r i c r e l a t i o n s h i p was f o u n d b e t w e e n t h e i n c r e a s e i n t h e d i o l 2 f r a c t i o n and t h e d e c r e a s e i n q u i n o n e f r a c t i o n 2 w h i c h c o n t a i n s m a i n l y BP 4 , 5 - o x i d e . Direct confirm a t i o n t h a t a r e n e o x i d e s o f BP a r e s u b s t r a t e s f o r t h e p u r i f i e d e p o x i d e h y d r a s e i s shown i n T a b l e I I . BP 4 , 5 - , 7,8and 9 , 1 0 oxides are a l l metabolized to the corresponding dihydrodiols a t c o m p a r a b l e r a t e s u s i n g t h e p u r i f i e d e n z y m e w h i l e BP 1 1 , 1 2 o x i d e i s a r e l a t i v e l y poor s u b s t r a t e f o r t h e enzyme. No d e t e c t a b l e BP 1 1 , 1 2 - d i h y d r o d i o l i s formed by t h e p u r i f i e d cytochrome P-448 c o n t a i n i n g monoxygenase system i n the presence of epoxide hydrase. T h e r e s u l t s o f o u r s t u d i e s o n t h e m e t a b o l i s m o f BP i n the presence or absence of epoxide hydrase demonstrate t h a t t h e d i h y d r o d i o l s a n d p h e n o l i c m e t a b o l i t e s o f BP s h a r e a r e n e o x i d e s a s common precursors. Requirements f o r t h e M e t a b o l i c A c t i v a t i o n o f Benzol a Ipyrene t o M u t a g e n i c P r o d u c t s by a P u r i f i e d Monoxygenase System. Mutag e n i c i t y t e s t s u t i l i z i n g m i c r o o r g a n i s m s o r c u l t u r e d mammalian c e l l s have been used w i t h i n c r e a s i n g f r e q u e n c y t o i d e n t i f y b i o activated metabolites of carcinogens. Metabolic activation of c h e m i c a l s t o m u t a g e n i c m e t a b o l i t e s has been most commonly p e r f o r m e d b y a p r o c e d u r e d e v e l o p e d b y Ames a n d h i s a s s o c i a t e s (23, 24). G e n e r a l l y , t h e c h e m i c a l , b a c t e r i a and a p p r o p r i a t e c o f a c E o r s are i n c u b a t e d w i t h microsomes or a 9000 xg s u p e r n a t a n t f r a c t i o n o f l i v e r i n a s e m i s o l i d agar g e l f o r 48 h o u r s . When l i t t l e is known a b o u t t h e m e t a b o l i s m o f a p a r t i c u l a r compound, o r a l a r g e number o f d i v e r s e c h e m i c a l s a r e b e i n g e v a l u a t e d f o r m u t a g e n i c a c t i v i t y , r e l a t i v e l y c r u d e t i s s u e homogenates s h o u l d be used as t h e s o u r c e o f enzymes t o e n s u r e t h a t a l l p o s s i b l e m e t a b o l i c pathways are being e v a l u a t e d . W h i l e t h i s p r o c e d u r e has been u s e d s u c c e s s f u l l y t o a c t i v a t e a number o f c a r c i n o g e n s t o b a c t e r i a l mutagens, i t has l i m i t a t i o n s f o r i d e n t i f y i n g t h e m u t a g e n i c m e t a b o l i t e s f o r m e d f r o m a compound w h i c h u n d e r g o e s oxidation v i a m u l t i p l e pathways. The 9000 xg s u p e r n a t a n t f r a c t i o n i s r e l a t i v e l y c r u d e and c o n t a i n s many e n z y m a t i c and s t r u c t u r a l p r o t e i n s , n u c l e i c a c i d s and numerous n u c l e o p h i l i c and e l e c t r o p h i l i c groups which could i n t e r a c t with the b i o a c t i v a t e d metabolites before they reach the b a c t e r i a . Moreover, regardless of the source of the monoxygenase a c t i v i t y , long i n c u b a t i o n times can r e s u l t i n spontaneous or enzymatic breakdown o f p r i m a r y m e t a b o l i t e s t o as y e t i l l - d e f i n e d p r o d u c t s ( 2 5 , 2 6 ) . An e x a m i n a t i o n o f t h e m e t a b o l i t e p r o f i l e o b t a i n e d f r o m BP w h e n l i v e r m i c r o s o m e s a r e i n c u b a t e d f o r 30 m i n u t e s w i t h l i m i t i n g s u b s t r a t e c o n c e n t r a t i o n s r e v e a l e d t h a t a l l p r i m a r y o x i d a t i v e p r o d u c t s o f BP ( d i h y d r o d i o l s , q u i n o n e s and p h e n o l s ) undergo e x t e n s i v e s e c o n d a r y m e t a b o l i s m by t h e monoxygenase system (26). Thus, use of prolonged i n c u b a t i o n times f o r m e t a b o l i c a c t i v a t i o n s t u d i e s i n agar gel would a l l but e l i m i n a t e the p o s s i b i l i t y of o b t a i n i n g a p r o f i l e of the m e t a b o l i t e s formed under t h e c o n d i t i o n s which induce mu-

6.

LEVIN ET A L .

Activation

and Detoxification

Table

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

Oxides

445

BP

321

BP 9 , 1 0 - o x i d e

BP

11,12-oxide

by P u r i f i e d

Epoxide

D i h y d r o d i o l Formed (nmol/mg p r o t e i n / m i n )

BP 4 , 5 - o x i d e

7,8-oxide

103

II

Metabolism o f Benzolajpyrene Arene "Hydrase

Substrate

of Benzolajpyrene

390

31

Incubation m i x t u r e s c o n t a i n e d 2-6 y g o f p u r i f i e d epoxide hydrase, 30 y g o f p h o s p h a t i d y l c h o l i n e , 12.5 ymol o f T r i s b u f f e r and 10-25 nmol o f t r i t i u m l a b e l l e d s u b s t r a t e i n a f i n a l v o l u m e o f 8 0 y l .

DRUG M E T A B O L I S M C O N C E P T S

104

tations. F i n a l l y , t h e a b i l i t y t o m a n i p u l a t e t h e amount o f v a r i o u s BP m e t a b o l i t e s f o r m e d t h r o u g h a l t e r a t i o n o f t h e r a t i o o f e p o x i d e hydrase t o t h e monoxygenase system ( c f T a b l e I) should be o f v a l u e i n d e t e r m i n i n g t h e n a t u r e o f t h e b i o a c t i v a t e d m e t a b o l i t e s formed from BP. We, t h e r e f o r e , s o u g h t t o d e v e l o p an e n z y m a t i c a l l y w e l l - d e f i n e d monoxygenase system which would m e t a b o l i z e BP a n d BP d e r i v a t i v e s t o m u t a g e n i c p r o d u c t s u n d e r c o n d i t i o n s w h i c h w o u l d p e r m i t t h e a n a l y s i s and i d e n t i f i c a t i o n o f the metabolites (27). Except f o r tRê presence of b a c t e r i a (Salmonella typhimurium s t r a i n T A 9 8 ) i n t h e r e a c t i o n m i x t u r e , t h e m e t a b o l i s m o f BP t o m u t a g e n i c p r o d u c t s by t h e r e c o n s t i t u t e d s y s t e m was p e r f o r m e d e s s e n t i a l l y as d e s c r i b e d f o r m e t a b o l i t e i d e n t i f i c a t i o n . Bacteria (2 χ 1 0 c e l l s ) were suspended i n a t o t a l i n c u b a t i o n volume o f 0 . 5 ml c o n t a i n i n g 2 . 5 y m o l o f s o d i u m p h o s p h a t e , 75 y m o l o f sodium c h l o r i d e , 0 . 0 8 y m o l (50 y g ) o f p h o s p h a t i d y l c h o l i n e , 150 u n i t s o f N A D P H - c y t o c h r o m e c r e d u c t a s e , 0 . 0 2 - 0 . 2 nmol o f c y t o c h r o m e P - 4 5 0 o r P - 4 4 8 , 25 nmol o f BP ( i n 1 2 . 5 y 1 a c e t o n e ) a n d 0 . 1 y m o l o f NADPH. T h e f i n a l pH o f t h e i n c u b a t i o n m i x t u r e was 6 . 8 . After i n c u b a t i o n a t 37 f o r 5 m i n u t e s , 9 nmol o f m e n a d i o n e was a d d e d t o s t o p t h e r e a c t i o n f o l l o w e d i m m e d i a t e l y by 2.0 ml o f m o l t e n t o p a g a r , and t h e s t a n d a r d Ames p o u r p l a t e p r o c e d u r e ( 2 4 ) was performed. S t u d i e s on t h e r e q u i r e m e n t s f o r t h e enzymaTTc acti­ v a t i o n o f BP b y t h e r e c o n s t i t u t e d c y t o c h r o m e P - 4 4 8 s y s t e m i n ­ dicated that optimal metabolic a c t i v a t i o n to mutagenic metabolites r e q u i r e d t h e p r e s e n c e o f NADPH, N A D P H - c y t o c h r o m e c r e d u c t a s e , c y t o c h r o m e P-448 and p h o s p h a t i d y l c h o l i n e ( T a b l e I I I ) . A 5 minute i n c u b a t i o n w i t h 0.1 nmol o f c y t o c h r o m e P - 4 4 8 and s a t u r a t i n g a m o u n t s o f N A D P H - c y t o c h r o m e c r e d u c t a s e , p h o s p h o l i p i d , NADPH a n d BP i n d u c e d a p p r o x i m a t e l y a 1 5 - f o l d i n c r e a s e i n h i s t i d i n e i n d e p e n d e n t c o l o n i e s i n s t r a i n TA 9 8 . An a b s o l u t e r e q u i r e m e n t was o b s e r v e d f o r a l l c o m p o n e n t s o f t h e s y s t e m e x c e p t p h o s p h a t i d y l ­ c h o l i n e which i s i n agreement w i t h t h e r e q u i r e d components for t h e m e t a b o l i s m o f BP t o p h e n o l i c m e t a b o l i t e s ( 1 9 ) . In o t h e r e x p e r i m e n t s ( 2 7 ) , i t was e s t a b l i s h e d t h a t t h e number o f m u t a t i o n s i n d u c e d i n S a l m o n e l l a t y p h i m u r i u m s t r a i n TA 9 8 was p r o p o r t i o n a l t o t h e amount o f added c y t o c h r o m e P - 4 4 8 and t o t h e t i m e o f i n ­ cubation (Figure 1).

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

B

M e t a b o l i s m o f Benzol a j p y r e n e t o M u t a g e n i c M e t a b o l i t e s by Various Pur i f i e d T o r m s of Cytochrome P-450. Γη t h e l a s t d e c a d e , numerous l a b o r a t o r i e s have i n v e s t i g a t e d t h e p o s s i b i l i t y t h a t m u l t i p l e hydroxylase systems e x i s t i n l i v e r microsomes. The p u r i f i c a t i o n and r e c o n s t i t u t i o n o f t h i s enzyme s y s t e m has p r o ­ v i d e d t h e means t o s t u d y i n d e t a i l t h e p h y s i c a l p r o p e r t i e s o f e a c h o f t h e components and t h e c a t a l y t i c a c t i v i t y o f c y t o c h r o m e P-450. These s t u d i e s have p r o v i d e d d i r e c t e v i d e n c e f o r t h e e x ­ i s t ance of m u l t i p l e forms of cytochrome P-450, each of which have d i f f e r e n t , but o v e r l a p p i n g , s u b s t r a t e s p e c i f i c i t i e s ( 4 - 7 ) . T a b l e I V s h o w s a c o m p a r i s o n o f t h e m e t a b o l i s m o f BP t o m u t a g e n i c

LEVIN E T A L .

6.

Activation

and Detoxification

Table

of Benzo[a]pyrene

105

III

Requirements f o r t h e M e t a b o l i c A c t i v a t i o n o f BenzoI a)pyrene t o mutagenic"ProducTs i n Salmonella Typïïimuriûm"Strain TA""98

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

Addition

His

Revertants/Plate

None

36

BP

37

Benzolajpyrene Metabolism (% A c t i v i t y )

-

440

100

30

0

32

1

32

2

-Phosphatidylcholine

74

18

-NADPH

34

0

Complete Monoxygenase

System

-BP -NADPH-cytochrome -Cytochrome

c

P-448

reductase

The c o m p l e t e monoxygenase system c o n s i s t e d o f 50 y g o f p h o s p h a t i d y c h o l i n e , 150 u n i t s o f NADPH-cytochrome c r e d u c t a s e , 0 . 1 nmol o f c y t o c h r o m e P - 4 4 8 , 0 . 1 y m o l o f NADPH a n d 2 5 n m o l o f B P i n a f i n a l volume o f 0 . 5 ml c o n t a i n i n g 2 χ 1 0 b a c t e r i a . Incubations were a t 3 7 ° f o r 5 m i n . 8

DRUG M E T A B O L I S M C O N C E P T S

106

Table

Metabolism

of

Benzolajpyrene Purified

Forms

to Mutagenic of

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

Source of Cytochrome P-450 Species Pretreatment of Animals

Rat

Rabbit

Products

by

Various

P-450

BenzolaJpyrene Metabolism (pmol f o r m e d / pmol h e m e p r o t e i n )

Mutations in S t r a i n TA 9 8 (His Revertants/pmol hemeprotein) +

38.0

1254

13.0

26.5

Phénobarbital

0.7

Aroclor

Rat

Cytochrome

15.0

3-Methylcholanthrene

Rat

IV

0.55

3-Methylcholanthrene

0.7

0.25

Phénobarbital,

0.5

0.05

0.1

0.05

Mouse

Fraction Mouse

A

2

Phénobarbital, Fraction C 2

A l l i n c u b a t i o n m i x t u r e s c o n t a i n e d t h e complete monoxygenase s y s t e m a n d 2 χ IQr b a c t e r i a i n a f i n a l v o l u m e o f 0 . 5 m l a s d e s c r i b e d in Table III. C y t o c h r o m e P - 4 5 0 was p u r i f i e d f r o m t h e l i v e r s o f r a t s ( 5 , 2 9 ) , r a b b i t s (6) o r m i c e (7) as p r e v i o u s l y d e s c r i b e d . The f i n a l s u b s t r a t e c o n c e n t r a t i o n w a s 75 y M . After incubation for 5 min at 37°C, t h e r e a c t i o n s were t e r m i n a t e d by t h e a d d i t i o n of 9 nmol o f m e n a d i o n e . B e n z o l a j p y r e n e h y d r o x y l a t i o n was m e a s u r e d as fluorescent phenols (28). f t

6.

LEVIN E T A L .

Activation

and Detoxification

of Benzo[a]pyrene

107

m e t a b o l i t e s by t h e p u r i f i e d and r e c o n s t i t u t e d monoxygenase system using cytochrome P-450 p u r i f i e d from animals t r e a t e d w i t h d i f f e r e n t i n d u c e r s as w e l l as from s e v e r a l d i f f e r e n t animal s p e c i e s . These r e s u l t s c l e a r l y demonstrate t h e marked d i f f e r e n c e s i n t h e m e t a b o l i s m o f BP t o f l u o r e s c e n t p h e n o l s b y v a r i o u s p u r i f i e d f o r m s of cytochrome P-450. T h e m e t a b o l i c a c t i v a t i o n o f BP t o p r o d u c t s m u t a g e n i c t o s t r a i n TA 9 8 o f S. t y p h i m u r i u m b y v a r i o u s p u r i f i e d c y t o c h r o m e P - 4 5 0 s was s i m i l a r t o t h e r a t e o f m e t a b o l i s m o f BP to fluorescent phenols (Table IV). Cytochrome P-450 i s o l a t e d from rats pretreated with e i t h e r 3-methylcholanthrene or A r o c l o r 1254 were t h e most e f f i c i e n t h e m e p r o t e i n s f o r t h e m e t a b o l i s m o f BP t o f l u o r e s c e n t p h e n o l s a n d m u t a g e n i c p r o d u c t s w h i l e t h e o t h e r p u r i f i e d c y t o c h r o m e P - 4 5 0 s w e r e much l e s s e f f e c t i v e . 1

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

1

E f f e c t o f Epoxide Hydrase on t h e M e t a b o l i c A c t i v a t i o n o f Benzol a lpyrene t o Mutagenic MetâEoTTtes. Epoxide hydrase i s an i m p o r t a n t enzyme i n t h e m e t a b o l i s m o f BP s i n c e i t c o n v e r t s t h e i n t e r m e d i a t e arene o x i d e s formed by t h e monoxygenase system to the corresponding trans dihydrodiols (15,16). In t h e absence of f u r t h e r metabolic a c t i v a t i o n , t h e d i h y d r o d i o l s produced from these arene oxides are e s s e n t i a l l y nontoxic (21,30-32). Addi t i o n o f h i g h l y p u r i f i e d e p o x i d e hydrase t o t h e monoxygenase s y s t e m d u r i n g t h e m e t a b o l i s m o f BP d e c r e a s e d t h e m u t a t i o n f r e q u e n c y b y a maximum o f 3 0 % i n S. t y p h i m u r i u m s t r a i n TA 9 8 ( F i g u r e 2 ) , i n d i c a t i n g t h a t a t l e a s t some o f t h e m u t a g e n i c m e t a b o l i t e s o f BP a r e a r e n e o x i d e s . In c o n t r a s t t o these r e s u l t s , mutations i n d u c e d b y BP 4 , 5 - o x i d e w e r e c o m p l e t e l y a b o l i s h e d b y t h e a d d i t i o n of 5 units o f epoxide hydrase (Figure 2 ) . Thus, t h e i n a b i l i t y of epoxide hydrase t o completely abolish the metabolic a c t i v a t i o n o f BP t o m u t a g e n i c p r o d u c t s s u g g e s t e d t h a t n o n - a r e n e o x i d e m e t a b o l i t e s o f BP m a y a l s o h a v e m u t a g e n i c a c t i v i t y o r t h a t some m u t a genic epoxide m e t a b o l i t e s a r e poor substrates f o r epoxide hydrase. Metabolic Activation of Benzolajpyrene Phenols. Phenols, formed e i t h e r by spontaneous i s o m e r i z a t i o n o f arene oxides (15) or d i r e c t oxygen i n s e r t i o n r e a c t i o n s ( 3 3 ) , a r e t h e p r i m a r y o x i d a t i v e p r o d u c t s when BP i s m e t a b o l i z e c T E y t h e p u r i f i e d m o n o x y genase system i n t h e absence o f epoxide hydrase ( c f Table I ) . Of t h e t w e l v e p o s s i b l e i s o m e r i c p h e n o l s o f B P , o n l y 6 - and 1 2 HOBP h a v e s i g n i f i c a n t i n t r i n s i c m u t a g e n i c a c t i v i t y i n s e v e r a l s t r a i n s o f S. t y p h i m u r i u m ( 3 2 ) . H o w e v e r , b a s e d on t h e amount o f phenols produced from B l H ï y t h e p u r i f i e d monoxygenase system (cf Table I ) , i t i s u n l i k e l y that these phenols contribute s i g n i f i c a n t l y t o t h e m u t a g e n i c i t y o b s e r v e d f r o m BP m e t a b o l i s m . S i n c e s e v e r a l s t u d i e s h a v e shown t h a t p r i m a r y o x i d a t i v e m e t a b o l i t e s o f BP, i n c l u d i n g phenols, can be f u r t h e r m e t a b o l i z e d b y t h e m o n o x y g e n a s e s y s t e m ( 2 6 , 3 4 ) , we u t i l i z e d a l l t w e l v e o f t h e phenols as s u b s t r a t e s f o r t h e p u r i f i e d monoxygenase system to determine i f f u r t h e r o x i d a t i v e metabolism would r e s u l t i n the formation o f mutagenic products. T a b l e V shows t h e m u t a g e n i c

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

108

DRUG M E T A B O L I S M

CONCEPTS

0.1 nmol CYTOCHROME

Figure I. Effect of cytochrome P-448 concentration and time of incubation on the metabolism of BP to products mutagenic to strain TA 98 of Salmonella typhimurium. Reaction mixtures were similar to those described in Table III. Each value represents the mean ± S.D. from three replicate incubation mixtures.

Figure 2. Effect of epoxide hydrase on mutations induced bu the metabolism of BP by the purified monoxygenase system (left) and on the mutagenic activity of BP 4,5-oxide (right). Incubation mixtures for the metabolic activation of BP were similar to those described in Table 111. The effect of epoxide hydrase on the mutagenic activity of BP 4,5-oxide was assayed using 0.4 nmole of BP 4,5-oxide in 0.5 ml containing 2 X 10* bacteria. All samples were incubated for 5 min at 37° before addition of the top agar.

10

Î 2 " 2 0

UNITS OF EPOXIOE HYDRASE

•i-^—k

6.

LEVIN ET A L .

Activation

and Detoxification

Table

of Benzo[a]pyrene

109

V

Metabolism o f Benzo|aIpyrene Phenols t o Mutagenic Products by a Pur i f i e T t y t o c h r o m e P-448""Dependen£ Monoxygenase System

Substrate

Cytochrome 0

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

Hi s

None BP 1-H0BP 2-H0BP 3-H0BP 4-HOBP 5-H0BP 6-HOBP 7-H0BP 8-HOBP 9-HOBP 10-H0BP 11-H0BP 12-H0BP

17 18 43 17 32 15 19 94 35 15 33 14 11 219

P-448 (pmol) 25 50 Revertants/Plate

-

280 109 67 117 22 19 116 43 35 93 22 21 302

17 670 140 73 158 29 24 261 49 45 180 21 21 372

I n c u b a t i o n m i x t u r e s c o n t a i n e d t h e complete monoxygenase system and t y p h i m u r i u m s t r a i n TA 98 a s d e s c r i b e d i n T a b l e I I I u s i n g f i x e d c o n c e n t r a t i o n s o f a l l components e x c e p t cytochrome P-448. The f i n a l c o n c e n t r a t i o n o f BP o r BP p h e n o l s was 25 μ Μ .

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

110

DRUG M E T A B O L I S M

CONCEPTS

a c t i v i t y o f t h e t w e l v e i s o m e r i c p h e n o l s o f BP i n s t r a i n T A 9 8 b e f o r e and a f t e r m e t a b o l i c a c t i v a t i o n by v a r i o u s amounts o f c y t o chrome P-448. The number o f r e v e r t a n t c o l o n i e s i n t h e a b s e n c e of the cytochrome i s a measure o f the i n t r i n s i c mutagenic a c t i v i t y of the phenols s i n c e the r e c o n s t i t u t e d system i s i n a c t i v e in the absence of the cytochrome. Of t h e t w e l v e p o s s i b l e p h e n o l s o f B P , o n l y 1 - , 2 - , 3 - , 6 - , 9 - and 12-HOBP w e r e m e t a b o l i c a l l y a c t i v a t e d to mutagenic products. However, s i n c e none o f t h e p h e n o l s was as e f f e c t i v e l y c o n v e r t e d t o m u t a g e n i c p r o d u c t s as B P , i t a p p e a r s t h a t f u r t h e r m e t a b o l i s m o f BP p h e n o l s t o m u t a g e n i c products is not responsible f o r the mutagenic a c t i v i t y of metab o l i c a l l y a c t i v a t e d BP. This data, together with the lack of i n h e r e n t m u t a g e n i c a c t i v i t y o f s i x p o s s i b l e BP q u i n o n e s (32), s t r o n g l y s u g g e s t s t h a t n o n e p o x i d e d e r i v a t i v e s o f BP a r e n o t r e s p o n s i b l e f o r t h e m u t a t i o n s o b s e r v e d w h e n BP i s m e t a b o l i z e d t o mutagenic p r o d u c t s i n t h e p r e s e n c e of t h e p u r i f i e d monoxygenase s y s t e m and e p o x i d e h y d r a s e . Metabolic A c t i v a t i o n of Benzolajpyrene Dihydrodiols. Since a d d i t i o n of e p o x i d e hydrase t o t h e p u r i f i e d monoxygenase system was u n a b l e t o b l o c k t h e f o r m a t i o n o f m u t a g e n i c m e t a b o l i t e s o f B P , i t w a s p o s s i b l e t h a t a n a r e n e o x i d e o f BP w a s c o n v e r t e d t o a d i h y d r o d i o l b y a d d i t i o n o f e p o x i d e h y d r a s e w h i c h was t h e n f u r t h e r m e t a b o l i c a l l y a c t i v a t e d t o a potent mutagen. In t h i s r e g a r d , B o r g e n e t a l . (35) have shown t h a t m e t a b o l i c a c t i v a t i o n o f BP 7 , 8 - d i h y d r o d i o l " T 5 y l i v e r m i c r o s o m e s r e s u l t s i n a m u c h g r e a t e r b i n d i n g t o DNA t h a n d o e s s u c h m e t a b o l i c a c t i v a t i o n o f B P , BP 4 , 5 - d i h y d r o d i o l o r BP 9 , 1 0 - d i h y d r o d i o l . Sims e t a l . ( 3 6 ) have p r e s e n t e d e v i d e n c e t h a t a BP 7 , 8 - d i o l - 9 , 1 0 - e p o x " u i e ~ T s Tiïe b i o a c t i v a t e d m e t a b o l i t e o f BP 7 , 8 - d i h y d r o d i o l w h i c h b i n d s t o DNA. I t h a s s i n c e b e e n e s t a b l i s h e d t h a t t h e s t e r e o i s o m e r i c BP 7,8-diol-9,10-epoxides a r e among t h e m o s t p o t e n t m u t a g e n s y e t described (21,31,37-39). We h a v e u t i l i z e d t h e p u r i f i e d m o n o x y g e n a s e s y s t e m i n an a t t e m p t t o m e t a b o l i c a l l y a c t i v a t e t h e f o u r BP d i h y d r o d i o l s w h i c h h a v e b e e n s y n t h e s i z e d i n o u r l a b o r a t o r i e s (21). A s s h o w n i n F i g u r e 3 , BP 7 , 8 - d i h y d r o d i o l w a s a c t i v a t e d t o a t l e a s t a 4 - f o l d g r e a t e r e x t e n t t h a n was B P . In c o n t r a s t t o t h e s e r e s u l t s , t h e BP 4 , 5 - , 9 , 1 0 - a n d 1 1 , 1 2 - d i h y d r o d i o l s were not m e t a b o l i c a l l y a c t i v a t e d to mutagenic m e t a b o l i t e s . The h i g h m u t a g e n i c a c t i v i t y o f a m e t a b o l i t e ( s ) o f BP 7 , 8 - d i h y d r o d i o l t o w a r d s S. t y p h i m u r i u m s t r a i n TA 98 p r o m p t e d f u r t h e r s t u d i e s o n t h e m e t a b o l i c a c t i v a t i o n o f BP 7 , 8 - d i h y d r o d i o l b y t h e p u r i f i e d monoxygenase system. Microsomal epoxide hydrase converts the a r e n e o x i d e s f o r m e d by t h e c y t o c h r o m e P-450 monoxygenase system into trans, v i c i n a l dihydrodiols (15). T a b l e VI shows t h a t ç i s BP 7 , 8 - d i h y d r o d i o l c a n a l s o b e m e t a i ï o l i c a l l y a c t i v a t e d t o a p o t e n t mutagen(s) by t h e monoxygenase s y s t e m , a l t h o u g h i t i s s l i g h t l y less a c t i v e than the trans isomer. These r e s u l t s i n d i c a t e that the r e l a t i v e p o s i t i o n of the hydroxyl groups i s not a c r i t i c a l f a c t o r in determining the mutagenic a c t i v i t y of the

BP 7 , 8 -

2±1

( ± ) - t r a n s BP 7 , 8 dihydroxy-7,8,9, 10-H BP

76+10

130±8

9±2

2

34±3

6±4

320+18

10±3

60±5

20

9±2

120±10

(pmol)

ΉΓ

Revertants7P1ate

522±45

His

+

Cytochrome P-448 5 80

Products

29±4

800±45

t o Mutagenic

I n c u b a t i o n m i x t u r e s c o n t a i n e d t h e c o m p l e t e monoxygenase system as d e s c r i b e d i n T a b l e I I I . The f i n a l s u b s t r a t e c o n c e n t r a t i o n was 25 y M . Background mutation f r e q u e n c i e s have been s u b t r a c t e d . V a l u e s r e p r e s e n t t h e mean + S . E . f o r t h r e e determinations.

4

1±0.5

6±1

1±0.6

( t ) - c i s BP 7 , 8 dihydrodiol

dihydrodiol

(±)-trans

BP

VI

A c t i v a t i o n o f Benzol aipyrene 7 , 8 - D i h y d r o d i o l

Substrate

Metabolic

Table

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

112

DRUG M E T A B O L I S M CONCEPTS

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

bioactivated metabolite(s). However, 7,8-dihydroxy-7,8,9,10t e t r a h y d r o B P , a c o m p o u n d r e l a t e d t o BP 7 , 8 - d i h y d r o d i o l b u t w i t h t h e d o u b l e bond removed f r o m t h e 9 , 1 0 - p o s i t i o n o f t h e m o l e c u l e , c a n n o t be m e t a b o l i c a l l y a c t i v a t e d t o mutagenic m e t a b o l i t e s by t h e monoxygenase system. These r e s u l t s suggest t h a t the mutag e n i c p r o d u c t f o r m e d o n m e t a b o l i c c o n v e r s i o n o f BP 7 , 8 - d i h y d r o d i o l i s a BP 7 , 8 - d i o l - 9 , 1 0 - e p o x i d e s i n c e t h e d o u b l e b o n d i n t h e 9,10-position of the molecule is a requirement f o r the formation o f an e p o x i d e a t t h a t p o s i t i o n . Metabolic A c t i v a t i o n of Benzolajpyrene 7,8-Oxide. Since BP 7 , 8 - d i h y d r o d i o l w a s r e a d i l y m e t a b o l i z e d t o a m u t a g e n i c p r o d u e t t s ) b y t h e c y t o c h r o m e P - 4 4 8 m o n o x y g e n a s e s y s t e m , we e x a m i n e d t h e e f f e c t o f t h e monoxygenase s y s t e m on t h e m u t a g e n i c a c t i v i t y o f i t s a r e n e o x i d e p r e c u r s o r , BP 7 , 8 - o x i d e ( T a b l e V I I ) . In t h e a b s e n c e o f a d d e d e p o x i d e h y d r a s e , t h e weak i n t r i n s i c m u t a g e n i c a c t i v i t y o f BP 7 , 8 - o x i d e ( 4 0 ) w a s u n a f f e c t e d b y a d d i t i o n o f t h e p u r i f i e d monoxygenase system. Addition of epoxide hydrase to t h e monoxygenase system r e s u l t e d i n a marked i n c r e a s e i n the m u t a t i o n f r e q u e n c y i n S. t y p h i m u r i u m s t r a i n TA 9 8 . These r e s u l t s d e m o n s t r a t e t h a t BP 7 , ï ï - o x i d e i s h y d r a t e d t o t h e c o r r e s p o n d i n g d i h y d r o d i o l w h i c h i n t u r n i s o x i d a t i v e l y m e t a b o l i z e d t o an a c t i v e m u t a g e n ( s ) , p r e s u m a b l y t h e s t e r e o i s o m e r i c BP 7,8-diol-9,10-epoxides. Metabolism of Benzolajpyrene 7,8-Dihydrodiol b^ the P u r i f i e d Monoxygenase System. Based on t h e s t u d i e s o f Borgen e t a l . ( 3 5 ) , Sims e t a l . ( 3 6 ) p r o p o s e d a BP 7 , 8 - d i o l - 9 , 1 0 - e p o x i d e a s t h e DTOa c t i v a t e ï ï " m e T â b o l i t e o f B P 7 , 8 - d i h y d r o d i o l w h i c h b i n d s t o DNA. T h e o x i d a t i v e m e t a b o l i s m o f BP 7 , 8 - d i h y d r o d i o l a t t h e 9 , 1 0 - p o s i t i o n o f t h e m o l e c u l e c o u l d r e s u l t i n t h e f o r m a t i o n o f two p o s s i b l e stereoisomers of the d i o l epoxide (Figure 4). Recently o u r l a b o r a t o r i e s (41) s y n t h e s i z e d and u n e q u i v o c a l l y assigned t h e r e l a t i v e s t e r e o c h e m i s t r y o f t h e t w o s t e r e o i s o m e r s o f BP 7 , 8 d i o l - 9 , 1 0 - e p o x i d e ( d i o l e p o x i d e s 1 and 2 ) , b o t h o f w h i c h a r e p o t e n t mutagens ( 3 1 , 3 7 - 3 9 ) . A d e t a i l e d study of the metabolism of BP-7,8-dihydrodiol is thus necessary in order to determine w h e t h e r one o r b o t h o f t h e s t e r e o i s o m e r s a r e r e s p o n s i b l e f o r t h e m u t a g e n i c a c t i v i t y o f m e t a b o l i c a l l y a c t i v a t e d BP 7 , 8 - d i h y d r o diol. R e c e n t l y , h i g h p r e s s u r e l i q u i d chromatography has been u s e d t o s e p a r a t e m e t a b o l i t e s o f BP 7 , 8 - d i h y d r o d i o l (42«43). Although a d i r e c t demonstration of the formation of the d i o l e p o x i d e s has n o t been p o s s i b l e due t o t h e i r e x t r e m e i n s t a b i l i t y i n aqueous s o l u t i o n s , t e t r a o l s have been i d e n t i f i e d as m e t a b o l i t e s f r o m BP 7 , 8 - d i h y d r o d i o l ( F i g u r e 4 ) . D i o l e p o x i d e s 1 and 2 u n d e r go c i s and t r a n s a d d i t i o n o f w a t e r a t t h e 1 0 - p o s i t i o n o f t h e oxirane (42,43) to form stereoisomeric p a i r s of (±)-7,8,9,10tetrahydroxy-/,8,9,10-tetrahydrο BP d e r i v a t i v e s i n w h i c h t h e r e l a t i v e s t e r e o c h e m i s t r y o f t h e 7 - and 8 - h y d r o x y l group i s a l w a y s t r a n s and t h e r e l a t i v e s t e r e o c h e m i s t r y b e t w e e n t h e 8 - and 9 -

6.

