Genetic Response to Metals

Genetic Response toMetals

Proceedings of the First International Symposium

Metals and Genetics held May 24-27, 1994, in Toronto, Ontario, Canada, under the auspices of the Hospital for Sick Children, Toronto, Canada, and the International Association of Environmental Analytical Chemistry, Basel, Switzerland

Cover illustration adapted k m

R e m 3 in Genetics ZO, 1994; used with permission.

Genetic HesDonse I

edited by

Bibudhendra Sarkar The Hospital for Sick Children Toronto, Ontario, Canada

Marcel Dekker, Inc. New York.Basel.Hong

Kong

Library of Congress Cataloging-in-Publication Data

Genetic Response to metalsl edited by Bibudhendra Sarkar cm. p. Includes bibliographical references and index. ISBN 0-8247-9615-2 (hbk.) 1 . DNA-ligand interactions--Congresses. 2. Metals-Carcinogenicity--Congresses. E. Mutagenesis--Congresses. 4. Zinc fmgerproteins--Congresses. 5. GeneticToxicology--Congresses. I.Sarkar,Bibudhendra. II. InternationalSymposiumon"Metalsand Genetics"(1st : 1994 : Toronto,Ont.) QP624.7.G46 1995 615.9'253--d~20 95-7010 CIP

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Preface The past few years have seen scientific discoveries greatly contribute to our understanding of the relationships between metals and DNA. The fields which have contributed to this area range from genetics to biochemistry and chemistry. This book provides a forum for investigators from these diverse fieldsto reflect on the broad impactof the direct and indirect interactions of metals and DNA. This volume contains29 chapters contributed by scientists who are known for their special expertise and outstanding contributions to the current state of knowledge in the field. Although chapters have been chosen from various subject areas,the general theme of the book is on metals and genetics. The book begins by discussing the latest advances in our knowledgeof the molecular mechanisms of metal-induced mutagenicityandcarcinogenicity.Thisdiscussionisfundamental to our understanding of how cancer may be caused by various metals as a result of occupationalandenvironmentalexposure.Further,recentinvestigations have demonstrated that various metal compounds are able to cleaveDNA.Therearechapters thatdealwith the designofsuch agents, their mechanisms of action, as well as possible applications for site-specific DNA cleavage. There are also exciting developments inthe area of DNA-binding proteins having zinc-finger motif. Aspectsof these proteins’ DNA recognition, effect of metal replacement, and their ture and function are also presented. In the area of genetics and biochemistry of metal-related diseases the identification of genes for Menkes andWilsondiseaseshasbeenamajorbreakthroughinrecenttimes. There are chapters written by the authors who discovered these genes. Advances in the understanding of the pathophysiology as well as advances in new therapies for Menkes and Wilson diseases are presented as well. Finally, there are chapters devoted to major advances in the area of gene regulation involving metals. Certainly,it is difficult to comprehensivelycoverallareas of metals and genetics. An attempt has been made, nonetheless, to highlight recent important advances. This book will be of infinite value to researchersinthefieldsofbiochemistry,environmentalchemistry, inorganic chemistry, genetics, molecular biology, physiology, pharmacology, toxicology, and medicine. iii

iv

Preface

ACKNOWLEDGMENTS This volume contains the proceedings of the First International Symposium on Metals and Genetics, held May24-27, 1994, at the Hospital for Sick Children, Toronto, Ontario, Canada. We are indebted to Laura Faiczak, who assisted in the planning and operation of the Symposium, andto the following for financial support: the Hospital for Sick Children Foundation, the Research Institute of the Hospital for Sick Children,the Ministry of Environment and Energy, the Province of Ontario, the Samuel Lunenfeld Charitable Foundation, the municipalityofmetropolitanToronto, the NationalInstituteofEnvironmental Health Sciences (National Institutes of Health), the Nickel Producers Environmental Research Association (NipERA), the Nickel Development Institute(NiDI), the National Cancer Institute of Canada, Sandoz Canada, Inc.,Allelix Biopharmaceuticals, Falconbridge Limited, DiaMed Lab Supplies, Life Technologies, Inniskillin Wines, Hillebrand Estates,HenryofPelhamEstates,MarynissenEstates,CaveSpring Cellars, and Brick Brewery.

Bibudhendra Sarkar

Contents iii

ir Molecular Mechanismof Metal-Induced Mutagenicity and Carcinogenicity 1. Regulation of Nuclear Calcium and Zinc Interference by Toxic Metal

Ions

Stefan Hechtenbergand Detmar Beyersmann 2.

Effects of Antioxidants. Zinc, and Chelators on Free Radical Status of Children Living in the Chernobyl Area Ljudmila G. Korkina and Igor B. Afmas’ev

3. The Roleof Ascorbate in Metabolism and Genotoxicityof

Chromium(VI) Karen E. Wetterhahn, DianeM. Steam, Manoj Misra, Paloma H. Giangrande, Laura S. PhiGer, Laura J. Kennedy. and Kevin D. Courtney

1

21

37

4. Inactivation of Critical Cancer-Related Genes by Nickel-Induced

DNA Hypermethylation and Increased Chromatin Condensation: A New Model for Epigenetic Carcinogenesis Max Costa 5. Oxidative Mechanisms of Nickel@) and Cobalt@) Genotoxicity Kazimierz S. Kaspnak 6. The Antimutagenic Effects of Metallothionein May Involve Free

Radical Scavening E. I. Goncharova and T,G. Rossman

53 69

87

7. Protection from Metal-Induced DNA Damage by Metallothionein

in an in Vino System Lu a i , Jim Koropatnick, and M. George Cherian

101

8. DNA Strand Breakage and Lipid Peroxidation as Possible

Mechanisms of Selenium Toxicity

J. Kitahara, Y. Seko, N. Imura, H. Utsumi, and A. Hamada

v

121

vi

Contents

9. Role of Metal in Oxidative DNA Damage by Non-mutagenic

Carcinogen Shosuke Kawanishi, Shinji Oikawa. and Sumiko Inoue

131

Metal-DNA, DNA Cleavage, and Zinc-Finger Proteins

10. Sequence-Selective Cleavage of DNA by Cationic Metalloporphyrins

153

Genevi2ve Pratviel, Pascal Bigey, Jean Bernadou. and Bernard Meunier

11. LanthanidemI) Complexes as Synthetic Nucleases: Hydroxyalkyl Group Participation in Catalysis Janet R. Morrow, K. 0. Aileen Chin, and Kelly Awes

173

12. Initiation of DNA Strand Cleavage by Iron Bleomycin: Key Role of DNA in Determining the Pathway of Reaction David H. Petering, Patricia Fulmer, Wenbao Li, QunkaiMao,

185

and William E. Antholine

13. Nickel Compiexes in Modification of Nucleic Acids

201

Steven E. Rokita. Ping Zheng, Ning Tang, Chien-Chung Cheng, Ren-Hwa Yeh. James G. Muller, and Cynthia J. Burrows

14. New Methods for Determining the Structure of DNA and DNAProtein Complexes Based on the Chemistry of I r o n 0 EDTA

217

Thomas D. Tullius

15. DNA Recognition by Steroid Hormone Receptor Zinc Fingers: Effects of Metal Replacement and Protein-Protein Dimerization Interface Bibudhendra Sarkar

16. HIV-1Tat Protein Forms a Zinc-Finger-like Structure Jean-Pierre Laussac, Honord Mazarguil. Dani2le P r o d ,

237 255

Monique Erard, and Manh-Thong Cung

Metal-Related Genetic Diseases:Menkes and Wilson Diseases

17. Menkes Disease: From Patients to Gene

S. Packman. C. Vulpe, B. Levinson. S. Das, S. Whimey, and J. Gitschier

275

Contents

vii

18. Variability in Clinical Expression of an X-Linked Copper

Disturbance, Menkes Disease Nina Horn, Tonne Tonnesen,and Zeynep Tiimer 19. Development of Copper-Histidine Treatmentfor Menkes Disease Bibudhendra Sarkar

285 305

20. Copper-Histidine Therapy in Menkes Disease: Clinical, Biochemical, and Molecular Aspects Stephen G. Kaler

317

21. Biochemical and Clinical Benefits of Copper-Histidine Therapy in Menkes Disease J. Kreuder. A. Otten. A. Borkhardt, F. Lampert, K. Baerlocher, H. J. BGhles. and A . D6rries

323

22. The Wilson Disease Gene: A Copper-Binding ATPase Homologous to the Menkes Disease Gene Diane W.Cox and Gordon R. Thomas

343

23. Zinc Therapy: An Advance in the Treatment of Wilson’s Disease Tjaard U. Hoogenraad

361

Metals and Gene Regulation 24. Transcriptional Regulation and Functionof Yeast Metallothioneh Genes Zhiwu Zhu, Mark S. Szczypka. and Dennis J. Thiele

379

25. Transcriptional Regulationof the Metallothionein Gene: MetalRegulatory 397 Responsive Zinc Factor Element and Shinji Koizumiand Fuminori Otsuka

26. Gene Disruption of the Transcription FactorMTF-l Leads to Loss of Metal Regulation of Mouse Metallothionein Genes 1 41 Freddy Radtke, Rainer Heuchel, Oleg Georgia? Gerlinde Stark, Michel Aguet, and Walter S c m e r 27. Characterization and Purification of MEP-l, a Nuclear Protein Which Binds to the Metal Regulatory Elements of Genes Encoding Metallothioneins Simon Labbi, Lucie Larouche, Jacinth Pr&ost, Paolo Remondelli, and CarlSkguin

425

viii

Liver

Contents

28. CopperAccumulation, and MetallotbioneinStability and Developmental Regulation, in the Toxic MiZk Mouse Jim Koropatnick, Greg Stephenson, and M. George cherian

443

29. Metallothionein Synthesis Is Selectively Enhanced by Copper in the of LEC Rats Kazuo T. Suzuki

467

I&

485

Contributors Igor B.Afanas’ev 117820, Russia

VitaminResearchInstitute,Nauchnypr.14A,Moscow

Michel Aguet Institut f i r Molekularbiologie I, UniversitiitZurich,CH-8093 Zurich, Switzerland William E. Antholie Medical College of Wisconsin, Milwaukee, W1 53226 Kelly Awes ChemistryDepartment,AchesonHall,StateUniversity York,Buffalo,NY14214

of New

K. Baerlocher Children’sHospital,St.Gallen,Switzerland Jean Bernadou Labomtoire de Chimie de Coordination, Centre Nationalde la Recherche Scientifique,.205, routede Narbome, 31077 Toulouse cedex,France

Detrnar Beyersmann DepartmentofBiologyandChemistry,Universityof Bremen NW2, D-W-2800Bremen 33, Germany

Pascal Bigey Laboratoire de Chmie deCoordination,CentreNationaldela Recherche Scientifique, 205, route de Narbome, 31077 Toulouse cedex, France Department of Pediatrics, Johann Wolfgang Goethe University, DdOOO Frankfurt, Germany

H. J. Bohles

A. Borkhardt DepartmentofPediatrics,Children’sHospital, University, Feulgenstrasse 12, D-35385 Giessen, Germany

Justus Liebig

Cynthia J. Burrows Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY 11794

Lu Cai

DepartmentofPathology,UniversityofWesternOntario,London, Ontario N6A 5C1, Canada Chien-Chung Cbeng Department of Chemistry, State Universityof New York at Stony Brook, Stony Brook, NY11794

M. GeorgeCherian

Department of Oncology,LondonRegionalCancer Centre, 790 Commissioners Road East, London, Ontario N6A 4L6, Canada ix

X

Contributors

K. 0 . Aileen Chin Chemistry Department, Acheson Hall, State University of NewYork,Buffalo,NY14214 Max Costa Nelson Instituteof Environmental Medicine,N W Medical Center,

LongMeadowRoad,Tuxedo,NY10967 Kevin D. Courtney NH 03766

DartmouthCollege,6128BurckeLaboratory,Hanover,

Diane W. Cox Department of Genetics, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1x8, Canada Manh-Thong Cung Laboratoire de Chimie Physique Macromolhlaire, C Nancy, France

N S ,

S. Das

Department of MedicineandtheHowardHughesMedicalInstitute, University of California, San Francisco, CA 94143

A. Diirries Department of Pediatrics, University Clinics, Wilrzburg, Gennany CNRS,

MoniqueErard InstitutdeBiologieCellulaireetdeGhnetique, route de Narbonne, 31077 Toulouse cedex, France Patricia Fulmer Department of Chemistry, University Milwaukee,Milwaukee, WI 53201

205,

of Wisconsin-

Oleg Georgiev Institut fir Molekularbiologie 11, Universitiit Zurich, Winterthurestrasse 190, CH-8057 Zurich, Switzerland Paloma H. Giangrande Hanover, NH 03766

Dartmouth College, 6128 Burcke Laboratory,

J. Gitschier DepartmentofMedicineandtheHowardHughesMedical Institute, University of California, San Francisco, CA 94143

E. I. Goncharova Nelson Institute of Environmental Medicine, NYU Medical Center,LongMeadowRoad,Tuxedo,NY10967 A. Hamada Japan

SchoolofPharmaceuticalSciences,ShowaUniversity,

Tokyo,

StefanHechtenberg Department of BiologyandChemistry,University Bremen NW2, D-W-2800 Bremen 33, Germany Rainer Heuchel Institut fiir Molekularbiologie 11, Universitiit Zurich, Winterthurestrasse 190, CH-8057 Zurich, Switzerland

of

xi

Contributors

Tjaard U. Hoogenraad Department of Neurology, University Hospital, 4584 CX Utrecht, The Netherlands Nina Horn Denmark