Activation

LEVIN E T A L .

and Detoxification

Table

Metabolic

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

BP

7,8-oxide

(25 μΜ)

Benzo[z]pyrene

113

VII

A c t i v a t i o n o f BP 7 , 8 - o x i d e

Substrate

of

t o Mutagenic

Metabolites

Monoxygenase System

Epoxide Hydrase

-

-

91±4

+

-

132±10

+

+

514±22

+

His

Revertants/Plate

The c o m p l e t e monoxygenase s y s t e m c o n s i s t e d o f 0 . 0 5 nmol o f c y t o ­ chrome P-448, 150 u n i t s o f NADPH-cytochrome c r e d u c t a s e , 50 ug o f p h o s p h a t i d y l c h o l i n e , 0 . 1 p m o l o f NADPH a n d 2 χ 1 0 b a c t e r i a i n a f i n a l volume o f 0.5 m l . Four u n i t s o f p u r i f i e d epoxide h y d r a s e was added t o t h e a p p r o p r i a t e r e a c t i o n m i x t u r e s ( s e e Table I f o r d e f i n i t i o n of u n i t s of epoxide hydrase activity). The m u t a t i o n s o b s e r v e d i n t h e absence o f t h e monoxygenase system and e p o x i d e h y d r a s e a r e a r e s u l t o f t h e i n h e r e n t m u t a g e n i c i t y o f BP 7 , 8 - o x i d e . Background m u t a t i o n f r e q u e n c i e s have been s u b ­ tracted. V a l u e s r e p e s e n t t h e mean + S . E . f o r t h r e e d e t e r m i n a t i o n s . 8

DRUG M E T A B O L I S M

114 80

CONCEPTS

r

φ

! » Ul g

60

t υ

50

1

40

I

30

χ ο

α

<

Î

ω ω

20

α:

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

*

Figure 3. Metabolic activation of (±)trans BP dihydrodiols. Samples were incubated with the purified monoxy­ genase system as described in Table III using 25 μΜ substrate concentration and 0.075-4.10 nmol cytochrome P-448.

SUBSTRATE BP

10 0

B P 4,5 - 0IHY0R0DI0L B P 7,8-DIHYDRODIOL BP9.IO-0IHY0RO0IOL BPII.I2-0IHY0R00I0L

OH

trons 2

Figure 4. Metabolism of BP 7,8-dihydrodiol to the stereoisomeric BP 7,8-diol-9,10-epoxides. Tetraols are formed upon cis and trans addition of water at the 10-position.

6.

LEVIN ET A L .

Activation

and

Detoxification

of

Benzolajpyrene

115

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

h y d r o x y l groups i s d e t e r m i n e d by t h e s t a r t i n g d i o l e p o x i d e ( F i g ure 4). When a u t h e n t i c d i o l e p o x i d e s 1 a n d 2 a r e i n c u b a t e d i n p o t a s s i u m p h o s p h a t e b u f f e r (pH 7.4) a t 3 7 ° , d i o l e p o x i d e 1 f o r m s 8 5 % c i s 1 a n d 1 5 % t r a n s 1 w h i l e d i o l e p o x i d e 2 f o r m s 7% cis 2 and 93% t r a n s 2 by r e s p e c t i v e c i s and t r a n s a d d i t i o n o f w a t e r (43). D i o l e p o x i d e s i n c u b a t e d ~ T n t h e p r e s e n c e o f enzymes g i v e s T T g h t l y d i f f e r e n t r a t i o s , p r o b a b l y as a r e s u l t o f t h e h y d r o p h o b i c i t y o f t h e enzyme s y s t e m . M e t a b o l i s m o f ( + ) - t r a n s - B P 7 , 8 - d i h y d r o d i o l was s t u d i e d u s i n g t h e p u r i f i e d monoxygenase system c o n t a i n i n g e i t h e r r a t l i v e r cytochrome P-450 or P-448 (Table V I I I ) . When t h e s y s t e m w a s r e c o n s t i t u t e d w i t h c y t o c h r o m e P - 4 4 8 , d i o l e p o x i d e 2 was t h e m a j o r m e t a b o l i t e , a c c o u n t i n g f o r a s much as 5 5 % o f t h e t o t a l m e t a b o l i s m w h i l e d i o l epoxide 1 accounted f o r 35-40% of the t o t a l metabolism. O t h e r unknown m e t a b o l i t e s a r e f o r m e d w h i c h i n c l u d e a p h e n o l i c d e r i v a t i v e ( 4 3 , 4 4 ) and t r i o l s w h i c h a r e f o r m e d v i a n o n e n z y m a t i c r e d u c t i o n o f t e t r a o l s b y NADPH ( 4 2 ) . Cytochrome P-450 from phenob a r b i t a l - t r e a t e d rats is markedly less e f f e c t i v e in metabolizing BP 7 , 8 - d i h y d r o d i o l , a s i s a l s o t h e c a s e when BP i s t h e s u b s t r a t e f o r the enzymes. The a d d i t i o n o f e p o x i d e hydrase t o t h e p u r i f i e d m o n o x y g e n a s e s y s t e m has no e f f e c t on t h e p r o f i l e o f m e t a b o l i t e s f o r m e d f r o m BP 7 , 8 - d i h y d r o d i o l , i n d i c a t i n g t h a t t h e d i o l epoxides o f BP a r e n o t s u b s t r a t e s f o r t h i s e n z y m e . Mutagenesis studies ( 2 7 « 3 9 ) h a v e a l s o s h o w n t h a t t h e s t e r e o i s o m e r i c BP 7,8-diol-9,10epoxides are poor substrates f o r epoxide hydrase (Figure 5). The l a c k o f e f f e c t o f e p o x i d e h y d r a s e on t h e d i o l e p o x i d e s is probably not a r e s u l t of t h e i r extreme i n s t a b i l i t y (39), since H -9,10-epoxide i s m e t a b o l i c a l l y i n a c t i v a t e d by t h e enzyme and t h i s compound has a h a l f - l i f e i n b u f f e r w h i c h i s s i m i l a r (39) to that for diol epoxide 2 (Figure 5). T h u s , t h e enzyme e p o x i d e h y d r a s e , l o n g c o n s i d e r e d t o be a d e t o x i f y i n g enzyme, can p l a y a v a r i e t y o f r o l e s i n t h e m e t a b o l i c i n a c t i v a t i o n and a c t i v a t i o n o f BP t o m u t a g e n i c m e t a b o l i t e s : (1) BP 4 , 5 - o x i d e i s a s u b s t r a t e f o r e p o x i d e h y d r a s e and i s i n a c t i v a t e d by t h i s enzyme. (2) BP 7 , 8 - o x i d e i s m e t a b o l i c a l l y a c t i v a t e d t o a m u t a g e n i c m e t a b o l i t e ( s ) i n t h e p r e s e n c e o f b o t h e p o x i d e h y d r a s e and t h e monoxyg e n a s e s y s t e m and (3) t h e s t e r e o i s o m e r i c BP 7,8-diol-9,10-epo x i d e s are not s u b s t r a t e s f o r t h e enzyme. 4

S i n c e s e v e r a l forms o f h i g h l y p u r i f i e d cytochrome P-450 ( P - 4 4 8 ) h a v e b e e n shown t o m e t a b o l i z e BP a t s i g n i f i c a n t l y d i f f e r e n t r a t e s , i t was o f i n t e r e s t t o d e t e r m i n e t h e a b i l i t y o f the various p u r i f i e d cytochromes to metabolize (+)-trans-BP 7,8d i h y d r o d i o l to mutagenic m e t a b o l i t e s . T a b l e IX shows t h a t t h e cytochrome P-450*s i s o l a t e d from 3-methylcholanthrene or A r o c l o r 1 2 5 4 - p r e t r e a t e d r a t s were t h e most e f f e c t i v e hemeproteins f o r t h e m e t a b o l i s m o f BP 7 , 8 - d i h y d r o d i o l t o m u t a g e n i c p r o d u c t s . The v a r i o u s o t h e r forms o f p u r i f i e d cytochrome P-450 m e t a b o l i z e d BP 7 , 8 - d i h y d r o d i o l t o m u t a g e n i c m e t a b o l i t e s b u t a t a m u c h l o w e r rate. I n t e r e s t i n g l y , t h e r e s u l t s o b t a i n e d w i t h BP 7,8-dihydrod i o l as s u b s t r a t e p a r a l l e l t h e r e s u l t s o b t a i n e d f o r t h e s e heme-

-

+

-

+

3

0.004

0.005

0.17

0.14

1

(nmol

Trans

1

0.03

0.02

0.60

0.61

0.06

0.06

1.00

1.03

2

Unknown

by

a

0.06

0.004

0.08

0.08

0.000

0.005

0.28

0.18

hemeprotein/minute)

Metabolites Trans 1 Cis

System

7,8-Dihydrodiol

formed/nmol

Cis

R e c o n s t i t u t e d Monoxygenase

(±)-trans-BenzolaJpyrene

VIII

0.10

0.10

2.77

2.69

Total

2

I n c u b a t i o n m i x t u r e s c o n t a i n e d 0 . 2 nmol o f c y t o c h r o m e P - 4 4 8 o r 0 . 4 nmol of cytochrome P-450, 225-450 u n i t s of NADPH-cytochrome c r e d u c t a s e , 3 0 - 5 0 ug o f p h o s p h a t i d y l c h o l i n e , 34 u n i t s o f e p o x i d e h y d r a s e , 0 . 5 pmol o f NADPH, 3 p m o l o f M g C l , 100 p m o l o f p o t a s s i u m p h o s p h a t e b u f f e r (pH 6.8) and 40 nmol o f l H J - ( ± ) - t r a n s - B P 7,8-dihydrodiol in a final volume o f 1 m l . S a m p l e s w e r e i n c u b a t e d f o r 10 m i n u t e s a t 3 7 ° .

P-450

P-450

P-448

P-448

Cytochrome

of

Epoxide Hydrase

Metabolism

Table

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

6.

LEVIN ET A L .

Activation

2

and Detoxification

4

6

8

UNITS O F E P O X I D E

of Benzolajpyrene

10

12

14

HYDRASE

Figure 5. Effect of epoxide hydrase on the mutagenic activity of the stereoisomeric BP 7,8-diol-9,10'epoxiaes (diol epoxide 1 and 2) and H 9,10'epoxide. Incubation mixtures consisted of 0.5 ml containing diol epoxide 1 (0.1 nmol), diol epoxide 2 (0.1 nmol) or H 9,10-epoxide (0.05 nmol), 2 Χ 10 bacteria, and the indicated amount of purified epoxide hydrase. After a 5-min incubation at 37°, top agar (2.0 ml) was added, and the plates were poured. The number of revotants induced per nmol of each derivative in the absence of epoxide hydrase was: diol epoxide 1, 3780; diol epoxide 2, 2650; H 9,10-epoxide, 6100. r

r

8

r

117

DRUG M E T A B O L I S M C O N C E P T S

118

Table

IX

Metabolism of (±)-trans-Benzol alpvrene 7,8-Pihydrodiol to Mutagenic P r o d u c t s by V a r i o u s P u r i f i e d Forms or Cytochrome

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

Source Species

of Cytochrome Prelreatment

His pmol

P-450 of Animals

Revertants/ Hemeprotein

Rat

3-Methylcholanthrene

435

Rat

Aroclor

1254

320

Rat

Phénobarbital

17

Rabbit

3-Methylcholanthrene

19

Mouse

Phénobarbital,

Fraction

A

Mouse

Phénobarbital,

Fraction

Cg

2

A l l i n c u b a t i o n m i x t u r e s c o n t a i n e d 50 g of phosphatidylcholine, 150 u n i t s o f NADPH-cyrochrome c r e d u c t a s e , 1 . 5 - 1 5 pmol o f p u r i f i e d cytochrome P-450, 2 χ 10 b a c t e r i a and 25 μ Μ s u b s t r a t e i n a f i n a l volume of 0.5 m l . I n c u b a t i o n s w e r e a t 37 for 5 min. Background m u t a t i o n f r e q u e n c i e s were s u b t r a c t e d . 8

6.

LEVIN ET A L .

Activation

and

Detoxification

of

Benzolajpyrene

119

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

p r o t e i n s u s i n g BP a s a s u b s t r a t e t o g e n e r a t e m u t a g e n i c m e t a b o l i t e s (cf Table IV), indicating that the substrate s p e c i f i c i t y of the p u r i f i e d c y t o c h r o m e s i s v e r y s i m i l a r f o r BP a n d BP 7,8-dihydro­ diol. I n a l l c a s e s , BP 7 , 8 - d i h y d r o d i o l was m e t a b o l i c a l l y a c t i ­ v a t e d t o a m o r e p o t e n t m u t a g e n ( s ) t h a n BP when b o t h substrates w e r e m e t a b o l i z e d b y t h e same h e m e p r o t e i n . Stereoselective Metabolism of Benzolajpyrene 7,8-Dihydrodiol by t h e Cytochrome P-448 Monoxygenase System. Among t h e w i d e array of f a c t o r s which influence the metabolism of xenobiotic compounds by t h e m i c r o s o m a l monoxygenase s y s t e m , e n a n t i o m e r s o f o p t i c a l l y a c t i v e compounds have been shown t o u n d e r g o differ­ e n t i a l rates of metabolism (45). A s t u d y by Huberman e t a l . ( 3 1 ) o n t h e m e t a b o l i s m o f r a d i o a c t i v e b i o s y n t h e t i c BP 778ΗΙΊn y c T r o d i o l w h i c h was d i l u t e d w i t h u n l a b e l l e d r a c e m i c d i o l c o n ­ c l u d e d t h a t g r e a t e r than 95% of the d i o l e p o x i d e formed by l i v e r microsomes from 3-methylcholanthrene-pretreated r a t s corresponded t o d i o l e p o x i d e 2 w h i l e Thakker e t a l . (43) showed t h a t s i g n i f i ­ c a n t amounts o f d i o l e p o x i d e s 1 a n d " ? were formed f r o m r a c e ­ m i c BP 7 , 8 - d i h y d r o d i o l w i t h l i v e r m i c r o s o m e s a s w e l l a s b y a h i g h l y p u r i f i e d monoxygenase system c o n t a i n i n g e i t h e r cytochrome P-450 or P-448 ( c f T a b l e V I I I ) . These c o n t r a s t i n g r e s u l t s prompt­ e d s t u d i e s o n t h e m e t a b o l i s m o f t h e o p t i c a l i s o m e r s o f BP 7 , 8 dihydrodiol. The r e s u l t s p r e s e n t e d i n T a b l e X d e m o n s t r a t e t h e marked s t e r e o s p e c i f i c i t y i n the metabolism of o p t i c a l l y pure (+)-and ( - ) - e n a n t i o m e r s o f t h e d i h y d r o d i o l by t h e p u r i f i e d c y t o ­ chrome P - 4 4 8 - c o n t a i n i n g monoxygenase system. The h i g h e s t s t e r e o s p e c i f i c i t y in the formation of diol epoxide 1 r e l a t i v e to diol e p o x i d e 2 was o b s e r v e d w i t h t h e ( + ) - e n a n t i o m e r o f BP 7 , 8 - d i h y d r o ­ diol. The r a t i o o f d i o l e p o x i d e 1 t o d i o l e p o x i d e 2 f o r m e d was f o u n d t o be 22:1 w i t h t h e ( + ) - e n a n t i o m e r and 1:4 f o r t h e (-)enantiomer. B o t h e n a n t i o m e r s o f BP 7 , 8 - d i h y d r o d i o l w e r e a l s o m e t a b o l i z e d t o a p h e n o l i c d e r i v a t i v e (unknown I I ) , tentatively i d e n t i f i e d as 6 , 7 , 8 - t r i h y d r o x y - 7 , 8 - d i h y d r o b e n z o U J p y ^ n e , which accounted f o r 5-10% o f t h e t o t a l m e t a b o l i t e s f o r m e d . Similar r e s u l t s were a l s o o b t a i n e d u s i n g microsomes from 3 - m e t h y l c h o l anthrene-pretreated rats (44). A d d i t i o n a l s t u d i e s have r e v e a l e d t h a t B P 7 , 8 - d i h y d r o d i o l f o r m e d f r o m BP b y l i v e r m i c r o s o m e s f r o m 3-methylcholanthrene-treated rats is p r i m a r i l y the (-)-enantiomer w h i c h r a t l i v e r m i c r o s o m e s and t h e p u r i f i e d monoxygenase system convert mainly into d i o l epoxide 2 (42,44). Examination of the a b i l i t y of the p u r i f i e d cytochrome P-448-containing monoxygenase system to convert (+)-and (-)-BP 7 , 8 - d i h y d r o d i o l i n t o mutagenic p r o d u c t s i n d i c a t e d t h a t b o t h o p t i c a l i s o m e r s o f BP 7 , 8 - d i h y d r o ­ d i o l a r e m e t a b o l i c a l l y a c t i v a t e d t o p o t e n t mutagens (43). Concluding Remarks. One o f t h e p r i m a r y r e a s o n s mining the mutagenic a c t i v i t y of chemicals is to i d e c o m p o u n d s w h i c h may c a u s e c a n c e r . Mutagenic potency t h e e x t e n t t o w h i c h compounds a r e c a p a b l e o f b i n d i n g

for detern t i f y those may r e f l e c t t o DNA.

of

(-)-and

0.46

(+)-Dihydrodiol

Metabolites —

Unknown

1.52

0.65 0.05

3.34 0.00

0.19 0.05

0.05 0.28

0.23

1 C i s 1 Trans" 2 C i s 2 I_ Γ Γ T n m o 1 formeïï/mnol~hemeprote i n / m i n u t e )

Tetraols

0.18

0.05

III

7,8-dihydrodiol

System

(+)-trans-BP

2.57

4.64

Total

a

Unknowns I and III c o n t a i n t r i o l - 2 and t r i o l - 1 , r e s p e c t i v e l y , w h i c h r e s u l t f r o m n o n e n z y m a t i c r e d u c t i o n o f d i o l e p o x i d e 2 and d i o l epoxide 1 at the 10-position (42,44). Unknown II c o n t a i n s a p h e n o l i c m e t a b o l i t e , t e n t a t i v e l y i d e n t i f i e d as 6 , 7 , 8 - t r i h y d r o x y - 7 , 8 - d i h y d r o b e n z o [ a J p y r e n e (44).

2

I n c u b a t i o n m i x t u r e s c o n t a i n e d 0 . 2 nmol o f c y t o c h r o m e P - 4 4 8 , 225 u n i t s o f NADPH-cytochrome c r e d u c t a s e , 30 y g o f p h o s p h a t i d y l c h o l i n e , 0.5 ymol o f NADPH, 3 y m o l M g C l , 100 y m o l o f p o t a s s i u m p h o s p h a t e b u f f e r (pH 6 . 8 ) and 4 0 n m o l o f r a d i o a c t i v e BP 7 , 8 - d i h y d r o d i o l i n a f i n a l v o l u m e o f 1 m l . S a m p l e s w e r e i n c u b a t e d f o r 10 m i η a t 3 7 ° .

0.14

Trans

(-)-Dihydrodiol

Substrate

Metabolism

Χ

b^ a R e c o n s t i t u t e d Monoxygenase

Stereoselective

Table

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

6.

LEVIN E T A L .

Activation

and Detoxification

of Benzolajpyrene

121

In t h i s s y s t e m , compounds w h i c h a r e h i g h l y s u s c e p t i b l e t o n u c l e o p h i l i c a t t a c k b y DNA r e l a t i v e t o o t h e r t y p e s o f r e a c t i o n s w o u l d be t h e most p o t e n t mutagens. S i n c e t h e m e t a b o l i t e s o f BP w h i c h are r e s p o n s i b l e f o r i t s mutagenic and c a r c i n o g e n i c a c t i v i t y a r e a n t i c i p a t e d t o have high chemical r e a c t i v i t y ( l o w s t a b i l i t y ) and may b e f o r m e d i n o n l y t r a c e a m o u n t s , t h e f o r m a t i o n o f s u c h p r o d u c t s may n o t b e r e a d i l y d e t e c t e d b y m e t a b o l i s m s t u d i e s . Because o f t h e s e c o n s i d e r a t i o n s , one a s p e c t o f o u r work t o i d e n t i f y t h e u l t i m a t e m u t a g e n i c a n d c a r c i n o g e n i c m e t a b o l i t e s o f BP has c e n t e r e d on t h e enzymes and mechanisms i n v o l v e d i n t h e m e t a b o l i c a c t i v a t i o n o f BP a n d BP d e r i v a t i v e s t o m u t a g e n i c p r o d u c t s . We, t h e r e f o r e , d e v e l o p e d an e n z y m a t i c a l l y w e l l - d e f i n e d m o d e l system u s i n g t h e p u r i f i e d monoxygenase system and e p o x i d e hydrase to help e l u c i d a t e the nature of the u l t i m a t e mutagenic metabol i t e ( s ) o f B P (27). These s t u d i e s have not o n l y demonstrated t h e complex i n t e r r e l a t i o n s h i p o f both t h e monoxygenase system and e p o x i d e h y d r a s e i n t h e m e t a b o l i c a c t i v a t i o n and i n a c t i v a t i o n o f BP a n d BP d e r i v a t i v e s b u t h a v e a l s o y i e l d e d v a l u a b l e i n f o r mation concerning the nature of the metabolites responsible f o r the m u t a g e n i c i t y o f BP. In t h e absence o f e p o x i d e h y d r a s e , t h e p u r i f i e d cytochrome P - 4 4 8 c o n t a i n i n g m o n o x y g e n a s e s y s t e m m e t a b o l i z e s BP t o a r e n e o x i d e s , p h e n o l s and q u i n o n e s ( c f T a b l e I ) . O f t w e n t y - o n e known a n d p o t e n t i a l p r i m a r y o x i d a t i v e m e t a b o l i t e s o f BP w h i c h h a v e been t e s t e d f o r m u t a g e n i c i t y i n S. t y p h i m u r i u m ( 2 1 , 3 2 , 4 0 ) , o n l y BP 4 , 5 - o x i d e i s a p o t e n t m u t a g e n . T h u s , BP 4 , 5 - o x i d e p r o b a b l y a c c o u n t s f o r m o s t o f t h e m u t a t i o n s i n d u c e d when BP i s m e t a b o l i c a l l y a c t i v a t e d by t h e p u r i f i e d monoxygenase system i n t h e absence o f epoxide hydrase. A s m a l l , b u t s i g n i f i c a n t number o f m u t a t i o n s ( £ 2 0 % o f t h e t o t a l m u t a t i o n s o b s e r v e d ) may be d u e t o t h e o t h e r p r i m a r y o x i d a t i v e m e t a b o l i t e s o f BP w h i c h i n c l u d e 6-HOBP. A d d i t i o n o f e p o x i d e hydrase t o t h e p u r i f i e d monoxyg e n a s e s y s t e m d e c r e a s e s m u t a t i o n s i n S. t y p h i m u r i u m s t r a i n TA 9 8 b y a maximum o f 3 0 % . T h e m u t a t i o n s o b s e r v e d when BP i s m e t a b o l i c a l l y a c t i v a t e d i n the presence of epoxide hydrase are not due t o BP 4 , 5 - o x i d e s i n c e s m a l l amounts o f e p o x i d e h y d r a s e c o m p l e t e l y i n a c t i v a t e BP 4 , 5 - o x i d e a s a b a c t e r i a l m u t a g e n ( c f F i g u r e 2). However, a d d i t i o n o f e p o x i d e hydrase t o t h e monoxygenase s y s t e m r e s u l t s i n t h e f o r m a t i o n o f BP 7 , 8 - d i h y d r o d i o l f r o m BP 7,8-oxide which i n t u r n i s m e t a b o l i c a l l y a c t i v a t e d by t h e p u r i f i e d monoxygenase system t o t h e h i g h l y mutagenic s t e r e o i s o m e r i c BP 7 , 8 - d i o l - 9 , 1 0 - e p o x i d e s w h i c h a r e n o t s u b s t r a t e s f o r e p o x i d e hydrase. Thus, t h e e f f e c t o f e p o x i d e h y d r a s e on t h e m e t a b o l i c a c t i v a t i o n o f BP t o m u t a g e n i c m e t a b o l i t e s a p p e a r s t o b e a c o m p o s i t e o f t h e i n a c t i v a t i o n o f BP 4 , 5 - o x i d e a n d t h e a c t i v a t i o n o f BP 7 , 8 - o x i d e v i a BP 7 , 8 - d i h y d r o d i o l t o BP 7 , 8 - d i o l - 9 , 1 0 - e p oxide. If t h i s i s indeed the case, then metabolic a c t i v a t i o n of 9,10-dihydrobenzolaJpyrene t o mutagenic metabolites should be r e a d i l y quenched upon a d d i t i o n o f e p o x i d e h y d r a s e s i n c e t h i s hydrocarbon cannot form a 7,8-diol-9,10-epoxide. T a b l e XI shows 2

3

DRUG M E T A B O L I S M C O N C E P T S

122

Table

XI

E f f e c t o f E p o x i d e H y d r a s e on t h e M e t a b o l i c A c t i v a t i o n o f Benzo (a)pyrene and 9 , 1 0 - D i h y d r o b e n z o i a l p y r e n e by a P u r i f i e d C y t o c h r o m ê T - 4 4 8 - C o n t a i n i ng MonoxygenaseSystem Epoxide

y

Substrate

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

His

Benzolajpyrene

9,10-DihydrobenzolaJpyrene

+

Hydrase

y

(units)

y

îo.o

Revertants/Plate

587

517

534

510

510

411

626

506

471

373

251

114

The c o m p l e t e monoxygenase s y s t e m c o n s i s t e d o f 50 y g o f p h o s p h a t i d y l c h o l i n e , 150 u n i t s o f NADPH-cytochrome c r e d u c t a s e , 0.1 nmol o f c y t o c h r o m e P - 4 4 8 , 0.1 y m o l o f NADPH, 1 2 . 5 nmol o f s u b s t r a t e and 0 - 1 0 u n i t s o f p u r i f i e d e p o x i d e h y d r a s e i n a f i n a l volume o f 0.5 ml c o n t a i n i n g 2 χ 1 0 b a c t e r i a of s t r a i n TA98. One u n i t o f e p o x i d e h y d r a s e i s d e f i n e d as 1 nmol s t y r e n e g l y c o l f o r m e d p e r miη from s t y r e n e o x i d e . 8

6.

LEVIN E T A L .

Activation

and

Detoxification

of Benzo[a]pyrene

123

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that 9,10-dihydrobenzolaJpyrene is metabolically activated to m u t a g e n i c m e t a b o l i t e s by t h e p u r i f i e d monoxygenase s y s t e m and t h a t a d d i t i o n of epoxide hydrase r e s u l t s i n a g r e a t e r than 85% i n h i b i t i o n in the mutations observed. F u t u r e s t u d i e s w i t h t h e p u r i f i e d monoxygenase system and e p o x i d e hydrase are aimed at p r e d i c t i n g t h e u l t i m a t e mutagenic metabolite(s) of other p o l y c y c l i c aromatic hydrocarbons. Indeed, p r e l i m i n a r y r e s u l t s i n o u r l a b o r a t o r y (47) have shown t h a t b e n z o (ajanthracene 3,4-dihydrodiol is metaboTTcally activated to a more p o t e n t mutagen(s) by t h e p u r i f i e d cytochrome P-448 c o n t a i n ­ ing monoxygenase system than i s benzol a J a n t h r a c e n e , o r b e n z o (ajanthracene 1,2-, 5,6-, 8,9- or Ι Ο , Π - d i h y d r o d i o l . A 1,2-epo x i d e o f t h e 3 , 4 - d i h y d r o d i o l w o u l d be i n t h e " b a y r e g i o n " o f the hydrocarbon. Such "bay r e g i o n " e p o x i d e s have been p r e d i c t e d t o be t h e most b i o l o g i c a l l y a c t i v e m e t a b o l i t e s o f p o l y c y c l i c a r o m a t i c h y d r o c a r b o n s b a s e d on c o r r e l a t i o n s o f known c a r c i n o ­ g e n i c i t y ( 4 8 ) and on q u a n t u m m e c h a n i c a l c a l c u l a t i o n s ( 4 9 ) . We a r e now e v a l u a t i n g t h e p o s s i b i l i t y t h a t t h e s t e r e o i s o m e r i c b e n z o l a j a n t h r a c e n e 3 , 4 - d i o l - l , 2 - e p o x i d e s may b e t h e u l t i m a t e c a r c i n o ­ genic m e t a b o l i t e s formed from benzolaJanthracene.

ance

Acknowledgment. We t h a n k M s . C a n d a c e C a s o f o r in the preparation of t h i s manuscript.

her

assist­

Footnotes 1

2

3

Abbreviations used: BP, b e n z o l a j p y r e n e ; 1-H0BP, 1 - h y d r o x y b e n z o l a J p y r e n e ; 2 - t o 1 2 - H 0 B P , o t h e r i s o m e r i c p h e n o l s ; BP 7,8-dihydrodiol, (±)-trans-7,8-dihydroxy-7,8-dihydrobenzolajp y r e n e ; BP 4 , 5 - , 9 , 1 0 - a n d 1 1 , 1 2 - d i h y d r o d i o l , o t h e r d i h y d r o ­ d i o l s o f B P ; BP 7 , 8 - d i o l - 9 , 1 0 - e p o x i d e , e i t h e r o r b o t h s t e r e o ­ isomers of (±)-7,8-dihydro-9,10-epoxy-7,8,9,10-tetrahydro BP; d i o l e p o x i d e 1, (±)-73,8a-dihydroxy-93,lO0-epoxy-7,8,9, 1 0 - t e t r a h y d r o BP; d i o l e p o x i d e 2, (±)-73,8a-dihydroxy-9a, 10a-epoxy-7,8,9,10-tetrahydro BP; H.-9,10-epoxide, (±)-9, 10-epoxy-7,8,9,10-tetrahydro BP. 6-H0BP o x i d i z e s n o n e n z y m a t i c a l l y t o 1,6-, 3 , 6 - and 6 , 1 2 quinone (46). I f a l l o f q u i n o n e f r a c t i o n 1 ( c f T a b l e 1) were due t o f o r m a t i o n o f 6-HOBP, t h e n a p p r o x i m a t e l y 10-15% o f t h e t o t a l m u t a t i o n s o b s e r v e d when BP i s m e t a b o l i z e d b y t h e cytochrome P - 4 4 8 - c o n t a i n i n g monoxygenase system c o u l d be due t o 6-HOBP. B a s e d o n t h e o b s e r v a t i o n t h a t BP i s m e t a b o l i z e d p r e d o m i ­ nantly to the (-)enantiomer of trans-BP 7,8-dihydrodiol by l i v e r microsomes f r o m 3 - m e t h y l c h o l a n t h r e n e - t r e a t e d r a t s ( 4 2 , 4 4 ) and t h e ( - ) e n a n t i o m e r i s m e t a b o l i z e d p r e d o m i n a n t l y t o d i o l e p o x i d e 2 ( c f T a b l e V I I I ) , most o f t h e mutagenic a c t i v i t y o f m e t a b o l i c a l l y a c t i v a t e d BP 7 , 8 - d i h y d r o d i o l must be due t o d i o l e p o x i d e 2 u s i n g t h e r e c o n s t i t u t e d monoxygenase system.

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CONCEPTS

Literature Cited 1. 2. 3. 4. 5.

6.

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7. 8. 9. 10. 11. 12. 13.

14.

15. 16. 17. 18.

19. 20. 21.

Conney, A. H. Pharmacol. Rev. (1967), 19: 317. Omura, T., Sato, R . , Cooper, D. Y., Rosenthal, O., and Estabrook, R. W. Fed. Proc. (1965), 24: 1181. G i l l e t t e , J . R . , Davis, D. C., and Sasame, H. A. Ann. Rev. Pharmacol. (1972), 12: 57. Haugen, D. Α., van der Hoeven, Τ. Α., and Coon, M. J . J. B i o l . Chem. (1975), 250: 3567. Ryan, D . , Lu, A. Y. H., Kawalek, J., West, S. B . , and Levin, W. Biochem. Biophys. Res. Commun. (1975), 64: 1134. Kawalek, J. C., Levin, W., Ryan, D . , Thomas, P. E., and Lu, A. Y. H. Mol. Pharmacol. (1975), 11: 874. Huang, M.-T., West, S. B . , and L u , Α. Y. H. J . B i o l . Chem. (1976), 251: 4659. Thomas, P. Ε., Lu, Α. Y. H., Ryan D . , West, S. B . , Kawalek, J., and Levin, W. J. B i o l . Chem. (1976), 251: 1385. Thomas, P. E., Lu, Α. Y. H., Ryan, D . , West, S. B . , Kawalek, J., and Levin, W. Mol. Pharmacol. (1976), 12: 746. World Health Organization, Prevention of Cancer, Tech. Rept. Serial 276 (1964), Geneva. Boyland, E . Prog. Exp. Tumor Res. (1969), 11: 222. Epstein, S. S. Cancer Res. (1974), 34: 2425. M i l l e r , E . C., and M i l l e r , J. A. in "Molecular Biology of Cancer," (Ed. Bush, H.), pp. 377-402, Academic Press, N.Y. (1974). Committee on the Biological Effects of Atmospheric P o l ­ lutants in "Particulate Polycyclic Organic Matter," pp. 13-35, Natl. Acad. Sci., Washington, D.C. (1972). Jerina, D. M . , and Daly, J. W. Science (1974), 185: 573. Sims, P . , and Grover, P. L . Adv.Cancer Res. (1974), 20: 165. Gelboin, H. V., Kinoshita, Ν . , and Wiebel, F . J. Fed. Proc. (1972), 31: 1298. Bend, J. R . , Ben-Ziui, Z., Van Anda, J., Dansette, P. M . , and Jerina, D. M. in "Polynuclear Aromatic Hydrocar­ bons: Chemistry, Metabolism and Carcinogenesis" (Ed. Freudenthal, R . , and Jones, P. W.), pp. 63-75, Raven Press, New York (1976). Lu, Α. Y. H., and Levin, W. Biochim. et Biophys. Acta (1974), 344: 205. L u , Α. Υ. H., Ryan, D . , Jerina, D. M . , Daly, J. W., and Levin, W. J. B i o l . Chem. (1975), 250: 8283. Jerina, D. M . , Yagi, H., Hernandez, O. Dansette, P. M . , Wood, A. W., Levin, W., Chang, R. L., Wislocki, P. G., and Conney, A. H. in "Polynuclear Aromatic Hydrocarbons: Chemistry, Metabolism and Carcinogenesis" (Ed. Freudenthal, R., and Jones, P. W.), pp. 91-113, Raven Press, New York (1976).

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22.

23. 24. 25. 26.

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27.

28. 29. 30. 31. 32.

33. 34. 35.

36. 37. 38. 39.

40.

41. 42.

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Benzo[a]pyrene

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Holder, G . , Yagi, H . , Dansette, P . , Jerina, D. M . , Levin, W., Lu, A. Y. H . , and Conney, A. H. Proc. Nat. Acad. S c i . U.S.A. (1974), 71: 4356. Ames, Β. Ν . , Durston, W. Ε., Yamasaki, Ε . , and Lee, F. D. Proc. Nat. Acad. S c i , U.S.A. (1973), 70: 2281. Ames, Β. Ν . , McCann, J., and Yamasaki, E. Mut. Res. (1975), 31: 347. Levin, W., and Conney, A. H. Cancer Res. (1967), 27: 1931. Holder, G. M . , Yagi, H . , Jerina, D. M . , Levin, W., Lu, Α. Y. H . , and Conney, A. H. Arch. Biochem. Biophys. (1975), 170: 557. Wood, A. W., Levin, W., Lu, Α. Y. H . , Yagi, H . , Hernandez, O., Jerina, D. M . , and Conney, A. H. J . B i o l . Chem. (1967), 251: 4882. Nebert, D. W., and Gelboin, H. V. J . B i o l . Chem. (1968), 243: 6242. Ryan, D . , Thomas, P. E., Lu, Α. Y. H . , West, S., and Levin, W. The Pharmacologist (1976), 18: 241. Malaveille, C . , Bartsch, H . , Grover, P. L., and Sims, P. Biochem. Biophys, Res. Commun. (1975), 66: 693. Huberman, E., Sachs, L., Yang, S. K . , and Gelboin, H. V. Proc. Nat. Acad. S c i . U.S.A. (1976) 73: 607. Wislocki, P. G . , Wood, A. W., Chang, R. L., Levin, W., Yagi, H . , Hernandez, O., Dansette, P. M . , Jerina, D. M . , and Conney, A. H. Cancer Res. (1976) 36: 3350. Tomaszewski, J . E., Jerina, D. M . , and Daly, J. W. BioChemistry (1975), 14: 2024. Wiebel, F. J. Archiv. Biochem. Biophy. (1975), 168: 609. Borgen, Α . , Darvey, H . , Castagnoli, N . , Crocker, T. T., Rasmussen, R. E., and Wang, I. Y. J . Med. Chem. (1973), 16: 502. Sims, P . , Grover, P. L., Swaisland, Α . , P a l , K . , and Hewer, A. Nature (1974), 252: 326. Wislocki, P. G . , Wood, A. W., Chang, R. L., Levin, W., Yagi, H . , Hernandez, O., Jerina, D. M . , and Conney, A. H. Biochem. Biophys. Res. Commun. (1976), 68: 1006. Newbold, R. F., and Brookes, P. Nature (1976), 261: 52. Wood, A. W., Wislocki, P. G . , Chang, R. L., Levin, W., Lu, Α. Y. H., Yagi, H . , Hernandez, O., Jerina, D. M . , and Conney, A. H. Cancer Res. (1976), 36: 3358. Wood, A. W., Goode, R. L., Chang, R. L., Levin, W., Conney, A. H . , Yagi, H . , Dansette, P. M . , and Jerina, D. M. Proc. Nat. Acad. S c i . U.S.A. (1975), 72: 3176. Yagi, H . , Hernandez, O., and Jerina, D. M. J . Amer. Chem. Soc. (1975), 97: 6881. Yang, S. K . , McCourt, D. W., Roller, P. P . , and Gelboin, H. V. Proc. Nat. Acad. S c i . U.S.A. (1976), 73: 2594.

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48. 49.

DRUG METABOLISM CONCEPTS

Thakker, D. R., Yagi, H., Lu, A. Y. H., Levin, W., Conney, A. H. and Jerina, D. M. Proc. Nat. Acad. Sci. U.S.A. (1976), 73: 3381. Thakker, D. R., Yagi, H., Akagi, H., Koreeda, M., Lu, A. Y. H., Levin, W., Wood, A. W., Conney, A. H., and Jerina, D. M. Chem.-Biol. Interactions (1976), in press. Jenner, P., and Testa, B. Drug Metabolism Reviews (1973), 2: 117. Lorentzen, R. J., Caspary, W. J., Lesko, S. Α., and Ts'o, P. O. P. Biochemistry (1975), 14: 3970. Wood, A. W., Levin, W., Lu, A. Y. H., Ryan, D., West, S. B., Lehr, R. E . , Schaefer-Ridder, M., Jerina, D. M., and Conney, A. H. Biochem. Biophys. Res. Commun. (1976), 72: 680. Jerina, D. M., and Daly, J . W. in "Oxidation at Carbon in Drug Metabolism" (Ed. Parke, D. V., and Smith, R. L.) pp. 15-33, Taylor and Francis, Ltd., London, (1976). Jerina, D. M., Lehr, R. E . , Yagi, H., Hernandez, O., Dansette, P. M., Wislocki, P. G., Wood, A. W., Chang, R. L . , Levin, W., and Conney, A. H. in "The Role of Metabolic Activation in Producing Carcinogenic Chemicals" (Ed. deSerres, F. J., and Fouts, J . R.) North Holland, Amsterdam, in Press (1976).

7

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch007

In vitro Reactions of the Diastereomeric 9,10-Epoxides of (+) and (—)-trans-7,8-Dihydroxy-7,8-dihydrobenzo[α]pyrene with Polyguanylic Acid and Evidence for Formation of an Enantiomer of Each Diastereomeric 9,10-Epoxide from Benzo[α]pyrene in Mouse Skin P. D. MOORE and M. KOREEDA—Department of Chemistry, The Johns Hopkins University, Baltimore, MD 21218 P. G. WISLOCKI, W. LEVIN, and A. H. CONNEY—Department of Biochemistry and Drug Metabolism, Hoffmann-La Roche Inc., Nutley, NJ 07110 H. YAGI and D. M. JERINA—Laboratory of Chemistry, NIAMDD, National Institutes of Health, Bethesda, MD 20014

Benzo[a]pyrene (BP) is a potent carcinogen which is wide­ spread in the environment. The precise mechanism by which BP and other polycyclic aromatic hydrocarbons (PAH's) induce neoplasia is not presently known. There is, however, some correlation between the extent of binding of PAH's to cellular macromolecules (DNA, RNA, and protein) and their carcinogenicity (1-3). Since carcino­ genic PAH's are also mutagenic in mammalian and bacterial test systems in which they undergo metabolic activation (3-5), their interactions with nucleic acids are of obvious biological impor­ tance. In addition, these interactions with nucleic acids are prospective initiation events in chemical carcinogenesis. BP is irreversibly bound to DNA in vitro when a cytochrome P-450 mono­oxygenase system is present (6,7). A number of reactive metabo­ lites have been considered. These include various epoxides and arene oxides (3,8,9), free radicals (10,11), radical cations (12, 13), and "non-K-region" diol epoxides. Recent studies on the binding of trans-7,8-dihydroxy-7,8-dihydro-BP (BP 7,8-dihydrodiol) to DNA in vitro (14) and evidence that this binding is me­ diated by a 7,8-dihydrodiol-9,10-epoxide (9) have led to the syn­ thesis of the two possible diastereomers (15,16) and to extensive study of their interactions with nucleic acids (9,14,17-26). The two possible racemic diastereomers in which the benzylic hydroxyl group and the oxirane oxygen are either c i s , 1 or trans, 2 are highly reactive (Figure 1). In water, these diol epoxides are rapidly hydrolyzed by cis and trans addition of water to the epoxides at C-10 (27-30). That 1 and/or 2 are probable ultimate carcinogenic forms of BP is suggested by the high carcinogenicity of their metabolic precursors, BP 7,8-oxide and BP 7,8-dihydro127

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128

d i o l (31,32) as w e l l as by t h e i r h i g h m u t a g e n i c i t y toward mamma­ l i a n and b a c t e r i a l c e l l s (30,33-36). The h i g h chemical r e a c t i v i t y and the mutagenic a c t i v i t y p r o v i d e c l e a r evidence o f the a b i l i t y o f these d i o l epoxides t o damage n u c l e i c a c i d s . M o d i f i c a t i o n o f n u c l e i c a c i d s w i t h carcinogens and mutagens has been e x t e n s i v e l y s t u d i e d (37). For example, almost every p o s i t i o n on the n u c l e o s i d e s as w e l l as the phosphate backbone i s known t o be a l k y l a t e d t o some extent. Common r e a c t i o n s are at N o f guanine 6 a (Figure 2) and N o f adenine (38) (see Table I ) . Recent q u a n t i t a t i v e s t u d i e s have r e v e a l e d that the amount o f 0a l k y l a t i o n at 0 o f guanine (39,40) and a t phosphate groups t o form p h o s p h o t r i e s t e r s (41,42) c o r r e l a t e s b e t t e r w i t h the b i o l o g ­ i c a l a c t i v i t y o f the a l k y l a t i n g agents than does the t o t a l extent o f a l k y l a t i o n . P r e s e n t l y i t i s u n c l e a r whether a s p e c i f i c a l k y l a t i o n i s important t o c a r c i n o g e n i c i t y o r whether o t h e r f a c t o r s such as d i f f e r e n c e s i n the r a t e o f DNA r e p a i r ( N > 0 - a l k y l ) are r e s p o n s i b l e f o r t h i s e f f e c t (43). Extensive m e t h y l a t i o n o f DNA at N o f guanine by tf-methy1-tfn i t r o s o u r e a i s t y p i c a l o f m e t h y l a t i n g agents. R e a c t i v e aromatic compounds o f h i g h e r molecular weight show d i f f e r e n t s e l e c t i v i t y . For example, tf-acetoxy-2-acetylaminofluorene 3 reacts selectively at C o f guanosine 6 b (44) and t o a l e s s e r degree at the 2-amino group o f guanosine (45). 7-Bromomethylbenζ[a]anthracene 4 (46) and 7,12-dimethylbenz[a]anthracene 5,6-oxide 5 (47) a l k y l a t e pre­ dominantly at the e x o c y c l i c 2-amino group o f guanosine. P r i o r o r i e n t a t i o n o f the p l a n a r r e a c t a n t by i n t e r c a l a t i o n between the base p a i r s may account f o r s e l e c t i v e a l k y l a t i o n o f the 2-amino group (48). I n t e r c a l a t i o n i n t o n u c l e i c a c i d s a l s o tends t o dra­ m a t i c a l l y i n c r e a s e the s o l u b i l i t y o f PAH s i n aqueous s o l u t i o n s (49,50). Thus, i n t e r c a l a t i o n o f PAH d e r i v a t i v e s may serve the dual f u n c t i o n o f i n c r e a s i n g the e f f e c t i v e c o n c e n t r a t i o n o f the r e a c t i v e form o f the PAH near the n u c l e i c a c i d and o f o r i e n t a t i n g t h i s r e a c t i v e form f o r s p e c i f i c s i t e s o f the polymer. D i o l epoxides 1 and 2 have been shown t o a t t a c k the e x o c y c l i c 2-amino group o f guanosine i n n u c l e i c a c i d s . D i o l epoxide 2, on r e a c t i o n w i t h p o l y g u a n y l i c a c i d (poly (G)) i n 75% acetone/water, has been shown t o form a p a i r o f d i a s t e r e o m e r i c adducts by t r a n s opening o f the epoxide at C-10 (22). One o f these diastereomers and other u n i d e n t i f i e d products were d e t e c t e d i n UNA i s o l a t e d from bovine b r o n c h i a l e x p i a n t s which had been exposed t o [ H]-BP (23). D i o l epoxide 1 has been shown t o form both c i s and t r a n s adducts at C-10 w i t h the 2-amino group o f guanosine i n p o l y (G) (50% ace­ tone/water) as w e l l as a l k y l a t e the phosphate backbone t o form p h o s p h o t r i e s t e r s (26). The products formed from the in vitro r e ­ a c t i o n s o f the d i o l epoxides w i t h DNA have been s t u d i e d (9,18,23, 25). We d e s c r i b e here d e t a i l s o f the r e a c t i o n s o f d i o l epoxides 1 and 2 w i t h p o l y (G) and p r o v i d e evidence f o r the s t r u c t u r e o f the RNA adducts which form when [ H]-BP i s p a i n t e d on mouse s k i n . Adducts from both d i o l epoxides 1 and 2 are formed i n t h i s in vivo 7

3

6

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch007

7

6

7

8

1

3

3

MOORE ET A L .

Diastereomeric

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch007

g,

9,10-Epoxides

from

c/s-2

Benzo[a]pyrene

trans-Z

Figure 1. Hydrolysis of diol epoxides to tetraoh. Unless specifically designated, diol epoxide 1 and diol epoxide 2 refer to racemic compounds. The structures 1 and 2 are intended to show the rehtive stereochemistry between the two sets of diastereomers and to show the absolute stereochemistry of the diol epoxide products derived from the (—) enantiomer of BP 7,8-aihydrodiol.

DRUG M E T A B O L I S M C O N C E P T S

130

experiment w i t h a t i s s u e toward which BP i s c a r c i n o g e n i c . These adducts are o p t i c a l l y a c t i v e and t h e i r absolute stereochemistry has been assigned through the use o f the o p t i c a l l y pure enantio­ mers o f d i o l epoxides 1 and 2. M a t e r i a l s and Methods D i o l Epoxides. D i o l epoxide 1 was synthesized from BP 7,8d i h y d r o d i o l v i a a b r o m o - t r i o l , w h i l e d i o l epoxide 2 was obtained by m-chloroperbenzoic a c i d o x i d a t i o n o f the same BP 7,8-dihydro­ d i o l (15,16). T r i t i a t e d d i o l epoxides were s y n t h e s i z e d from [9, 10- H]-BP 7,8-dihydrodiol as p r e v i o u s l y d e s c r i b e d (27) and were used at a s p e c i f i c a c t i v i t y o f 2.3 pCi/ymol. O p t i c a l l y a c t i v e d i o l epoxides 1 and 2 were s y n t h e s i z e d from (+) and (-)-BP 7,8d i h y d r o d i o l which had been r e s o l v e d v i a the d i a s t e r e o m e r i c b i s (-)-a-methoxy-a-trifluoromethylphenylacetates (51-53). B i n d i n g o f D i o l Epoxides t o Poly (G). The potassium s a l t o f p o l y g u a n y l i c a c i d ( 5 ) (mol. wt. > 150,000) was obtained from Sigma. [ 8 - H ] - P o l y g u a n y l i c a c i d was purchased from M i l e s Research Products and used at a s p e c i f i c a c t i v i t y o f 0.31 yCi/ymol o f phos­ phorous. For a t y p i c a l b i n d i n g experiment, 1.7 mg o f p o l y (G) was d i s s o l v e d i n 0.5 ml o f d i s t i l l e d water and the pH was adjusted by a d d i t i o n o f d i l u t e HC1 o r KOH. A pH o f 7.0 (monitored w i t h a pH e l e c t r o d e ) was used i n a l l cases except those s p e c i f i c a l l y d e s i g ­ nated. An equal volume o f s p e c t r o s c o p i c grade acetone was added and the s o l u t i o n was t r e a t e d w i t h 0.4 mg o f e i t h e r d i o l epoxide i n 100 μΐ o f dry t e t r a h y d r o f u r a n (THF). The s o l u t i o n s were incubated at 37 C f o r 18 hours. T e t r a o l s which r e s u l t from h y d r o l y s i s were removed by e x t r a c t i o n w i t h t h r e e equal volumes o f e t h y l a c e t a t e . The modified polymer was i s o l a t e d by a c i d i f i c a t i o n o f the s o l u t i o n w i t h 0.1 volume o f 2.5 M sodium acetate (pH 5.0) and p r e c i p i t a t i o n o f the polymer w i t h 3 volumes o f e t h a n o l . The mixture was s t o r e d at 0°C overnight and then c e n t r i f u g e d f o r 15 min. at 3,000 rpm. The supernatant was removed and the p r e c i p i t a t e d polymer was wash­ ed w i t h 2.0 ml o f ethanol. The polymer could be r e d i s s o l v e d i n water and the p r e c i p i t a t i o n procedure was repeated without s i g n i ­ f i c a n t change i n the amount o f bound hydrocarbon absorbing at 350 nm (54). H y d r o l y s i s o f P o l y (G) t o Nucleosides. M o d i f i e d p o l y (G) was d i s s o l v e d i n 1.0 Ν KOH and incubated f o r 18 hours at 37°C. The s o l u t i o n was n e u t r a l i z e d w i t h 2 N HCIO4 which r e s u l t e d i n p r e c i p i ­ t a t i o n o f KCIO4. A f t e r c e n t r i f u g a t i o n the s a l t was separated by decanting the aqueous s o l u t i o n . Tris-(hydroxymethyl)-aminomethane was added t o the aqueous supernatant t o produce a f i n a l concentra­ t i o n o f 1.0 mg/ml and the pH was adjusted t o 8.4. Bacterial alka­ l i n e phosphatase (1 unit/mg o f p o l y (G)) was added and the s o l u ­ t i o n was incubated f o r 24 hours at 37°C, which a f f e c t e d complete h y d r o l y s i s t o n u c l e o s i d e s as monitored by paper chromatography. Removal o f Ribose by N - M e t h y l a t i o n w i t h Dimethyl S u l f a t e . A f t e r base h y d r o l y s i s , the modified n u c l e o t i d e mixture from 50 mg o f p o l y (G) was d i s s o l v e d i n 10 ml o f 2 M KH2PO4 at pH 7.0 and

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch007

3

f

3

e

7

7.

MOORE E T A L .

Diastereomeric

9,10-Epoxides

from Benzofajpyrene

131

t r e a t e d w i t h three 100 y l a l i q u o t s o f dimethyl s u l f a t e a t 2 hour i n t e r v a l s . The pH was maintained > 6.0 by o c c a s i o n a l a d d i t i o n o f d i l u t e KOH. When the uv spectrum o f the mixture showed the spec­ t r a l s h i f t s w i t h pH which are c h a r a c t e r i s t i c o f 7-methylguanosine (55), the pH was adjusted t o 6.0 and the mixture was r e f l u x e d f o r 1 hour t o remove the r i b o s e from the methylated base. The d i o l epoxide-7-methylguanine base was s e l e c t i v e l y e x t r a c t e d w i t h t h r e e equal volumes o f e t h y l acetate. Treatment o f Mice w i t h [ H]-BP. The backs o f eleven C57BL/6J female mice were shaved and, two days l a t e r each mouse was p a i n t e d w i t h 100 ]xg o f [ H]-BP (Amersham S e a r l e ) (1.4 mCi/mouse) i n 100 y l o f acetone. Twenty f o u r hours l a t e r the mice were s a c r i f i c e d , and the epidermal l a y e r was i s o l a t e d and homogenized as d e s c r i b e d (56) The RNA was, i s o l a t e d by a p h e n o l - c r e s o l method (57). The 1.3 mg o f RNA thus obtained contained % 2.8 χ 1 0 dpm/mg. High-pressure L i q u i d Chromatography. The n u c l e o s i d e adducts from 1 and 2 were c o n v e n i e n t l y separated from unmodified guanosine w i t h a Poragel PN column (3/8" χ 3 ) e l u t e d w i t h 85% methanol i n water a t a f l o w r a t e o f 5.0 ml/min. For high r e s o l u t i o n a n a l y s i s o f the n u c l e o s i d e adducts, a Waters yC^g-Bondapak column (1/4" χ l ) was e l u t e d a t a constant r a t e o f 1.2 ml/min w i t h 39% methanol/ water f o r 50 minutes f o l l o w e d by a 60 minute l i n e a r gradient t o 50% methanol/water. 3

3

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch007

5

f

f

Results At pH 7.0 d i o l epoxides 1 and 2 were found t o b i n d t o p o l y (G) t o about the same extent. Binding was c h a r a c t e r i z e d by the uv a b s o r p t i o n p a t t e r n o f the 7,8,9,10-tetrahydro-BP moiety i n the 320 - 355 nm r e g i o n (Figure 3). The a b s o r p t i o n maximum at 352 nm r e ­ presents a bathochromic s h i f t o f 9 nm and a s u b s t a n t i a l decrease i n the e x t i n c t i o n c o e f f i c i e n t which would be expected f o r the 7, 8,9,10-tetrahydro-BP chromophore i n t h i s r e g i o n . These changes i n the chromophore l a r g e l y disappear upon a l k a l i n e h y d r o l y s i s o f the modified p o l y (G) ( i n s e t F i g u r e 3 ) . This c l e a r l y i n d i c a t e s t h a t the hydrocarbon i s c l o s e l y a s s o c i a t e d w i t h the polymer (50). Evidence p r o v i n g t h a t t h i s a s s o c i a t i o n i n v o l v e s covalent bond formation i s presented l a t e r . E f f e c t o f pH on Rate and Extent o f Binding. The r a t e and extent o f b i n d i n g were explored w i t h [9,10-^H]-diol epoxides s i n c e t o t a l b i n d i n g could be a c c u r a t e l y determined r a d i o c h e m i c a l l y . B u f f e r s o l u t i o n s were avoided s i n c e the d i o l epoxides r e a c t w i t h phosphate i o n s . For example, from 5 - 10% o f the hydrocarbon becomes non-extractable due t o a l k y l a t i o n o f 0.05 M phosphate a t pH 7.0 (26). When these phosphate-diol epoxide products were heated f o r 1 hour on a steam bath, a l l o f the hydrocarbon was l i b ­ erated from i t s phosphate e s t e r s . When d i o l epoxide was added t o pure water o n l y a t r a c e ( « 1%) o f hydrocarbon remained a f t e r e x t r a c t i o n , which i n d i c a t e s t h a t very l i t t l e non-extractable s o l v o l y s i s products are formed.

DRUG M E T A B O L I S M C O N C E P T S

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch007

132

Figure 3. Ultraviolet spectra of poly (G), diol epoxide 1, and poly (G) modified by diol epoxide 1. The following c values were observed, poly (G) in water € o = 9,000 and diol epoxide 1 in dry THF € s = 46,500. The inset shows repetitive scans of poly (G)-diol epoxide 1 in 0.5N KOH at 25°C. 26

Si

7. MOORE E T A L .

Diastereomeric

9,10-Epoxides

from Benzo[a]pyrene

133

The e f f e c t o f pH on t o t a l b i n d i n g i s shown i n F i g u r e 4. Maximum b i n d i n g takes p l a c e i n s l i g h t l y a c i d i c media. Both d i o l epoxides showed s i m i l a r pH-dependent r a t e s o f b i n d i n g t o p o l y (G). Binding o f d i o l epoxide 1 was s l i g h t l y f a s t e r but q u a l i t a t i v e l y the same as t h a t o f d i o l epoxide 2. Figure 5 shows t h e r a t e o f b i n d i n g f o r d i o l epoxide 2 a t v a r i o u s pH v a l u e s . The r a t e o f b i n d i n g a t pH 4.0 i s a t l e a s t twenty times g r e a t e r than t h a t a t pH 7.0. Even though b i n d i n g a t pH 4.0 i s much f a s t e r than t h a t a t pH 7.0, t h e t o t a l amount bound a f t e r 2 hours o f i n c u b a t i o n i s s l i g h t l y l e s s . These observations a r e c o n s i s t e n t w i t h a c i d c a t a l y z e d a l k y l a t i o n o f the polymer competing w i t h a c i d c a t a l y z e d hyd r o l y s i s o f the d i o l epoxide t o t e t r a o l s .

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch007

Reaction on the Phosphate Backbone o f P o l y (G) When p o l y (G) was modified by e i t h e r d i o l epoxide and p u r i f i e d by s o l v e n t e x t r a c t i o n and m u l t i p l e p r e c i p i t a t i o n , non-bound t e t r a o l s were completely removed. The uv spectrum o f t h i s p u r i f i e d modified p o l y (G) showed a d i s t i n c t v a l l e y a t 343 nm. When the modified polymer was heated t o 100°C a t n e u t r a l pH a small amount o f f r e e t e t r a o l s was l i b e r a t e d and could be detected by t h e i r uv spectrum. The l i b e r a t i o n o f t e t r a o l s was confirmed by co-chromatography w i t h standards and by the mass spectrum o f t h e t e t r a a c e t y l d e r i v a t i v e (M a t m/e 488). A f t e r e x t r a c t i o n o f these t e t r a o l s prolonged h e a t i n g (4 hours) o f the modified p o l y (G) f a i l e d t o l i b e r a t e f u r t h e r q u a n t i t i e s o f t e t r a o l s . Base hydrolys i s o f modified p o l y (G) which had not been heated i n water a l s o l e d t o the formation o f t e t r a o l s . Q u a n t i t a t i v e measurements i n d i cated t h a t 10 - 15% o f the t o t a l bound hydrocarbon could be r e leased as t e t r a o l s . Since p o l y c y c l i c aromatic hydrocarbons, such as t h e t e t r a o l s or the d i o l epoxides themselves, can p h y s i c a l l y i n t e r c a l a t e i n t o n u c l e i c a c i d s t h e p o s s i b l e presence o f r e s i d u a l non-covalently1inked hydrocarbon d e r i v a t i v e s cannot, a priori, be e l i m i n a t e d . A d d i t i o n o f p o l y (G) modified by 1 t o 0 enriched water (18%) f o l l o w e d by h e a t i n g f o r 15 minutes a t 100°C generated t e t r a o l s which i n c o r p o r a t e d 0.96 atom % o f s o l v e n t oxygen. When c i s - 1 and trans-l t e t r a o l s were added t o t h i s same p o l y (G) s o l u t i o n and s i m i l a r l y t r e a t e d , no i n c o r p o r a t i o n o f 0 i n the r e i s o l a t e d t e t r a o l s was observed. This r e s u l t s t r o n g l y argues against the presence o f p h y s i c a l l y bound t e t r a o l s . Since the h a l f - l i f e f o r b i n d i n g i s l e s s than 10 minutes a t pH 7.0, i t seems u n l i k e l y t h a t i n t a c t d i o l epoxides could have s u r v i v e d d u r i n g the i n c u b a t i o n w i t h out e i t h e r b i n d i n g t o the p o l y (G) o r undergoing h y d r o l y s i s t o ext r a c t a b l e t e t r a o l s . I n a d d i t i o n , i t i s u n l i k e l y t h a t t h e d i o l epoxides could s e l e c t i v e l y r e s i s t e x t r a c t i o n compared t o t e t r a o l s . R e l a t i v e amounts o f c i s and t r a n s t e t r a o l s which form i n v a r i o u s c o n d i t i o n s are shown i n Table I I . Base h y d r o l y s i s o f d i o l epoxide 1 leads t o s u b s t a n t i a l trans-l t e t r a o l w h i l e the t e t r a o l s gener+

i 8

1 8

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch007

DRUG M E T A B O L I S M C O N C E P T S

Ο·

4.0

,

1

eo

ao

1—

ιαο

PH

Figure 4. Extent of binding as a function of pH. Poh (G) solutions of 3.4 mg/ml were adjusted to pH 4.0, 6.0, 7.0, 8.0, ana 10.0, then diluted with an equal volume of acetone. 2.95 ftCi [9,10H] diol epoxide I or 2 was added to 1.0 ml of the stock poly (G) solu­ tions at 37°C, and 50-μΙ aliquots of the reaction mixture were removed at convenient time intervals. Each aliquot was added to a tube containing 1.0 ml of H 0 and 5.0 ml of ethyl acetate. This mixture was shaken vig­ orously, and the ethyl acetate was removed. The aqueous solution of poly (G) was then extracted twice with 5.0 ml of ethyl acetate. Since tetraols are readily extracted from aqueous poly(G) solutions the [ H] counts present were assumed to be bound to poly (G). Results obtained when uv absorbance at 350 nm was used to quantitate binding indicated that the above extraction procedure was correct to ±10%. Diol epoxide 1 was examined after 60 min of reaction and diol epoxide 2 after 50 min of reaction. Points represent the average of two separate determinations. 3

2

3

7.