The John F. KennedyInstitute, G1. Lmdevej7,Glastrup2600,

N. h u r a School of PharmaceuticalSciences,Kitasat0university,5-9-1, Shirokane, Minato-h, Tokyo 108, Japan SumikoInoue Department of PublicHealth,Faculty University, Kyoto 606, Japan

of Medicine,Kyoto

Stephen G. Kaler Human Genetics Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 10, Room 8C429, 9OOO Rockville Pike, Bethesda, MD 20892 Kazimien S. Kaspnak Department of HumanHealth,FrederickCancer Research & Development, Building 538, Room 205E, Frederick, MD 217021201 Shosuke Kawanishi Department of Public Health, Facultyof Medicine, Kyoto University, Kyoto 606, Japan Laura J. Kennedy Dartmouth College, 6128 Burcke Laboratory, Hanover, NH 03766 J. Kitahara School of PharmaceuticalSciences,KitasatoUniversity,5-9-1, Shirokane, Minato-ku, Tokyo 108, Japan Shinji Koizumi Department of Experimental Toxicology, National Institute of Industrial Health, 6-21-1 Nagao, Tama-Ju, Kawasaki 214, Japan Ljudmila G. Korkina Lab, Cell Biophysics and Biochemistry, Russian Institute of Pediatric Hematology, Leninskii pr. 117, Moscow 117513, Russia

Jim Koropatnick Department of Oncology, London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario N6A 4L.6, Canada J. Kreuder Department of Pediatrics,Children’sHospital, University, Feulgenstrasse 12, D-35385 Giessen, Germany

Justus Liebig

Simon Labbe CentredeRechercheenCandrologiedel’Universit6Laval, l’H6tebDieu de Qu6bec. 11 C6te du Palais, Quebec GlR 2J6, Canada Department of Pediatrics,Children’sHospital,JustusLiebig University, Feulgenstrasse 12, D-35385 Giessen, Germany

F. Lampert

Contributors

LucieLarouche Centre de Recherche en Canc6rologie de l’Universit6 Laval, l’H6tel-Dieu de Qu6bec, 11 CBte du Palais, Quebec GlR W6, Canada Jean-Pierre Laussac Laboratoire de Chimie de Coordination, C N R S , 205,route de Narbonne, 31077 Toulouse &ex, France

B. Levinson Department of MedicineandtheHowardHughesMedical Institute, University of California, San Francisco, CA 94143 WenbaoLi Department of Chemistry, University of Wisconsin-Milwaukee, Milwaukee. WI 53201 Qunkai Mao Department of Chemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53201 Honor6 Mazarguil Institut de Pharmacologie et de Biologie Structurale, CNRS,

205,route de Narbonne, 31077 Toulouse cedex, France Bernard Meunier Laboratoire de Chimie de Coordination, Centre National de laRechercheScientifique, 205, routedeNarbonne, 31077 Toulousecedex, France ManojMisra 03766

DartmouthCollege, 6128 BurckeLaboratory,Hanover,

NH

Janet R Morrow ChemistryDepartment,AchesonHall,StateUniversity NewYork,Buffalo, NY 14214

of

James G. Muller Department of Chemistry, State University of New York at 11794 StonyBrook,StonyBrook,NY

ShinjiOikawa Department of PublicHealth,Faculty University, Kyoto 606,Japan

of Medicine,Kyoto

A. W e n

Department of Pediatrics, Children’s Hospital, Justus Liebig University, Feulgenstrasse 12. D-35385 Giessen, Germany FuminOri

Otsuka

TeikyoUniversity,Kanagawa,Japan

S. Packman

Department of Pediatrics,DivisionofGenetics,University California, Box 0748,San Francisco, CA 94143

of

David H. Petering Department of Chemistry, University Wisconsin-Milwaukee,Milwaukee, WI 53201

of

xiii

Contributors

Laura S. Phieffer Dartmouth College, 6128 Burcke Laboratory, Hanover, 03766 GeneviBvePratviel Laboratoirede ChmiedeCoordination,CentreNational de la Recherche Scientifique, 205. route de Narbonne, 31077 Toulouse cedex, France

Jacinthe Pr6vost Centre de Recherche en Candrologie de l’Universit6 Laval, 1’HBtel-Dieu de Quebec, 11 CBte du Palais, Quebec G1R 276, Canada Dani&le Prom6Institut de Phannacologie et de Biologie Structurale, CNRS, 205, route de Narbonne, 3 1077Toulouse cedex, France Freddy Radtke Institut fir Molekularbiologie II, Universittit Zurich, Winterthurestrasse 190, CH-8057 Zurich, Switzerland Paolo Remondelli Centre de Recherche en Candrologie de l’Universit6 Laval, 1’HBtel-Dieu de Q u C b e c , 11 CBte du Palais, Quebec GlR 276, Canada

Steven E. Rokita Department of Chemistry, State University of New York at StonyBrook,StonyBrook,NY11794

T. G. Rossman

NelsonInstitute of EnvironmentalMedicine, N W Medical Center,LongMeadowRoad,Tuxedo,NY10967

Bibudhendra Sarkar Biochemistry Research, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario MSG 1x8, Canada Walter Schaffner Universitslt Zurich, Institut fiir Molekularbiologie Winterthurestrasse 190, CH-8057 Ziirich, Switzerland

11,

Carl S*in CentredeRechercheenCandrologiedeI’UniversitCLaval, 1’HBtel-Dim de Qu6bec, 1 1 CBte du Palais, Quebec GlR 256, Canada

Y. Seko

School of PharmaceuticalSciences, Shirokane, Minato-ku, Tokyo 108, Japan

Kitasato University,5-9-1,

Gerlinde Stark Institut fir MolekularbiologieI, Universitslt Zurich, CH-8093 Zurich, Switzerland Diane M. Stearns Dartmouth College, 6128 Burcke Laboratory, Hanover, NH 03766 Greg Stephenson Department of Oncology, London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario N6A 4M. Canada

xiv

Contributors

KazuoT. suzuki FacultyofPharmaceuticalSciences,ChibaUniversity, Yayoi, Inage, Chiba 263, Japan Mark S. Szczypka DepartmentofBiologicalChemistry,TheUniversityof Michigan Medical School, M5416 Medical Science 1, 1301 Catherine Road, Ann Arbor, MI 48109-0606

Ning Tang Department of Chemistry, State University of New York at Stony Brook,StonyBrook, NY 11794 Dennis J. ThieleDepartmentofBiologicalChemistry,TheUniversityof Michigan Medical School,M5416 Medical Science1, 1301 Catherine Road,h Arbor, MI 48109-0606 Gordon R. Thomas Department of Genetics, The Hospital for Sick Children, S55 University Ave., Toronto, Ontario MSG 1x8,Canada TenneTannesenThe 2600,Denmark

John F. KennedyInstitute,G1.Landevej

7, Glastrup

Thomas D. Tullius Department of Chemistry, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218 Zeynep Tiimer The John F. Kennedy Institute, GI.Landevej 7, Glastmp2600, Denmark H. Utsumi Japan

SchoolofPharmaceuticalSciences,ShowaUniversity,Tokyo,

Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143

C. Vulpe

Karen E.Wetterhahn Dartmouth College, 6128Burcke Laboratory, Hanover, NH 03766 S. Whitney Departments of Pediatrics and Medicine, University of California,

San Francisco, CA 94143 Ren-HwaYehDepartmentofChemistry,StateUniversity StonyBrook,StonyBrook, NY 11794

of NewYork at

Ping Zheng Department of Chemistry, State University of New York at Stony 11794 Brook,StonyBrook,NY

Zhiwu Zhu Department of Biological Chemistry, The University of Michigan Medical School, M5416 Medical Science 1, 1301 Catherine Road, Ann Arbor, MI 48109-0606

1 Regulation of Nuclear Calcium and Zinc Interference by Toxic Metal Ions Stefan HechtenbergandDetmarBeyersmann Department of Biology and Chemistry, University of Bremen NW2, D-W-2800 Bremen 33, Germany

L

INTRODUCTION

The genetic toxicologyof metals is very complex, since toxic metal ions of bind to multiple cytoplasmic and nuclear target molecules. Low levels heavy metals interfer with crucial nuclear functions as DNA replication, DNA repair, and gene expression [l]. A pertinent mechanismof action of toxic metals may be the interference with nuclear uptake, homeostasis and function of essential metal ions. Calcium and zinc ions are believed of celldifferentiation,proliferationand toparticipateinthecontrol apoptosis. Calciumis involved directly or via calcium binding proteins in likecalmodulin in nearly all majorsignaltransductionchains multicellular organisms [2]. These include the transferof messages from of gene extracellularsignalsintothecellnucleusandthecontrol function. Table 1 lists some relevant findings about the role of Ca2+ or Ca2+/calmodulin in the regulation of gene expression. A comprehensive has review of the functionsof calcium and calmodulin in the cell nucleus been published by Carafolis Laboratory [3]. In at least one instance, the direct interaction of Ca2+/calmodulin with a subclass of transcription factors has been demonstrated, recently [4]. 1

2

Hechtenberg and Beyersmann

Table 1. Control of gene expressionby Ca2+ or Ca2+/calmodulin

1 Calcium specifically prolaction-gene expression

rsuda et al. 1986 [l91 Morgan k Curran 1986 [20] Sheng et al. 1990 [21] Sch6nthaI et al. 1991 [22]

Sheng et al. 1991 [23]

Can et al. 1991 [24]

Kapiloff et al. 1991 [25]

(h2+, PKC and CAMP regulate c-fos expression ~ a 2 flux + contro1s c-fos expression

Ca2+/calmodulin controls c-fos expression Mobilization ofCa2+ by inhibition of Ca2+ ATPase causesc-fos and c-jun expression Ca2+/calmodulin dependentkinases activate transcription factor CREB (CAMP responsive) Both Ca2+ and PKC regulate expression ofthe gene for the thyrotropin B-subunit

Ca2+/calmodulindependentprotein kinase activated the rat prolactin gene independently of PKC

Bartlett et al. 1991 [26]

Ca2+ ionophore A 23187 induces expression of the gadd153 gene (growth arrest and DNA damage

Liu et al. 1992 [27]

inducible) Transcription factors ATF-l and CREB mediate Ca2+ and CAMP-induced transcription

Corneliussen et al. 1994 [4]

Ca2+/calmodulin *%its directly DNA binding of helix-loophelix transciption factor domains -

Regulation of Nuclear Calcium and Zinc

3

Interference

Whereas the regulatory functions Ch2+ of are wellestablished, little hard data are available about the role of Zn2+ in cellular controls. Certainly, key zinc is a structural constituent of hundreds of proteins comprising enzymes involved in cellular controls and transcription factors, namely as well as protein kinase C, RNA polymerase and reverse transcriptase the zinc-finger transcription factors. The intracellular free Zn2+ concentration is probably regulated by metallothionein and other zincis binding proteins, and the expression of the metallothionein gene itself controlled by Zn2+ via regulatory zinc binding proteins[5].However, the evidence for the involvement of Zn2+ in reproduction andgrowth control is relatively indirect. Dietary zinc is rapidly taken up by cell nuclei [6], zinc deprivation inhibits specifically cell growth and differentiation [7] whereas zinc restoration seems to trigger DNA synthesis and mitosis [S]. The intracellular homeostasis of essential metal ions is a sensitive target of toxic metal ions. Especially the cellularfree Ca2+ is highly vulnerable by heavymetals [9,10].Lowconcentrationsof cadmium ionsinduce inositol1,4,5-trisphosphateand Ca2+ mobilization in fibroblasts [l l], submicromolarlevelsofHg2+ionsamplifyreceptor-mediatedCa2+ signals in PC12 cells [l21 and micromolar concentrations of lead ions cause a sustained increase in the basal free Ca2+ level but diminish the hormone-stimulatedrise in free Ca2+ inbonecells [13 . It may be presumed that the nuclear calcium homeostasis and the dependent nuclearactivitiesalso are susceptibletointerference by toxicmetals. Cadmium activates nuclear protein kinase C [14],it inhibits the Ca2+ activated DNA fragmentation [ 151 and it stimulates the transcription of theprotooncogenesc-jun,c-myc [l61 and c-fos [17]. It still has to be elucidated whether the latter effects are caused by interference withCa2+ dependent gene expression or by direct interactions of toxic metal with proteins controlling transcription. Last but not least, the genotoxicity of radiation and chemical mutagens may be enhanced if toxic metal ions DNA. interfer with the function of Ca2+ in the repair of damaged

CaJ+

IL

CONTROL OF FREE Ca2+ CONCENTRATIONS IN CELL NUCLEI

To accomplish its control of nuclear activities, the intranuclear free Ca2+ concentrationmust be finelyadjusted in timeandspace.Table2 free Ca2+ summarizes reports whichdemonstratethatthenuclear concentration is not simply following changes in cytoplasmic free ~ a 2 + but is undergoingspecificmodulationwhencellsarestimulated by

Table 2. Reports on the nuclear control of free Ca2+ c

S'mooth muscle cells Williams et al. 1987 [28] Neylon et al. 1990 [29] Himpens et al. 1992 [30] lveurons and neuralcells Lipscombe et al. 1988 [3 l]

-

Nuclear Ca2+ is shielded from cytoplasmic stimulation Nuclear Ca2+ is lower in resting cells, but exceeds cytoplasmic Ca2+ upon stimulation No difference between cytoplasmic and nuclear ~ a 2 +in resting cells

Hernandez-Cruz et al. 1990 [32] P r z y w a r a et al. 1991 [33] Nuclear Ca2+ signal is larger than cytoplasmic Birch et al. 1992 [34] Nuclear Ca2+ signal is larger than cytoplasmic during neurite regeneration M-Mohanna et al. 1994:[35] The neuroblastoma cell nucleusis insulated from large cytosolic ~a2+ changes, but follows small changes rapidly G r M h f d o r - and hormone-stimulatedc e k Tucker & Fay 1990 [36] Waybill et al. 1991[37] Yelamarty et al. 1990 [38]

PDGF-stimulated fibroblasts exhibit similar Ca2+ transients in nucleus and cytoplasma EGF-stimulated hepatocytes show higher Ca2+ peaks in cytoplasma than in nucleus Eryhropoetin-inducedincrease in nuclear Ca2+in erythroblasts

Ca2+ uutake and releasefiver in cell nuclei Nicotera et al. 1989 [39], Hechtenbergsi Beyersmann 1993 [40] ATp-stimulated uptake of Ca2+ into isolated nuclei Nicotera et al. 1990 [41] Ip3-induced releaseof Ca2+ from rat liver nuclei Malviya et al. 1990 [42] 1p3 mediates ~ a 2 +release from rat liver nuclei 4

Regulation of Nuclear Calcium

and Zinc Interference

5

variousextracellularfactors. This conclusion has beenreachedfor stimulatedsmoothmusclecells,triggeredneuralcellsorregenerating neurons and hormone or growth factor stimulated cells. With isolated liver cell nuclei it has been demonstrated that ATP elicits the uptake of Ca2+ whereasinositol1,4,5-trisphosphateinducesthereleaseofCa2+ from nuclei (Table 2). All these findings suggest that the nucleus has additional mechanismsto controlits free Ca2+ levels.