M O O R E ET A L .

Diastereomeric

9,10-Epoxides

from Benzolajpyrene

135

Table I . M e t h y l a t i o n o f Salmon Sperm DNA by tf-Methyl-tfn i t r o s o u r e a . Data o f Lawley and Shah (38).

Site

Base

Guanine

% Total i d e n t i f i e d m e t h y l a t i o n o f bases

7

Ν 0 N3

75.7 7.3 1.1

6

3 Adenine

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch007

Thymidine

i N N

Source

1

7

N 0

3

N

3

Cytidine

Table I I .

11.2 1.4 2.5

N

0.3 0.1

4

0.6

Percent c i s and t r a n s T e t r a o l s from D i o l Epoxides 1 and 2 and M o d i f i e d Poly (G).

Conditions

50% acetone/water, pH 7.0 ( c o n d i t i o n s of binding)

c i s - 1 trans-l

85

trans-2

15

50% acetone/water, pH 7.0 ( c o n d i t i o n s of binding) 75

cis-2

15

85

39

61

2

98

25

P o l y (G)

100°C, pH 7.0, 15 minutes

Poly (G)

100°C, pH 7.0, 15 minutes

1

1.0 Ν KOH, 20% THF/H 0, 37°C, 24 h r s .

18

81

Poly (G)

1.0 Ν KOH, 37°C, 24 h r s .

95

5

P o l y (G)

1.0 Ν KOH, 37°C, 24 h r s .

2

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136

DRUG M E T A B O L I S M C O N C E P T S

ated from base h y d r o l y s i s o f p o l y (G) modified by 1 are almost ex­ c l u s i v e l y c i s - 1 t e t r a o l s . T h i s provides another argument t h a t the l a b i l e hydrocarbon products are not due t o r e s i d u a l d i o l epoxides. These r e s u l t s are c o n s i s t e n t w i t h the presence o f a heat and a l k a l i n e l a b i l e covalent d i o l epoxide adduct. The formation o f phosphate-diol epoxide monoesters which could be cleaved by simple h e a t i n g at pH 7.0 together w i t h the chemical and thermal s t a b i l i t y o f the guanosine adducts d i s c u s s e d below s t r o n g l y suggest t h a t the l i b e r a t e d t e t r a o l s are d e r i v e d from a l k y l phosphates o f p o l y (G) (37,40-42,58). Reaction on the Guanine Base o f P o l y (G). When modified p o l y (G) was hydrolyzed i n base, the t e t r a o l s r e l e a s e d from the phosphate adducts were r e a d i l y removed by e x t r a c t i o n o f the neu­ t r a l i z e d h y d r o l y s a t e w i t h e t h y l a c e t a t e . A f t e r a l k a l i n e phospha­ tase treatment, the n u c l e o s i d e adducts became s u f f i c i e n t l y nonp o l a r t o a l l o w e x t r a c t i o n i n t o organic s o l v e n t s . However, the n u c l e o s i d e adducts were more c o n v e n i e n t l y i s o l a t e d by reverse phase chromatography o f the h y d r o l y s a t e on a Poragel PN high-pres­ sure l i q u i d chromatography column (Figure 6 ) . Guanosine, nucleo­ s i d e adducts, and t e t r a o l s are a l l separated. The h i g h c a p a c i t y o f the column a l l o w s s e p a r a t i o n o f 10 - 100 mg samples w i t h a s i n g l e i n j e c t i o n . The n u c l e o s i d e adduct f r a c t i o n contains > 95% o f a l l non-tetraol-hydrocarbon products. High r e s o l u t i o n chromatography on yC -Bondapak separated the n u c l e o s i d e adduct f r a c t i o n i n t o a number o f peaks. Each d i o l ep­ oxide formed f o u r products which, on the b a s i s o f peak a r e a , ap­ peared t o c o n s i s t o f two groups. N u c l e o s i d e - d i o l epoxide 1 prod­ u c t s were e l u t e d i n the approximate r a t i o o f 1:2:1:2. Products from d i o l epoxide 2 were o v e r a l l s l i g h t l y more p o l a r and e l u t e d i n r a t i o o f 3:1:1:3 (Figure 7 ) . The CD s p e c t r a (Figure 8) o f the i n d i v i d u a l peaks e s t a b l i s h e d t h a t the products o f equal peak area were a c t u a l l y p a i r s o f diastereomers which r e s u l t e d from the r e ­ a c t i o n o f racemic d i o l epoxides w i t h o p t i c a l l y pure p o l y (G). F u r t h e r p r o o f t h a t the p a i r s o f products are diastereomers was ob­ t a i n e d through the use o f o p t i c a l l y pure d i o l epoxides 1 and 2, s i n c e o n l y one component o f each p a i r was obtained from each o f the d i o l epoxide enantiomers (Table I I I ) . Thus, when ( + ) - d i o l ep­ oxide 1 d e r i v e d from (+)-BP 7,8-dihydrodiol was reacted w i t h p o l y (G), o n l y n u c l e o s i d e adducts F and D were formed. S i m i l a r l y , (+)d i o l epoxide 2 from (-)-BP 7,8-dihydrodiol produced o n l y nucleo­ s i d e adducts Β and G. S t r u c t u r e o f the Guanosine Adducts. Normally the p o s i t i o n at which a p u r i n e base i s a l k y l a t e d can r e a d i l y be e s t a b l i s h e d by uv spectroscopy. C h a r a c t e r i s t i c changes i n the uv s p e c t r a o f the a l k y l a t e d bases as a f u n c t i o n o f pH g e n e r a l l y p r o v i d e s s u f f i c i e n t evidence f o r assignment o f the s i t e o f a l k y l a t i o n (37). This ap­ proach proved t o be o f l i m i t e d value i n the present study s i n c e the uv s p e c t r a o f the n u c l e o s i d e adducts as a f u n c t i o n o f pH showed l i t t l e change. The a b s o r p t i o n spectrum o f the 7,8,9,1018

Diastereomenc

MOORE ET AL,

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch007

7.

40

9,10-Epoxides

from Benzo[a]pyrene

137

Figure 5. Rate of binding of diol epoxide 2 to poly (G) as a function of pH. Conditions were the same as in Figure 4 with the exception that the binding was examined at the indicated time intervals.

60

Time (min)

guanosine

nucleoside adducts

tetraols 5

10

15

20

25

Figure 6. Purification of guanosinediol epoxide 1 products via Poragel PN chromatography (see Materiats and Methods" for details) u

Tîme(min)

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch007

138

DRUG M E T A B O L I S M

CONCEPTS

E; Diol Epcoode 2

10

20

30

40

50 60 Time (min)

70

80

90

100

110

Figure 7. High resolution chromatography of the guanosine adducts from diol epoxides 1 ana 2. Conditions are as described in "Materials and Methods." The peak with a retention time of 39 min in the lower trace is caused by trans-2 tetraol from diol epoxide 2 which was a contaminate in this sample.

MOORE ET

AL.

Diastereomeric

9,10-Epoxides

from

Benzo[a]pyrene

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch007

•125-1

-I25H

Figure 8. CD spectra of the major pairs of guanosine adducts from diol epoxide 1(D and H) and diol epoxide 2 (A and G). Spectra were determined in methanol, and Δε is based on e = 37,000. Both sets of products were isolated as in Figure 7 and will be shown later to arise from trans addition of the 2-amino group of guanosine at C-10 of the respective diol epoxide. The two pairs of diastereomeric cis addition products show almost identi­ cal mirror image CD spectra simUar to Ό and H. sil

DRUG M E T A B O L I S M

140

Table I I I .

R e l a t i o n between t h e Nucleoside Adducts and t h e O p t i c a l l y A c t i v e BP 7,8-Dihydrodiol and D i o l Epoxide Products from Which They Are Derived.

BP 7 , 8 - D i h y d r o d i o l

a

Diol Epoxide

a

Nucleoside Adduct" (+)- c i s '

C+) - d i o l epoxide 1

S y m b o l

m n

F

(-)-trans

79

D

(-)- c i s

70

C

108

H

(+)-cis

89

Ε

(-)-trans

56

A

64

Β

93

G

d

0

(-)- d i h y d r o d i o l

(+)- d i h y d r o d i o l

(-)- d i o l epoxide 1

(-)- d i o l epoxide 2

(+)-trans

(-)-cis° (-)- d i h y d r o d i o l

(+)- d i o l epoxide 2

(•)-trans

e

r i \e 92

c

(+)- d i h y d r o d i o l

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch007

CONCEPTS

cd

* Sign o f [o] . Sign o f CD Cotton e f f e c t a t 250 nm. Nucleoside adducts i d e n t i f i e d i n the RNA i s o l a t e d from t h e s k i n of mice which had been t r e a t e d w i t h BP. Due t o co-chromatography, n u c l e o s i d e products F and G can not be d i s t i n g u i s h e d . Chromatographic p r o f i l e o f n u c l e o s i d e adducts (see F i g u r e 7 ) . D

b

c

e

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch007

7.

Diastereomeric

MOORE ET AL.

9,10-Epoxides

from Benzolajpyrene

141

tetrahydro-BP chromophore, whose e x t i n c t i o n c o e f f i c i e n t a t 270 nm i s almost 10 times as l a r g e as t h a t o f guanosine (Figure 9 ) , masked changes t h a t presumably occur i n the p u r i n e chromophore on changing pH. I n a d d i t i o n , the a b s o r p t i o n spectrum near 340 nm, which i s due e n t i r e l y t o the hydrocarbon p o r t i o n o f the adduct, was observed t o change w i t h pH. D i f f e r e n c e uv s p e c t r a between the n u c l e o s i d e adducts and the t e t r a o l s p r o v i d e d l i t t l e h e l p s i n c e the a b s o r p t i o n p a t t e r n i n the n u c l e o s i d e adducts i s s l i g h t l y s h i f t e d compared t o t h a t o f t e t r a o l s . Without the conventional s p e c t r a l method f o r assignment o f the p o s i t i o n o f a l k y l a t i o n , chemical methods were used t o l o c a t e the s i t e o f s u b s t i t u t i o n on the guanine base. A c i d H y d r o l y s i s o f the Nucleoside Adducts. When the d i a s t e r eomeric mixture o f p u r i f i e d n u c l e o s i d e adducts from 1 was heated i n 0.1 N HC1, two t e t r a o l s (cis-1 and trans-1) and guanosine were r a p i d l y generated. At 85°C the h a l f - l i f e f o r r e l e a s e o f t e t r a o l s i s approximately 15 minutes. When these n u c l e o s i d e adducts were t r e a t e d w i t h 0.1 N HC1 i n 1 0 enriched water (18%) a t 85°C, mass s p e c t r a l a n a l y s i s o f the t e t r a o l mixture a f t e r a c e t y l a t i o n showed i n c o r p o r a t i o n o f 0.96 atom % 0 i n t o the i s o l a t e d t e t r a o l s . O v e r a l l recovery o f the t e t r a o l s exceeded 80%. Under these cond i t i o n s the t e t r a o l s , c i s - 1 and trans-1, i n c o r p o r a t e d 0,86 atom % o f one s o l v e n t oxygen (26). Since a carbon-carbon bond would not be expected t o be a c i d - l a b i l e , the experiment suggest an oxygen o r n i t r o g e n t o carbon bond i n the s i t e o f attachment. Reaction o f D i o l Epoxides 1 and 2 w i t h [ 8 - H ] - P o l y (G)• At pH 7.0 the most r e a c t i v e s i t e on the guanosine base towards a l k y l a t i n g reagents i s g e n e r a l l y the N p o s i t i o n (37). A f t e r methyla t i o n a t N the hydrogen a t C becomes r e a d i l y exchangeable w i t h water (59). Thus, treatment o f [ 8 - H ] - p o l y (G) w i t h d i o l epoxides 1 o r 2 would p r o v i d e a s e n s i t i v e t e s t f o r C o r N a l k y l a t i o n . Binding o f the d i o l epoxides 1 and 2 w i t h [ 8 - H ] - p o l y (G) was performed i n 50% acetone/water a t pH 7.0. A f t e r i n c u b a t i o n overn i g h t , the s o l u t i o n s were d i l u t e d w i t h 5 volumes o f pH 7.0 phosphate b u f f e r , and the water was recovered by d i s t i l l a t i o n a t 25°C under h i g h vacuum. Release o f t r i t i u m above the blank o f 0.5% was not observed f o r e i t h e r d i o l epoxide d e s p i t e m o d i f i c a t i o n o f 10% o f the guanosine s i t e s . The r e s i d u a l m o d i f i e d p o l y (G) was hydrol y z e d t o n u c l e o s i d e s which were p u r i f i e d by Poragel PN chromatography. These n u c l e o s i d e adducts had a s p e c i f i c a c t i v i t y which was > 80% o f t h a t o f the s t a r t i n g guanosine i n [ 8 - H ] - p o l y (G). T h i s r e s u l t i s c o n s i s t e n t w i t h a l k y l a t i o n a t a s i t e other than a t C o r N o f guanosine. However, exchange o f the C hydrogen o f 7-methylguanosine r e l i e s on the s t a b i l i t y o f the 7-methylimmonium i o n . The s t a b i l i t y o f the h i g h l y hindered immonium i o n which would form i f the d i o l epoxides r e a c t e d a t N i s p r e s e n t l y unknown. The h y p o t h e t i c a l pathway i n F i g u r e 10 f o l l o w s the p r o posed r o u t e o f a l k a l i n e (pH 9 - 10) decomposition o f 7-methylguanosine and would r e s u l t i n r e t e n t i o n o f t r i t i u m a t C . Strong 8

1 8

3

7

7

8

3

8

7

3

3

8

7

8

7

8

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch007

DRUG M E T A B O L I S M

CONCEPTS

nm

Figure 9. Ultraviolet spectra of nucleoside adducts from diol epox­ ide 1 in methanol as a function of pH. Neutral ( ); alkaline (— ); acidic ( ). The c values of the adduct mixture are based on the specific activity of the [9J.0~ H] diol epoxides. S

no exchange of

Ή

Figure 10. A possible decomposition pathway of"N -substitutedguano­ sine leading to retention of tritium in the product. DEBP represents diol epoxide lor 2 after opening of the 9,10-epoxide at C-10. 7

7.

MOORE

Diastereomeric

ET AL.

9,10-Epoxides

143

from Benzofajpyrene

base treatment o f the mixture o f formamides would produce the non­ v o l a t i l e s a l t o f [ l - H ] - f o r m i c a c i d (60). To t e s t f o r t h i s pos­ s i b i l i t y , the p u r i f i e d n u c l e o s i d e adducts from [8- H]-poly (G) were heated a t 100°C i n 2 Ν KOH, c o n d i t i o n s where [8- H]-guanosine exchanges. Most o f the t r i t i u m d i s t i l l e d from t h i s b a s i c s o l u ­ t i o n . A f t e r a c i d i f i c a t i o n and r e d i s t i l l a t i o n v o l a t i l e [ 1 - H ] formic a c i d was not found i n the d i s t i l l a t e . The f a i l u r e t o r e ­ lease v o l a t i l e [1- H]-formic a c i d upon a c i d i f i c a t i o n e l i m i n a t e d a l k y l a t i o n a t N and the mechanism i n Figure 10. Taken t o g e t h e r , these r e s u l t s completely exclude N and C as the s i t e o f a t t a c h ­ ment o f the hydrocarbon. Base S t a b i l i t y o f Nucleoside Adducts. I n 1.0 Ν KOH a t 100°C c e r t a i n (Table IV) s u b s t i t u t e d p u r i n e r i b o s i d e s r a p i d l y degrade. This degradation can proceed by a t t a c k o f hydroxide on the i m i d ­ azole r i n g a t C o r a t v a r i o u s s i t e s o f the p y r i m i d i n e r i n g . However, p u r i n e r i b o s i d e s w i t h a hydrogen on the N p o s i t i o n , except N d e r i v a t i v e s , are deprotonated t o y i e l d a monoanion and become s t a b i l i z e d towards f u r t h e r r e a c t i o n w i t h base (60). A c c o r d i n g l y , Ν , Ν , N , and 0 s u b s t i t u t e d guanosines are known t o be degraded (Table IV) w h i l e N s u b s t i t u t e d guanosine i s s t a b l e towards base treatment (61,62). Treatment o f n u c l e o s i d e adducts w i t h 1 Ν KOH a t 100°C f o r 2 hours r e s u l t e d i n o n l y minor degra­ d a t i o n and allowed h i g h recovery o f unchanged adducts. This s t r o n g l y suggests the presence o f an amidic proton i n the nucleo­ s i d e adducts. Determination o f the p K Values o f the Nucleoside Adducts. Measurement o f the p K s o f the n u c l e o s i d e adducts could e s t a b l i s h whether o r not a f r e e amidic proton i s present a t N . Spectrophotomeric t i t r a t i o n was p o s s i b l e but subject t o u n c e r t a i n t y due t o the s m a l l change i n the uv spectrum t h a t occurred upon i o n i z a ­ t i o n . Since the sample s i z e precluded d i r e c t t i t r a t i o n , a more s e n s i t i v e and r e l i a b l e method was sought. The n u c l e o s i d e adducts are s o l u b l e i n water but can be e x t r a c t e d i n t o p o l a r o r g a n i c s o l ­ vants. Since the i o n i z e d form o f guanosine i s much more s o l u b l e i n water than the n e u t r a l form, a p K should be d e t e c t a b l e by a change i n p a r t i t i o n c o e f f i c i e n t w i t h pH (26,63) (Figure 11). The a c i d i c pKa a t 9.8 i n d i c a t e s deprotonation from n e u t r a l n i t r o g e n and the b a s i c pKa a t 1.5 i n d i c a t e s p r o t o n a t i o n o f n e u t r a l n i t r o ­ gen. Although the pKa values obtained are approximate due t o the s e l e c t i v e removal o f one component from the acid-base e q u i l i b r i u m , the presence o f a f r e e N proton i s c l e a r l y demonstrated. This r e s u l t , i n combination w i t h the s t a b l e nature o f the n u c l e o s i d e adducts towards base treatment, e l i m i n a t e s N i , N , and 0 as pos­ s i b l e s i t e s o f attachment. Since N and C s u b s t i t u t i o n had pre­ v i o u s l y been e l i m i n a t e d , an N s u b s t i t u t e d guanosine becomes the s o l e p o s s i b l e s t r u c t u r e f o r the n u c l e o s i d e adducts. 3

3

3

3

3

7

7

8

8

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch007

1

7

1

3

7

6

2

a

f

a

1

a

1

3

7

2

8

6

144

DRUG M E T A B O L I S M C O N C E P T S

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch007

Table IV. E f f e c t o f 1.0 Ν KOH a t 100°C on S u b s t i t u t e d Guanosines.

7.

MOORE

ET AL.

Diastereomeric

9,10-Epoxides

from Benzo[a]pyrene

145

Nmr and Mass S p e c t r a o f the Adducts. F i n a l d e t e r m i n a t i o n o f the s t r u c t u r e s o f t h e n u c l e o s i d e adducts from 1 by mass s p e c t r o ­ metry and p r o t o n nmr was g r e a t l y handicapped by the added mass and the i n t e r f e r i n g resonances o f the r i b o s e moiety. S i n c e a c i d treatment cleaved the hydrocarbon-guanosine bond much more r e a d i l y than the g l y c o s i d i c l i n k a g e , the normal method o f d i r e c t d e p u r i n a t i o n w i t h a c i d was not u s e f u l . An a l t e r n a t i v e m i l d method f o r cleavage o f the g l y c o s i d i c l i n k a g e was sought. S i n c e the N p o s i ­ t i o n was known not t o be s u b s t i t u t e d and s i n c e dimethyl s u l f a t e shows a marked p r e f e r e n c e f o r a l k y l a t i o n o f N , the mixture o f f o u r n u c l e o t i d e adducts o b t a i n e d from d i o l epoxide 1 was t r e a t e d w i t h dimethyl s u l f a t e t o l a b i l i z e the r i b o s e l i n k a g e (55) (Figure 12). Mass spectrometry o f the d e r i v e d t r i a c e t a t e o f the methylated adducts by chemical i o n i z a t i o n w i t h methane showed t h a t 7-methylguanine-tetrahydro-BP t r i a c e t a t e s had been formed. Chromatography on ODS allowed the i s o l a t i o n o f two products (Figure 13) 8 and 9 w i t h i d e n t i c a l mass s p e c t r a (26). Only two adducts were expected s i n c e l o s s o f the o p t i c a l l y pure r i b o s e moiety reduces each p a i r o f o p t i c a l l y a c t i v e diastereomers w i t h m i r r o r image CD s p e c t r a i n t o s i n g l e racemic compounds. F o u r i e r t r a n s f o r m 220 MHz p r o t o n nmr o f the major product showed t h r e e a c e t y l s i n g l e t s a t 1.98, 2.04, and 2.25, one broad N-methyl s i n g l e t a t 3.38, the f o u r one proton s i g n a l s from p o s i t i o n s 7,8,9, and 10 o f the tetrahydro-BP r i n g a t 6.71, 5.52, 5.58, and 6.14, r e s p e c t i v e l y , and the remain­ i n g C and pyrene s i g n a l s from 7.95 t o 8.28 ppm. Comparison o f the chemical s h i f t s and c o u p l i n g constants o f t h i s product ( ^7 « = 5.2, J q = 5.4, J q i o = 2.6 Hz) w i t h those eq, eq eq, eq ' eq, eq 7

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch007

7

8

3

3

7

3

8f t

8

9

y

l u

o f the 9 , 1 0 - t r a n s - a n i l i n e adduct (--NHC H a t C-10 o f 8) from d i o l epoxide 1 (28) c o n v i n c i n g l y e s t a b l i s h e d t h a t the major prod­ u c t was d e r i v e d from t r a n s a d d i t i o n o f the 2-amino group o f gua­ n i n e t o the C-10 p o s i t i o n o f 1 . S i m i l a r l y , the p r o t o n nmr o f the minor product showed t h r e e a c e t y l s i n g l e t s a t 1.95, 2.00, and 2.13, one N-methyl s i n g l e t a t 3.93, the f o u r one p r o t o n s i g n a l s from p o s i t i o n s 7,8,9, and 10 o f the tetrahydro-BP r i n g a t 6.93, 6.18, 5.61, and 6.29, r e s p e c t i v e l y , and the remaining C and pyrene s i g n a l s from 7.95 t o 8.40 ppm. Comparison o f the c o u p l i n g con­ s t a n t s o f t h i s product ( J =8.0, J 9 =12.0, 6

5

8

3

3

7

3 j

o

y

8

ax,

8

ax

ax,

ax

ι λ = 4.0 Hz) w i t h those o f the 9,10-cis-phenol adduct ax, eq i U

(--0C H a t C-10 o f 9) from d i o l epoxide 1 (28) showed t h a t 9 r e s u l t e d from c i s opening o f the o x i r a n e r i n g o f 1 by the 2-amino group o f guanine. A r e c e n t p u b l i c a t i o n (22) has assigned the major product from d i o l epoxide 2 and p o l y (G) as a t r a n s N s u b s t i t u t e d guanosine based s o l e l y on the nmr and mass s p e c t r a o f the m o d i f i e d nucleo­ s i d e . S t r u c t u r a l i n t e r p r e t a t i o n o f the mass s p e c t r a a t t h e 6

5

2

146

DRUG M E T A B O L I S M

0

I—«

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch007

10

,

•

,

,

•

,

.

•

20

30

4.0

50

60

70

80

ao

CONCEPTS

ι IQO

IIJO

rI2J0

PH

Figure 11. Estimation of pK by change in partition coefficient with pH. Solutions (pH 2.0-9.0) were prepared by mixing 0.05M citric acid pH 1.8, 0.05M KJ/POj 7.0, and 0.05M NaHCO pH 9.0. Solutions outside the range of pH 2.0-9.0 were obtained adding HCl or KOH to 0.05M citric acid or NaHC0 . Nucleoside adducts from 1 or 2 wer partitioned with 25% n-butanol in ethyl acetate. Guanosine triacetate was partitioned wi n-butanol in ethyl ether. Distribution between the two phases was determined spectro metrically as described (26,63). a

s

3

Figure 12. Labilization of the glycosidic linkage by N methylation. Diol epoxide 1 (DEBP) guanosine products were treated with dimethyl sulfate as described in materials and methods. 7

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n u c l e o s i d e l e v e l i s somewhat d i f f i c u l t s i n c e s t r u c t u r e 7 (Figure 14) which has a mass 18 u n i t s h i g h e r than the N s u b s t i t u t e d anal o g might be expected t o g i v e a s i m i l a r cleavage p a t t e r n a f t e r e l i m i n a t i o n o f water. A major argument presented f o r N s u b s t i t u t i o n was an observed s p i n - s p i n c o u p l i n g between the 2-amino hydrogen and the hydrogen a t C-10. A s i m i l a r c o u p l i n g between the hydrogen a t C-10 and the amino hydrogen a t N i n 7 would be expected. The formamide s i g n a l i n 7 would be obscured by the pyrene resonances (64). Although the data presented by these authors (22) does not exclude s t r u c t u r e 7 , the present t r i t i u m r e l e a s e s t u d i e s w i t h [ 8 - H ] - p o l y (G) and d i o l epoxide 2 does. Thus, both d i o l epoxides 1 and 2 a t t a c k the e x o c y c l i c 2-amino group o f guanosine (22,26). Assignment o f the A b s o l u t e C o n f i g u r a t i o n o f the Guanosine Adducts from D i o l Epoxides 1 and 2 . For d i o l epoxide 1 , the minor p a i r o f diastereomers were shown t o r e s u l t from c i s opening and the major p a i r o f diastereomers by t r a n s opening o f the o x i r a n e r i n g a t C-10. Each o f the enantiomers o f d i o l epoxide 1 leads t o the formation o f a c i s and t r a n s adduct. The CD s p e c t r a o f t h i s c i s / t r a n s p a i r are almost m i r r o r images w i t h o p p o s i t e signs a t t h e i r s t r o n g e s t t r a n s i t i o n s (Figure 8 ) . The c i s and t r a n s adducts from a given enantiomer o f d i o l epoxide 1 d i f f e r o n l y i n t h e i r c o n f i g u r a t i o n a t C-10. Since each enantiomer o f d i o l epoxide 2 a l s o produces a p a i r o f adducts w i t h o p p o s i t e CD s p e c t r a and s i n c e the major adduct from each enantiomer o f d i o l epoxide 2 c o n s t i t u t e a p a i r o f t r a n s diastereomers (22), i t i s reasonable t o conclude on the b a s i s o f the CD s p e c t r a t h a t the minor p a i r o f adducts from d i o l epoxide 2 c o n s t i t u t e a p a i r o f c i s diastereomers. S i n c e the a b s o l u t e c o n f i g u r a t i o n o f the enantiomeric BP 7,8d i h y d r o d i o l s and the f o u r p o s s i b l e corresponding d i o l epoxides has been assigned (53) and s i n c e these d i o l epoxides have been s e p a r a t e l y r e a c t e d w i t h p o l y (G), the assignments i n Table I I I are p o s s i b l e . A l l o f these assignments are based on the e x c i t o n c h i r a l i t y CD spectrum o f the Jbis-(p-N,i\r-dimethylaminobenzoate) ester o f o p t i c a l l y active trans-7,8-dihydroxy-4,5,7,8,9,10,11,12octahydro-BP. 2

2

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Formation o f RNA-Nucleoside Adducts from [ H]-BP on Mouse S k i n Once the s t r u c t u r e s and the chromatographic p r o p e r t i e s o f the adducts which form when d i o l epoxides 1 and 2 a l k y l a t e p o l y (G) had been e s t a b l i s h e d , i t became p o s s i b l e t o determine whether these products form from BP in vivo. Since we have determined the c a r c i n o g e n i c i t y o f BP 7,8-oxide and BP 7 , 8 - d i h y d r o d i o l on the s k i n o f C57BL/6J mice, we examined t h e . s k i n epidermis o f t h i s mouse s t r a i n f o r the formation o f d i o l epoxide-nucleoside adducts a f t e r t o p i c a l a p p l i c a t i o n o f [ H]-BP t o the mouse. Mice were p a i n t e d w i t h [ H]-BP and the i s o l a t e d RNA was hyd r o l y z e d t o a mixture o f n u c l e o s i d e s . A f t e r a d d i t i o n o f c a r r i e r amounts o f the e i g h t p o s s i b l e guanosine adducts, the r a d i o a c t i v e 3

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8, 9, \0-trans

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Figure 13. Structure of the guanosine adducts of diol epoxide 1 after methylation at N 7

Figure 14. Possible alternative structure of guanosine adducts from trans opening at C-10 of diol epoxide 2 (DEBP)

Ribose

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CONCEPTS

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n u c l e o s i d e s were p u r i f i e d by Poragel PN chromatography t o remove both t e t r a o l s and unmodified n u c l e o s i d e s . The r a d i o c h e m i c a l y i e l d of the products e l u t e d i n the guanosine adduct f r a c t i o n was > 30%. Most o f the remaining r a d i o a c t i v i t y was e l u t e d i n the f i r s t peak (Figure 6) and i s o f unknown s t r u c t u r e . On t h i s Poragel PN column n u c l e o s i d e adducts from d i o l epoxides 1 and 2 and p o l y (A) a r e e l u t e d i n the t e t r a o l f r a c t i o n which contained < 5% o f the t o t a l radioactivity. When the g u a n o s i n e - d i o l epoxide f r a c t i o n which had been pur­ i f i e d by Poragel PN chromatography was examined by HPLC on μΟχβ" Bondapak (Figure 15), r a d i o a c t i v e guanosine adducts w i t h chromato­ graphic r e t e n t i o n times i d e n t i c a l t o adduct peaks B , D, F, and 6 were found. F u r t h e r c o n f i r m a t i o n o f the nature o f the i n d i v i d u a l r a d i o a c t i v e peaks was obtained by d e t e r m i n a t i o n o f t h e i r e x t r a c ­ t i o n versus pH p r o f i l e (as i l l u s t r a t e d i n F i g u r e 11) (26,63). A l l t h r e e r a d i o a c t i v e peaks showed a change i n p a r t i t i o n co­ e f f i c i e n t i n the range o f pH 9.0 - 11.0 i n d i c a t i n g t h a t each i s a N o r C s u b s t i t u t e d guanosine adduct. Adducts a t C are pre­ sumed not t o be present based on the model experiments w i t h p o l y (G). The p a i r o f adducts Β and D a r i s e from d i o l epoxide 2 and 1 , r e s p e c t i v e l y , and p r o v i d e the f i r s t evidence t h a t these d i o l ep­ oxides are formed and b i n d t o RNA i n mouse s k i n . The formation of both o f these d i o l epoxides was f u r t h e r confirmed by a c i d hy­ d r o l y s i s o f the t r i t i a t e d n u c l e o s i d e adducts t o r a d i o a c t i v e t e t r a ­ o l s r e l a t e d t o d i o l epoxides 1 and 2. The formation o f at l e a s t t h r e e o f the e i g h t d i a s t e r e o m e r i c guanosine d e r i v a t i v e s ( B and D along w i t h e i t h e r o r both o f F and G) p r o v i d e s i n s i g h t i n t o the metabolism o f BP i n mouse s k i n . Peak Β i s d e r i v e d from ( + ) - d i o l epoxide 2 and guanosine by c i s a d d i t i o n a t C-10 w h i l e peak D i s d e r i v e d from ( + ) - d i o l epoxide 1 by t r a n s a d d i t i o n a t C-10. (+)-Diol epoxide 2 i s formed from (-)BP 7 , 8 - d i h y d r o d i o l , w h i l e ( + ) - d i o l epoxide 1 i s formed from the (+)-enantiomer. Furthermore, the c i s / t r a n s counterpart o f both peaks Β and D (peaks F and G ) are known t o co-chromâtograph w i t h the t h i r d r a d i o a c t i v e peak. These r e s u l t s imply t h a t the monooxygenases i n mouse s k i n which produce the 9,10-epoxides are h i g h l y s t e r e o s e l e c t i v e i n t h a t predominantly ( + ) - d i o l epoxide 1 from (+)-BP 7 , 8 - d i h y d r o d i o l and ( + ) - d i o l epoxide 2 from (-)-BP 7,8-dihydrodiol form d e t e c t a b l e RNA adducts. 2

8

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Conclusions We have observed t h a t both d i o l epoxides 1 and 2 r e a c t s i m i l a r l y w i t h p o l y (G) i n 50% acetone/water. A l k y l a t i o n occurs a t phosphate s i t e s (10 - 15%) and a t the e x o c y c l i c 2-amino group o f guanosine. The formation o f both c i s and t r a n s a d d i t i o n products to the o x i r a n e r i n g s o f d i o l epoxides 1 and 2 suggests t h a t these r e a c t i o n s may proceed a t l e a s t i n p a r t by Sjjl mechanism. T h i s r e a c t i v i t y under n e u t r a l c o n d i t i o n s i s a l s o demonstrated by a l k y l -

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10

20

30

40

50

60

70

80

90

HO

100

Fraction Number

Figure 15. Coinjection of Poragel PN-purified nucleoside adducts from [ H] BP on mouse skin and 2-amino guanosine-diol epoxide adducts from both diol epoxides 1 and 2 on high resolution μϋ Bondapak. Refer to Figure 7 and Table III for definition of the nucleoside symbols. Cochromatography of the longest retained radioactive peak with uv markers F and G suggests that very little radioactive Ε was present. 3

18

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ation of inorganic phosphate. The relative biological importance of 2-amino substitution and phosphate alkylation can not as yet be estimated. Little is known about the effect of 2-amino substitu­ tion by large aromatic groups on factors such as the conformation of the polymer or the fidelity of transcription. Alkylation at phosphate, however, has been suggested to have high biological significance (41,42). This report describes the structural assignment of the covalently bound guanosine adducts derived from RNA, which are form­ ed when mouse skin is exposed to BP. Both (+)-diol epoxide 1 and (+)-diol epoxide 2 from the (+)- and (-)-enantiomers of BP 7,8dihydrodiol, respectively, are produced in vivo and react with cellular RNA on the 2-amino group of guanosine by cis and trans addition. When bovine bronchial expiants were exposed to BP (23) only diol epoxide 2 was observed to form an adduct at the 2-amino group of guanosine. The differences in these two results may be explained by a difference in metabolism of BP between mouse skin and bovine bronchial expiants or by the fact that only about half of the radioactivity in the chromatographic region for diol ep­ oxide adducts of guanosine was characterized in the latter study. Whether or not the enantiomers of diol epoxides 1 and 2 are impor­ tant in the binding of BP to the DNA of mouse skin is presently under study. Acknowledgment This research has been supported in part by Grant CA 18580 from the National Cancer Institute of the US Public Health Service (to M. K.). Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Brookes, P. and Lawley, P. D., Nature (London) (1964) 202, 781. Miller, J., Cancer Res. (1970) 30, 559. Sims, P. and Grover, P. L . , Adv. Cancer Res. (1974) 20, 165. Berwald, Y. and Sachs, L . , J. Natl. Cancer Inst. (1965) 35, 641. Ames, Β. N . , Durston, W. E . , Yamasaki, E . , and Lee, F. D., Proc. Natl. Acad. Sci. U.S.A. (1973) 70, 2281. Grover, P. L. and Sims, P., Biochem. J. (1968) 110, 159. Gelboin, H. V., Cancer Res. (1969) 29, 1272. Jerina, D. M. and Daly, J . W., Science (1974) 185, 573. Sims, P., Grover, P. L . , Swaisland, Α., Pal, K., and Hewer, Α., Nature (London) (1974) 252, 326. Ts'o, P. O. P., Caspary, W. J., Cohen, Β. I., Leavitt, J. C., Lesko, S. Α., Lorentzen, R. J., and Schechtman, L. M. in "Chemical Carcinogenesis, Part A", Ts o, P. O. P. and DiPaolo, J . Α., eds., p.113, Marcel Dekker, New York, Ν. Υ., 1974. '

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11. Nagata, C., Tagashira, Y . , and Kodama, Μ., ibid., p. 87. 12. Fried, J., ibid., p. 197. 13. Cavalieri, E., Roth, R., and Rogan, E. G. in "Carcinogenesis", Vol. 1; Polynuclear Aromatic Hydrocarbons: Chemistry, Meta­ bolism, and Carcinogenesis, Freudenthal, R. and Jones, P. W., eds., p. 181, Ravern Press, New York, Ν. Υ., 1976. 14. Borgen, Α., Darvey, Η., Castagnoli, Ν., Crocker, T. T., Rasmussen, R. E., and Wang, I. Y . , J. Med. Chem. (1973) 16, 502. 15. Yagi, H., Hernandez, O., and Jerina, D. M., J. Am. Chem. Soc. (1975) 97, 6881. 16. McCaustland, D. J., Fischer, D. L . , Kolwyck, K. C., Duncan, W. P., Wiley, J . C., Jr., Menon, C. S., Engel, J. F., Selkirk, J . K., and Roller, P. P. in ref. 13, p. 349. 17. Daudel, P., Duquesne, M., Vigny, P., Grover, P. L . , and Sims, P., FEBS Lett. (1975) 57, 250. 18. Nebert, D. W., Kouri, R. E., Yagi, H., Jerina, D. M., and Boobis, A. R. in "Reactive Intermediates: Formation, Toxicity, and Inactivation", Jollow, D., Kocsis, J., Snyder, R., Vainio, Η., eds., Plenum Press, New York, Ν. Y . , in press. 19. Meehan, T., Warshawsky, D., and Calvin, Μ., Proc. Natl. Acad. Sci. U.S.A. (1976) 73, 1117. 20. Osborne, M. R., Thompson, M. H., Tarmy, E. M., Beland, F. Α., Harvey, R. G., and Brookes, P., Chem.-Biol. Interact. (1976) 13, 343. 21. Osborne, M. R., Beland, F. Α., Harvey, R. G., and Brookes, P., Int. J . Cancer (1976) 18, 362. 22. Jeffrey, A. M., Jennette, K. W., Blobstein, S. Η., Weinstein, I. Β., Beland, F. Α., Harvey, R. G., Kasai, H., Miura, I., and Nakanishi, K., J. Am. Chem. Soc. (1976) 98, 5714. 23. Weinstein, I. Β., Jeffrey, Α. Μ., Jennette, K. W., Blobstein, S. H., Harvey, Ri G., Harris, C., Autrup, Η., Kasai, H., and Nakanishi, Κ., Science (1976) 193, 592. 24. King, H. W. S., Thompson, Μ. Η., Tarmy, Ε. Μ., Brookes, P., and Harvey, R. G., Chem.-Biol. Interact. (1976) 13, 349. 25. King, H. W. S., Osborne, M. R., Beland, F. Α., Harvey, R. G., Brookes, P., Proc. Natl. Acad. Sci. U.S.A. (1976) 73, 2679. 26. Koreeda, M., Moore, P. D., Yagi, H., Yeh, H. J. C., and Jerina, D. M., J. Am. Chem. Soc. (1976) 98, 6720. 27. Thakker, D. R., Yagi, H., Lu, Α. Y. H., Levin, W., Conney, A. H., and Jerina, D. Μ., Proc. Natl. Acad. Sci. U.S.A. (1976) 73, 3381. 28. Yagi, H., Thakker, D. R., Hernandez, O., Koreeda, Μ., and Jerina, D. M., J. Am. Chem. Soc. (1977) 99, in press. 29. Yang, S. K., McCourt, D. W., Roller, P. P., and Gelboin, H. V., Proc. Natl. Acad. Sci. U.S.A. (1976) 73, 2594. 30. Wood, A. W., Wislocki, P. G., Chang, R. L . , Levin, W., Lu, Α. Y. H., Yagi, Η., Hernandez, O., Jerina, D. Μ., and Conney, A. H., Cancer Res. (1976) 36, 3358.

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31. Levin, W., Wood, A. W., Yagi, H., Dansette, P., Jerina, D. M., and Conney, A. H., Proc. Natl. Acad. Sci. U.S.A. (1976) 73,

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32. Levin, W., Wood, A. W., Yagi, Η., Jerina, D. M., and Conney, A. H., ibid. (1976) 73, 3867. 33. Conney, A. H., Wood, A. W., Lu, A. Y. H., Chang, R. L . , Wislocki, P. G., Holder, G. M., Dansette, P., Yagi, H., and Jerina, D. M. in ref. 18, in press. 34. Wislocki, P. G., Wood, A. W., Chang, R. L . , Levin, W., Yagi, H., Hernandez, O., Jerina, D. M., and Conney, A. H., Biochem. Biophys. Res. Commun. (1976) 68, 1006. 35. Newbold, R. F. and Brookes, P., Nature (London) (1976) 261, 52. 36. Huberman, E., Sachs, L . , Yang, S. K., and Gelboin, H. V., Proc. Natl. Acad. Sci. U.S.A. (1976) 73, 607. 37. Singer, B., Prog. Nucleic Acid Res. Mol. Biol. (1975) 15, 219 and references cited therein. 38. Lawley, P. D. and Shah, S. Α., Chem.-Biol. Interact. (1973) 7, 115. 39. 40. 41.

Loveless, Α., Nature (London) (1969) 223, 206. Lawley, P. D. and Thatcher, C. J . , Biochem. J . (1970) 116, 693. Singer, B. and Fraenkel-Conrat, H., Biochemistry (1975) 14, 772.

Singer, B., Nature (London) (1976) 264, 333. 43. Goth, R. and Rajewsky, M. F . , Proc. Natl. Acad. Sci. U.S.A.

42.

(1974) 71, 639.

44. Kriek, E., Miller, J . Α., Juhl, U., and Miller, E. C., Biochemistry (1967) 6, 177. 45. Westra, J . G., Kriek, E., and Hittenhausen, H., Chem.-Biol. Interact. (1976) 15, 149. 46. Dipple, Α., Brookes, P., Mackintosh, D. S., and Rayman, M. P., Biochemistry (1971) 10, 4323. 47. Jeffrey, A. M., Blobstein, S. H., Weinstein, I. B., Beland, F. Α., Harvey, R. G., Kasai, Η., and Nakanishi, K., Proc. Natl. Acad. Sci. U.S.A. (1976) 73, 2311. 48. Brown, D. M. in "Basic Principles of Nucleic Acid Chemistry", Ts'o, P. O. P., ed., Vol. II, p. 66, Academic Press, New York, Ν. Y . , 1974. 49. Boyland, E. and Green, B., Brit. J . Cancer (1962) 16, 507. 50. Lesko, S. Α., Smith, Α., Ts'o, P. O. P., and Umans, R. S., Biochemistry (1968) 7, 434. 51. Dale, J . Α., Dull, D. L . , and Mosher, H. S., J . Org. Chem. (1969) 34, 2543.

52. Thakker, D. R., Yagi, H., Akagi, H., Koreeda, M., Lu, Α. Υ. Η., Levin, W., Conney, Α. Η., and Jerina, D. M., Chem.-Biol. Interact. (1977) in press. 53. Yagi, H., Akagi, H., Mah, H. D., Koreeda, Μ., and Jerina, D. Μ., submitted. 54. Blobstein, S. Η., Weinstein, I. Β., Dansette, P., Yagi, H., and Jerina, D. Μ., Cancer Res. (1976) 36, 1293.

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55. Haines, J. Α., Reese, C. Β., and Todd, L . , J. Chem. Soc. (1962) 5281. 56. Bowden, G. T., Shapas, B. G., and Boutwell, R. Κ., Chem.-Biol. Interact. (1974) 8, 379. 57. Irving, C. C. and Veazey, R. Α., Biochem. Biophys. Acta (1968) 166, 246. 58. Holy, A. and Scheit, Κ. H., ibid. (1967) 138, 230. 59. Tomasz, Μ., ibid. (1970) 199, 18. 60. Kochetkov, Ν. K. and Budovskii, Ε. I., "Organic Chemistry of Nuclei Acids", Part B, p. 381, Plenum Press, New York, Ν. Y . , 1972. 61. Holmes, R. E. and Robins, R. K., J. Org. Chem. (1963) 28, 3483. 62. Chambers, R. W., Moffatt, J . G., and Khorana, H. G., J . Am. Chem. Soc. (1957) 79, 3747. 63. Moore, P. D. and Koreeda, Μ., Biochem. Biophys. Res. Commun. (1976) 73, 459. 64. Hecht, S. Μ., Adams, B. L . , and Kozarich, J. W., J. Org. Chem. (1976) 41, 2303.

8 Role of Metabolic Activation in Chemical-Induced Tissue Injury SIDNEY D. NELSON, MICHAEL R. BOYD, and JERRY R. M I T C H E L L

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, Bethesda, MD 20014

An important result of metabolism studies in recent years has been the realization that many chemical compounds are metabolized by the liver and various other tissues to potent alkylating and arylating intermediates (1-12). Such studies demonstrate how chemically stable compounds can produce serious tissue lesions in man and experimental animals, including hepatic, renal, and pulmonary necrosis, bone marrow aplasia, neoplasia and other injuries. Although these lesions are rare, such toxic effects are of great c l i n i c a l concern because they often lead to irreversible failure of the liver, lungs, kidneys or other organs, and subsequent death of the patient. Many of the initial concepts of metabolic activation were developed during studies of chemical carcinogenesis; the work of the Millers in the United States (1,2) and of Magee and co-workers in England (3) has been especially illuminating. The realization that the enzyme pathways responsible for the conversion of certain chemicals to proximate carcinogens are the same microsomal mixed-function oxygenases that metabolize most drugs and other xenobiotics led to the concept that drug-induced tissue lesions might also be mediated through the covalent binding of reactive metabolites (6-11). The lack of reactivity of most chemically stable compounds and the frequent localization of tissue damage only in those organs or to those animal species having the necessary drug-metabolizing enzymes supported this view. Additionally, these studies frequently demonstrated a role for sulfhydryl-containing compounds, particularly glutathione, in protecting tissues from such toxic reactions. Most drugs and foreign compounds that enter the body are converted to chemically stable metabolites that are readily excreted into urine and b i l e , or are expired. Thus, it has become important to distinguish those toxicities that are mediated by chemically reactive metabolites and those reactions due to an exaggerated therapeutic effect or unwanted secondary effect caused by the drug or one of i t s stable metabolites. The toxicologic activity produced by the latter class of reactions usually can be monitored by 155

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DRUG M E T A B O L I S M C O N C E P T S

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

measuring the c o n c e n t r a t i o n of the compound and i t s m e t a b o l i t e s i n body f l u i d s * However, when the response i s t i s s u e damage caused by the covalent b i n d i n g of c h e m i c a l l y r e a c t i v e m e t a b o l i t e s t o t i s s u e macromolecules, r a r e l y can a r e l a t i o n s h i p between t i s s u e l e v e l s of the m e t a b o l i t e and the s e v e r i t y of the l e s i o n be d e t e r mined* Indeed, h i g h l y r e a c t i v e m e t a b o l i t e s may e x i s t f o r o n l y a few seconds o r l e s s and w i l l t h e r e f o r e never accummulate i n body fluids. Parameters f o r s t u d y i n g r e a c t i v e m e t a b o l i t e s . How then can the formation of such c h e m i c a l l y u n s t a b l e and r e a c t i v e m e t a b o l i t e s be studied? Based on s t u d i e s where an animal model has been developed f o r a p a r t i c u l a r chemical-induced t i s s u e l e s i o n , a r e l a t i o n s h i p can o f t e n be made between the s e v e r i t y of the t i s s u e l e s i o n and the amount of m e t a b o l i t e t h a t i s c o v a l e n t l y bound to the damaged t i s s u e . That i s , covalent b i n d i n g of the r e a c t i v e m e t a b o l i t e can be used as an index of formation of the m e t a b o l i t e . Furthermore, t h i s parameter might w e l l be the most r e l i a b l e e s t i mate of the a v a i l a b i l i t y of the m e t a b o l i t e i n s i t u f o r causing t i s s u e damage, s i n c e much of the m e t a b o l i t e o f t e n decomposes or i s f u r t h e r metabolized before i t can be i s o l a t e d i n body f l u i d s . Thus, one approach t o the problem i s t o determine whether r a d i o l a b e l e d drugs administered t o animals over a wide dose range are c o v a l e n t l y bound t o macromolecules i n t a r g e t t i s s u e s t h a t subsequently become n e c r o t i c . Pretreatment of animals w i t h inducers o f drug metabolism, such as phénobarbital, or w i t h i n h i b i t o r s of drug metabolism, such as p i p e r o n y l butoxide, c o b a l t c h l o r i d e , o r ©(-naphthylisothiocyanate, s i m i l a r l y should a l t e r the r a t e of metabolism of t o x i n , the extent of covalent b i n d i n g of r e a c t i v e m e t a b o l i t e , and the s e v e r i t y of t i s s u e i n j u r y . I n c o n j u n c t i o n w i t h these s t u d i e s i n animals, experiments can be performed i n v i t r o w i t h microsomal enzymes i s o l a t e d from the t a r g e t organ t i s s u e . Covalent b i n d i n g of r e a c t i v e m e t a b o l i t e s may be one u s e f u l index of product formation when v a r i o u s a d d i t i o n s or d e l e t i o n s from the system are made, or when animals are p r e t r e a t e d w i t h v a r i o u s enzyme inducers and i n h i b i t o r s . Another u s e f u l index of r e a c t i v e product formation i n t h i s system i s the t r a p p i n g of e l e c t r o p h i l i c intermediates w i t h a l t e r n a t e n u c l e o p h i l e s such as c y s t e i n e or g l u t a t h i o n e . S t r u c t u r a l e l u c i d a t i o n of such i n t e r m e d i a t e s may o f t e n p r o v i d e i n s i g h t i n t o the s t r u c t u r e o f the i n i t i a l r e a c t i v e m e t a b o l i t e . U l t i m a t e l y , i s o l a t i o n and s t r u c t u r e e l u c i d a t i o n of the r a d i o l a b e l e d m a t e r i a l bound to the t i s s u e macromolecules (RNA, DNA, p r o t e i n ) can be c a r r i e d out. The approach d e s c r i b e d has been used to i m p l i c a t e t o x i c m e t a b o l i t e s as mediators of the t o x i c i t i e s caused by s e v e r a l drugs. Hepatic n e c r o s i s has been a s s o c i a t e d w i t h the use of hydrazides i s o n i a z i d ( I ) , a t u b e r c u l o s t a t i c agent, and i p r o n i a z i d ( I I ) , an a n t i d e p r e s s a n t . Both h e p a t i c and r e n a l i n j u r y are a s s o c i a t e d w i t h the use of h i g h doses of two s u b s t i t u t e d aminophenol a n a l g e s i c s , acetaminophen ( I I I ) and phenacetln ( I V ) . The f u r a n - c o n t a i n i n g d i u r e t i c agent, furosemide (V), and the thiophene-containing

8.

NELSON E T A L .

Chemical-Induced

Tissue

Injury

157

a n t i b i o t i c , c e p h a l o r i d i n e ( V I ) , are a s s o c i a t e d w i t h r e n a l i n j u r y i n man. Ipomeanol ( V I I ) , a f u r a n - c o n t a i n i n g d e r i v a t i v e produced by moldy sweet potatoes, i s an example o f a chemical t o x i n which produces pulmonary l e s i o n s v i a r e a c t i v e m e t a b o l i t e formation. These and other experimental s t u d i e s w i t h model compounds w i l l be presented t o i l l u s t r a t e the concepts which u n d e r l i e the r o l e o f metabolic a c t i v a t i o n i n chemical-induced t i s s u e i n j u r y and the parameters used t o e s t a b l i s h these concepts. Hydrazines and Hydrazides

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

I s o n i a z i d . A good example of t o x i c drug r e a c t i o n s caused by metabolic a c t i v a t i o n i s i s o n i a z i d - i n d u c e d l i v e r i n j u r y . This drug provides a unique o p p o r t u n i t y to show how a study can be pursued from a c l i n i c a l l y manifest t i s s u e l e s i o n t o the proposal o f a r a t i o n a l chemical mechanism f o r the t o x i c i t y . C l i n i c a l f i n d i n g s . Three c l i n i c a l s t u d i e s (13-15) provided evidence t h a t metabolic a c t i v a t i o n was i n v o l v e d i n the s e r i o u s h e p a t i t i s caused by i s o n i a z i d when t h i s drug was administered i n t h e r a p e u t i c doses. F i r s t was a p r o s p e c t i v e study c a r r i e d out i n 1972 (13). SGOT and serum b i l i r u b i n concentrations were examined monthly i n 250 p a t i e n t s r e c e i v i n g i s o n i a z i d f o r one year. These b i o c h e m i c a l i n d i c e s i n d i c a t e d that i s o n i a z i d was hepatotoxic i n a l a r g e p r o p o r t i o n o f i n d i v i d u a l s but most adapted t o the i n s u l t and recovered r a t h e r than developing severe h e p a t i t i s . Measurement of plasma concentrations o f i s o n i a z i d i n these p a t i e n t s , f a i l e d t o show a c o r r e l a t i o n between plasma l e v e l s o f i s o n i a z i d and l i v e r i n j u r y . I n t h i s study, no a n t i - i s o n i a z i d a n t i b o d i e s were found and no c o r r e l a t i o n was seen between h e p a t i c i n j u r y and a n t i n u c l e a r a n t i b o d i e s measured a t the end o f the study. The seoncd study was a r e t r o s p e c t i v e a n a l y s i s o f 114 p a t i e n t s w i t h i s o n i a z i d - r e l a t e d h e p a t i t i s (14). Some o f the important f i n d i n g s were t h a t : 1) i s o n i a z i d - r e l a t e d l i v e r i n j u r y was c l i n i c a l l y i n d i s t i n g u i s h a b l e b i o c h e m i c a l l y and m o r p h o l o g i c a l l y from i p r o n i a z i d - i n d u c e d l i v e r damage or from other causes o f acute h e p a t o c e l l u l a r i n j u r y such as v i r a l h e p a t i t i s ; 2) no c l i n i c a l evidence such as r a s h , f e v e r , a r t h r a l g i a s o r e o s i n o p h i l i a was found f o r h y p e r s e n s i t i v i t y mechanism; 3) about 30% o f the p a t i e n t s w i t h h e p a t i c r e a c t i o n s were r e s i d e n t s o f Honolulu and o f O r i e n t a l a n c e s t r y ; on g e n e t i c b a s i s , 90% or more o f these p a t i e n t s would be expected t o be r a p i d a c e t y l a t o r s o f i s o n i a z i d i n c o n t r a s t t o b l a c k and w h i t e populations i n whom 45% are r a p i d a c e t y l a t o r s (16). I n the t h i r d study (15), 21 n o n - O r i e n t a l p a t i e n t s who had recovered from i s o n i a z i d h e p a t i t i s were g e n e t i c a l l y phenotyped as r a p i d o r slow a c e t y l a t o r s o f i s o n i a z i d u s i n g the sulfamethazine method. E i g h t y - s i x percent o f them d i s p l a y e d the r a p i d a c e t y l a t o r phenotype f o r i s o n i a z i d metabolism. Metabolism s t u d i e s i n man. Based on these c l i n i c a l f i n d i n g s , we re-examined the metabolism of i s o n i a z i d and i d e n t i f i e d the

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

158

DRUG M E T A B O L I S M C O N C E P T S

m e t a b o l i t e s by co-chromatography, reverse i s o t o p e d i l u t i o n w i t h s y n t h e s i z e d standards and by mass s p e c t r a l a n a l y s i s (15). T r i t i u m r i n g - l a b e l e d i s o n i a z i d and a c e t y l i s o n i a z i d , the major primary m e t a b o l i t e of i s o n i a z i d , were administered to human v o l u n t e e r s i n s i n g l e 300 mg doses and u r i n a r y m e t a b o l i t e s were c o l l e c t e d f o r 24 h r s . As shown i n F i g u r e 2, about 55% of a dose of a c e t y l i s o n i a z i d was metabolized by h y d r o l y s i s t o i s o n i c o t i n i c a c i d and f r e e a c e t y l h y d r a z i n e r e g a r d l e s s of g e n e t i c phenotype of the p a t i e n t s f o r a c e t y l a t i n g i s o n i a z i d * I n c o n t r a s t , p a t t e r n of m e t a b o l i t e s a f t e r a d m i n i s t r a t i o n of i s o n i a z i d was very dependent upon the r a t e a t which i s o n i a z i d was a c e t y l a t e d * On the b a s i s of the r e l a t i v e amounts of a c e t y l i s o n i a z i d and i s o n i c o t i n i c a c i d excreted i n t o the u r i n e , we c a l c u l a t e d t h a t almost a l l of the i s o n i c o t i n i c a c i d was formed by way of a c e t y l i s o n i a z i d * We a l s o c a l c u l a t e d that p a t i e n t s who were f a s t m e t a b o l i z e r s of i s o n i a z i d converted about 94% of an i s o n i a z i d dose to a c e t y l i s o n i a z i d ; o n l y 2.8% of the drug was exc r e t e d unchanged i n the u r i n e and 3.6% as hydrazone conjugates. Slow a c e t y l a t o r s , on the other hand, excreted almost 37% of the drug i n the u r i n e e i t h e r f r e e or as a hydrazone. Thus, o n l y 63% was converted t o a c e t y l i s o n i a z i d and subsequently to i s o n i c o t i n i c a c i d and a c e t y l h y d r a z i n e . We concluded, t h e r e f o r e , that f a s t a c e t y l a t o r s are exposed t o much more a c e t y l i s o n i a z i d and a c e t y l hydrazine than are slow a c e t y l a t o r s . Hepatic n e c r o s i s i n animals. A c e t y l i s o n i a z i d and i s o n i a z i d were given t o r a t s , mice and hamsters to see i f they could produce h e p a t i c n e c r o s i s (17,18). These hydrazines were given i n a doseresponse manner to s e v e r a l hundred animals. I s o n i a z i d d i d not cause n e c r o s i s i n any of the animals. However, a c e t y l i s o n i a z i d produced o c c a s i o n a l s i n g l e c e l l n e c r o s i s i n r a t s and mice. Moreover, as shown i n Table I , pretreatment of r a t s w i t h phénobarbital, which i s known to i n c r e a s e drug m e t a b o l i z i n g enzymes, g r e a t l y p o t e n t i a t e d the n e c r o s i s . The l i v e r damage was prevented by p r e treatment of r a t s w i t h c o b a l t c h l o r i d e , which i n h i b i t s s y n t h e s i s of cytochrome P-450 m e t a b o l i z i n g enzymes. S i m i l a r l y when hydrol y s i s of a c e t y l i s o n i a z i d was i n h i b i t e d by pretreatment of r a t s w i t h b i s - p a r a - n i t r o p h e n y l phosphate (BNPP), the n e c r o s i s was prevented. The e f f e c t on the l i v e r of the h y d r o l y s i s product, a c e t y l h y d r a z i n e , was t h e r e f o r e examined. This hydrazine i s a very potent hepatotoxin which produces h e p a t i c n e c r o s i s i n phenobarbital-pret r e a t e d r a t s a f t e r s i n g l e doses of 10 mg/kg. The n e c r o s i s was p o t e n t i a t e d by pretreatment w i t h phénobarbital and prevented by pretreatment w i t h c o b a l t c h l o r i d e (Table I ) . However, BNPP, which i n h i b i t e d the h y d r o l y s i s of a c e t y l i s o n i a z i d and prevented the n e c r o s i s , had no e f f e c t on n e c r o s i s produced by a c e t y l h y d r a z i n e . Thus, the metabolic a c t i v a t i o n of the l i b e r a t e d a c e t y l h y d r a z i n e moiety of a c e t y l i s o n i a z i d to a t o x i c m e t a b o l i t e s a t i s f a c t o r i l y accounts f o r the h e p a t i c n e c r o s i s produced by i s o n i a z i d . Subsequently, i s o n i a z i d i t s e l f was shown to produce acute h e p a t i c n e c r o s i s i n p h e n o b a r b i t a l - t r e a t e d r a t s . The p r o p o r t i o n or the i s o n i a z i d that i s a c e t y l a t e d i n r a t s decreases markedly

8.

NELSON

C-N-NH

Chemical-Induced

ETAL.

S H H

Tissue

Injury

159

/C 3 H

C-N-N-CH

2

Ô

ô

un

(I)

9

HN-C-CH

HN—C—CH

5

3

0-CH -CH

OH (III)

2

3

(IV)

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

HOOC-"

HOOC

OAc

(VI)

(V)

CH,^CH,

HO'' ΓΗ

Figure 1. Structures of the compounds discussed in the text

(VII)

X Of DOSE DRUG

PATIENTS ACETYLATIOtl RATE 00

AcIMH

FAST (2)

AcINH

INH HTDRAZONES

AcINH

INA DERIVATIVES

ESTIMATED ACETYL HYDRAZINE

...

54.912.2

45.112.7

45.112.7

...

SLOW (3)

—

53.811.2

46.211.1

46.211.1

...

INH

FAST (3)

2.810.4

3.610.4

49.211.9

44.413.9

41.013.8

3.410.1

INH

SLOW (4)

10.910.8 26.5±4.8

32.111.2

30.513.5

26.813.3

3.7*0.2

INH

ESTIMATED HYDRAZINE

JLS-CCH CH C-

o -

3

ACETYLISONIAZID

ISONIAZID

ISONIAZID HÏDRAZONES

ACETYLHYDRAZINB

ISONICOTINIC ACID

Figure 2. Twenty-four hour urinary excretion of metabolites after administration of 300 mg of acetylisoniazid- H-ring-labeled (AcINH) or isoniazid- H-ring-labeled (INH) to male volunteers (See Ref. 15) s

3

160

DRUG M E T A B O L I S M C O N C E P T S

Table I

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

ACUTE HEPATIC NECROSIS IN RATS PRODUCED BY ISONIAZID (INH), ACETYLISONIAZID (AcINH), ACETYLHYDRAZINE (AcHz), IPRONIAZID (IpINH), AND ISOPROPYLHYDRAZINE (IpHz)

Treatments

INH 100 mg/kg*

AcINH 200 mg/kg

AcHZ 20 mg/kg

0 or +

IpINH 200 mg/kg

IpHz 20 mg/kg

+

+

None

0

0 or +

Phénobarbital

+

++

-H+

Phénobarbital + CoCl

0

0

0

0 or+

0 or +

Phénobarbital + BNPP

0

0

-H-+

0 or+

+4+4-

2

*Admlnisterd every hour for 6 hours* +CoCl • cobalt chloride. 2

ÎBNPP • bls-para-nitrophenyl phosphate.