EL

FURTHER CHARACTERIZATION OF THE CALCIUM UPTAKE AND RELEASE FROM LIVER NUCLEI

Bovine liver nuclei were loaded with the fluorescent metal chelator Fura2 in its acetoxymethyl ester form. The measurement of intranuclearfree Ca2+ was feasible since (i) the Fura-2 ester was taken up and (ii) it was hydrolyzed to the free dye by intranuclear esterase(s). The fluorescence was calibratedtogivethe intensityoftheCa2+-Fura-2complex Ca2+ intranuclearfree Ca2+ concentration.Figure1Ashowsthatthe uptake into liver nuclei is stimulated by ATP, whichis in agreement with nM extranuclear Ca2+, an results of other laboratories [39]. With 200 intranuclear Ca2+ concentration of 500 n M was reached.ATP also stimulated an increaseintotalnuclear C@+. I ~ M ATP causedthe intranuclear Ca2+ levelto rise to 150 nmoVmg DNA, i.e.,twicethe amount taken upin the absence of ATP. Also shown in Figure 1A is the Ca2+ rapidlossofaccumulatednuclearfreeCa2+afteradditionof ionophoreA23187.Subsequentapplication of thedetergentNP40 brought the nuclearCa2+ level down to the equilibrium concentration. TheATP-stimulated Ca2+ uptakeintolivernuclei was prevented by thapsigargin, an antagonistofthe Ca2+ pumpsof theendoplasmic with further reticulum (Figure 1B). Figure 2 summarizes results obtained an inhibitors.Vanadate, a typicalATPaseinhibitorandtributyltin, antagonist of membrane ion transport,also inhibited the uptake of Ca2+ into liver nuclei. At variance verapamil, a blocker of plasma membrane Ca2+ channels, was not effective as inhibitor of nuclear Ca2+ uptake. All these results are consistent with the assumption that the nuclear ~a2+ pump is related to that of the endoplasmic reticulum. In concordance, Lanini et al. did not find any difference in molecular weight, antibody Ca2+ ATPases of the binding and phosphoenzyme formation between the nucleus and the endoplasmic reticulum from rat[43]. liver

6

Hechtenberg and Beyersmann 600 500

E +

400

(Y

G

g 300

t L 8

j 3

200

2

E 100 Y

0

100

200

300

400

500

600

Time (S) 600

g

500

+ N

d

3

400

.m

U

G v)

300

2 m 200 5:

E

.z

100 I

0

200

400

600

800

Time (S)

Figure lk ATP-stimulated increaseof intranuclear free Ca2+. B. Inhibition of ATP-stimulated Ca2+ accumulation by thapsigargin.

Isolated bovine liver nuclei were treated with the following additions at thetimes indicated:200nM free Ca2+, 1 m M ATP, 250 nM thapsigargin (TG), 2 p M A 23187, and 0,05% NP40.

Regulation of Nuclear Calcium and Zinc Interference

7

I

Figure 2. Efffeets of inhibitors on ATP-stimulated intranuclear free Ca2+ uptake.

Isolated bovine liver nuclei were treated with 200 IIM C@+, 1mMATP and the following concentration of inhibitors5 min: for 100 pMvanadate, 250 n M thapsigargin, 5 p M tributyltin chloride, 100 p M verapamil. Regarding the release of Ca2+ from liver nuclei, two laboratories have 1,4,5-trisphosphate stimulates Ca2+ demonstrated that inositol mobilization [41,42]. Since the same second messenger also causes Ca2+ release from endoplasmic reticulum stores, it may be concluded that the Ca2+ mobilization mechanisms of both nuclear and endoplasmic This conclusion is further reticulum compartments are similar. substantiated by our finding thatalso GTP triggers a part~alrelease from liver nuclei (Figure 3A). This effect is dependent on GTP concentrations and it can be further enhanced in the ph siological range(50 - 500 m, if the Ca& reuptake is inhibited by thapsigargin (Figure 3B). The effect of GTP on Ca2+ release from nucleiis again similar tothe effect of GTP on Ca2+ mobilizationfromendoplasmicreticulumvesicles[44,45]. However, the mobilization of Ca2+ from the nucleus seems tobe subject of the to additional control, since the inositol l,4,5-trisphosphate receptor nuclear membraneis activated by proteinkinase C [46].

700

IA

GTP

3 300 V

3

2 200

L

E

U

100 0 0

100

200

300 500 400

600

Time (S)

B "

0

0

100

200

300 400 600 500 GTp(IrM)

Figure 3A. ATP-stimulated uptake and GTP-induced release of intranuclear Ca2+. B. Dependence of Ca2+ release from nuclei on the concentration of GTP.

Isolated bovine liver nuclei were treated with 200 n~ free and 1 mM ATP. At the times indicatedGTP was added at the NP40 was added concentrations given. Subsequently the detergent to afinal concentration of O,O5%. In the experimentshown in Figure 3B, 250 nM thapsigargin was added prior tothe administration of GTP. C $ +

8

Regulation of Nuclear Calcium and Zinc

Interference

9

NUCLEAR UPTAKE OF ZINC, CADMIUM AND LEAD IONS

N.

The same fluorescent dye, Fura-2, which is used to estimate free W + concentrations, can be used for the determination of free Zn2+; CM2+ and &+[47]. Figure 4 shows the spectral titration curves with Fura-2 in

800

f

a

B

E;

"1 D 1.6

l .o

0.5

a

. m

. p

. o

. y

. o

. ~

r

y

)

.

y

.

Y

)

. . . . - 3 2 0 3 1 o m m m

Excitation wavelength (nm) Excitation wavelength (nm). Figure 4. Excitation spectraof Fura-2 in the presenceof various concentrations of free metal ions. A. Spectrawith Ca2+ C. Spectra with Cd2+ B. Spectrawith Zn2+ D. Spectrawith Pb2+

The fluorescence emissionwas recorded at 5 10 nm. Free Ca2+ and Zn2+ concentrations are given in n M , free CM2+ and P@+ concentrations inPMin increasing order from bottom to top curve.

10

Hechtenberg and Beyersmann

comparison. The Zn2+-Fura-2complex is excitated at a slightly longer complexwithanevenmore wavelength than therespective pronounced increase in fluorescence intensity. Similarly, Cd2+ and form fluorescent complexes with Fura-2. From the titration curves, the stoichiometry and the dissociation constants were calculated. the stoichiometry was 1:l in all cases, and the dissociation constants for the Ca2+, Zn2+, Cd2+, and P@+ complexes were 2.0.10-7 M, 1.5.10-9 M, 2.7*10-13M, and 2.1.10-12 M, respectively. The allinities of the metal ions forFura-2 are several ordersof magnitude greater that that of(h2+. To avoidfurther the interference of Ca2+ withtheZn2+-sensitive Ca2+ bywashingwith fluorescence,livernucleiweredepletedof chelatorcontaining bufferprior to the experiments with zinc. G & +

F @ +

Figure SA shows that the uptake of Zn2+ into liver nuclei was time- and of a membraneconcentrationdependent as with Ca2+. Addition impermeant metal chelator (DTPA) stopped the further uptake of Zn2+ butdidnotsigmiicantlychangetheZn2+dependentfluorescence. In a membrane-permanentchelator contrast,thesubsequentadditionof ("PEN) caused rapid a decrease in the Zn2+-Fura-2complex free Zn2+ fluorescence.Figure 5B demonstratesthattheintranuclear concentration is limited compared with thefree Zn2+ concentrationof the medium. this conclusion does not apply to the total nuclear zinc level whichincreased three- to fivefold over theextranucleartotalzinc 5A concentration, probably due to tight binding to nuclear protein. Figure further shows that the increase in nuclearfree Zn2+ is not stimulated by ATP. Furthermore, the intranuclear free Zn2+ level is not changed by thapsigargin andby the addition ofGTP to the medium(data not shown). These findings clearly demonstrate that the mechanism of Zn2+ uptake from that of uptake. into liver nuclei differs C @ +

Theuptake of Cd2+ and intoisolatedlivernuclei also can be measured by the Fura-2 fluorescence technique as described for Zn2+, still tighter than Zn2+. since the heavy metal ions are binding to the dye The nuclear uptake ofCd2+ and P$+ is shown in Figure 6 as a function AS withZn2+, 'the ofextranuclear free metalionconcentrations. intranuclear. free Cd2+ and &+concentrations stay well below the medium concentrations. Furthermore, as with Zn2+, no stimulation of Cd2+ or &+ uptake into nucleiby ATP was observed (datanot shown). In conclusion, thereis no activetransport system available to pumpCd2+ or &+ into cell nucleus, and the intranuclearfree ion concentrations of these heavy metals stay below those offered in the medium. p b z +

11

Regulation of Nuclear Calcium and Zinc Interference

:i 2

0 100 200

0

300 400 500

600

700

Time (S)

-9

-0

-7

-6

Extranuclear fm Zn2+ (log M)

figure 5. Uptake of zn2+ into liver nuclei A. ~acreasein nuclearfree zn2+ concentration. Isolated liver nuclei were incubated with 10 nM (lower trace), 100 nM (middle trace), and316 nM free Zn2+ (upper trace).1 mM ATP, 200 p M DTPA, and 200 @ TPEN l were each added at the times and to the samples indicated in the figure. B. Dependence of Zn2+ uptake on the extranuclear free Zn2+ concentration.

Isolated bovine liver nuclei were incubated5 for min with various concentrationsof free Zn2+. (From ref.40 with permission)

12

Hechtenberg and Beyersmann 0.20

0.15

0.10

0.05

I

-12

I

I

-11

-10

1

I

I

I

-8

-7

I

-9

I

I

Extranuclear freeCd2+ (log M) 8 -

7

6

"

B

--

5

"

4

"

3

"

2

"

1

"

0-? -12

I

I

I

-1 1

-1 0

-9

I

II

-8

-7

Extranuclear freePb2+ (log M)

Figure 6. Uptake of Cd2+ and Pt?+ into livernuclei. Isolated bovine liver nuclei were incubated for 5 min with various concentrations of free Cd2+(A) and P@+ (B).

Regulation of Nuclear Calcium and Zinc

V.

Interference

13

INHIBITION OF NUCLEAR Ca2+ TRANSPORT BY Cd2+ AND Pb2+ IONS

Since the heavy metal ions interfer with fluorimetric assay of intranuclear special experimentalrecautionshadtobeused.Isolatednuclei P$+ solutions, washed with chelator were preincubated with Cdg or Ca2+ and ATP thereafter. With containing buffer, and supplemented with thenuclear Ca2+ accumulationwasdiminished bothCd2+and (Figures 7 and 8). From the concentration dependence (Figures 7B and 8B) it is estimated that halfmaximal inhibition fo Ca2+ uptake is obtained at 8 n M free Cd2+ and 14 nM free P@+, respectively. These results are Ca2+ pumpsof the consistentwith a closerelationshipbetweenthe nuclearenvelopeandthatoftheendo-/sarcoplasmicreticulum.Ina Ca2+ uptakeinto previous s t u d y , wealsoobservedaninhibitionof sarcoplasmic reticulum vesicles by cadmium and lead and estimated the halfmaximal inhibitory concentrations to be 450 n M free Cd2+ and 60 n M free P@+, respectively [48]. Ca2+,

p b 2 +

M.

CONCLUSIONS AND PERSPECTIVES

Thenuclearlevelsoffree Ca2+ and free Zn2+donotfollowthe cytoplasmicconcentrationsoftheseionspassivelybutarecontrolled differentially. the uptake ofCa2+ into nuclei is an active, ATP-stimulated process,and Ca2+ mobilizationfromnuclei is stimulated by inositol 174,5-trisphosphate and GTP. These controls allow a distinct regulation of intranuclear Ca2+ and Ca2+'calmodulin dependent nuclear functions. The uptake of Ca2+ into nuclei is inhibited by nanomolar concentrations of F@+, and probably by Hg2+, too. This mechanism of heavy free Cd2+ and metal toxicity extends thelist of known interferences of toxic metals with signal transduction steps such as signal reception, mobilization of second messengers and gene expression. Whereas the regulatory role of nuclear free Ca2+ is well established, a corresponding function of free in cell nucleiis still speculative. our data show that the level of free nuclear Zn2+ is limited, suggesting a barrier function of the nuclear membrane. Up to now, no mechanism for the stimulation of Zn2+ mobilizationin nuclei has been detected. On the otherhand,there are distinctexamplesofZn2+andCd2+mediated [49] controls of gene expression, i.e., via the activation of metallothionein and heat shock protein [50] promotors. The observed induction of the early control genes c-fos, c-myc and c-jun by Cd2+ may be mediated by &+

14

Hechtenberg and Beyersmann

100

0

200

300

500

400

Time (S)

-12

-11

-10

-9

-8

-7

-6

Extranuclear free Cd2+ (log M)

Figure 7. Effect of Cd2+ on nuclearCa2+ uptake.

A. Time dependenceof inhibition by Cd2+. Nuclei were preincubated for10 min with l O n M free Cd2+, washed, and supplemented with200 nM free Ca2+ and 1 m M ATP at the times indicated. B. Dependence of the inhibition on the extranuclearfree Cd2+ concentration duringthe preincubation period.

Regulation of Nuclear Calcium and Zinc Interference

15

600

Control

500 400

300 200 100

100

0

200

300

500

400

Time (S) I L"

IB

T

100

0

I

I

I

I

I

I

-11

-10

-9

-8

-7

-6

Extmnnclerr f m P@+ (log M)

Figure'8. Effect of P$+ on nuclear uptakeof Ca2+. k Time dependenceof inhibition by Pp+ B. Dependence of inhibition on extranuclear free Pp+ concentration during the preincubation period.

Experimental conditions werethe same as described in the legend to Figure 7 .

inte~erence of the metal ion wi homeos~sis. Thus, cellular balance r e n ~ a ~ oton p r o l ~ e r a ~ oinn a nongenoto interac~on.

A. PreDaration of nuclei Cell nuclei were prepared from bovine liver by homogenization in 50 mM Tris-HC1, 120 mM KC1, 4 mM MgC12, 0.2 mM phenylmethylsulfonylfluoride, 1 mM dithiothreitol, 0.25 M sucrose adjusted to pH 7.5, and dfierential centrifugation as decribed elsewhere [40]. The nuclear fraction was virtually free of contamination by plasma membranes, microsomes and mitochondria, as checked by marker enzyme activities (5'-nucleotidase, alkaline phosphatase I, glucose-6-phosphatase7 succinate-IN" reductase). Purified nuclei were suspended in standard incubation medium containing 25 mM Hepes, 125 mM KCl, 2 mM K;?HPO4, 4 mM MgC12, 0.5 mM EGTA, 0.5 mM EDTA, 2 mM NTA, adjusted to pH 7.0.

C. Loading of nuclei with fura-2 and fluorescence measurements Isolated nuclei were preloaded with 7.5 jiM fbra-2 acetoxymethyl ester for 45 min at 4°C. The nuclear suspension was then washed twice in standard incubation medium. Fluorescence measurements were performed at 25°C in a dual-wavelength fluorescence spectrometer (Perkin-Elmer LS 50). Fluorescence intensity was monitored at an emission wavelength of 510 nm, by using a pair of excitation wavelengths at 340 nm and 380 nm. Free ion concentrations were calculated from ratio data as described by Grynkiewicz et al. [53], by using the Perkin-Elmer intracellular biochemistry application sohare. The apparent dissociation constants for the fura-2-complexes were determined as described by Kwan and Putney [54].

18

Hechtenberg and Beyersmann

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RD. Lohmann and D. Beyersmann, Biochem. Biophys. Res. 190: 1097-1103 (1993). CO-.

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M. Sheng, G. McFadden andM.E. Greenberg, Neuron4: 571-582 (1990)

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A. Schenthal, J. Sugarman, J. Heller-Brown, M.R Hanley andJ.R Feramisco, Proc. Natl. A d . Sci USA 88: 7096-7100 (1991).

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J.D. Bartlett, J.D. Luethy, S.G. Carlson,S. J. Sollott and N. J. Holbrook, J.Biol. Chem. 267: 20465-20470 (1992).

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C.B. Neylon, J. Hayland, W.T. Masonand RF. Irvine, Am. J. Physiol. 263: C95-C105 (1990).

Regulation of Nuclear Calcium and Zinc

Interference

30.

B. Himpens, H. desmedt, G.Droogmans andR.Casteels, Am.J. Physiol. 263: C95-ClO5 (1992).

3 1.

D.Lipscombe, D.V. Madison, M. Poenie, H. Reuter,RY. Tsien

19

and R.W. Tsien, Proc. Natl. Acad. Sci. USA85: 2398-2404 (1988) 32.

A. Hernandez-C-, F. Sala and

P.R Adams, Science 247 858-

862 (1990).

33.

D.A. Przywara, S.V. Bhave, A. Bhave, T.D. Wakade andA.R Wakade, FASEB J.5: 217-222 (1991).

34.

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38.

RV. Yelamarty, B.A. Miller,RC. Scaduto, Jr., F.T.S. Yu,D.L. Tillotson and J.Y. Cheung, J. Clin. Invest. 85: 1799-1809 (1990).

39.

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S. Hechtenberg andD. Beyersmann, Biochem. J.289: 757-760 (1993).

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2 Effects of Antioxidants, Zinc, and Chelators on Free Radical Status of ChildrenLivingintheChernobyl Area Ljudmila G . Korkina Lab, Cell Biophysics and Biochemistry, Russian Institute of Pediatric Hematology, Leninskii pr. 117, Moscow 117513, Russia

Igor B. Afanas’ev 117820, Russia

VitaminResearchInstitute,Nauchnypr.

14A, Moscow

L INTRODUCTION Is the exposure of a human organism to prolonged low level irradiation really dangerous? This question now is not a theoretical one but one of utmost.importance for the people (the adults and especially children) living in the areas contaminated with radioisotopes after the accidents at atomicpowerstations and atomicreactors(ChemobylinUkraine, Chelyabinsk in Russia, etc) and exposed to low-levelbutcontinuous irradiation..Untilnowtheattempts to answer it wereunsuccessful possibly due totwo major remons: (1) Many classical hematologicaland biochemical testsarenotspecificallydesignedfordetection of such effects; (2) To make statistically significant conclusions, it is necessary to study a sufficiently large human populationexposed long enough to lowlevel irradiationto detect any real changes. Itis nowwellestablishedthat damaging effects of irradiationare mediated by the active o&gen species formed as a result of the direct interaction of y- and p-rays with the molecules of oxygen, water, and organiccompounds [ 1,2] . Moreover,incorporatedradioisotopesmay influencetheendogenoussystemsproducingactive oxygen species, 21

22

and

Korkina

Afanas'ev

for example, blood leukocytes, which is thought to be a main endogenous sourceofthesespecies [3]. Thesespecies(theinorganicradicals superoxide ion and hydroxyl radical, oxygen-centered organic radicals and hydrogen peroxide) are ableto interact practically with all important biologicalsubstances(lipids,proteins,nucleicacids,polysaccharides etc.) changing their properties and causing irreversible damage. As long as radiation has always been the part of environment, human beings have adapted to some extent to the potential risk of irradiation. Therearepowerfulantioxidantsystems in humanorganismswhich protectthecellsandnon-cellularmaterialagainstfreeradicals.They consist of enzymes such. as superoxidedismutases (SOD), catalase, glulathioneperoxidase,andglutathionereductaseandvariousnonenzymatic compounds (reduced glutathione, iron-binding proteins, antioxidant vitamins,uric acid, and so on). It is believed that disbalance between the rates of fkee d i c a l production and their inactivation by antioxidant defense systems may lead to pathologiessuch as tumors, degenerative, age-associated, environment-induced diseases and inflammatory and allergic states [4]. Most of these pathologies may be initiated by high-level or chronic low-level irradiationof an organism[5]. Therefore, it is veryimportant to detectthe fust subtlesignsof prmxidant/antioxidant disbalance before the development of irradiationinduced disorders. The main aim of present work is to study comparatively large groups of children from irradiated areas in orderto evaluate the very first features of thedeleteriouseffectsofaccidentalirradiation, to obtainnew knowledgeofpossiblemechanismofthedevelopment of disorders associated with low-level irradiation, andto develop non-toxic long-term administration capable of preventing irradiation-induced disorders. We investigated the effects of irradiation on the children who had been living for 6 years now in the regions contaminated with radioisotopes aftertheChemobylaccident.Contaminationlevelsintheseregions ranged from 15 to 40 Gykm2. Several major parametem of the organism's free radicalstatus including oxygen radical generation in the wholebloodandbyisolatedleukocytes,theactivitiesofantioxidant enzymes, and the changes in glutathione metabolism have been determined. The frequency of spontaneous chromosome breakage in the blood lymphocytes as well as the amount of &-staining material in the lymphocyte nucleolus wasalso determined.

Free Radical Status of Chernobyl Children

23

IL SUBJECTSAND METHODS k Children 156 Chernobyl .children of both sexes (62 males and 94 females) were practically healthy without any serious disorders in the main biochemical and hematologicalparameters.Thechildren'agerangedfrom 8 to 14 years (an average agewas of 11.4-2.1 years). For comparison, 21 healthy childrenofthesameagerange f i o i the Moscow region were tested after their informed consents. After preliminary testing, 70 Chernobyl children with the enhanced level of spontaneous luminol-amplified CL in isolated leukocytes were selected for clinical trial and randomized into 5 groups: Group A:16 children were given 400 lipoic mg acid day. a Group B: 14 children were given 400 mg lipoic + acid 200 mg vitamin E a day. Group C: 14 children were given 200 mg vitaminE a day. Group D: 14 children were given 100 mg zinc aspartate a day. Group E: 12 children without any treatment (control group). h the beginning and after completing the alinical trial, pediatrician examinationandallstandardhematologicalandbiochemicalanalyses essential for the evaluation of general living functions have been carried out. It was shown that there were no any side effects, allergic reactions, and other adverse events induced by the drug administration.

B. Chemicals Vitamin E was t?om Henkel Co. (Germany), lipoic acid (Tioctacid)was fi-om ASTA Medica (Germany), zinc aspartate was from Dr. F.Kohler ChemieCO(Germany).Luminol,lucigenin, phorbol-12-myristate-l3acetate (F'MA), reduced (GSI-I) and oxidized glutathione (GSSG), glutathione reductase, latex particles of 1 pm diameter,andcolchicine werepurchased from SigmaChemical Co., USA. Bovineerythrocyte superoxidedismutase(CuZnSOD, EC 1.15.1 .l) and thymol free bovine liver catalase @C I .l 1.1.6) were from Serva, Germany. Dextran sulfate and sodium metrizoate were from Phamacia, Sweden.

24

Korkina and Afanas'ev

Phitogemagglutinin (PGA) was h m Difco-P, USA All commercialreagentsandsolventswere of the highest purity.

other

C. Leukocyte and erythrocyte preparation.

5.0 m1 venousbloodwas drawn .beforebreakfast at 7.30 a.m.by disposable syringes with20 U/ml of heparin as an anticoagulant. 1.5 m1 blood sample was layered on the equal volume dextranof metrizoate mixture ( 5 2 v/v of 6.2% dextran and 38% metrizoate) and sedimented at 25% for 30 min. The cell-rich Supernatant was centrifuged at 15% for 10 min, and cell pellets were washed twice with cold Hanks' balancedsaltsolution (HBSS). Finally,thecellswere resuspended in medium 199 endstoredat 4% duringexamination. Cell viability was assessed by exclusion of 0.1% trypan blue; usually, it exceeded 95%. Cell differential count was confimed microscopicallybyGiemza , & S a &l suspensionconsisted of PMNs (50-60%), monocytes (4-10%) andlymphocytes (3045%). The contaminationwithplateletsanderythrocytes was negligibleanddid notaffectchemiluminescenceparameters. Erythrocytes wereobtained after the blood sedimentation on dextran-metrizoate gradlent. and washed twice with a cold 0.1M potassium phosphate buffer (pH 7.2). Washed cells were resuspended in the same buffer and their amountwas ajusted to 2% of hematocrit.

D. Chemiluminescence measurement of oxygen radical production by blood celis.

Luminol-amplifiedchemiluminescence(CL) was measuredon a LKB luminometer(mod. 1251,Sweden)equippedwith a computer to control experimental conditions, the data registration and estimation. 20 pL whole blood diluted 10 times with HBSS or 20 pL leukocyte swpension ( 2 ~ 1 0cells/ml) ~ wereadded to 0.85 m1 HBSS containing 50 p M luminol and incubated in the lml polysterene cuvette of the CL unit at 37O C andcontinuousmixingfor 5 min.Stimuli (PM 100 ng/mlor0.1%latexsuspension)weredispensedintothe CL cuvette automatically, and the light emission was recorded each 10 sec. Maximal intensity ofspontaneousandactivated CL was registered,andresults were expressed in mV/ 106 PMNs.

Radical Free

25

Status of Chernobyl Children

E. Determination of glutathione metqbolism in erythrocytes. The contents of reduced (GSH) and oxidized (GSSG) glutathione were determined bythe Beutler method[6].

F. The measurement of CuZnSODandMnSODactivitiesin erythrocytes and leukocytes.

Superoxide dismutase (SOD) activity was determined by the adrenaline method [7], in which the rate of superoxide production WBS measured by lucigenin-amplified CL [8]. Heparinized venous blood (0.2 ml) was hemolyzed with ice-cold water. Lysate was added to the equal volume of ethanol-chloroform mixture (1:1 v/v) and centrifuged at 1 500 x g for 30 min. Protein content in supernatantwas determined by the Lowry method [g]. The total SOD activity was calculated fiom calibration curve using commercial SOD as a standard and expressed as U/mg protein. For the determination of MnSOD activity, CuZnSOD WBS inhibited bythe additionof NaCN (4 to the supernatant. After that, the CL measurement of WSOD activity was performed as described above. CuZnSOD activity was estimated as a differencebetween the total and &SOD activities.

m

G. Measurement of spontaneous chromosome aberrations in lymphocytes

The whole blood was cultured accordingto the standard procedures [lo] with culture medium containing 75% Minimal Essential Medium, 15% fetal calf serum, and 10% whole blood. Lymphocytes were stimulated to divide by the addition of PHA at 0 h o€ culture. Blood was cultured at 37% in the 25 mL culture flasks for 56 h. 2.5 h before cell fixation, colchicine was added to a final concentration of 0.25 mg/ml. Cell fixation and the cytogenetic analysis were carried out by standard methods [l l]. 200-300 cells were analyzed for each subject.

H. Determination of &-staining material

in the lymphocyte

nucleolus.

Blood smears were prepared,fmed with methanol,-andstained with silver nitrate accordmgto method described previously[121. The stained smears

26

Afanas'ev

Korkina and

were examined microscopically and the results were expressed as average number of granules for300-350 nuclei analyzed.

L Statistical evaluation Statistical analysis was performed using Student's t-test assumed 5% as a level of significant difference.

EL RESULTS All Chernobyl children exhibited the strong featuresofinternal irradiation: the individual doses of incorporated radioisotoS being equal to 3 m 4 0 nCi and the excretion of radioactive isotope 7Cs with urine was w b i the rangeof 12-180 B&. Cytogenetic analysis showed that in comparison with normal cells the circulating lymphocytes from Chernobyl childrenhadasignificantlyhigherlevel of chromosomeaberrations especially slngle and double chromosome breakages comparing with the control children (Table 1). We found a greater number of Ag-staining granules in the nucleolus of Chernobyl children' lymphocytes (82% of children had from 1 to 5 granules per a nucleolus, 15% - from 6 to 10 granules, and 3% - more than 10 granules per a nucleolus) comparing with normalgroup (97% ofchildrenhadfrom1 to 5 granules pera nucleolus and3% - from 6 to 10 granules).

r

TABLE

1 3romosome aberrations in the blood lymphocytes. Number of Number of Subject . aberrant cells metaphases scored (percentage) 526 18,568 Chernobyl children (2.83%) (n = 98) p 0.001 Control 65 5,604 children (1.16%) (n = 22)

"

Frequency of aberrations ".