+++

8.

NELSON

ET AL.

Chemical-Induced

Tissue

Injury

161

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

above 100 mg/kg i n d i c a t i n g a s a t u r a b l e mechanism. Thus, a s i n g l e l a r g e dose does not cause l i v e r n e c r o s i s , but the a d m i n i s t r a t i o n o f i s o n i a z i d i n s i x s i n g l e doses o f 100 mg/kg per hour caused acute h e p a t i c n e c r o s i s (Table I ) . Covalent b i n d i n g s t u d i e s i n v i v o . As f u r t h e r support f o r the hypothesis t h a t a c e t y l i s o n i a z i d i s converted i n the b o d y j j o a c h e m i c a l l y r e a c t i v e form v i a a c t i v a t i o n o f a c e t y l h y d r a i n e , Ca c e t y l i s o n i a z i d r a d i o l a b e l e d i n the a c e t y l moiety and C - a c e t y l hydrazine were given t o r a t s and evidence f o r covalent b i t i d i n g t o t i s s u e macromolecules was sought (18,19). A l a r g e amount o f cov a l e n t b i n d i n g was found upon d i g e s t i o n o f the p r o t e i n s i n the l i v e r , the t a r g e t organ f o r t o x i c i t y , but l i t t l e was found i n other t i s s u e s . This b i n d i n g was p r o p o r t i o n a l t o dose, was i n creased by pretreatment w i t h phénobarbital and was markedly decreased by pretreatment w i t h c o b a l t c h l o r i d e (Table I I ) . However, no c o v a l e n t l y bound r a d i o l a b e l e d m a t e r i a l was found when a c e t y l i s o n i a z i d r a d i o l a b e l e d i n the p y r i d i n e r i n g was administered. Thus, the r e a c t i v e m e t a b o l i t e came only from the a c e t y l h y d r a z i n e moiety. BNPP, which b l o c k s the h y d r o l y s i s o f a c e t y l i s o n i a z i d , decreased the covalent b i n d i n g o f ^ C - a c e t y l i s o n i a z i d |> t h a t o f C - a c e t y l h y d r a z i n e , p a r a l l e l i n g the e f f e c t o f BNPP on the hepatic necrosis. ut n

o

t

14

Covalent b i n d i n g s t u d i e s i n v i t r o . Based on the e f f e c t s o f mixed f u n c t i o n oxygenase inducers and i n h i b i t o r s on the h e p a t i c n e c r o s i s and covalent b i n d i n g found i n animals, experiments were c a r r i e d out u s i n g l i v e r microsomes i n v i t r o t o determine t h e enzyme requirements f o r the b i n d i n g r e a c t i o n . The r e s u l t s o f experiments w i t h a c e t y l h y d r a z i n e and r a t l i v e r microsomes under v a r i o u s c o n d i t i o n s (Table I I I ) showed t h a t a s u b s t a n t i a l amount o f covalent b i n d i n g occurred a t 37 C i n the presence o f l i v e r microsomes, a i r and NADPH. The b i n d i n g was almost a b o l i s h e d by l a c k o f NADPH, heat dénaturât i o n o f the enzymes, o r l a c k o f oxygen. A carbon monoxide: oxygen atmosphere, SKF-525A, p i p e r o n y l butoxide pretreatment, o r an antibody a g a i n s t NADPH cytochrome £ reductase i n h i b i t e d covalent b i n d i n g , thereby i n d i c a t i n g i n v o l v e ment o f a cytochrome P-450 mixed f u n c t i o n oxygenase. Furthermore, experiments w i t h h e p a t i c microsomes prepared immediately f o l l o w i n g the traumatic death o f a h e a l t h y young a d u l t male demonstrate t h a t the a c t i v a t i o n system i s present i n human t i s s u e s (19, Table III). — G l u t a t h i o n e and c y s t e i n e , n a t u r a l l y o c c u r r i n g s u l f h y d r y l compounds, s u b s t a n t i a l l y decreased covalent b i n d i n g i n v i t r o by formation o f the adducts, S - a c e t y l g l u t a t h i o n e and N - a c e t y l c y s t e i n e . The work o f Smith and G o r i n (21), which showed t h a t S - a c e t y l c y s t e i n e rearranges r a p i d l y a t n e u t r a l pH values t o the thermodynamically more s t a b l e N - a c e t y l c y s t e i n e , suggests t h a t the i n i t i a l product might have been S - a c e t y l c y s t e i n e which subsequently r e arranged t o the observed product, N - a c e t l y c y s t e i n e . Both N - a c e t y l c y s t e i n e and S - a c e t y l g l u t a t h i o n e were i s o l a t e d from e

162

DRUG M E T A B O L I S M

CONCEPTS

Table I I EFFECT OF TREATMENTS ON IN VIVO HEPATIC COVALENT BINDING OF 3

3

ISONIAZID- H-RING-LABELED (INH),* ACETYLISONIAZID- H-RING LABELED 14

(AcINH)*, ACETYLISONIAZID- C-ACETYL-LABELED (AçINH), ACETYL-HYDRA14

3

ZINE- C-ACETYL-LABELED (AcHz), IPRONIAZID- H-RING-LABELED

(IpINH),

3

IPR0NIAZID-2- H-IS0PR0PYL-LABELED (ΙηΙΝΗ), and ISOPROPYLHYDRAZINE3

2- H-IS0PR0PYL-LABELED (IpHz) IN RATS.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

R e s u l t s a r e expressed as means + standard e r r o r s o f 3 separate experiments u s i n g 3 animals i n each experiment.

Treatment

AcINH IpINH IDINH AcHz IpHz 200 200 20 200 20 mg/kg mg/kg mg/kg mg/kg mg/kg Covalent B i n d i n g Covalent B i n d i n g nmole/mg p r o t e i n nmole/mg p r o t e i n (6 h r a f t e r dose) (6 h r a f t e r dose)

None

0.20 + .021

0.15 + .012

0.09 + .015

0.28 + .029

0.35 + .023

Pb**

0.31 + .021

0.19 + .012

0.10 + .015

0.53 + .038

0.44 + .038

0.15 + .039

0.09 + .008

0.18 + .017

0.22 + .029

0.11 + .033

0.23 + .035

0.17 + .019

0.32 + .025

Pb + C o C l

2

Pb + ΒΝΡΡΦ

Covalent b i n d i n g f o r these two compounds was p r o t e i n f o r a l l treatments. + CoCl

β 2

< 0.05 nmole/mg

phénobarbital + c o b a l t c h l o r i d e

tpb + BNPP » phénobarbital + b i s - p a r a - n i t r o p h e n y l phosphate

8.

NELSON

ET

AL.

Chemical-Induced

Tissue

Injury

163

Table I I I 14

14

COVALENT BINDINGQIN VITRO OF ACETYL-( C)-HYDRAZINE ( C-AcHz) AND ISOPROPYL-(2-nQ-HYDRAZINE CH-IpHz) TO RAT LIVER MICROSOMES A

Conditions

X-AcHz (1 mM)

~Ή-ΙρΗζ (0.1 mM)

** % of C o n t r o l

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

A.

C o n t r o l * ( a i r atmosphere) B o i l e d microsomes - C o f a c t o r (NADPH generating system) +NADH (-NADPH generating system) 100% N atmosphere 2

6%

10%

7% 15%

7% 11%

atmosphere

92%

97%

C0:0

(9:1) atmosphere

37%

48%

64% 25% 35% 49%

70% 45% 58% 65%

2

: 0

2

+SKF 525-A (0.2 mM) P i p e r o n y l butoxide+ +GSH (1 mM) +Cysteine (1 mM)

C.

100% 12%

2

N

B.

100% 13%

$

Preimmune - g l o b u l i n

0.099

nmoles/mg/15 min 0.328

Immune Ï - g l o b u l i n (NADPH-cytochrome c reductase antibody)

0.048

0.159

C o n t r o l ( a i r atmosphere) - c o f a c t o r (NADPH generating system)

0.16

Human Microsomes 0.37

0.02

0.03

Microsomes were prepared from r a t l i v e r and human l i v e r , incubated as d e s c r i b e d i n Table IV and covalent b i n d i n g was determined ( 1 9 ) . The c o n t r o l b i n d i n g o f AcHz w i t h r a t l i v e r microsomes was 0.55 nmoles/mg/15 min and f o r IpHz was 0.58 nmoles/mg/15 min. ^Administered

(0.3 ml) i . p . 30 min p r i o r t o s a c r i f i c i n g

the animal.

"^Each i n c u b a t i o n contained 7 mg of p a r t i a l l y p u r i f i e d preimmune o r immune jf - g l o b u l i n per mg microsomal p r o t e i n , as p r e v i o u s l y described (20).

164

DRUG M E T A B O L I S M C O N C E P T S

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

i n c u b a t i o n mixtures by g e l f i l t r a t i o n on Sephadex f o l l o w e d by anion exchange chromatography* The products were then c h a r a c t e r i z e d by chemical i o n i z a t i o n mass spectrometry (22,23)* I p r o n i a z i d * Studies i n animals o f the metabolism of i p r o n i a z i d ( I I ) , an antidepressant drug removed from c l i n i c a l use because o f a h i g h i n c i d e n c e of h e p a t i t i s s i m i l a r to t h a t o f i s o n i a z i d r e v e a l e d t h a t i p r o n i a z i d a l s o r e q u i r e d enzymatic h y d r o l y s i s to produce the h e p a t i c l e s i o n (Table I ) . S p e c i f i c r a d i o l a b e l l n g and covalent b i n d i n g s t u d i e s showed that i s o p r o p y l h y d r a z i n e was r e leased by h y d r o l y s i s and then o x i d a t i v e l y a c t i v a t e d i n v i t r o to a potent hepatotoxin (19, Table I I ) . M e t a b o l i c a c t i v a t i o n of i s o p r o p y l h y d r a z i n e , the hepatotoxic m e t a b o l i t e of i p r o n a z i d , t o a r e a c t i v e intermediate showed enzyme requirements v i r t u a l l y i d e n t i c a l t o those f o r the a c t i v a t i o n of a c e t y l h y d r a z i n e (Table I I I ) . Thus, a cytochrome P-450 oxygenase mediated the covalent b i n d i n g o f i s o p r o p y l h y d r a z i n e to t i s s u e p r o t e i n . Trapping experiments w i t h c y s t e i n e and g l u t a t h i o n e showed t h a t S - i s o p r o p y l c y s t e i n e and S - i s o p r o p y l g l u t a t h i o n e were formed (23)· The a c t i v a t i n g enzyme system could be assessed k i n e t i c a l l y u s i n g covalent b i n d i n g o f r a d i o l a b e l e d m e t a b o l i t e as an index o f r e a c t i v e product formation* A d o u b l e - r e c i p r o c a l p l o t of the enzyme-dependent b i n d i n g o f a c e t y l h y d r a z i n e to microsomal p r o t e i n s ( F i g u r e 3A) shows t h a t the r e a c t i o n r a t e i s markedly increased by phénobarbital pretreatment, which p o t e n t i a t e d the h e p a t i c n e c r o s i s and b i n d i n g i n v i v o , whereas i t i s decreased by pretreatment o f the animals w i t h c o b a l t c h l o r i d e , which blocked the h e p a t i c n e c r o s i s and b i n d i n g i n v i v o . The same e f f e c t s were found f o r the b i n d i n g r e a c t i o n o f i s o p r o p y l h y d r a z i n e to r a t l i v e r microsomes, except that the Κ f o r b i n d i n g was 1/10 that f o r a c e t y l h y d r a z i n e (Figure 3B). This may account f o r the greater h e p a t o t o x i c i t y observed w i t h i s o p r o p y l ­ hydrazine when compared to t h a t of a c e t y l h y d r a z i n e . I n a d d i t o n , the e v o l u t i o n o f propane from microsomal r e a c t i o n s was determined by gas chromatography and gas chromatography-mass spectrometry .As shown i n Table IV, phénobarbital i n c r e a s e d both covalent b i n d i n g and propane formation, whereas c o b a l t c h l o r i d e decreased both. Double-isotope experiments w i t h a c e t y l i s o n i a z i d - a c e t y l h y d r a z i n e and i p r o n i a z i d - i s o p r o p y l h y d r a z i n e . In order to study the metabolic a c t i v a t i o n process f o r the covalent b i n d i n g of a c e t y l i s o n i a z i d and a c e t y l h y d r a z i n e i n more d e t a i l , we administered to r a t s a mixture o f a c e t y l i s o n i a z i d and a mixture of a c e t y l h y d r a z i n e l a b e l e d w i t h t r i t i u m and carbon-14 i n the a c e t y l moiety. The H/ r a t i o o f the c o v a l e n t l y bound m e t a b o l i t e from e i t h e r a c e t y l i s o n i a z i d or a c e t y l h y d r a z i n e was almost i d e n t i c a l to the ^H/^C r a t i o o f the administered mixture (19, Table V ) . This i n d i c a t e d 3r ij y ! group was bound. Moreover, Incubation of the ^H- and ^ C - a c e t y l h y d r a z i n e w i t h r a t l i v e r microsomes i n v i t r o gave the same r e s u l t s (19, Table V ) . 3

t h a t

t h e

e n t

e

a c e t

8.

Chemical-Induced

NELSON E T A L .

Tissue

Injury

165

200

PHENOBARBITAL + COBALTOUS CHLORIDE PRETREATMENT K^-0.98 aM V^-0.03 NMOLES/MG/MIN

150 1

(NMOLES/MG/MIN)"

NORMAL 1^-0.95^ V ax-0.06 NMOLES/MG/MIN B

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

PHENOBARBITAL 1^-1.13 «M V - _ - 0 . l l NMOLES/MG/MIN

1

1/S («Μ" ) 100

r

PHENOBARBITAL + CoCl, Κ - 0.07 m V ? " 0.04 naolM/ag/aln

NORMAL K^- 0.09 WÊL V_ • 0.07 naol*s/ag/aln

PHENOBARBITAL K_« 0·10 «M - 0.13 naoUs/ae/aln

-15

-10

-5

Figure S. Lineweaver-Burk plots of mixed function oxidase-dependent covalent binding of acetylhydrazine (A) ana isopropylhydrazine (B) to rat microsomal protein in vitro. For each incubation, rat microsomes were prepared, incubated under air with either Cacetylhydrazine (A) or isopropyl-2 ( H)-hydrazine (B) and a NADPH-generating system, and covalent binding was determined. 14

s

166

DRUG M E T A B O L I S M

CONCEPTS

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

TABLE IV CORRELATION OF PROPANE EVOLUTION WITH IN VITRO COVALENT BINDING OF ISOPROPYL-(2- H)-HYDRAZINE TO HEPATIC MICROSOMES Ice-cold incubation mixtures (3 ml) contained rat l i v e r microsomal protein (2 mg/ml) isolated from rats pretreaÇed as i n dicated; phosphate buffer, pH 7.4, 83 mM; isopropyl-(2- H)hydrazine, 0.1 mM; and a NADPH-generating system (NADP, 0.64 mM; glucose-6-phosphate, 15.5 mM; glucose-6-phosphate dehydrogenase, 2U/ml; MgC^, 10 mM). Reactions were incubated under a i r i n septum-sealed incubation vessels for 15 min with shaking (Dubnoff shaker incubator) at 37 C and covalent binding determined (19). The head-space gases were analyzed by GLC as described i n Ref. 19} the propane effluent was trapped in Aquasol s c i n t i l l a n t cooled i n dry ice-acetone, and radioa c t i v i t y was counted by s c i n t i l l a t i o n spectrometry. Results are expressed as means + standard deviations. Numbers i n parentheses are number of determinations.

Treatment

None Phénobarbital (75 mg/kg i.p.

I n v i t r o Covalent B i n d i n g (nmoles/mg p r o t e i n / 1 5 min)

0.58 + 0.051 (9)

χ 4 days)1.06 + 0.059 (6)

Phénobarbital (75 mg/kg i.p. χ 4 days) + cobalt chloride (30 mg/kg s.c. 12 hourly χ 4 doses) 0.33 + 0.006 (6)

Propane evolved (% o f t o t a l radioactivity i n 15 min)

13.0% + 1.00 (9)

19.5% + 0.75 (6)*

9.4% + 0.81 (6)*

*P jC 0.05 when compared to respective control values as determined by Student's t test.

8.

Chemical-Induced

NELSON E T A L .

Tissue

Injury

167

Table V 3

14

RATIOS ( H / C ) RADIOLABEL BOUND TO HEPATIC PROTEIN VERSUS THAT IN INITIAL SUBSTRATE MIXTURES Mixtures o f

14 3 C-carbonyl- and H-methyl-labeled t/

i s o n i a z i d (AcINH; 200 mg/kg; sp. a c t .

acetyl*x

C, 0.15 mCi/mmole;

H,

0.53 mCi/mmole) and s i m i l a r mixtures o f a c e t y l h y d r a z i n e (AcHz, 1 4

3

20 mg/kg; sp. a c t . C , 0.36 mCi/mmole; H , 1.01 mCi/mmole) were administered t o male F i s c h e r r a t s . I n a d d i t i o n , mixtures o f 14 3 s p e c i f i c a l l y l a b e l e d i s o p r o p y l - ( 2 - C ) - and i s o p r o p y l - ( 2 - H ) l a b e l e d i p r o n i a z i d (IpINH. 200 mg/kg; sp. a c t C , 0.50 mCi/ 1 4

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

3

mmole; sp. a c t .

H, 1.43 mCi/mmole) and i s o p r o p y l h y d r a z i n e 14 3 (IpHz, 20 mg/kg; sp. a c t . C, 0.30 mCi/mmole; sp. a c t . H,

0.87 mCi/mmole) were administered t o F i s c h e r r a t s . I n other experiments, mixtures o f H- and C-acetylhydrazine (1 mM) 3 14 and i s o p r o p y l - ( 2 - H)- and i s o p r o p y l - ( 2 - C ) - hydrazine (0.1 mM) were incubated i n a i r w i t h an NADPH-generating system and w i t h microsomes i s o l a t e d from F i s c h e r r a t l i v e r .

Covalent b i n d i n g o f

r a d i o l a b e l t o h e p a t i c t i s s u e p r o t e i n was determined by methods p r e v i o u s l y d e s c r i b e d (19) and found t o be 0.20 nmoles/mg ( i n v i v o , AcHz), 0.28 nmoles/mg ( i n v i t r o , IpHz). 3

Values a r e r e p o r t -

4

ed as H/^* C r a t i o s o f the c o v a l e n t l y bound r a d i o l a b e l , as determined by the c h a n n e l s - r a t i o method u s i n g i n t e g r a l counting, 3

14

d i v i d e d by the H / C r a t i o o f the i n i t i a l s u b s t r a t e mixture as determined by the same method. R e s u l t s a r e expressed as means + standard e r r o r s o f 4 such determinations.

Conditions

Substrate

AcINH

AcHz

IpINH

IpHz

In vivo (6 h r a f t e r dosing) 0.90 + .055 0.92+0.021 0.92+. 032 0.96+0.060 In v i t r o (15 min i n c u b a t i o n s )

-

0.94+0,012

-

0.98+0.022

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

168

DRUG M E T A B O L I S M

CONCEPTS

L i v e r microsomes and NADPH were a l s o incubated w i t h c y s t e i n e and approximately equimolar amounts o f a c e t y l - and t r i d e u t e r o a c e t y l h y d r a z i n e (22). N - a c e t y l c y s t e i n e was i s o l a t e d from i n cubation mixtures c o n t a i n i n g NADPH, oxygen, c y s t e i n e and a c e t y l hydrazine. Chemical i o n i z a t i o n mass spectrometry showed q u a s i molecular i o n s (QM ) a t m/e 164 and 167 f o r the non- and t r i d e u t e r a t e d N - a c e t y l c y s t e i n e . These i o n s were monitored and found t o have t h e same H/D r a t i o as t h e quasimolecular ions o f the a c e t y l hydrazine s u b s t r a t e mixture (m/e 75 and 78, F i g u r e 4 ) . This study e l i m i n a t e d ketene as the r e a c t i v e i n t e r m e d i a t e . S i m i l a r experiments w i t h i p r o n i a z i d and i s o p r o p y l h y d r a z i n e , l a b e l e d w i t h t r i t i u m and carbon-14 i n the methine carbon o f the i s o p r o p y l group, showed t h a t e q u i v a l e n t amounts o f t r i t i u m and carbon-14 were bound both i n v i v o and i n v i t r o , demonstrating t h a t methine hydrogen was r e t a i n e d and t h e r e f o r e e l i m i n a t i n g acetone as t h e intermediate i n the b i n d i n g r e a c t i o n (19). T h i s was confirmed by a t w i n - i o n study u s i n g s p e c i f i c a l l y C-2 deuterated i s o p r o p y l h y d r a z i n e . The r e a c t i v e m e t a b o l i t e was trapped from microsomal i n c u b a t i o n s i n v i t r o w i t h c y s t e i n e . Mass s p e c t r a l a n a l y s i s o f t h e i s o l a t e d c y s t e i n e d e r i v a t i v e showed t h a t S - i s o p r o p y l c y s t e i n e was formed and t h a t no deuterium was l o s t from the i s o p r o p y l group (23). M e c h a n i s t i c i m p l i c a t i o n s f o r the metabolic a c t i v a t i o n o f t o x i c m e t a b o l i t e s o f i s o n i a z i d and i p r o n i a z i d . From the r e s u l t s i n v i v o showing c o r r e l a t i o n s between t i s s u e n e c r o s i s and covalent b i n d i n g , s t u d i e s i n v i t r o showing a microsomal P-450 oxygenase requirement, and t r a p p i n g experiments w i t h c y s t e i n e and g l u t a t h i o n e , we propose the f o l l o w i n g r e a c t i o n scheme (Figure 5) f o r formation o f t o x i c m e t a b o l i t e s from i s o n i a z i d and i p r o n i a z i d . I s o n i a z i d i s a c e t y l a t e d t o i t s major m e t a b o l i t e a c e t y l i s o n i a z i d . I n man, r a p i d a c e t y l a t o r s convert a t l e a s t 35% more i s o n i a z i d t o a c e t y l i s o n i a z i d than slow a c e t y l a t o r s . A c e t y l i s o n i a z i d i s then e f f i c i e n t l y hydrolyzed t o i s o n i c o t i n i c a c i d and a c e t y l hydrazine. A c e t y l h y d r a z i n e i s f u r t h e r metabolized by a P-450 oxygenase, p o s s i b l y t o a N-hydroxy hydrazine. T h i s intermediate would probably dehydrate t o a c e t y l d i a z e n e which could be t h e e l e c t r o p h i l i c a c y l a t i n g s p e c i e s . However, mono-substituted d i a zenes are known t o fragment i n the presence o f oxygen most l i k e l y t o r a d i c a l s (24) and t h i s could be t h e t o x i c i n t e r m e d i a t e . Ketene has been e l i m i n a t e d as the r e a c t i v e a c y l a t i n g s p e c i e s o f a c e t y l hydrazine by chemical i o n i z a t i o n mass s p e c t r a l t w i n - i o n study w i t h t r i d e u t e r o - a c e t y l h y d r a z i n e , which i n d i c a t e d t h a t t h e e n t i r e a c e t y l group was bound (22). By mechanisms t h a t are not understood t h e r e a c t i v e m e t a b o l i t e i n i t i a t e s processes t h a t l e a d t o h e p a t i c necrosis. I p r o n i a z i d i s hydrolyzed t o i s o n i c o t i n i c a c i d and i s o p r o p y l hydrazine. Isopropylhydrazine i s then f u r t h e r o x i d i z e d t o a r e a c t i v e a l k y l a t i n g agent. Since the e v o l u t i o n o f propane p a r a l l e l s covalent b i n d i n g , we suspect t h e two r e a c t i o n s d e r i v e from common

NELSON E T A L .

Chemical-Induced

Injury

0AC75

100

I

Tissue

S

ΐθΜ,+78

80

Η R-C-N-NH

2

R-CH *CD3 3

60

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

•Ι

40 20

100

90

80 M/E

70

100 Ο H Il H I R-C-N-C-CH2-SH

δ

1 C

80

QM+167

COOH R=CH +CD 3

60

3

40 '22 UO 121 1 2 3 |

20

1

80

100

120 M/E

3

3

140

160

180

Figure 4. Chemical ionization mass spectra (isobutane reactant gas) of a sample of the substrate mixture of acetyl- and trideuteroacetylhydrazine (A) and of the cysteine adduct iso­ lated from a microsomal incubation containing the substrate mixture, NADPH and cysteine (B) (See Ref. 22)

170

DRUG M E T A B O L I S M

CONCEPTS

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

i n t e r m e d i a t e . Furthermore, a t w i n - i o n study w i t h s p e c i f i c a l l y C-2 deuterated i s o p r o p y l h y d r a z i n e , s i m i l a r t o the study c a r r i e d out with trideuteroacetylhydrazine, indicated that the e n t i r e isopro­ p y l group was r e t a i n e d i n t h e bound m e t a b o l i t e . A r e a c t i o n scheme ( F i g u r e 5 ) compatible w i t h these r e s u l t s i s the formation o f the i s o p r o p y l r a d i c a l o r c a t i o n from i s o p r o p y l d i a z e n e . These r e a c t i v e a l k y l a t i n g agents then c o v a l e n t l y b i n d t o t i s s u e macromolecules. Whatever t h e intermediate may be, i t i s c l e a r t h a t o x i d a t i v e a c t i v a t i o n o f these hydrazines by microsomal enzymes mediates l i v e r n e c r o s i s i n animals. Since these enzymes a r e present i n human l i v e r t i s s u e , these intermediates probably cause the s e r i o u s and o c c a s i o n a l l y l e t h a l h e p a t i t i s seen w i t h i s o n i a z i d and i p r o n i a z i d therapy i n man.

t Covalent Binding to Macromolecules Λ

Propane

* •

Expired Propane

Hepatic Necrosis

Figure 5.

Proposed metabolic activation pathways for isoniazid, acetyliso­ niazid, and isopropylisoniazid (iproniazid)

8.

NELSON

ET AL.

Chemical-Induced

Tissue Injury

171

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

Acetaminophen S t u d i e s i n v i v o i n man and l a b o r a t o r y animale» Acetaminophen ( p - h y d r o x y a c e t a n i l i d e , I I I ) i s a commonly used m i l d a n a l g e s i c which i s a p p a r e n t l y q u i t e s a f e when taken i n normal t h e r a p e u t i c doses. However, l a r g e overdoses cause l i f e - t h r e a t e n i n g l i v e r l e s i o n s i n man (2J5,26), r a t s (27,28), mice (28), and hamsters (29). P r i o r treatment o f animals w i t h inducers o f drug metabolism, such as phénobarbital o r 3-methylcholanthrene, g r e a t l y p o t e n t i a t e s t h e s e v e r i t y o f t h e n e c r o s i s (28,30). I n c o n t r a s t , pretreatments w i t h i n h i b i t o r s o f drug metabolism, such as p i p e r o n y l b u t o x i d e , c o b a l t c h l o r i d e , o r «-naphthylisothiocyanate, prevent t h e n e c r o s i s (28,30). A l a c k o f c o r r e l a t i o n between acetaminophen t i s s u e l e v e l s and acetaminophen-induced h e p a t i c n e c r o s i s i n d i c a t e s t h a t a t o x i c m e t a b o l i t e r a t h e r than acetaminophen i t s e l f causes the h e p a t i c t i s s u e i n j u r y . Acetaminophen r a d i o l a b e l e d w i t h t r i t i u m o r w i t h carbon-14 was g i v e n t o normal mice and mice p r e t r e a t e d w i t h compounds t h a t a l t e r e d acetaminophen-induced h e p a t i c n e c r o s i s . The animals were k i l l e d a t v a r i o u s times and t h e l i v e r s examined f o r c o v a l e n t l y bound m e t a b o l i t e s o f acetaminophen. Autoradiograms showed covalent b i n d i n g o f acetaminophen p r e f e r e n t i a l l y i n t h e n e c r o t i c c e n t r i l o b u l a r area o f t h e l i v e r , i . e . , there was a d i r e c t c o r r e l a t i o n between the two measurable parameters t i s s u e n e c r o s i s and c o v a l e n t b i n d i n g (31). Pretreatment w i t h an inducer o f microsomal metabolism, phénobarbital, i n c r e a s e d b i n d i n g , whereas p r e t r e a t ments w i t h d i f f e r e n t i n h i b i t o r s o f metabolism decreased b i n d i n g . Thus, t h e e f f e c t o f treatment on covalent b i n d i n g c o r r e l a t e d d i r e c t l y w i t h treatment e f f e c t s on h e p a t i c n e c r o s i s . Evidence f o r the c o v a l e n t nature o f t h e b i n d i n g was obtained by d i g e s t i o n o f s o l v e n t - e x t r a c t e d l i v e r p r o t e i n w i t h protease and i s o l a t i o n o f the r a d i o l a b e l bound t o amino a c i d and peptide fragments. These s t u d i e s i n d i c a t e d t h a t acetaminophen was converted by microsomal enzymes, t o a r e a c t i v e a r y l a t i n g agent which c o v a l e n t l y bound t o macromolecules i n the t a r g e t t i s s u e f o r damage, t h e l i v e r . Concept o f a dose-threshold f o r t o x i c i t y . Because o f t h e s t r i k i n g c o r r e l a t i o n between t h e s e v e r i t y o f h e p a t o t o x i c i t y and the extent o f covalent b i n d i n g by t h e a r y l a t i n g m e t a b o l i t e o f acetaminophen, i t was s u r p r i s i n g t h a t s i g n i f i c a n t b i n d i n g d i d not occur u n t i l over 60% o f t h e drug had been e l i m i n a t e d from t h e l i v e r . G l u t a t h i o n e i s depleted from t h e l i v e r o f animals r e c e i v i n g acetaminophen because i t combines w i t h a minor m e t a b o l i t e o f the drug and forms a r e a d i l y excreted mercapturic a c i d (4,30,32, 33). Thus t h e p o s s i b i l i t y a r i s e s t h a t t h e a r y l a t i n g m e t a b o l i t e o f acetaminophen i n t i a l l y i s d e t o x i f i e d by r e a c t i n g p r e f e r e n t i a l l y w i t h g l u t a t h i o n e (Figure 6 ) . A f t e r t h e major r o u t e s o f acetaminophen metabolism ( s u l f a t i o n and g l u c u r o n i d a t i o n pathways) become s a t u r a t e d , and a f t e r t h e l i v e r i s depleted o f g l u t a t h i o n e , t h e r e a c t i v e m e t a b o l i t e can combine w i t h l i v e r macromolecules and by undefined mechanisms cause c e l l death.

DRUG M E T A B O L I S M

172

CONCEPTS

ACETAMINOPHEN HNCOCH

HNCOCH,

HHCOCH-

3

0

OH P-450 MIXEDJFUNCTION OXIDASE HO-NCOCH "

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

3

•

NCOCH,

0 -

POSTULATED TOXIC INTERMEDIATES

NUCLEOPHILIC CELL MACROMOLECULES HNCOCH,

HNCOCH,

OLUTATHIONE

0 =

CELL MACROMOLECULES

•

ERCAPTURIC ACID

• CELL DEATH

Figure 6. Pathways of acetaminophen metabolism

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

8.

NELSON E T A L .