0.030 p 0.001 0.013

Radical Free

27

Status of Chernobyl Children

To evaluate the oxygen radical productionbybloodleukocytes, the luminol-amplified CL ,a sensitive and specific methodof measuring therelease of reactiveoxygenspecies @OS) bystimulated and nonstimulated phagocytic cells [131 was applied. We measured spontaneous CL and the CL producedby theleukocytesstimulated with PMA (thesolubleactivatoractingthroughprotein kinase C receptor) and latex particles (an activator of phagocytosis). As is seen from Table 2, bothnonstimulatedandstimulatedleukocytesisolated from the blood of Chernobyl children produced a significantly greater amount of ROS than those of Moscow children.

TABLE 2

CuZnSOD activity

133&21

(24)

61k5

MnSOD activity (Wmg protein)

3139

(24)

15G

(Uhg protein)

*) Standard deviation

(22)

28

and

Korkina

Afanas'ev

Thus, the initial spontaneous CL in isolated leukocytes nearly doubled that in noma1 children Thesametendency m observed for ROS production in the whole blood: the intensities of spontaneous and latexstimulated CL were equal to 10 and 210 mV/lO3 cells, respectively, for irradiated children andto 3 and 70 mV/lO3 cells for donors.We found a good correlation between spontaneous CL intensities and the number of Ag-staining granules in Chernobylchildren'lymphocyteswiththe correlationcoeffkient being equal to 0.83 and 0.80 forisolated lymphocytes and the whole blood, respectively. There was no significant difference in the erythrocyte GSH content between Chernobyl and Moscow children, however, the GSSG concentration and,Correspondingly, the GSSG/GSH ratio was 1.S times higher for Chemobyl children (Table 2). It was also found that both CuZnSOD andMnSOD activities were two times higherin the blood of Chemobyl children (Table 2) than in the blood of Moscow children Surprismgly, SODS activitiesdid not correlate with the intensities of spontaneous or activated CL as well as with the content of Ag-staining material in the nucleolus. Administration of the combinationof lipoic acid + vitamin E (Group C), zincaspartate (Group D), and a-lipoic acid .(Group A) led to significant changes in spontaneous CL of isolated leukocytes: its values diminished by4.67, 3.42, and 1.59 times, respectively (Fig. 1). It is seen that the levels of spontaneous CL achieved normal values in groups C and D that was confirmed statistically. In contrast, neither the combination of vitamin E + a-lipoic acidnor its componentstaken separately affected significantly the levelsof P M - and latex-stimulated CL @8ta are not shown). At the s m e time, Zn aspartate adminiStr3tiOn resulted in a decrease in PMA- and latex-stimulated CL by 40% and 25%, respectively. Thecombination of vitamin E and a-lipoicacid as well as Zn aspartate turned out to be very effective inhibitorsof PMA-activated CL in the wholeblood(Fig. 2). The same antioxidants significantly improvedglutathionemetabolism by decreasingthe GSSG level and, correspondingly, lowering theGSSG/GSH ratio down to its normal value (Data are notshown).Neitherlipoicacidnovitamin E takenalone affected the GSSG content.

Free Radical Status of Chernobyl Children

Group Healthy Group Group Group B C D donors A

29

Group E

Fig. 1. Spontaneous chemiluminescencein the isolated blood leukocytes before ( 0 ) and after ( H ) the clinical trial. Group A:children were given 400 mg a-lipoic acid a day for 28 days; Group B: 400 mg a-lipoic acid + 200 mg vitamin E; Group C: 200 mg vitamin E, G~oupD. 100mg zinc aspartate a day, Group E: control.

TV.DISCUSSION Our results show thatcontinuouslow-levelirradiationdoesaffect the Gee radical status of children living in the areas contaminated with radioisotopes. There are three different important parameters characterizing the intensity of the organism's oxidative stress, which were affected by low-level irradiation. 1. Spontaneousluminol-amplified CL of nonstimulated("dormant") leukocytesin the absence of anystimulusand CL of theleukocytes stimulated with either a soluble receptor-associated agonist (Pm)or a

30

Korkina and Afanas'ev

3004

v

Healthy Group Group Group Group Group

donors

A

B

C

D

E

Fig. 2. PMA-activated chemiluminescence in the whole blood after the clinical trial.

particular activator acting through an unreceptor pathway (latex particles) were enhanced by 2.2,5.0, and 1.8 times, respectively. The same tendency was observed for the whole blood. 2. The GSSG level andthe GSSG/GSH ratio in erythrocytes increased by 1.5times. 3. There was a significant (approx.200%) increase in the CuZnSOD and

MnSOD activities. It is known that under physiological conditions the level of oxygen radicals released' by "dormant" i.e. nonstimulated leukocytes normally is very low, but it sharply increases during a respiratory burst triggered by endogenous and exogenous stimuli such as microorganisms, tumor cells, immunecomplexes,complementcomponents,lymphocytes,etc.[ 1l]. Certain pathological condltions may drastically enhance oxygen radcal production by blood leukocytes through the so-called "priming"effect, which can be induced by inflammatory agents, allergenes, y-interferon,

Radical Free

Status of Chernobyl Children

31

tumor necrosis factor,growth factors, and some other biologically active compounds [123. In this study we have found for the fmt time that continuous low-level irradiation couldbe the in vivo 'priming" agent enhancing the production of active oxygen species by circulating blood leukocytes (and perhaps tissue phagocytes and bone marrow cells). It is a potentially dangerous symptom because "primed" cells can continue the inflammatory process which in turn can be the cause of the enhanced frequency of mutations, tumor initiation and promotion [13]. Thus, a significantly higher than normal level of chromosome breakage, which was found for Chernobyl children (Tablel), may be partly dueto the oxidative stress mediated by c i r c u l w leukocytes (Table2). The occurence of a pennanent source of oxygenradicaloverproductionwithmutageniccapacityapparently significantly enhances therisk of tumor development. It should be noted that occasional CL measurements, which were perfomed by us 3 years ago, did not show a large increase in the oxygen radical production by leukocytes of Chernobyl children. Therefore, the possibility of developing "free radical" pathologies obviously becomes higher with the prolongation of living in the contaminated areas. An increaseintheGSSGlevel(Table 2) is directevidence of the beginning of antioxidant depletionin the blood of Chernobyl children.It is known that free radicals fmtly attack the low-molecular antioxidants such as glutathione reduced, ascorbic acid (vitamin C), and a-tocopherol (vitamin E). A is virtually impossible to detect the effect of low-level irradiation on ascorbic acid and a-tocopherol because the amount of these vitamins in the organism dependson the qualityof the food consumed. It isalsoimpossible to detectsmallchanges in GSHconcentration; however, there was a significant increase in the GSSG concentration and the GSSG/GSH ratio. It is known that GSSG is a toxic compound andits formation is specificfortheconditions of oxidativestress [l 31. Therefore, the GSSG/GSH ratio is apparently one of the most important. an parameterscharacterizingtheprooxidantlantioxidantbalancein organism,and itsincreaseadditionallyindicatestheprevalence of oxidative processesin an irradiated organism. An increase in SODS activities (Table 2) in the blood of Chernobyl children maybeinterpreted as theresponseof an organism to the radiation-inducedoxygenradicaloverproduction.Indeed,it has been shown [ 14,151 that human and animal organisms as well as

32

and

Korkina

Afanas'ev

microorganismsexposedtoradiationareable to enhanceantioxidant defense systems via the induction of antioxidantenzymes,whichare thought to beregulatedbythelevel of ROS. It was shown by us previously [161 and in present work that Chemobyl children exhibited ROS overproduction.Actually,theexpressionof growing number of genesencoded SODS, DNA repair' enzymes,proto-oncoproteins,heat stress proteins, and others is now known to be modulated by oxidative stress [17]. This gene overexpression leadsto enhanced FWAdependent proteinsynthesis[l8]. It has beenreportedpreviously[l9]thatthe variability of the amount of nucleolar Ag-staining material reveals the degree of nucleolar, mainly transcriptional activity. Therefore, it seems to be of importance that there is a strong correlation between intensities of. oxygenradicalproductionandtheamountof &-staining materials, because this fact may reflect the process of adaptationto oxidative stress, which is talung place in Chernobyl population. Our results apparently suggest the benefits of longterm administration of nontoxic compounds topreventthedevelopmentof"freeradical"pathologies in subjects (children and adults) living in the areas contaminated with radioactive materials.Thesepreventingandtherapeuticagentsarelikely to be classicalantioxidants(such as vitamin E), zinc-containing drugs, and transition metal chelators.To the bestof our knowledge, there areat least four major parthways of suppressing free radical-initiated processes and reducingoxidative stress: a) the scavenging of freeradicals ; b)the stimulation of the antioxidantenzyme activities; c)the suppression of the activities of enzymescatalyzingtheoxygenradicalformation;d)the chelation and inactivation of active transition metalions, which catalyze the productionof hydroxyl and hydroxyl-like radicals. There is a Jarge body of evidence that shows lipoic acid exhibits both antioxidant a d chelatingactivities[20,211,and that vitamin E is a powerfid natural lipid-soluble antioxidant[22]. It was also sugested that non-transitionmetalsarecapableofindirectlyaffectingfreeradical processes.Atpresent,aspecialattention is drawn to theantioxidant activity of zinc compounds. Thus it wasfound that zinc inhibited the formation of free radicals in the in w'tro systems containing iron ions and cysteine, decreased the level of peroxidation products formed in erythrocyte membranes exposed to x a n h e oxidase, suppressed damagng free radical processes in cultured hepatocytes and superoxide production by human neutrophils, and protected E.coZi bacteria against

Free Radical Status of Chernobyl Children

33

copper-mediated paraquat-induced damage [23-271. Animal studies confirm the antioxidant capacity of zinc in vivo [28]. We found that short-term admjnistration of the combination of lipoic acid with vitamin E and zinc aspartate substantiallydecreased the level of spontaneous oxygen radical production by isolated blood leukocytes.At the same time, the combination of vitamin E and lipoic acid failed to inhibit PMA- orlatex-activatedoxygenradicalgenerationbyisolated cells. h contrast, zinc aspartate inhibited both spontaneous and activated oxygen radical production (Fig. 1 and 2). However, both the combination of lipoicacid with vitamin E andzincaspartatewereveryeffective inhibitors of spontaneous andactivated oxygen radical productionin the whole blood. Theabovefmdmgs could be explainedbythe direct inhibition of leukocyte NADPH-oxidase by zinc cations (our unpublished d ata)but this is not the case for vitamin E or a-lipoic acid. Apparently, vitamin E and a-lipoic acid acted as effectivescavengemof free radicals, while zinc aspartate is believed to be a regulator for cellular oxygen radicalproducing systems [27]. Thus our results show that vitamin E and lipoic acid are effective inhibitors of oxygen radical production by "primed" leukocytes but practicallydo not influenceNADPH oxidase activity and leukocyte activation. At the same time, vitaminE + lipoic acid and zinc aspartate significantly enhance the oxygen radical-scavenging activity of bloodplasma,that is apossiblecause of theinhibitionoflatexstimulated CL in the whole bloodof Chemobyl children.

ACKNOWLEDGMENTS Dr. F. Kohler Chemie GmbH company provided financialsupport for the clinical trial. We appreciate very much the advice of Elena Samochatova

in themedicalpartoftheclinicalstudy.TatianaSnigireva, Galina Ibragimova, and Irina Deeva provided valuable technical assistance in the laboratory work. REFERENCES 1. C.L.Greenstock, in Free Radicals, Aging, and Degenerative Diseuses, Alan R. Liss, Inc., 1986, pp.509-526.

34Afanas'ev

and

Korkina

2. J.F.Ward, A h . Radiat. BioL 5: 181, (1975). 3. B.M.Babior, Blood 64: 959 (1984). 4. B.Halliwel1and J.M.C.Gatterige, Free Radicals in Biology and Medicine. W o r d University Press, 1984. 5. C.L.Greenstock, Advances in Radiation Biology 11: 269 (1982). 6. E.Beutler, Red Cell Metabolism,Grune & Stratton, N.Y.1975, pp. 6971 and 112-1 17 7. H.Misra andL Fridovich, J. BioL Chem. 247: 3170 (1972). 8. H. Gyllenhanunar, J. ImmunoL Methods 97: 209 (1987). 9.O.H.Low1-y~ N.J.Rosebrough,AL.Fm, and R.J.hdal1, LBioLChem 193s 265 (1951). 10. RC-Allen and L.D.Loose, Biochern Biophys Res Commun 69: 245 (1976). 11. I.B.Afanas'ev, Superoxiide Ion: Chemistry artd Biological Implicatiom CRC Press, Bocs Raton, 1991; v. 2: pp. 87-134. 12. T.HFinkel, MJ.Rabst, RSuzulu, L.AGuthrie, J.RForehand, W.APhillips, and RB.Johnston, JBiolChem 262: 12589 (1987). 13. C.W.White and J.E.Repine, in Superoxide and Superoxide (G.Rotilio, Ed.), Dismutase in Chemistry,BiologyandMedicine Elsevier Sci. Publ., Amsterdam, 1986:pp. 524-527. 14. IFridovich,HorizBiochemBiophys., 1: 1 (1974). 15.T.Nakata, K.Sumki, J.Fujii, M.khikawa, H.Tatsumi,T.Sugiyama, T-Nishida,TShimizu, M.Yakushiji, and N.Taniguchi, Carcinogenesis 13: 1941 (1992). 16. L.G.Korkma, LB.Afanas'ev, and AT.Diplock, BiochemSoc.Trans 21: 314s (1993). 17. HSies, Amer: . l Mea! 91(3C): 31S(1991). 18. G.Storz,L-ATartaglia,and 'B.N.Ames, Science 248: 89 (1990). 19. D.Hemandez-Verdun,J. Cell S&. 9 9 : 465 (1991). 20. Y .J . S d , MTsuchiya, and L.Packer, Free RadRes. Comms. 15: 255 (1991). 21. L.Mullerand H.Menzel, Biochim Biopkys. Acta 1052: 386 (1990). 22. G.W.Burton, AJoyce, and K.U.Ingold, ArchBiochemBiophys. 221: 281 (1983). 23. C.Coudray, S.Rachidi, and AFavier, Biol Trace Element Res. 38: 273 (1993). 24. AW.Girotti,J.P.Thomas, and J.E.Jordan, Free Rad BioL Med 1: 395 (1 985).

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Free Radical Status of Chernobyl Children

25. P.H.Bemrik, P.C.Branneh and S.S.Hurles,J.Clin

Lab.ImrnunuL 21:

71 (1986).

26. M.-J.Richard,P.Guiraud, U-T.Lessia, J.-C.Beani, and AFavier, Bioi!

Trace Element Res. 37: 187 (1993). 27. M.Chvapi1, L.Stankova, C.Zukoski LLAClinMed 89: 135 (1977).

W, and C.Zukiski III,

28. D.E.Coppen, D.E.Richar&on, and R.J. Cousins, Proc. Soc. Exper.

Bioi! Med. 189: 100 (1988).

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3

The Role of Ascorbate in Metabolism and Genotoxicity of Chromium(VI) Karen E. Wetterhahn,DianeM. Stearns, Manoj Misra,Paloma H. Giangrande, Laura S. Phieffer, Laura J. Kennedy, and Kevin D. Courtney Dartmouth College, 6128 Burcke Laboratory, Hanover, NH 03766

I.

INTRODUCTION

Epidemiological studies have shown that chromium(VI) [Cr(VI)] compoundscause cancer, chromeulcersand allergiccontactdermatitisinpopulationsexposedto chromiumintheworkplace [1,2]. In addition to occupational exposure, significant environmental exposure to carcingenic chromium has been shown to occur because of landfill and waste sites contaminated with Cr(VI) [3,4]. For example, over 130 different sites in HudsonCounty,NewJerseyhavebeenfoundto contaminatedwithwastefrom Cr(V1) manufacturing plants [3]. Topical solutions of ascorbate have been used to prevent skin problems, i.e., ulcers and contact dermatitis, in workers exposed to Cr(VI) [2,5]. However, there is a considerable lack of information regarding the role of 37

38

Wetterhahn et al.

ascorbate (vitamin C) in Cr(VI)-induced genotoxicity both in vitro and in vivo. Intracellular reduction of the carcinogen Cr(VI) is widely considered to be a prerequisite for Cr(VI)-induced DNA damage [6]. Ascorbate has been shown to be the major reductant of Cr(VI) in rat liver, kidney and lung tissues [A. In vitro EPR studies indicate that reaction of Cr(VI) with ascorbate produces Cr(V), Cr(IV), ascorbate radical and othercarbon-centered radicals species [8]. Spectroscopic studiesindicatethatthefinal chromium(II1) product complexes with ascorbate and oxidized forms of ascorbate that are produced in the reaction [g]. Although Cr(VI) itself is unreactive toward DNA, chromium binding to DNA and DNA strand breaks are observed when DNA is reacted with Cr(VI) in the presence of ascorbate [lo]. The level of DNA damage induced by Cr(VI) in the presence of ascorbate appears to correlate with the levels of Cr(V) and radicals detected by EPR. In contrast to the glutathionemediated reaction of Cr(VI) with DNA which results in glutathione-chromium-DNA adducts [ll], it appears that the ascorbate-mediated reaction does not result in crosslinking of ascorbate to DNA by chromium. Ascorbate is required as a vitamin in the diet for the Osteogenic Disorder Shionogi (ODS) rat, making it an ideal in vivo animal model in which to study the effect of ascorbate on Cr(VI)-induced genotoxicity. Ascorbate was required for binding of chromium to DNA in liver of ODS rats treated with Cr(VI). Little hepatic Cr-DNA binding occurred upon Cr(VI) treatment of ascorbate-deficient ODS rats fed 5 ppm ascorbate in the diet. High Cr-DNA binding was observed in liver of ODS rats fed200 ppm ascorbate in the diet, a level which supports normal growth, andlower but significant Cr-DNA binding was observed at800 pprn dietary ascorbate. The hepatic Cr-uptake and glutathione

Ascorbate in Metabolism and Genotoxicity

of Chromium(V1)

39

levels were similar at 5, 200, and 800 ppm ascorbic acid in the diet. Depletion of glutathione caused a decrease in hepatic Cr-DNA binding in the ODs rats fed 200 and 800 ppm dietaryascorbate,suggesting thatglutathione is involved in Cr-DNA binding in the presence of normal ranges of ascorbate concentrations. These results indicate that ascorbate plays a critical role in activation of Cr(VI) to produce Cr-DNA binding in vivo. 11.

SPECTROSCOPICCHARACTERIZATION OF SPECIES FORMED IN THE REACTIONOF CHROMIUM(VI) WITH ASCORBATE

Thegeneralaim of the invitro studieshasbeento determine the types of species that are produced during the reaction between Cr(VI) and ascorbate, and to evaluate the factors which may influence the reactivity of these species. Varying the buffer or the ascorbate concentration relative to Cr(VI) was found to produce intermediates in differentrelativeamounts.Oncethesespecieswere identified and their relative stabilities established, their reactivity with DNA could be studied in vitro. A.

Reactive intermediates observed by EPR spectroscopy

Thereaction of Cr(V1) (1.8 mM, from K2Cr207) with (0, 0.5, 1.0, 1.5, 3.0, 5.0, and 10 sodiumascorbate stoichiometric equivalents) was studied by EPR spectroscopy in twodifferent buffers, N-[2-hydroxyethyl]piperazine-N"[2-ethanesulfonic acid] (HEPES, 0.10 M) and tris(hydroxymethy1)aminomethane hydrochloride (Tris. HC1, 0.050 M) at pH 7.0, at room temperature. Unstable free radicals were detected by reaction with 5,5-dimethyl-lpyrroline-l-oxide, (DMPO, 0.10 M). Chromium(V)and free radical EPR signals were quantitated as described [8]. Representative spectra havebeen published [8,10].

40

Wetterhahn et al.

A Cr(V) EPR signal was observed in HEPES buffer at g = 1.980 at the 0.5:l and 1:l reaction ratios of ascorbate to Cr(VI). Chromium(V) levels corresponded to 0.3 k 0.1 per cent of chromiumasCr(V)forthesereactionratios, No Cr(V) was respectively at a 1.3 minreactiontime. detected above the 1:l ratio in HEPES, or at any reaction ratio in Tris-HC1buffer.

Chromium(IV) levels were estimated by reaction of Cr(VI) (1.8 mM) with ascorbate (0 - 18 mM) in the presence of 3.6 mM manganese(I1) (from MnC12.4H20) through quantitation of the decrease of themanganese(I1) EPR signal after a 1.3 min reaction [8]. Concentrations ofCr(IV) were found toincrease with increasing ascorbate to Cr(VI) reaction ratio and ranged from 15 k 5 to 37 k 8 per cent of chromium as Cr(IV) for the 0.5:l to 10:l reaction ratios in both buffers. Carbon-based radicals were observed as DMPO-radical adducts in both HEPES and Tris-HC1 buffers [8]. These radicalswerepresumedtobeproducts of the Cr(V1)induced fragmentation of ascorbate. Carbon-based radicals were observed only at low reaction ratios (0.5:l and 1:l) of ascorbate to Cr(VI). Unlike the Cr(V) intermediate, levels of carbon-based radicals were lower in HEPES than in Tris-HC1buffer by approximately 3-fold, corresponding to 0.06 per cent starting ascorbate for the 1:l reaction ratio in HEPES buffer and 0.2 per cent starting ascorbate for the 1:l reaction in TrissHCI buffer [g]. The ascorbate radical anion was observed in both buffers during reaction of Cr(VI) with ascorbate [g], and was not trappedby DMPO. Levels of ascorbateradical anion increased with increasing reactant ascorbate concentration

Ascorbate in Metabolism and Genotoxicity of Chromium(VI1

41

for the 1.3 min reaction and ranged from 0.5 to 1 pM for the 0.5:l to 10:l ratios in HEPES and Tris-HC1buffer. B.

Finalchromium(II1)product observed by Whisible spectroscopy

Chromium(II1)levels weredeterminedbyUV/visible spectroscopy for the reaction of 1.8 mM Cr(VI) with 0 - 18 mM ascorbate in HEPES and Tris-HC1buffers (pH 7.0) at 1.3 min reaction and 30 minreactiontimes atroom temperature [9]. For all reaction ratios the spectra over time showed a decrease in the Cr(VI) charge transfer band at 370 nm and an increase in chromium(II1) visible bands at -400 nmand 570-580 nm. Levels of chromium(II1) increasedwithincreasingascorbateto Cr(V1) reaction ratios. Concentrations of chromium(II1) for the 1.3 min reaction ranged from 37 to 92 per cent of total chromium for the 0.59 to 1O:l ratios in HEPES, and 37 to 87 per cent in Tris-HC1. The finalconcentrations of chromium(II1) afterreactionshadgonetocompletion at 30 min correspondedto 43 to 100 percentchromiumas chromium(II1)forthe 0.5:l to 1O:l reactionratios, respectively, in bothbuffers. The final km,, for chromium(III) varied between buffers and reaction ratios. For 30 min reactions in HEPES buffer the 0.5:l reaction showed a visible absorbance at 568 nm spectrum with an whilethe 1 O : l reactiongaveafinal absorbance at 584 nm.Reactions in TrisaHCl buffer showed a final kmax of 558 nm and 580 nm for 0.5:l and 1O:l reactions, respectively. These differences likely reflect of thefinalchromium(II1) a differentcoordination productdependingonthe relativeascorbatereaction concentration.Inthepresence of excess ascorbatethe chromium(II1)wouldpresumablybecoordinatedby ascorbate, whereas in reactions with excess Cr(VI) the only

42

Wetterhahn et al.

potential ligands for chromium(II1) would be dehydroascorbate or, more accurately, its ring open form diketogularic acid [12]. Spectra of reactions of 10-fold ascorbate or dehydroascorbate withchromium(III) support this hypothesis. Differences between HEPES and Tris-HC1 bufferssuggestinteraction of theTrisbufferin chromium(II1) coordination. C.

Summary of intermediates and products produced from reaction of chromium(V1) with ascorbate

To summarize, the reaction of Cr(VI) with ascorbate at pH 7.0 produced Cr(V), Cr(IV), chromium(III), carbon-based radicals, andascorbateradical anion. The relative concentrations of these intermediates could be altered by changing the reaction ratio and/or buffer. Chromium(V) and carbon-basedradicalswereonlyobserved at low reaction ratios in HEPES buffer, and carbon-based radicals butnoCr(V)wereobservedin Tris.HC1 buffer. Chromium(IV),chromium(II1)andascorbateradical predominatedathigh reactionratios inbothbuffers. These differences allowed for evaluation of the reactivity of these intermediates toward DNA. 111.

DNA DAMAGE RESULTING FROM THE REACTION OF DNA WITH CHROMIUM(VI) AND ASCORBATE

Unliketheothergenotoxicmetalsknowntomediate indirect oxidative damage of DNA[l31 Cr(VI)-induced DNA damage also includes direct chromium binding to DNA, as well as single-strand breaks, DNA interstrand cross-links and DNA-protein cross-links [14]. The role of theabovedescribedintermediatesintheformation of chromium-DNAadductsandsingle-standbreakswas evaluated in calf thymus and pBR322 plasmid DNA.

Ascorbate in Metabolism and Genotoxicity of Chromium(V1)

A.

43

Direct chromium binding to calf thymus DNA

Binding of chromium to calf thymus DNA was determined for DNA (1.8 mM DNA-P) incubated with Cr(VI) (1.8 mM, from K2Cr207) and ascorbate (0 - 18 mM) in HEPES and Tris-HC1 buffers (pH 7.0) after a 30 min incubationat 37°C. Non-covalently boundchromium was separated from DNA by NENsorb chromatography following published procedures [1l]. Concentrations of chromium and DNA in reaction samples were determined by atomic absorption spectroscopy and the diaminobenzoicacid (DABA) fluorometricassay [15], respectively, from which a chromium to DNA ratio was calculated (Cr/DNA-P). Levels of chromiumboundto DNA werehighestfor reactions of 1:l ascorbate to Cr(VI) in HEPES buffer and correspondedto 29.3 & 6.1 x 10-3 Cr/DNA-P. The 1:l reactioninTris-HC1bufferresulted in 8-fold lower binding, or 3.7 f 1.1x 10-3 Cr/DNA-P [lo]. Preincubation of Cr(VI) with ascorbatefor 30 min at 37°C priorto incubationwith DNA resultedindecreasedlevels of chromium bound to DNA, as did reaction of calf thymus DNAwithsolutions of chromium(II1) andvarying ascorbate that had been preincubated for 30 min at 37°C. Based on the above spectroscopic studies, these results are consistent with the interpretation that Cr(V) is the form of chromium reacting with calf thymus DNA. B.

No formation of ascorbate-chromium-DNAadducts

The reduction ofCr(V1) by GSH in the presence of calf thymus DNA was shown to result in a GSH-Cr-DNA of adduct [ll]. In order to evaluate the analogous role ascorbate in chromium-DNA adduct formation the DNA bindingexperimentsdescribedabovewerecarried out

44

Wetterhahn et al.

with 14C(l)-labelled ascorbic acid. Calf thymus DNA (1.8 mM) was incubated with Cr(VI) (1.8 mM) and varying Wascorbic acid (0 - 9.0 mM) in either 0.10 M HEPES or 0.050 M Tris.HC1 buffers at pH 7.0 for 30 min at 37°C. Amounts of chromium and DNA-P were determined as described above, and levels of W-ascorbate were determined by scintillationcounting. The presence of Cr(V1) didnot result in ascorbate binding to DNA above control levels. C.