Chemical-Induced

Tissue

Injury

173

I n support o f t h i s view, covalent b i n d i n g and l i v e r n e c r o s i s occurred o n l y a f t e r doses o f acetaminophen s u f f i c i e n t l y l a r g e t o exceed t h e a v a i l a b i l i t y o f g l u t a t h i o n e f o r d e t o x i f i c a t i o n (Figure 7). S i m i l a r l y , when g l u t a t h i o n e concentrations i n the l i v e r were compared w i t h t h e extent o f covalent b i n d i n g a t v a r i o u s times a f t e r t h e a d m i n i s t r a t i o n o f acetaminophen, s i g n i f i c a n t b i n d i n g had occurred o n l y a f t e r g l u t a t h i o n e was s e v e r e l y depleted (30,32). I n accord w i t h t h i s view, p r i o r a d m i n i s t r a t i o n o f d i e t h y l maleate, which decreases t h e g l u t a t h i o n e c o n c e n t r a t i o n i n l i v e r without causing l i v e r n e c r o s i s , markedly p o t e n t i a t e s t h e l i v e r damage caused by acetaminophen (30,32), and d i e t s t h a t lower the conc e n t r a t i o n o f g l u t a t h i o n e enhance the t o x i c i t y as w e l l (34). On the other hand, the a d m i n i s t r a t i o n o f the a l t e r n a t e s u l f h y d r y l compounds, c y s t e i n e o r cysteamine, prevented t h e l i v e r n e c r o s i s (6,32). Recent s t u d i e s w i t h acetaminophen have supported t h e view t h a t a g l u t a t h i o n e t h r e s h o l d i s o p e r a t i v e i n man as w e l l as l a b o r a t o r y animals (33.35,36). Therefore, s u l f h y d r y l reagents such as c y s t e i n e , cysteamine, d i m e r c a p r o l , and g l u t a t h i o n e i t s e l f a r e being s u c c e s s f u l l y used i n the therapy o f acetaminophen-overdosed p a t i e n t s (37). T h i s emphasizes t h e importance o f understanding b i o c h e m i c a l mechanisms o f t o x i c i t y before r a t i o n a l approaches t o treatment can be made. Phenacetin Phenacetin ( p - e t h o x y a c e t a n i l i d e , IV) has been i m p l i c a t e d i n r e n a l i n j u r y i n man (38). Therefore, we considered t h e p o s s i b i l i t y t h a t t h i s s p e c i a l type o f n e p h r i t i s , c a l l e d a n a l g e s i c nephropathy, was r e l a t e d t o metabolic a c t i v a t i o n . Although no c o n s i s t e n t l y r e p r o d u c i b l e l e s i o n could be obtained i n l a b o r a t o r y animals t r e a t e d w i t h l a r g e doses o f phenacetin, l i v e r n e c r o s i s was observed, e s p e c i a l l y i n hamsters (39)» a species u n u s u a l l y s u s c e p t i b l e t o acetaminophen-induced h e p a t i c n e c r o s i s (29,30). As w i t h acetaminophen, phenacetin-induced l i v e r n e c r o s i s i n hamsters i s p o t e n t i a t e d by pretreatment w i t h 3-methylcholanthrene but not by phénobarbital. For example, phenacetin doses o f 400 mg/kg produce massive c e n t r i l o b u l a r n e c r o s i s i n 3-methylcholanthrene-treated animals. Moreover, t h e s e v e r i t y o f n e c r o s i s p a r a l l e l s t h e magnitude o f t h e covalent b i n d i n g o f r a d i o l a b e l e d phenacetin t o h e p a t i c p r o t e i n s and t h e d e p l e t i o n o f h e p a t i c g l u t a t h i o n e (39). L i t t l e b i n d i n g o r h e p a t i c n e c r o s i s occurs a t doses that deplete h e p a t i c g l u t a t h i o n e l e s s than 80%. However, c o n s i d e r a b l e b i n d i n g and n e c r o s i s occur at doses t h a t deplete g l u t a t h i o n e more than 80%. Pretreatment o f hamsters w i t h 3-methylcholanthrene i n c r e a s e s d e p l e t i o n o f h e p a t i c g l u t a t h i o n e , t h e covalent b i n d i n g , and the s e v e r i t y o f n e c r o s i s a f t e r phenacetin, whereas pretreatment w i t h cobaltous c h l o r i d e o r p i p e r o n y l butoxide decreases them. These f i n d i n g s i n d i c a t e t h a t g l u t a t h i o n e i n t h e l i v e r prevents covalent b i n d i n g and n e c r o s i s by combining w i t h a r e a c t i v e a r y l a t i n g m e t a b o l i t e o f phenacetin.

DRUG M E T A B O L I S M

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

IN v m

2* SO OOSC OF^-ACCTAMINOPMCNfmc/kt,

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Figure 7. Relationship in vivo between hepatic glutathione concentration, the formation of an acetaminophen-glutathione conjugate (measured in unne as acetaminophen mercapturic acid), and covalent binding of an acetaminophen metabolite to liver proteins (See Ref. 33)

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

8.

NELSON E T A L .

Chemical-Induced

Tissue

Injury

175

M e c h a n i s i t i c I m p l i c a t i o n s f o r the metabolic a c t i v a t i o n o f acetaminophen and phenacetin based on s t u d i e s u s i n g hamster microsomal enzymes* Although the i d e n t i t y of the a r y l a t i n g m e t a b o l i t e s o f acetaminophen and phenacetin are u n c e r t a i n , the involvement o f N-hydroxy d e r i v a t i v e s o r arene oxides as r e a c t i v e m e t a b o l i t e s has been p o s t u l a t e d (30, 40). Experiments i n v i t r o w i t h hamster l i v e r microsomes suggests t h a t the a r y l a t i n g m e t a b o l i t e s o f a c e t aminophen and phenacetin are d i f f e r e n t , a t l e a s t i n t h i s microsoma l system (41)* The evidence i s as f o l l o w s : l ) T h e maximum v e l o c i t y of covalent b i n d i n g f o r phenacetin exceeds t h a t f o r acetaminophen, showing t h a t phenacetin i s not f i r s t deethylated to acetaminophen which i s then a c t i v a t e d * 2)Pretreatment o f hamsters w i t h 3-methylcholanthrene i n c r e a s e s the r a t e o f covalent b i n d i n g f o r acetaminophen but decreases the r a t e of b i n d i n g f o r phenacetin* 3) Phénob a r b i t a l pretreatment i n c r e a s e s the r a t e o f covalent b i n d i n g f o r phenacetin without a f f e c t i n g the r a t e of b i n d i n g f o r acetaminophen* 4) When covalent b i n d i n g was prevented by t r a p p i n g o f the r e a c t i v e m e t a b o l i t e s w i t h g l u t a t h i o n e d u r i n g i n c u b a t i o n s c a r r i e d out under atmospheres o f oxygen-18, r e d u c t i o n by Raney-nickel o f the g l u t a t h i o n e conjugates formed from e i t h e r acetaminophen or phenacetin y i e l d e d acetaminophen; however, the acetaminophen conjugate formed d u r i n g i n c u b a t i o n w i t h phenacetin i n c o r p o r a t e d 50% oxygen-18 i n t o the 4 - p o s i t i o n , whereas the acetaminophen-glutathione conjugate d u r i n g i n c u b a t i o n w i t h acetaminophen i n c o r p o r a t e d v i r t u a l l y no oxygen-18. Mechanisms based on these r e s u l t s are presented i n F i g u r e 8. The l a c k of i n c o r p o r a t i o n of oxygen-18 i n t o the g l u t a t h i o n e conjugate d e r i v e d from acetaminophen i s c o n s i s t e n t w i t h e i t h e r an N-hydroxylation o r 2,3-epoxidation mechanism. I n d i r e c t evidence i n v i v o supports an N-hydroxy l a t i o n mechanism f o r the metabolic a c t i v a t i o n of acetaminophen. Masking of the amide n i t r o g e n , as i n N-methyl-4-hydroxy a c e t a n i l i d e , b l o c k s h e p a t o t o x i c i t y (39). Pretreatment o f hamsters w i t h 3-methylcholanthrene, which i n creases the h e p a t o t o x i c i t y of acetaminophen, correspondingly i n creases the N - h y d r o x y l a t i o n of 4 - c h l o r o a c e t a n i l i d e and 2 - a c e t y l a minofluorene and the covalent b i n d i n g o f acetaminophen (20, 42, 43) By c o n t r a s t , phénobarbital pretreatment n e i t h e r a l t e r s the r a t e o f N-hydroxylation o f 4 - c h l o r o a c e t a n i l i d e nor the covalent b i n d i n g of acetaminophen (43). I t seems l i k e l y t h a t i f an N-hydroxylated m e t a b o l i t e were formed i t would subsequently undergo dehydration t o a c h e m i c a l l y r e a c t i v e imidoquinone before a r y l a t i n g t i s s u e macromolecules (Figure 8 ) . The i n c o r p o r a t i o n of 50% oxygen-18 i n t o the 4 - p o s i t i o n of the a r y l a t i n g m e t a b o l i t e of phenacetin i s p e r p l e x i n g . The s t r o n g imp l i c a t i o n i s t h a t the carbon atom i n t h i s p o s i t i o n (C-4 o f the a r o matic r i n g ) becomes t e t r a h e d r a l , b i n d i n g e q u i v a l e n t oxygen-18, der i v e d from molecular oxygen, and oxygen-16. Mechanisms c o n s i s t e n t w i t h t h i s i n t e r p r e t a t i o n are presented i n F i g u r e 8. Although these mechanisms are c o n s i s t e n t w i t h the r e s u l t s obt a i n e d i n v i t r o w i t h phenacetin, they may have l i t t l e b e a r i n g on the s i t u a t i o n i n v i v o . The major m e t a b o l i t e of phenacetin i n v i v p

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176

DRUG M E T A B O L I S M

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CONCEPTS

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Figure 8.

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Possible reaction mechanisms for reactive metabolite formation from acetaminophen and phenacetin using hamster liver microsomes

8.

NELSON E T A L .

Chemical-Induced

Tissue

Injury

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Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

i s acetaminophen and those pretreatments i n hamsters which i n crease h e p a t i c n e c r o s i s and c o v a l e n t b i n d i n g f o r acetaminophen i n c r e a s e the h e p a t i c n e c r o s i s and c o v a l e n t b i n d i n g f o r phenacetin (39). A d d i t i o n a l s t u d i e s i n v i v o w i t h phenacetin, s p e c i f i c a l l y deuterated i n theot-methylene carbon atom o f the 4-ethoxy group, a l s o i n d i c a t e d t h a t d e e t h y l a t i o n o f phenacetin t o acetaminophen i s a t least p a r t i a l l y rate-determining f o r hepatic tissue i n j u r y <44> 45). Renal i n j u r y w i t h a c e t a n i l i d e s . R e a c t i v e i n t e r m e d i a t e s o f acetaminophen, phenacetin, and other a c e t a n i l i d e s may a l s o mediate the r e n a l i n j u r y caused by these compounds. Nery (46) has suggested t h a t the N-hydroxylated d e r i v a t i v e o f phenacetin i s an i n t e r m e d i a t e f o r minor u r i n a r y m e t a b o l i t e s o f phenacetin. Calder e t a l . (47) subsequently examined the n e p h r o t o x i c i t y o f N-hydroxyphenacetin, 4-aminophenol, hydroquinone, and p-benzoquinone and found acute r e n a l t u b u l a r n e c r o s i s i n r a t s . I t t h e r e f o r e seemed p o s s i b l e t h a t acetaminophen and phenacetin c o u l d be n e p h r o t o x i c through t h e i r N-hydroxy m e t a b o l i t e s and u l t i m a t e l y through the common r e a c t i v e d e r i v a t i v e , N-acetyl-4-benzoimidoquinone. Both acetaminophen and phenacetin c o v a l e n t l y b i n d t o a s m a l l extent t o F i s c h e r r a t kidney and acetaminophen causes r e n a l tubul a r n e c r o s i s (39). Both compounds d e p l e t e r e n a l g l u t a t h i o n e , w i t h acetaminophen much more potent than phenacetin. Experiments i n v i t r o w i t h r a t kidney microsomes show t h a t these compounds can be a c t i v a t e d t o r e a c t i v e m e t a b o l i t e s . A l t e r n a t i v e l y , acetaminophen and phenacetin may be N-hydroxylated i n the l i v e r and t r a n s ported t o the k i d n e y , p o s s i b l y as N-O-glucuronides. The g l u c u r o n i d e s might then be h y d r o l y z e d under the a c i d i c c o n d i t i o n s i n the u r i n e o r by glucuronidases i n the kidney o r u r i n e t o cause the observed r e n a l i n j u r y . However, 3-methylcholanthrene pretreatment i n c r e a s e s the h e p a t i c n e c r o s i s , but s l i g h t l y decreases the r e n a l n e c r o s i s produced by acetaminophen, suggesting t h a t the acetaminophen a c t i v a t i o n r e s p o n s i b l e f o r r e n a l i n j u r y occurs i n the kidney i t s e l f . Furans and Thiophenes Furosemide T o x i c i t y i n v i v o o f furosemide. Furosemide (V), a f r e q u e n t l y used d i u r e t i c drug, i s c o n t r a i n d i c a t e d i n pregnancy because o f i t s recognized t e r a t o g e n i c p o t e n t i a l (48). The drug a l s o has been reported t o p o t e n t i a t e r e n a l i n j u r y when used i n combination w i t h c e p h a l o r i d i n e (49, 50). Furosemide produces massive h e p a t i c n e c r o s i s i n mice and the n e c r o s i s i s prevented when metabolism i s i n h i b i t e d by pretreatment o f mice w i t h p i p e r o n y l b u t o x i d e , c o b a l t c h l o r i d e and «c-naphthylisothiocyanate (51). Covalent b i n d i n g o f the drug t o h e p a t i c t i s s u e i n mice i s a l s o blocked by these p r e treatments and occurs a few hours b e f o r e h i s t o l o g i c a l l y r e c o g n i s a b l e n e c r o s i s . Thus, the formation o f a r e a c t i v e furosemide metab o l i t e i s c a u s a l l y r e l a t e d t o the development o f furosemideinduced h e p a t i c n e c r o s i s .

DRUG M E T A B O L I S M C O N C E P T S

178

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As w i t h acetaminophen and phenacetin, the h e p a t i c n e c r o s i s produced by furosemide a l s o e x h i b i t s a dose-threshold f o r t o x i c i t y . No covalent b i n d i n g o r n e c r o s i s occurs u n t i l a dose of 100 mg/kg i s exceeded. U n l i k e the dose t h r e s h o l d f o r acetaminophen and phen e a c e t i n h e p a t o t o x i c i t y , the furosemide t h r e s h o l d i s not due t o a p r o t e c t i v e r o l e of g l u t a t h i o n e , s i n c e furosemide does not deplete h e p a t i c g l u t a t h i o n e . Studies of metabolism, d i s t r i b u t i o n , and r e v e r s i b l e plasma p r o t e i n b i n d i n g of furosemide a f t e r t o x i c and nont o x i c doses i n d i c a t e t h a t the dose-threshold f o r t o x i c i t y r e s u l t s from e i t h e r s a t u r a t i o n o f the organic anion b i n d i n g s i t e s on plasma p r o t e i n s a f t e r t o x i c doses, o r p o s s i b l y s a t u r a t i o n of b i l i a r y or r e n a l e x c r e t i o n of the drug (52). P o s s i b l e mechanism of metabolic a c t i v a t i o n . The h e p a t i c i n j u r y produced by furosemide apparently r e s u l t s from the metabolic a c t i v a t i o n of the f u r a n r i n g , p o s s i b l y by an e p o x i d a t i o n s i m i l a r t o t h a t proposed i n F i g u r e 9. Furosemide, r a d i o l a b e l e d w i t h t r i tium i n i t s f u r a n moiety, i s bound c o v a l e n t l y t o h e p a t i c microsomes i n the presence of oxygen and NADPH t o the same extent as furosemide r a d i o l a b e l e d s p e c i f i c a l l y w i t h s u l f u r - 3 5 i n i t s sulfonamide moiety, demonstrating t h a t the bound m e t a b o l i t e cont a i n s both p a r t s of the furosemide molecule. To determine where the b i n d i n g occurred on the molecule, the m e t a b o l i t e - p r o t e i n conj u g a t e s i s o l a t e d from the l i v e r were hydrolyzed under m i l d a c i d c o n d i t i o n s t h a t s p l i t furosemide i n t o i t s methylfuran and s u l f a m y o l a n t h r a n i l i c a c i d p o r t i o n s . The b i n d i n g of f u r a n - r a d i o l a b e l e d ^ r o s e m i d e t o p r o t e i n was unchanged, whereas the b i n d i n g of S-labeled furosemide was l o s t . The r e s u l t s suggested t h a t the f u r a n r i n g was being m e t a b o l i c a l l y a c t i v a t e d (51, 52). A d d i t i o n a l s t u d i e s i n v i t r o reported by W i r t h e t a l . (53) u s i n g ^ r f - H] furosemide, [35S] furosemide], k ~ H] furosemide, and H ] furosemide i n d i c a t e d t h a t formation of a p o s s i b l e e l e c t r o p h i l i c imine i n t e r m e d i a t e was u n l i k e l y and t h a t theoC-carbon was not a s i t e of metabolic a c t i v a t i o n . This f u r t h e r i m p l i c a t e d the f u r a n r i n g . Since covalent b i n d i n g was enhanced by an epoxide hydrase i n h i b i t o r and d i d not occur when tetrahydro [35S] furosemide was used as s u b s t r a t e , the authors speculated t h a t an arene oxide i n t e r m e d i a t e of the f u r a n moiety was i n v o l v e d i n the b i n d i n g . 4-Ipomeanol T o x i c i t y i n v i v o of 4-ipomeanol. Chemicals which r e p r o d u c i b l y produce an acute, s p e c i f i c pulmonary t o x i c i t y by r o u t e s of admini s t r a t i o n other than i n h a l a t i o n are r a r e and t h e i r mechanisms of a c t i o n are p o o r l y understood. 4-Ipomeanol ( V I I ) , a 3 - s u b s t i t u t e d f u r a n and the major component of "lung edema f a c t o r " produced i n sweet potatoes (Ipomoea b a t a t a s ) i n f e c t e d w i t h a common mold, has provided a v a l u a b l e t o o l w i t h which t o probe chemical-induced lung disease(54).The i n g e s t i o n of mold-damaged sweet potatoes has been i m p l i c a t e d f o r many years i n outbreaks of p o i s o n i n g i n c a t t l e . A f f e c t e d animals s u f f e r severe and o f t e n f a t a l r e s p i r a t o r y d i s t r e s s .

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ET AL.

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The same k i n d o f lung damage observed i n c a t t l e can be produced i n l a b o r a t o r y animals by a d m i n i s t r a t i o n o f s y n t h e t i c 4-ipomeanol (55» 56). The lung i s t h e primary t a r g e t organ i n most species* P a t h o l o g i c a l changes, such as p l e u r a l e f f u s i o n s , i n t r a a l v e o l a r and p e r i v a s c u l a r edema a r e apparent w i t h i n 6-24 hours a f t e r administration of the t o x i n . Studies were undertaken t o determine t h e p o s s i b l e formation of a c h e m i c a l l y r e a c t i v e m e t a b o l i t e i n t h i s pulmonary t o x i c i t y . Rats have been used as t h e experimental species i n a l l experiments d e s c r i b e d here. Pretreatments o f animals w i t h metabolic i n h i b i t o r s such as p y r a z o l e , p i p e r o n y l butoxide, and c o b a l t c h l o r i d e a l l markedly reduced the t o x i c i t y o f 4-ipomeanol i n the lung (57, 58). Phénobarbital, an inducer o f mixed f u n c t i o n oxygenase a c t i v i t y d i d not a l t e r t h e nature o f the lung t o x i c i t y but s i g n i f i c a n t l y increased the LD50 v a l u e , p o s s i b l y by i n c r e a s i n g d e t o x i f i c a t i o n pathways more than t o x i c pathways (59). Another type o f inducer, 3-methylcholanthrene showed a s t r i k i n g phenomenon when i t was used t o p r e t r e a t r a t s . M o r t a l i t y was decreased because o f marked decrease i n lung damage. I n c o n t r a s t , t i s s u e i n j u r y increased d r a m a t i c a l l y i n t h e l i v e r w i t h appearance o f widespread c e n t r i l o b u l a r n e c r o s i s (60). Thus, the t a r g e t organ f o r t o x i c i t y had switched from t h e lung t o the l i v e r . Covalent b i n d i n g i n v i v o and i n v i t r o . I n non-pretreated r a t s , r a d i o a c t i v i t y from 14C-4-ipomeanol becomes c o v a l e n t l y bound, p r e f e r e n t i a l l y t o t h e lungs, a f t e r a l l routes o f a d m i n i s t r a t i o n . Pretreatments w i t h mixed-function oxygenase i n h i b i t o r s , which decreased lung t o x i c i t y , caused a p a r a l l e l decrease i n covalent b i n d i n g t o lung t i s s u e i n v i v o and t o lung microsomes i n v i t r o (57, 58, 61). Phénobarbital pretreatment a l s o decreased the covalent b i n d i n g o f t o x i n t o both lung and l i v e r t i s s u e i n v i v o . However, t h e maximal r a t e o f b i n d i n g t o l i v e r microsomes i n v i t r o was increased whereas no change occurred i n b i n d i n g t o lung microsomes. The a l t e r a t i o n o f t a r g e t organ s p e c i f i c i t y f o r t i s s u e damage observed w i t h 3-methylcholanthrene pretreatment was p a r a l l e l e d by s i m i l a r a l t e r a t i o n o f covalent b i n d i n g o f r a d i o l a b e l e d 4-ipomeanol i n v i v o (60). The l e v e l o f c o v a l e n t l y bound t o x i n was markedly e l e v a t e d i n l i v e r s o f 3-methylcholanthrene-induced r a t s , whereas b i n d i n g t o lung was s i g n i f i c a n t l y reduced. The r a t e o f b i n d i n g t o l i v e r microsomes i n v i t r o was a l s o markedly i n c r e a s e d , whereas the r a t e o f b i n d i n g t o lung microsomes was unchanged (60). An important c o n c l u s i o n can be drawn from the experiments i n 3-methylcholanthrene p r e t r e a t e d animals. The t o x i c m e t a b o l i t e o f 4- ipomeanol i s so r e a c t i v e t h a t l i t t l e , i f any, o f i t escapes t h e organ i n which i t i s formed. I t t h e r e f o r e appears that i n normal animals the s p e c i f i c lung t o x i c i t y produced by 4-ipomeanol r e s u l t s p r i m a r i l y from pulmonary metabolism o f t h e agent; t h e l i v e r i s not a s i g n i f i c a n t source o f t h e r e a c t i v e m e t a b o l i t e t h a t binds t o and damages t h e lungs. The i n c r e a s e i n h e p a t i c t o x i c i t y i n 3-methylcholanthrene p r e t r e a t e d animals i s due t o an increased h e p a t i c metabolism o f 4-ipomeanol; t h e r e d u c t i o n i n pulmonary t o x i c i t y

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probably i s due t o an e l e v a t e d r a t e o f h e p a t i c clearance o f t o x i n . Nature o f the c h e m i c a l l y r e a c t i v e m e t a b o l i t e o f 4-ipomeanol. Studies i n v i v o and i n v i t r o i n d i c a t e t h a t the r e a c t i v e m e t a b o l i t e formed by mixed f u n c t i o n oxygenase-catalyzed metabolism o f 4-ipomeanol i s a h i g h l y e l e c t r o p h i l i c species (57, 58). A d d i t i o n o f the n u c l e o p h i l i c t r i p e p t i d e , g l u t a t h i o n e , markedly i n h i b i t e d covalent b i n d i n g o f 4-ipomeanol i n v i t r o , presumably by a c t i n g as an a l t e r n a t e n u c l e o p h i l e . D e p l e t i o n of endogenous g l u t a t h i o n e i n v i v o by d i e t h y l m a l e a t e pretreatment s i g n i f i c a n t l y increased t o x i c i t y and covalent b i n d i n g o f 4-ipomeanol i n v i v o . Analogs o f 4-ipomeanol i n which the f u r a n moiety was replaced by phenyl o r methyl s u b s t i t u e n t s were not metabolized t o t o x i c e l e c t r o p h i l e s i n v i v o o r i n v i t r o (58). Thus, the furan r i n g appears t o be e s s e n t i a l f o r the observed t o x i c i t y and covalent b i n d i n g . Based on these r e s u l t s and those found f o r the metabolic a c t i v a t i o n o f furosemide, F i g u r e 10 r e v e a l s a p o s s i b l e scheme f o r f u r a n a c t i v a t i o n . Since f u r a n has l e s s a r o m a t i c i t y than benzene, i t i s not u n l i k e l y t h a t t h i s heteroaromatic nucleus could form an epoxide. T h i s epoxide would probably be q u i t e r e a c t i v e , and could y i e l d other e l e c t r o p h i l e s by spontaneous r e arrangement o r r i n g s c i s s i o n r e a c t i o n s . This arene oxide could a l s o be d e a c t i v a t e d by an epoxide hydrase, g l u t a t h i o n e , o r a g l u t a t h i o n e t r a n s f e r a s e . R e l a t i v e a c t i v i t i e s o f the v a r i o u s pathways i n t h e v a r i o u s t i s s u e s would modulate s u s c e p t i b i l i t y t o t h e toxin. H e p a t o t o x i c i t y , r e n a l t o x i c i t y , and pulmonary t o x i c i t y a r e a l s o caused by other f u r a n compounds, some of which show a g l u t a t h i o n e t h r e s h o l d , and others which show no such t h r e s h o l d (51). Furan, 2-hydroxymethylfuran, and 2 - a c e t y l f u r a n a r e hepatot o x i c . Furosemide, f u r a n , 2,3-benzofuran and c e r t a i n other furans produce acute r e n a l t u b u l a r n e c r o s i s . Other simple furans such as 2-methylfuran, 3-methylfuran, and furan i t s e l f produce lung damage, and pulmonary edema (58). Thus, a v a r i e t y of t i s s u e l e s i o n s seen a f t e r the use o f f u r a n - c o n t a i n i n g compounds probably r e s u l t s from metabolic a c t i v a t i o n s i m i l a r t o t h a t proposed f o r 4-ipomeanol and furosemide. Cephaloridine Extension o f the f u r a n s t u d i e s t o thiophene, another h e t e r o aromatic d e r i v a t i v e , has shown t h a t s e v e r a l thiophene-containing compounds produce h e p a t i c and r e n a l n e c r o s i s (39). Cephaloridine ( V I ) , a w i d e l y p r e s c r i b e d cephalosporin a n t i b i o t i c , has i t s use l i m i t e d p r i m a r i l y because o f r e n a l n e c r o s i s a s s o c i a t e d w i t h such therapy (49). Pretreatments o f mice w i t h p i p e r o n y l butoxide and c o b a l t c h l o r i d e decrease r e n a l n e c r o s i s caused by c e p h a l o r i d i n e . F u r t h e r s t u d i e s a r e i n progress t o determine i f t h i s s p e c i f i c r e n a l n e c r o s i s i s mediated by a r e a c t i v e m e t a b o l i t e .

8.

NELSON E T A L .

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F«rf«r«M«liyd«.

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Injury

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Frmm Wmre—mié* A

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Figure 9. Possible reaction pathways for the metabo­ lism of furosemide (See Réf. 52)

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Figure 10. Possible mechanisms for the metabolism offurans(See Réf. 54)

182

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Conclusion The g e n e r a l concepts d i s c u s s e d i n t h i s chapter which u n d e r l i e the r o l e of m e t a b o l i c a c t i v a t i o n i n chemical-induced t i s s u e i n j u r y are the f o l l o w i n g : 1) F i r s t i s the concept of r e a c t i v e m e t a b o l i t e f o r m a t i o n and whether the m e t a b o l i t e i s important i n m a n i f e s t i n g a t o x i c r e a c t i o n . 2) Second i s the concept of t a r g e t organ. Given t h a t t h e r e i s t o x i c i t y t o a c e r t a i n organ, i s t h i s due to metabol i t e s formed i n t h a t t i s s u e o r elsewhere. Embodied i n t h i s concept i s the important phenomenon, t a r g e t organ s w i t c h i n g . 3) F i n a l l y i s the concept of dose-threshold f o r a t o x i c drug r e a c t i o n . Some compounds such as acetaminophen, phenacetin and furosemide, as w e l l as c e r t a i n other furans and thiophenes, are r e l a t i v e l y i n nocuous u n t i l a t h r e s h o l d dose i s reached. Other compounds such as a c e t y l i s o n i a z i d show evidence f o r t i s s u e i n j u r y a t almost a l l l e v e l s of dosage. The parameters t h a t we u t i l i z e to study such phenomena are t i s s u e n e c r o s i s i n the t a r g e t organ, the i r r e v e r s i b l e b i n d i n g of drug m e t a b o l i t e s t o the t a r g e t organ - something we c a l l covalent b i n d i n g , the t r a p p i n g of r e a c t i v e m e t a b o l i t e s and a n a l y s i s of the p a t t e r n o f drug m e t a b o l i t e s . To p r o v i d e a b i o c h e m i c a l b a s i s f o r the t o x i c r e a c t i o n s , we a l t e r the amounts and/or a c t i v i t i e s of enzymes r e s p o n s i b l e f o r the formation of r e a c t i v e m e t a b o l i t e s and l o o k f o r c o r r e l a t i o n s i n the f o u r parameters. I t should be emphasized t h a t w i t h both inducers and i n h i b i t o r s of metabolism i t i s d i f f i c u l t t o p r e d i c t a p r i o r i t h e i r a c t u a l e f f e c t on a given t o x i c i t y i n v i v o , s i n c e they may a f f e c t both t o x i f y i n g as w e l l as d e t o x i f y i n g pathways. The s i t u a t i o n i s e s p e c i a l l y complex when more than one t i s s u e can serve as a s i t e f o r both t o x i f i c a t i o n and d e t o x i f i c a t i o n , and when each t i s s u e may respond d i f f e r e n t l y to a given inducer o r i n h i b i t o r . Whatever e f f e c t a p a r t i c u l a r pretreatment produces, however, the c o r r e l a t i o n between the s e v e r i t y of the t o x i c i t y and the degree of covalent b i n d i n g of the t o x i n to the t a r g e t t i s s u e should be maint a i n e d i f the two phenomena are r e l a t e d . Thus, m a n i f e s t a t i o n of chemical-induced t i s s u e i n j u r y depends on s e v e r a l c r i t i c a l k i n e t i c parameters - namely, r a t e of f o r m a t i o n , the i n h e r e n t r e a c t i v i t y , the r a t e of d e t o x i f i c a t i o n (e.g. by g l u t a t h i o n e conjugation) of t o x i c m e t a b o l i t e s , and the r a t e of t i s s u e r e p a i r by the i n j u r e d organ. While t h i s work has as i t s g o a l the development of an i n t e g r a t e d approach f o r determining b i o c h e m i c a l mechanisms i n v o l v e d i n the pathogenesis of chemical-induced t i s s u e i n j u r y , s e v e r a l questions remain unresolved. The phenomenon of metabolic a c t i v a t i o n of chemi c a l s t o r e a c t i v e i n t e r m e d i a t e s which c o v a l e n t l y b i n d t o t i s s u e macromolecules i s w e l l e s t a b l i s h e d as an i n i t i a l step i n l e a d i n g t o c e l l damage. But there i s no e x p l a n a t i o n f o r why a p a r t i c u l a r r e a c t i o n such as N - o x i d a t i o n l e a d s to t i s s u e n e c r o s i s a f t e r the a d m i n i s t r a t i o n of one compound and n e o p l a s i a f o l l o w i n g another.