Single-strand breaks in pBR322 plasmid DNA

Single-strand breaks in pBR322. DNA were measured by gelelectrophoresisafterreaction of plasmid (0.36 mM DNA-P, 82 nMplasmid)with Cr(V1)(1.8mM, from K2Cr207) and ascorbate (0 - 18 mM) in HEPES and TrisoHC1 buffers (pH 7.0). Plasmidwasincubated with reaction solutions for 30 min at 37°C. Relaxation of supercoiled plasmid was visualized by ethidium bromide staining and quantitated by scannirig densitometry. Amounts of relaxedplasmidwerehighest for the 1:l reaction ratios and were 2-fold higher for reactions in Tris-HC1comparedto HEPES buffer. Based onthe spectroscopic studies presented above, this observation suggested that carbon-based radicals rather than Cr(V) were responsible for the plasmid relaxation. This hypothesis was tested by carrying out reactions at the 1:l ascorbate to Cr(VI) ratio in the presence of 0.10 M DMPO whichwasshownby EPR spectroscopytoreactwith carbon-based radicals but not Cr(V). Levels of plasmid nicking were found to decrease in both buffers in the presence of DMPO. Fromtheseexperimentsitwas concludedthatplasmidrelaxationwaspredominantly caused by the carbon-based radicals produced during the reduction of Cr(VI) by ascorbate.

Ascorbate in Metabolism and Genotoxicity of Chromium(V1)

IV.

45

IMPLICATIONSFOR IN W O GENOTOXICITY

The results of the in vitro spectroscopic and DNA damage experiments with Cr(VI) and ascorbate are summarized in Table I.

Effect of the ascorbate concentrationon reaction intermediates andDNA damage produced during the in vitro reduction of chromium(V1) by ascorbate.

TABLE I.

Asc : Cr(VI) Reaction Ratio

1O:l

1:l

Chromium(V) Chromium(1V) Chromium(II1) Carbon-Based Radicals Ascorbate Radical

none

++

++ + + ++ +

Cr-DNA Adducts Plasmid Relaxation

none none

max max

++ ++

none

Chromium(III) was found toreact with DNA quite slowly when it was fully coordinated by ascorbate or oxidized products compared to "uncoordinated" hexaaquo chromium(II1). Fullycoordinatedchromium(II1)isthe likely form that will be produced by the in vivo reduction of Cr(VI); therefore, our results are consistent with the hypothesis that the more reactive intermediates Cr(V) and carbon-basedradicalsareresponsible for the Cr-DNA adducts and plasmid relaxation in vitro, and that these types of intermediates will likely be the genotoxic agents in vivo. Theformationand/ordecay of the reactive intermediateswasstrongly affected bythereactant ascorbate concentration. The hypothesis that the in vivo

et

46

Wetterhahn

al.

Cr(VI)-induced DNA damage would also be affected by intracellular ascorbate concentration was tested in a rat model. V.

EFFECTS OF ASCORBATE ONCHROMIUM(V1)-INDUCED DAMAGE IN ODSRATS IN W O

A.

Animal model

Most in vivo experimentalanimals,includingnormal species of rats, biosynthesize L-ascorbic acid. Humans cannot synthesize L-ascorbic acid and thus require it as a vitamininthe diet. In order tomimicthe range of vitaminCfoundinhumans,ananimalrequiring Lascorbic acid as a vitaminis needed. The ODS rat is a mutantWistarratwhich lacks Lgulonolactone oxidase (GLO, EC1.1.3.8), the enzyme which catalyzes the last step in the biosynthesis of ascorbic acid [16,17]. Studies by Horio et al. [l81 have shown that the ODs rat grows normally and shows nosigns of vitamin C deficiency when maintained on a diet containing -300 ppm ascorbic acid. TheODS rat was used to study the effects of ascorbate on Cr(VI)-induced genotoxicity in vivo. B.

Diets andtreatments

The ODS rats were initially purchased from Clea Japan, Inc., Japan and bred at Veterans Administration Medical and RegionalResearchServiceCenter,Vermont.The ascorbic acid-free (basal) and supplemented diets were purchased from Zeigler Bros.,Inc., Gardners, PA, USA. Six-eight week old male ODS rats were usedin the present study. The rats were kept at 24°C with a 12-h light cycle (from 0800-2000 h) and dark. All ratswerehousedin duplicate and were provided feed and water ad libitum.

Ascorbate in Metabolism and Genotoxicity of Chromium(V1)

47

Before the experiment, rats were divided into six groups each consistingof 6-8 rats and two of each group were fed abasaldietsupplementedwith 5, 200, and 800 pprn ascorbate for 14-days. On the 14th day, groups of each diet were injected intraperitoneally (i.p.) with 10 mg of sodium dichromate/kg body weight for 4 h with or without 5 h pretreatment of 4 mmol of L-buthionine-S,R-sulfoximine (L-BSO)/kg body i.p. Before diets were fed to rats, diets supplemented with ascorbic acid were tested for actual ascorbic acid content by the HPLC-ECD method described below. C.

Dietary ascorbate and hepatic ascorbate levels

Ascorbatelevelsweremeasuredbyhighperformance liquidchromatographywithelectrochemical detection (HPLC-ECD) using a modified method of Berger et al. [19]. The hepatic ascorbate level was about 10-fold higher in rats fed 200 pprn ascorbate and about 16-fold higher in rats fed 800 pprn ascorbate than ratsfed 5 ppm ascorbate in diet for 14 days (0.05 f 0.007, 0.51 k 0.072, and 0.93 k 0.1 pm01 ascorbate/g tissue in rats fed with 5, 200, and 800 ppm ascorbate in diet for 14-days, respectively). BSO treatment hadno effect onhepaticascorbate levels although glutathione (GSH) levels were decreased 60-65%, suggesting that there is no direct relationship between GSH and ascorbate levels in ODS rats. Cr(VI)-treatment in BSO-pretreated rats fed 800 pprn ascorbate increased the ascorbate level as compared to saline or Cr(VI)-treated rats suggesting asynergistic effect of Cr and GSH-depletion on ascorbate levels. However, no effect of Cr(VI) on ascorbate 5, 200, and 800 ppm levels wasobservedinratsfed ascorbate and in BSO-treated rats fed with 5 and 200 ppm ascorbate in the diet.

48

D.

Wetterhahn et al.

Ascorbate levels and Cr-uptake

Pre-weighed liver was digested with nitric acid and H 2 0 2 essentiallyasdescribed by Veillon and Patterson [20]. Chromiumwasdeterminedbyatomicabsorption spectroscopy on a Perkin-Elmer 503 atomic absorption spectrophotometer with an HGA-2100 graphite furnace. Different levels of hepatic ascorbate did not affect Cruptake levels. In rats fed 5, 200, and 800 pprn ascorbate, the levels of hepatic Cr-uptake were0.54-0.75 pm01 of Cr/g tissue in Cr(V1)-treated rats. Also, BSO-pretreatment did not affect the levels of Cr taken up in livers of Cr(V1)treated rats. This result suggests that hepatic Cr-uptake is not dependent on ascorbate or glutathione levels. E.

,

Ascorbate and hepatic Cr-DNA binding

Liver nuclei were isolated [21] and DNA was isolated and analyzed using the fluorescent dye Hoechst 33258 method [22]. Chromiumwasdetermined by atomicabsorption spectroscopy as described above. The level of Cr-DNA binding in liver was about 9-fold higher in Cr(VI)-treated rats fed 200 pprn ascorbate than rats fed 5 pprn ascorbate, but Cr-DNA binding was about 2.5-fold lower in Cr(V1)treated rats fed 800 ppm ascorbate than rats fed 200 ppm ascorbate. BSO-pretreatment decreased Cr-DNA binding in rats fed 200 and 800 pprn ascorbate in diet. A significant 2.8-fold decrease in hepatic Cr-DNA binding was observed 200 pprn inCr(V1)-treatedBSO-pretreatedratsfed ascorbate. These results indicate concentrationa dependent role of ascorbate in Cr-DNA binding in liver and also suggest that glutathione is involved in Cr-DNA binding in the presence of normal ranges of ascorbate concentrations.

Ascorbate in

G.

Metabolism and Genotoxicity of Chromium(VI1

49

Summaryof in vivo results

This is thefirstworkinanintactanimalsystem demonstrating the effect of dietary ascorbate on Cr-DNA binding in vivo. The results of the in vivo experiments with Cr(VI) in ODs rats are summarized in Table II.

II. Effect of different levels of ascorbate in diet on liver ascorbate, hepatic Cr-uptake and Cr-DNA binding in ODS rats after 4 h treatment with sodium dichromate.

TABLE

Ascorbate in Ascorbate diet (ppm)

5 200 800

+ ++

+++

Cr-DNA binding Cr-uptake -BSO -BSO +BSO +BSO

+ l +

+ +

+ +

+

+++ ++

I none

++ +

The availability of the ODS rats, which like humans, do not biosynthesize ascorbate, allowed us to evaluate the effect of dietary ascorbate levels on Cr-DNA binding in liver. Despite similar Cr-uptake at all levels of ascorbate tested (5, 200, and 800 ppm), Cr-DNA bindingwas significantly different. We found that dietary supplementation of ascorbate in amounts that supported normalgrowth of theanimals (200 ppm)weremost potent in inducing Cr-DNA binding inliver,whereas (5 ppm) little Cr-DNA binding was observed at lower dietary ascorbate, and higher (800 ppm) ascorbate levels caused less Cr-DNA binding than was induced by 200 ppm.Our resultsalsosuggestarole for glutathione, another endogenous antioxidant and reductant of Cr(VI), in influencing Cr-DNA binding. Thus for the first time, we have identified a function of ascorbate in facilitating

50

Wetterhahn et al.

Cr-DNA bindinginanintactanimal. These results indicate that ascorbate plays a critical role in activation of Cr(VI) to produce Cr DNA binding in vivo. ACKNOWLEDGEMENTS

This work was supported by PHS Grant No. CA34869, National Cancer Institute, DHHS (KEW). DMS was supported bya postdoctoral fellowship from the Norris Cotton Cancer Center, DartmouthHitchcock Medical Center, and an NRSA Fellowship (CA59292) from the National Cancer Institute, DHHS.PHG wassupported bya HowardHughesUndergraduate Biological Science Research Internship. LSP was supported by a Dartmouth College Presidential ScholarResearchAssistantship and a Waterhouse Research Grant. LJK and KDC were supported by the NSF Research Experiences for Undergraduates Program (NSF CHE-9100493).

REFERENCES 1. 2. 3. 4. 5. 6. 7.

8. 9.

S. Langard, Am. J. Ind. Med. 17189-215 (1990). N.B. Pederson, Top. Environ. Health 5:249-274 (1982). T. Burke, J. Fagliano, M. Goldoft, R. Hazen, R. Iglewicz, and T. McKee, Environ. HealthPerspect. 92:131-137 (1991). C.D. Palmer, and P.R. Wittbrodt, Environ. Health Pwspecf. 92:2540 (1991). D. Burrows in Chromium:Metabolism and Toxicity, CRC Boca Raton, Fla, 1983, pp. 137-163. P. H. Connett, and K. E. Wetterhahn, Structure and Bonding 5493124 (1983). (a) Y. Suzuki, and K. Fukuda, Arch. Toxicol. 64169-176 (1990). (b) A. M. Standeven, and K. E. Wetterhahn, Carcinogenesis 12:1733-1737 (1991). (c) A. M. Standeven, and K. E. Wetterhahn, Carcinogenesis 13:1319-1324 (1992). D.M. Steams, and K. E. Wetterhahn, C h . Res. Toxicol. 7219230 (1994). D. M. S t e m , L. J. Kennedy, K. D. Courtney, P. H. Giangrande, L. S. Phieffer, and K. E. Wetterhahn, Biochemistry, submitted for

publication.

Ascorbate in Metabolism and Genotoxicity of Chromium(VI1

51

10. D. M. Steams, K. D. Courtney, P. H. Giangrande, L. S. Phieffer, and K. E. Wetterhahn, Environ. Health Perspect. 202(Suppl 3):2125 (1994). 11. K. M. Borges, and K. E. Wetterhahn, Carcinogenesis 10:2165-2168 (1989). 12. B. M. Tolbert, and J. B. Ward, (1982) in Ascorbic Acid: Chemistry,

Metabolism and Uses (P. A. Seib, and B. M. Tolbert, Eds.), 200, Washington, DC., 1982, pp. Advances in Chemistry Series 116-118. 13. K. S. Kasprzak, Chem. Res. Toxicol. 4:604-615(1991). Z16914. (a) S. De Flora, and K. E. Wetterhahn, LifeChem.Reports 244 (1989). (b) S. De Nora, M. Bagnasco, D. Serra,and P. Zanacchi, Mutat. Res. 23899-172(1990). 15 J. M. Kissane, and E. Robins, J. Biol. Chem. 233984-188 (1958). 16. (a) S. Makino, and K. Katagiri, Exp. Anim. (inJapanese) 29:374375 (1980). (b) Y. Mizushima, T. Harauchi, T. Yoshizaki, and S. Makino, Experientia, 40: 359-361 (1984). 17. N. Nishikimi, T. Koshizaka, H. Mochizuki, H. Iwata, S. Makino, Y. Hayashi, T. Ozawa, and K. Yagi, Biochem. Int., 16:615-621 (1988). 18. F. Horio, K. Ozaki, A. Yoshida, S. Makino, and Y. Hayashi, J. Nutr. 115~1630-1640(1985). 19. J.Berger, D. Shepard, F. Morrow, and A. Taylor, J. Nutr. 119:734740 (1989). 20. C. Veillon and K.Y. Patterson in Environmental carcinogens

.

selected methods of analysis. Vol. 8--Some metals: As, Be, Cd, Cr, Ni, Pb, Se, Zn. (ONeill, I.K., Schuller, P., and Fishbein, L., Eds.) M C ,1986, pp 433-439. 21. H.S. Lilja, E. Hyde, D.S. Longnecker, and J.D. Yager, Jr. Cancer Res. 373925-3931 (1977). 22. C.Labarca and K. Paigen, Anal. Biochem. 102:344-352 (1980).

This Page Intentionally Left Blank

Inactivation of Critical Cancer-Related Genes by Nickel-InducedDNAHypermethylation and Increased Chromatin Condensation: A NewModelfor EpigeneticCarcinogenesis Max Costa Nelson Instituteof Environmental Medicine, N W Medical Center, Long MeadowRoad, Tuxedo, NY 10967

I.