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Until the chemical-tissue interactions c r i t i c a l to particular ne­ crotic or neoplastic processes are defined, a f u l l assessment of meaning of the covalent binding of metabolites to tissue macromolecules cannot be made. For the present, it seems necessary to accept the fact that we cannot have new advances in drug therapy, for example, without some risk of causing structural tissue lesions, and to concentrate on assessing benefit/risk ratios. Literature Cited 1. 2.

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3. 4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18. 19.

Miller, J . A . , Cancer Res. (1970), 30, 559. Miller, E.C. and Miller, J . A . , Physiological Rev. (1966), 18, 805. Magee, P.N. and Barnes, J.M., Adv. Cancer Res. (1967), 10, 163. Recknagel, R.O., Pharmacol. Rev. (1967), 19, 145. Traiger, G.J. and Plaa, G.L., J. Pharmacol. Exp. Therap. (1972), 183, 481. Mitchell, J.R., Jollow, D . J . , Gillette, J.R. and Brodie, B.B., Drug Metab. Dispos. (1973), 1, 418. Gillette, J.R., Mitchell, J.R. and Brodie, B.B., Ann. Rev. Pharmacol. (1974), 14, 271. Mitchell, J.R., Jollow, D.J. and Gillette, J.R., Israel J . Med. Sci. (1974), 10, 339. Mitchell, J.R. and Jollow, D . J . , Israel J . Med. Sci. (1974) 10, 312. Mitchell, J.R. and Jollow, D . J . , Gastroenterology (1975), 68, 392. Mitchell, J.R., Potter, W.Z., Hinson, J . A . , Snodgrass, W.R., Timbrell, J.A. and Gillette, J.R., "Toxic Drug Reactions", in "Concepts i n Biochemical Pharmacology: Handbook of Experi­ mental Pharmacology", p. 383, v o l . 28/3, Gillette, J.R. and Mitchell, J.R., eds., Springer-Verlag, New York, 1975. Jerina, D.M. and Daly, J.W., Science (1974), 185, 573. Mitchell, J.R., Long, M.W., Thorgeirsson, U.P., and Jollow, D . J . , Chest (1975), 68, 181. Black, Μ., Mitchell, J.R., Zimmerman, H . J . , Ishak, K . , and Epler, G.R., Gastroenterology (1975), 69, 289. Mitchell, J.R., Thorgeirsson, U.P., Black, Μ., Timbrell, J.Α., Snodgrass, W.R., Potter, W.Z., Jollow, D.J. and Keiser, H.R., Clin. Pharmacol. Therap. (1975), 18, 70. Kalow, W.P., "Pharmacogenetics, Heredity and the Response to Drugs", p. 99, W.B. Saunders, Philadelphia, 1962. Snodgrass, W.R., Potter, W.Z., Timbrell, J.Α., Jollow, D.J. and Mitchell, J.R., Clin. Res. (1974), 22, 323 A. Nelson, S.D., Snodgrass, W.R. and Mitchell, J.R., Fed. Proc. (1975), 34, 784. Nelson, S.D., Mitchell, J.R., Snodgrass, W.R., Timbrell, J.A. and Corcoran, G.B., Science (1976), 193, 901

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20. Thorgeirsson, S.S., Jollow, D . J . , Sasame, H.A., Green, I . and Mitchell, J.R., Molec. Pharmacol. (1973), 9, 398. 21. Smith, H.A. and Gorin, G., J. Org. Chem. (1961), 26, 820. 22. Nelson, S.D., Hinson, J.A. and Mitchell, J.R., Biochem. Biophys. Res. Commun. (1976), 69, 900. 23. Nelson, S.D., Pohl, L.R. and Mitchell, J.R., "Application of Chemical Ionization Mass Spectrometry and the Twin-Ion Technique in the Analysis of Reactive Intermediates in Drug Metabolism", i n "Proceedings of the First International Symposium on the Use of Mass Spectrometry in Drug Metabolism Studies", June 1976, Milan, Italy, in press. 24. Huang, P.C. and Kosower, E.M., J . Amer. Chem. Soc. (1967), 89, 3910. 25. Prescott, L . F . , Wright, Ν., Roscoe, P. and Brown, S.S., Lan­ cet (1971), 1, 519. 26. Clark, R., Borirakchanyavat, V . , Davidson, A.R., Thomphson, R.P.H., Widdop, Β., Gouldine, R. and Williams, R., Lancet (1973), 1, 66. 27. Boyd, E.M. and Bereczky, G.M., Br. J. Pharmacol. (1966), 26, 606. 28. Mitchell, J.R., Jollow, D . J . , Potter, W.Z., Davis, D.C., Gillette, J.R. and Brodie, B.B., J . Pharmacol. Exp. Therap. (1973), 187, 185. 29. Davis, D.C., Potter, W.Z., Jollow, D.J. and Mitchell, J.R., Life Sci. (1974), 14, 2099. 30. Potter, W.Z., Thorgeirsson, S.S., Jollow, D.J. and Mitchell, J.R., Pharmacology (1974), 12, 129. 31. Jollow, D . J . , Mitchell, J.R., Potter, W.Z., Davis, D.C., Gillette, J.R. and Brodie, B.B., J . Pharmacol. Exp. Therap. (1973), 187, 175. 32. Mitchell, J.R., Jollow, D . J . , Potter, W.Z., Gillette, J.R. and Brodie, B.B., J . Pharmacol. Exp. Therap. (1973), 187, 211. 33. Jollow, D . J . , Thorgeirsson, S.S., Potter, W.Z., Hashimoto, M. and Mitchell, J.R., Pharmacology (1974), 12, 251. 34. McClean, A.E.M. and Day, P.Α., Biochem. Pharmacol. (1975), 24, 37. 35. Jagenburg, O.R. and Toczko, Κ., Biochem. J. (1964), 92, 639. 36. Mitchell, J.R., Thorgeirsson, S.S., Potter, W.Z., Jollow, D.J. and Keiser, Η., Clin. Pharmacol. Therap. (1974), 16, 676. 37. Wright, N. and Prescott, L . F . , Scot. Med. J. (1973), 18, 56. 38. Spuhler, O. and Zollinger, H.V., Z. Klin. Med. (1953), 151, 1. 39. Mitchell, J.R. et al., unpublished results. 40. Andrews, R.S., Bond, C.C., Burnett, J., Saunders, A. and Watson, Κ., J. Internat. Med. Res. (1976), 4, (Supplement 4), 34. 41. Hinson, J . A . , Nelson, S.D. and Mitchell, J.R., The Pharma­ cologist (1976), 18, 241.

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

8.

NELSON ET AL.

Chemical-Induced Tissue Injury

185

42. Potter, W.Z., Davis, D.C., Mitchell, J.R., Jollow, D . J . , Gillette, J.R. and Brodie, B.B., J . Pharmacol. Exp. Therap. (1973), 187, 203. 43. Hinson, J.Α., Mitchell, J.R. and Jollow, D . J . , Molec. Pharm­ acol. (1975), 11, 462. 44. Garland, W.A., Nelson, S.D. and Sasame, H.A., Biochem. Biophys. Res. Commun. (1976), 72, 539. 45. Nelson, S.D. et al., unpublished results. 46. Nery, R., Biochem. J. (1971), 122, 317. 47. Calder, I . C . , Creek, M.J. and Williams, P.J., J. Med Chem. (1973), 16, 499. 48. Physicians Desk Reference (1973), Medical Economics Co., Orad e l l , N . J . , p. 774. 49. Foord, R.D., "Cephaloridine and the Kidney", in "Proceedings of the Sixth International Congress of Chemotherapy", p.597, Vol 1, University Park Press, Baltimore, 1970. 50. Dodds, M.G. and Foord R.D., Br. J. Pharmacol. (1970), 40, 227. 51. Mitchell, J.R., Potter, W.Z., Hinson, J . A. and Jollow, D.J. Nature (1974), 251, 508. 52. Mitchell, J.R., Nelson, W.L., Potter, W.Z., Sasame, H.A. and Jollow, D.J., J . Pharmacol. Exp. Therap. (1976), in press. 53. Wirth, P . J . Bettis, C.J. and Nelson, W.L., Molec. Pharmacol. (1976), 12, 759. 54. Boyd, M.R., in "Environmental Health Perspectives", (1976), in press. 55. Boyd, M.R., Wilson, B . J . and Harris, T.M., Nature (1972), 236, 158. 56. Boyd, M.R., Burka, L . T . , Harris, T.M. and Wilson, B.J., Biochim. Biophys. Acta (1974), 337, 184. 57. Boyd, M.R., Burka, L . T . , Neal, R.A., Wilson, B . J . and Holscher, M.M., Fed. Proc. (1974), 33, 234. 58. Boyd, M.R. et al., unpublished results. 59. Gillette, J.R., Biochem. Pharmacol. (1974), 23, 2927. 60. Boyd, M.R., Tox. Appl. Pharmacol. (1975), 33, 132. 61. Boyd, M.R., Burka, L.T. and Wilson, B . J . , Tox. Appl. Pharma­ col. (1975), 32, 147. Acknowledgement: Dr. Boyd is a staff fellow, Pharmacology Research Associate Program, National Institute of General Medical Sciences.

INDEX

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ix001

A Absorption spectra 38 Acetaminophen (p-hydroxyacetanilide) 171 -glutathione conjugate 174 metabolism, pathways of 172 reactive metabolite formation from 176 Acetanilide hydroxylase 75,79 Acetanilides, renal injury with 177 Acetylhydrazine 160,169 double-isotope experiments with acetylisoniazid 164 to rat microsomal protein, binding of 165 Acetylisoniazid 160 acetylhydrazine, double-isotope experiments with 164 H-ring-labeled 159 metabolic activation pathways for .. 170 Acid hydrolysis of the nucleoside adducts 141 Activation of benzo(a)pyrene 99 metabolic (see Metabolic activation) oxygen 1 Activity of PB 7,8-diol-9,10 epoxides, mutagenic 117 Activity in the reconstituted enzyme system 49 Alkylating intermediates 155 2-Amino guanosine-diol epoxide 150 Animals, hepatic necrosis in 158 Animal studies, laboratory 171 Anion complex, peroxide 5 Anticoagulant warfarin, oral 82 Anticytochrome P-450 Antimycin A 1 Aromatic compounds of guanosine, reactive 129 Arylating intermediates 155 3

Benzo ( a ) pyrene ( continued ) 7,8-dihydrodiol metabolism by purified monoxygenase system 3-hydroxylase in mouse skin, formation of an enantioner of each diastereo­ meric 9,10-epoxide from to mutagenic metabolites purified forms of cytochrome P450, metabolism of metabolic activation of 7,8-oxide, metabolic activation of .... phenols, metabolic activation Benzphetamine N-demethylase Binding of acetylhydrazine and isoproplhydrazine to rat microcosmal rotein

119 112 79 127 103 101 112 107 79

Sart

165

161,179 of diol epoxides to poly(G) 130,137 effect of pH on rate and extent of .. 131 as a function of pH, 134 of the reactive metabolite, covalent 156 Bis-para-nitrophenyl phosphate (BNPP) 158 BNF, (0-naphthoflavone) 47 induced microsomes 53 BP, (see also Benzo(a)pyrene) 99 dihydrodiols, metabolic activation of + trans 108 7,8-dihydrodiol 140 7,8-diol-9,10 epoxides, mutagenic activity of 117 metabolism of 108 4,5 oxide, mutagenic activity of 108 treatment of mice with [ H] 131 C Camphor 32 hydroxylase P-450, bacterial 27 hydroxylating cytochrome of Pseudomonas putida, P-450 57 Carbon monoxide, inhibition of 9 Β Carbonyl stage D of P-450 38 Base stability of nucleoside adducts .. 143 Carcinogens 99,127,155 Benzo(a)pyrene (see also BP) 78,99 Catalytic activities of P-450 59 139 activation and detoxification 99 C D spectra of guanosine adducts Cephaloridine 180 and benzo ( a ) pyrene arene oxides, metabolism of 100 Characterization of liver microsomal cytochrome, P-450 50 dihydriols, metabolic activation of .. 110 3

cam

LM4

189

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ix001

190

DRUG M E T A B O L I S M

Chemical-induced tissue injury, metabolic activation in 155 Chromatography of guanosine adducts, high resolution 138 high-pressure liquid 131 poragel PN 137 Configuration of the guanosine adducts, absolute 147 Crystal structure 42 Cumene hydroperoxide, oxidation of 20 Cyclic function of cytochrome P-450 3,4 Cysteine adduct 169 Cytochrome(s) immunological differences between the 73 metabolism of the substrates 76 P-448 concentration 108 induction 72 monoxygenase system 119 purified 99 P-450 catalyzed reactions 4 cyclic function of 3,4 enzymes, microsomal 81 immunochemical studies on forms of 57 liver microsomal 46-48 model complexes for the iron center in 27 monoxygenase system 127 in oxygen activation for drug metabolism 1 P-450 M, liver microsomal 47 peroxidase function of 15 purification 50,76,104 reactions attributed to liver microsomal 47 reductase, NADPH 65 resolution of multiple forms of ...72,77 specificity of reductase toward .... 59 substrate complex, ferrous 3 synthetic models for the reaction stages of 27 of Pseudomonas putida, camphorhydroxylating 57 purified 54 L

D Decomposition pathway of N substituted guanosine Demethylation, N- (see Ndemethylation) Detoxification of benzo(a)pyrene Diastereomeric 9,10-epoxide from enantiometer of each Difference spectra, n-octylamine 7

142 99 127 75

CONCEPTS

Diffusion analysis, Ouchterlony double 62 Dihydrodiols, of benzo ( a ) pyrene 110,112,119,140 Dihydroxy-7,8-dihydrobenzo ( a ) pyrene 127 Dimethyl sulfate, removal of ribose by N -methylation with 130 Diol epoxide(s) guanosine adducts of 148 to poly ( G ), binding of 130 to tetraols, hydrolysis of 129 uv spectra of nucleoside adducts from 132,142 1 products, guanosine 137 2-amino guanosine150 2 to poly ( G ), binding of 137 2, trans opening of 148 1 and 2, guanosine adducts from ... 138 1 and 2, tetraols from 135 Dissociation of the hydroxylated product 5 DNA 127,135 Dose-threshold for toxicity 171 Drug(s) metabolism biochemical studies on 46 cytochrome P-450 in oxygen activation for 1 microsomal 99 oxidations of 81 7

ε

Electron-transport reactions, microsomal 4 Electrophoresis polyacrylamide gel 56,65,77 Enantiomer (s) comparative hydroxylation of the .. 85 of each diastereomeric 9-10-epoxide from benzo(a)pyrene 127 of warfarin, microsomal metabolism of 87,89 Enantiomeric selectivity 81 Enzyme(s) activities, reconstituted 78 hamster microsomal 175 mechanistic studies with purified 66 multiplicity of microsomal 81 substrate complex 91 system, activity in the reconstituted 49 system of liver microsomal membranes 46 Epoxide from benzo ( a ) pyrene, diastereomeric 9-10 127 hydrase 99

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ix001

INDEX

191

Epoxidehydrase (continued) metabolism of benzo(a)pyrene and benzo ( a ) pyrene arene oxides 100 on mutations, effect of 108 to poly(G), binding of diol 130 of ( + ) and ( —)-trans-7,8-dihydroxy-7,8-dihydrobenzo (a) pyrene, diastereomeric 9-10 .... 127 EPR data for high spin ferric porphyrin complexes 35 signals of liver microsomes associated with NADPH oxidation .. 14 spectra for low spin ferric porphyrin complexes 33 studies 13 p-Ethoxyacetanilide (phenacetin) .... 173 Ethyl morphine, N-demethylation of 9,10,12,16

FePPIXDEE 38,40 Ferric P-450, substrate bonded high spin 35 porphyrin complexes 33,35 stage A, low spin 29 stage B, high spin 34 thiolate complex 29 Flavoproteins 2 Furans 177,181 Furosemide 177,181

Generation of hydrogen peroxide ...9,10,12 Glutathione concentration, hepatic .... 174 Glutathione conjugate, acetaminophen174 Glycoside linkage by N -methylation, labilization of 146 Guanine base of poly(G), reaction of the 136 Guanosine adducts absolute configuration of the 147 C D spectra of 139 of diol epoxide after methylation 148 from diol epoxides 1 and 2, of .... 138 structure of the 136 decomposition pathway of N substituted 142 diol epoxide 1 products, purification of 137 substituted 144 +7

7

H

[ H]-BP on mouse skin, RNA-nucleoside adducts from 147 Hamster liver microsomes 176 Hamster microsomal enzymes 175 Hemoprotein(s) 1 cytochrome P-450, iron center in reaction stages of the 27 ferric 3 structural characteristics of iron in 28 Hepatic necrosis in animals 158 High resolution chromatography of guanosine adducts 138 Hydrase, epoxide 107,108 Hydrazides 157 Hydrocarbons, polycyclic aromatic (PAH) 127 Hydrogen peroxide, generation of 7,9,10,12 Hydrolysis of diol epoxides to tetraols 129 of the nucleoside adducts, acid 141 of poly(G) to nucleosides 130 Hydroperoxide, oxidation of cumene .. 20 p-Hydroxyacetanilide (acetaminophen) 171 Hydroxylase, microsomal acetanilide .. 75 Hydroxylases, mixed-function 1 Hydroxylated product, dissociation of the 5 Hydroxylated products, formation of 84 Hydroxylation 4'90 6- and 888 789 benzylic 90 of the enantiomers, comparative .... 85 reaction, mechanisms of the 66 substrate 1 system of liver microsomal membranes 48 of R-warfarin 83 of S-warfarin 85 3

I Immobilization of low spin ferric porphyrin thiolate complexes Immunochemical properties of forms of P-450 · » Immunochemical studies on rorms or cytochrome P-450 Immunological differences between the cytochromes Incubation, microsomal Incubation, time of Inhibition of carbon monoxide Inhibition of microsomal acetanilide hydroxylase LM

J

e

£

38 57 57 73 169 108 9 75

DRUG M E T A B O L I S M C O N C E P T S

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ix001

192

Inhibitor metyrapone [2-methyl-l,2bis ( 3-pyridyl ) -1-propanone] 8 Injury with acetanilides, renal 177 Injury, metabolic activation in chemical-induced tissue 155 4-Ipomeanol, metabolite of 180 4-Ipomeanol, toxicity in vivo of 178 Iproniazid 160 -isopropylhydrazine, double-iso­ tope experiments with 164 metabolic activation of toxic metab­ olites of 168 Iron center in reaction stages of the hemoprotein cytochrome P-450, model complexes 27 in hemoproteins, structural characteristics of 28 porphyrins 28 Isoniazid 157,160 metabolic activation pathways for.. 170 H-ring-labeled 159 metabolic activation of toxic metabolites of 168 Isopropylhydrazine 160 -iproniazid, double isotope experiments with 164 to rat microsomal protein, oxidasedependent covalent binding of 165 metabolic activation pathways for .. 170 Isotope experiments with acetylisoniazid-acetylhydrazine and iproniazid-isopropylhydrazine, double 164 3

Κ

Kinetics of the oxidation of warfarin by normal microsomes from rat liver, comparative kinetics of the

83

L Labilization of glycosidic linkage by N -methylation Ligand, appended axial thio ether Liver comparative kinetics of the oxida­ tion of warfarin by normal microsomes from rat cytochrome P-450, resolution of multiple forms of rabbit microsomal cytochrome P-450 purification and characteriza­ tion of reactions attributed to substrates for +7

146 42

83 72 46 50 47 48

Liver (continued) P-450 47 microsomal membranes, enzyme system 46 membranes, hydroxylation system of 48 NADPH-cytochrome P-450 reductase 59 rabbit 62 microsomes associated with NADPH oxida­ tion, EPR signals of 14 hamster 176 oxidation of cumene hydro­ peroxide by rat 20 purification of P-450 M from ΡΒ-induced rabbit 52 purified reductase from rabbit .... 60 purified reductase from rat 63 rabbit 57 L M , purification of P-450 M 52 LM

L

4

2

L

2

M Man, studies in vivo in 171 Man, metabolism studies in 157 M C D spectra 40 Mechanistic studies with purified enzymes 66 Metabolic activation of benzo ( a ) pyrene dihydrodiols 110 to mutagenic products 102 7,8-oxide 112 phenols 107 in chemical-induced tissue injury .. 155 mechanism of 178 pathways for isoniazid, acetyl­ isoniazid, and isopropylisoniazid 170 of toxic metabolites of isoniazid and iproniazid 168 of +-trans BP dihydrodiols 114 Metabolism of benzo(a)pyrene and benzo(a)pyrene arene oxides 100,104,108 of benzo(a)pyrene, 7,8-di­ hydrodiol 112,119 of enantiomers of warfarin, microsomal 87,89 of furans 181 of furosemide 181 microsomal drug 99 pathways of acetaminophen 172 studies in man 157 of the substrates, cytochrome 76 Metabolite(s) covalent binding of the reactive 156 formation from acetaminophen and phenacetin, reactive 176

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ix001

INDEX

193

Metabolite ( s ) ( continued ) index of formation of the 156 of isoniazid and iproniazid, metabolic activation of toxic .... 168 purified forms of cytochrome P-450, metabolism of benzo(a)pyrene to mutagenic 104 studying reactive 156 urinary excretion of 159 Metalloenzymes 27 Membranes, enzyme system of liver microsomal 46 Membranes, hydroxylation system of liver microsomal 48 Methylation with dimethyl sulfate, removal of ribose by N 7 130 guanosine adducts of diol epoxide after 148 labilization of glycosidic linkage by N 146 of salmon sperm D N A by Nmethyl-N-nitrosourea 135 2- Methyl-l,2-bis ( 3-pyridyl ) -1propanone), inhibitor 8 3- Methylcholeanthrene 72 N-Methyl-N-nitrosourea, methyla­ tion of salmon sperm D N A by .... 135 Metyrapone 8,10 Mice with [ 3 H ] - B P , treatment of 131 Michaelis-Menten equation 91 Microsomal enzymes, multiplicity of 81 metabolism of enantiomers of warfarin, quantitative 87 metabolism of enantiomers of war­ farin, stereoselectivity of 89 mixed-function oxidation reactions 3 Microsomes associated with N A D P H oxidation, E P R signals of liver 14 BNF-induced 53 hamster liver 176 normal, P B and 3-MC induced hepatic 87 oxidation of cumene hydroperoxide by rat liver 20 PB-induced 53 purification of P-450 L M from PB-induced rabbit liver 52 purified NADPH-cytochrome P-450 reductase, PB-induced rat liver 65 purified reductase from rabbit + 7

2

liver

57,60

purified reductase from rat liver .... from rat liver, comparative kinetics of the oxidation of warfarin by normal

63 83

Mixed-function oxidases 1,48 oxidation reactions, microsomal 3 oxygenases, microsomal 1,155 Monoxygenase system cytochrome P-450 127 metabolic activation of benzo(a)pyrene by a purified 101 metabolism of benzo(a)pyrene and benzo(a)pyrene arene oxides by the purified 100 metabolism of benzo(a)pyrene ^ 7,8-dihydrodiol by the purified 112 stereoselective metabolism of benzo (a) pyrene 7,8-dihydro­ diol by the cytochrome P-448 .. 119 Mouse skin formation of an enantiomer of each diastereomeric 9,10-epoxide from benzo(a)pyrene in 127 injection in 150 RNA-nucleoside adducts from [ H]-BPon 147 Mutagenic activity of BP-4,5 oxide .... 108 Mutagenic products, metabolic activation of benzo(a)pyrene to 101 Mutations, effect of epoxide hydrase on 108 3

Ν

N -substituted guanosine, decompo­ sition pathway of 142 N-Demethylation of ethyl morphine, synergistic effect of N A D H or NADPHdependent 9,10,12,16 N A D H or NADPH-dependent N-demethylation of ethyl morphine, synergistic effect of 16 NADH synergism 13 NADPH cytochrome P-450 reductase 61 liver microsomal 59 PB-induced rat liver microsomes purified 65 rabbit liver microsomal 62 oxidation, EPR signals of liver microsomes associated with .... 14 oxidation, formation of hydrogen peroxide during 7 steady state concentrations of 10 0-Napthoflavone, BNF 47 Necrosis in animals, hepatic : 158 Nomenclature 50 Nucleoside(s) adducts acid, hydrolysis of the 141 base stability of 143 7

194

DRUG M E T A B O L I S M C O N C E P T S

Nucleoside adducts (continued) from diol epoxide 1, uv spectra of from [ H]BP from [ H]-BP on mouse skin, RNApK values of the hydrolysis of poly (G)

P-450 (continued) for PB-induced rabbit liver microsomes, purification of 52 slab polyacrylamide gel electro­ phoresis of 56 147 143 substrate specificity of forms of 58 130 P-450 and M , purification of 52 P-450M4> catalytic activities of 59 Partition coefficient with pH, Ο change in 146 n-Octylamine difference spectra 75 PB-induced microsomes 53,65 Ouchterlony double diffusion analysis 62 PB phénobarbital 47 Oxidase(s) Peroxidase function of cytochrome dependent covalent binding of P-450 15 acetylhydrazine and isopropyl­ Peroxide anion complex 5 hydrazine to rat microsomal pH, rate of binding of diol epoxide 2 protein 165 to poly (G) as a function of 137 hydroxylases or oxygenases, mixedpH on rate and extent of binding, function 1 effect of 131 system, mixed function 48 Phenacetin ( p-ethoxyacetanilide ) .. 173,176 Oxidation(s) Phénobarbital, PB 47 of cumene hydroperoxide by rat Phenols, metabolic activation of liver microsomes 20 benzo(a)pyrene 107 of drugs 81 Phosphate backbone of poly (G) 133 EPR signals of liver microsomes 42 associated with NADPH 14 Picket fence porphyrins pK , estimation of 146 reactions, microsomal mixedfunction 3 pK values of the nucleoside adducts 143 Poly (G) of warfarin by normal microsomes uv spectra of 132 from rat liver, comparative as a function of pH, rate of binding kinetics of the 83 of diol epoxide 2 to 137 7,8-Oxide, metabolic activation of to nucleosides, hydrolysis 130 benz(a)pyrene 112 phosphate backbone 133 Oxycytochrome P-450 5 tetraols from modified 135 Oxygen Polyacrylamide gel electrophoresis .65,77 activation for drug metabolism, cytochrome P-450 in 1 Polycyclic aromatic hydrocarbons (PAH's) 127 to water, stepwise reduction of 21 Oxygenases, mixed-function 1,155 Polyguanylic acid, diastereomeric 9,10-epoxides of and (-)-trans7,8-dihydroxy-7,8-dihydrobenzoΡ (a) pyrene with 127 P-448 concentration, cytochrome 108 Poragel PN chromatography 137 P-448 induction of cytochrome 72 Porphyrin(s) P-450 complexes, EPR data for ferric 33,35 anticytochrome 75 iron 28 bacterial camphor hydroxylase 27 picket fence 42 carbonyl stage D of 38 thiolate complexes, immobilization cytochrome (see Cytochrome of low spin ferric 38 P-450) 46 Product ratios 94 oxycytochrome 5 perturbation of 81,91 resting stage A of 32 Protein, rat microsomal 165 reductase, rabbit liver microsomal Pseudomonas putida, the camphorNADPH-cytochrome 62 hydroxylating cytochrome of, P-450 32,57 P-450c. 57 P-450 Purification immunochemical properties of cytochrome P-450 76 forms of 57 of liver microsomal cytochrome liver microsomal cytochrome 47 P-450 50 3

LM

142 150

S

a

LM2

L

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ix001

L

a

a

cam

LM

m

4

195

INDEX

Purification ( continued ) of guanosine-diol epoxide 1 products of P-450 from PB-induced rabbit liver microsomes of P-450 and L M Purified monoxygenase system LM2

LM2

4

7

137 52 50 108

R Rabbit liver cytochrome P-450, resolution of multiple forms of 72 microsomal NADPH-cytochrome P-450 reductase 62 microsomes 57 purification of P-450 M hom PB-induced 52 purified reductase from 60 Radioactivity 150 Rat(s) liver microsomes, oxidation of cumene hydroperoxide by .... 20 oxidation of warfarin by normal microsomes from 83 purified NADPH-cytochrome P-450 reductase, PB-induced 65 purified reductase from 63 microsomal protein, oxidasedependent covalent binding of acetylhydrazine and isopropylhydrazine to : 165 Sprague-Dawley 87 Wistar 88 Reaction stages of cytochrome P-450, synthetic models for 27 Reaction stages of hemoprotein cytochrome P-450, model complexes for iron center in Reductase toward cytochrome P-450, specificity of 59 PB-induced rat liver microsomes purified NADPH-cytochrome P-450 65 rabbit liver microsomal NADPHcytochrome P-450 62 from rabbit liver microsomes, purified 60 from rat liver microsomes, purified 63 trypsin-treated 65 Reduction of oxygen to water, stepwise 21 Renal injury with acetanilides 177 Resolution of multiple forms of rabbit liver cytochrome P-450 .... 72 Resting stage A of P-450 32

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ix001

L

2

Ribose removal by N -methylation with dimethyl sulfate of RNA-nucleoside adducts from [ H]BP on mouse skin

130

3

147

S Salmon sperm DNA by N-methylN-nitrosourea, methylation of 135 Salmonella typhimurium 105,108 SDS-polyacrylamide gel electrophoresis 65 Selectivity, enantiomeric 81 Skin, mouse formation of an enantiomer of each diastereomeric 9,10-epoxide from benzo(a)pyrene in 127 injection in 150 RNA-nucleoside adducts from [ H]-BPon 147 Slab polyacrylamide gel electrophoresis of P-450 56 Sodium dodecylsulfate, polyacrylamide gel electrophoresis in the presence of 77 Soret 36 Specificity of reductase toward cytochrome P-450 60 Stability of nucleoside adducts, base 143 Stage A, low spin ferric 29 Stage A of P-450, resting 32 Stage B, high spin ferric 34 Stage C 36 Stage D 36 of P-450, carbonyl 38 Stage E, oxygenated 39 Stages of the hemoprotein cytochrome P-450 model complexes for the iron center in reaction 27 Stereoselectivity of microsomal metabolism of enantiomer 89 Structural characteristics of iron in hemoproteins 28 Substrate(s) bonded high spin ferric P-450 35 complex, ferrous cytochrome P-450 3 hydroxylation 1 for liver microsomal cytochrome P-450 48 specificity of forms of P-450 M 58 uncoupling effect of 14 Superoxide anion 5 Synergistic effect of N A D H or NADPH-dependent N-demethylation of ethyl morphine 16 Synthetic models for the reaction stages of cytochrome P-450 27 3

LM

L

DRUG M E T A B O L I S M C O N C E P T S

196

Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ix001

Τ

2,3,7,8-Tetra-chlorodibeiizo-p-dioxin (TCDD) Tetraols from diol epoxides 1 and 2 and modified poly (G) Tetraols, hydrolysis of diol epoxides to Thiolate complex, ferric Thiophenes Tissue injury, chemical-induced Toxic metabolites of isoniazid and iproniazid Toxicities, potentially lethal Toxicity concept of a dose-threshold for in vivo of furosemide in vivo of 4-ipomeanol Trans opening of diol epoxide 2 Trideuteroacetylhydrazine Tritium retention Trypsin-treated reductase U

Uncoupling effect of substrates Urinary excretion of metabolites

72

UV spectra of nucleoside adducts from diol epoxide 1 U V spectra of poly ( G ) and diol epoxide

135 V 129 29 Velocity for formation of hydrox­ 177 ylated products, theoretical 155 W 168 81 Warfarin by normal microsomes from rat liver, oxidation of 171 oral anticoagulant 177 quantitative microsomal metabolism 178 of enantiomers 148 stereoselectivity of microsomal 169 metabolism of 142 65 R-Warfarin hydroxylation of S-Warfarin hydroxylation of 14 Water, stepwise reduction of oxygen to 159

142 132

84

83 82 87 89 94 83 94 85 21