INTRODUCTION

For a numberof years ithas been known that nickel carcinogenesisadds a unique component and is synergistic to the carcinogenesis process of other genotoxic carcinogens such as W, benzopyrene, and x-rays (1). The most carcinogenicnickelcompounds

are generally water insoluble andare

phagocytized by target cells (2). Phagocytosis permits large quantities of nickel to enter cells allowing rapid dissolution of Ni2+ by the acid pH of the phagocytic vacuoles (3). Solublenickel salts are generally not highly carcinogenic

in

experimental animals because the uptakeof soluble Ni*+is very poor, however, in tissue culture situations where extracellular exposure can be maintained, soluble nickelsalts are active (2). Crystalline NiS induces a number of genotoxic effects in cells including the production of strand breaks,DNA-protein crosshks, and generationof oxygen radicals that produce oxidized DNA bases (2). However, the majorityof these effects occurin heterochromatic DNA which

is not genetically active andthus has little mutagenic consequence (2).This may

explain why nickel compounds that are potently carcinogenic are generally not mutagenic in most systems (2). 53

54

Costa In the present report, we describe a new model forthe mechanism by

whichnickelmayproduce

its mostprominent

genetic effects during

that carcinogenesis. This model is based upon our previously reported findings

nickel inducesDNA hypermethylation and therebyturns off a senescence gene during the courseof nickel-induced transformation of Chinese hamster embryo cells (4). 11.

PHAGOCYTOSISAND DELIVERY OF NICKELCOMPOUNDS INTO THE CELL Using two model compounds, carcinogenic crystalline nickel sulfide and

non-carcinogenicamorphousnickel

sulfide that havebeen

studied by

Sunderman and colleagues for carcinogenic activity in experimental animals(5), we investigated the molecular mechanisms responsible for the differences in the carcinogenicity of these compounds(2). In cultured hamster cells, we found that the crystalline nickel sulfide particles w.ere actively phagocytized to a much greater extent than the amorphous nickel sulfide particles (3). Phagocytosis depended upon the surface charge, and we found that the crystalline nickel sulfide particles were negatively charged whereas amorphous nickel sulfide of particles were positively charged(6,7). When we changed the surface charge amorphous nickel sulfide particles by treating them with lithium aluminum hydride, we activated their phagocytosis and their cell transforming activity to levels similar to thatof crystalline nickel sulfide(8). This is shown in Tables 1 and 2. Water soluble nickel saltsare not potentially carcinogenic because they do not yieldhigh intracellular levelsof ionic nickel(2). Thus, the abilityof nickel is of obvious critical importance to its carcinogenic action (3). It to enter the cells

is puzzling though with nickel oxide that thelow temperature calcined black

form is more potentthan the high temperature green form, but it was difficult to

Gene Inactivation by Ni-Induced DNA Hypermethylation

55

Table 1 Phagocytosis of solvent washed andLiAlH4 reduced crystalline and amorphous NiS particles in CH0 Cells

Treatment compound Amorphous NiS Untreated Pyridine washed LiAlH4 reduced in pyridine

9.9 f 2.05 27.7 f 0.90

CrystallineaNiS Untreated Pyridine washed LiAlH4 reduced

29.2 f 252 33.3 f 9.8 53.1 f4.59

4.8 f 0.16

CH0 cells grownin monolayer culture were exposedin 60 mm diameter tissue

culture dishes to 10 pg/ml (1.78 pg/an3 surface growth area) of the particle preparations for 24 hr. Following treatment, cells were fixed, stained, and the percentage ofcells containing intracellular particles was detennined by light microscopy. The particle size of the preparations ranged from1p m to 3.6 p, and within this range there was little effect of particle size on phagocytosis. Each number shownin the tableis the meanof 4 plates where at least 500 cells were examined in each plate. No. of cells with phagocytized NiS particles/total no. of cells examined, meanf S.D.for 4 plates. Uptake differs from untreated particles. Pd.05 Student t-test

Reproduced with permission fromRef. 9.

study phagocytosis of the oxide particles. Perhaps there is some component involving membrane interactions that contributes to the carcinogenic process as well. This phenomenon has not been well investigated, however, there is evidence that signal transduction can substantially affect gene expression and one cannot rule out the possibility that these particles may perturb membrane function related to signal transduction. Following phagocytosis of insoluble particles, Ni2+ dissolves from the particle and high intracellular levels of nickel are generatedas modeled in Figure 1.

56

Costa

Table 2 Enhancement of cellular transformation following lithium hydride reductionof Crystalline and amorphous nickelsulfide particles Treatment compound

Transformed coloNes/total pg/d growth area

AmorphousNiS particles 1.0 p, (0.39) untreated 2/510 1.27

pyridine 2.66 pm,washed 1.27

17 5

10

6

1.27 2.07 pmwashed ,pyridine

0.13 0.63

(0.49)

10/2054

8

(0.41)

8/1949

9

1

0.13

5

5 10

0.63

8

1/636 (0.15)b 1/614 (0.16)b 3/248 (1.21)b

3

5/1088 (0.46)b

L i A l Q reduced 1 2.26 pm,reduced in pyridine 5 1.27 10

Crystalline aNiS particles 237 p, untreated

No.ofplatessurvivingcolonies(%.)a

0.13 3 (2.42)c 0.620/826

5

6 5

(1.47) 0.63 17/115!5

6

10 10/1784 1 6 5

10

6/183 (3.27)c

(2.67)

0.13 0.63 li7

5

4

27/1026 (2.63)c W268 (4.47)b

0.13 0.63 1.27

3 9 7

4/264 (151)d 24/972 (2.47)c 40/534 (7.49)C

(0.56)d

"

U A l Q reduced

1 3.76 pm,reduced in pyridine 5

10

The cell transformation assay was conducted as d e s c n i . ?he particle sizeof each preparation is shown with the treatment compound. aNo. of transformd colonies/total no. of surviving colonies bNot statistically significant from exposure to corresponding untreated particles CDiffers significantly from corresponding untreated particles: P 4 0 0 1x2test dcorresponding concentration not available for statistical analysis Reproduced withpermission from Ref. 9.

Gene Inactivation by Ni-Induced DNA Hypermethylation

57

Figure 1 111.

INTERACTION OF NICKEL WITH HETEROCHROMATIN

A promhent site for genetic damage induced by nickel compounds in

mammalian cellsis within heterochromatic chromosomal regions. This has been shownforbothChinesehamster

and mousecellswhichhave

different

organizations of their heterochromatin(10). In fact, the modeof delivery of Ni2+ into the cellis very important in inducing heterochromatin damage.If Ni2+ is delivered throughliposomesorby

particle phagocytosis, it damages

heterochromatin (11). If Ni2+ is presented to a cell as a soluble salt, it cannot enter the cell very well, andit also undergoes ligand exchange reactions such that most of the nickel ionsare not available for reactingwith nuclear proteins (11).

Figure 2 shows the damage that nickel selectively induces in

heterochromatin in Chinese hamster cells. This damage is illustrated by what appears to be decondensation of the heterochromatin, however, a mitotic chromosome is maximally condensed and this apparent decondensationis not relevant in a non-mitotic cell. In non-mitotic cells Ni*+ inducesan increase in

58

Costa .

...

d

Fig. 2 Chromosome damage inducedin Chinese hamster ovary cellsby NiC12 and NiS. Cells were treated for 16 h with 1 mM NiC12 and mitotic cells were collected following colcemid exposure. Figure 2A shows a cell where the heterochromatic long arm of the X chromosome was not properly condensed during mitosis. Figure 2B is a photograph of a Chinese hamster ovary cell X chromosome after the cells had been treated with crystalline NiS particles. Cells were treatedwith crystalline NiS particlesranging in concentration from10 to 20 pg/ml for 24 to 48 hr. The figure shows an increasing degreeof fragmentation of the long arm of the X chromosome with higher dosages and longer exposure times (a-i). Note the absence of fragmentation of the euchromatic short arm. from Refs. 11and 17. Reproduced with permission

Gene Inactivation Ni-Induced by

DNA Hypermethylation

59

A

i

=-

A

Fig. 3A C-banding of parental BALB/c 3T3 (1)and B200 cell (2) chromosomes. Arrows, regions of heterochromatic DNA.

Fig. 3B Localization of mouse satellite sequencesof BALB/c 3T3 (1) and B200 cells (2). Chromosomes of BALB/c 3T3 cells and B200 cells were hybridizedin situ with atritium-labeledmousesatellite DNA probelabeled by nicktranslation. The hybridized sequences were visualized by autoradiography and by Giemsastaining. Reproduced with permission from Ref. 13.

60

Costa

chromatin condensationas the resultof substituting for divalent metal ions, such as magnesium

(12).

The acquisition of nickel resistance in mousecells is

associated with fusionsof heterochromatin as shown in Figure3 (13). It should be noted that DNA-protein crosslinks were found to be induced selectively in heterochromatin in cells treated with nickel compounds (2) and that nickel bound selectively to magnesium insoluble fractions of heterochromatin (14,15). There is substantial evidence that nickel interacts with heterochromatin, and the selective interactionsof nickel with heterochromatin may explain why nicke! compounds are not generally mutagenic.

W.

INTERACTIONS OF NICKEL WITH MAGNESIUM BINDING

SITES IN CELLS

It has been known for some time that excess magnesium can antagoniz nickel-induced carcinogenesisin uivo (16). We have previously reported that magnesium can antagonize nickel-induced damage to heterochromatin (17) and that nickel-induced cell transformation can be inhibited by elevating the extracellular levelsof magnesium (17). Nickel and magnesium have very similar is thought to interact with magnesium binding in sites the atomic radii, and Ni2+

cell (17). This is particularly interesting with regard to heterochromatin iswhich maintained in an increased condensation state by several factors of which magnesium is thought tobe of great importance. Table3 shows the antagonism of nickel-inducedcelltransformation magnesium, and Table

4

by increasingtheconcentration

of

illustrates the antagonism of nickel-induced

heterochromatic damage by increasing the magnesium concentrations in the cell (17). Clearly, magnesium can inhibit nickel-induced heterochromatic damage

and its carcinogenesis. Therefore, there mustbe some relationship between the carcinogenesis. heterochromatic damage induced by nickelitsand

Gene Inactivation by Ni-Induced DNA Hypermethylation

61

Table 3 The effect ofM S 1 2 on NiC12 or K2CrOq-induced SHE cell transformationa

Treatment MgC12 Incidence morphological of survival SHE cell (mM) transformation

in SHE cells

(transformants/survivingcolonies)

0/680

0.8

0

5oow NE12

0.2 0.8 5 20

0/235 (0) (0) 0/130 (0)

94 100 71 59

0.2

11/901(1.2) 1/431(0.2) 1/565(0.2) 0/755 (0)

24 29 24 29

ND 21/149(1.4) 9/1039 (0.9) 1/1667 (O.l)b

ND 24 18 24

5/984(0.5) 10/1240 (0.8) 10/1028 (1.0)

18 24 18

5 20 0.2lOOOpM NE12

0.8

5 20 10W 0.8K2CrO4

("huntreated) of

0.2 20

om1

(0)

%HE cells were incubated for 2 h with Mg2+a "-free E M supplemented with 10% FBS and varying concentrations of MgC12. NiCl2 or K2CI04 were then addedanfor

additional 24 h. Following treatment,cultures were allowed to incubate for 14 days. Cultures were fixed, stained, and morphological transformation was determined. bp4.005 compared with 0.8 mM Mg&, Xi-ltest. Reproduced with permission from Ref. 17. How did nickel produce suchhigh a incidence of 6-TG resistance in G12 cells? There wasno structural mutations detected ingptthe gene, however,in 27 out of 31 "mutants" induced by nickel, there was hypermethylation in the coding and flanking regions of this gene (24). T h i s DNA methylation was associated

with the acquisition of decreased DNase1 sensitivity and decreased MSPl degradation of the gene (24). Additionally, thegpt gene was readily reactivated by treatment of cells with azacytidine which induces hypomethylation of DNA (24). It is clear that nickel can induce hypermethylation of genes that inactivates theirexpression.

This is alsoassociated

with increases in chromatin

is no longer expressed. condensation such that the gene

62

63

64 V.

Costa

NICKEL-INDUCEDOMDATIVEDNADAMAGE There is no question that nickel compounds can produce oxygen radicals

both in vi& and in vim. However, the way they produce oxygen radicals is very iron,cobalt different than the way most metals with active Fenton chemistry(e.g.,

and copper) produce oxygen radicals. Oxygen radicalsare very important in metal carcinogenesis processes (18). We have shown that nickel increases oxygen radical levels in intact cells (19). Kasprzak and others have shown that oxygen radicals can be produced by nickel in vitro (20). The interesting point about nickel-induced oxygen radical formationis that Ni2+ binds to certain peptide of Ni2+ from1.09 v to about0.79 ligands andthis lowers the oxidation potential v such that Ni2+ can now be oxidized by hydrogen peroxide to generate Fenton chemistry (21). Nickel may thus produce oxygen radicals at selected binding sites. However, the end result is that mostof the oxidative damage produced by nickel

OCCUTS

in heterochromatin which is genetically inactive DNA and,

therefore, nickel does not produce mutations in most cells. This may explain why the oxygenradicalsinducedbyNi2+

are of little direct mutagenic

consequence.

VI.

NICKELINDUCED DNA METHYLATION A number of years ago, in studies attempting to understand the

mechanism by which nickel induces carcinogenesis through its interaction with heterochromatin, we discovered

that male Chinese hamster embryo cells

transformed by nickel compounds exhibited the of losssenescence gene activity associated with the X chromosome (4,22). The

X chromosome contains the

longest contiguous regionof heterochromatin in the Chinese hamster genome and, therefore, nickel was targeted this to chromosome preferentially over other

Gene Inactivation by Ni-Induced DNA Hypermethylation

0

0.1

65

l .2

l .8

NiO Black (pglcrn')

Fig. 4 Comparative mutagenesisof NiS (A) and NiO black (B) in three cell lines -G12 (m), G10 (A) and V79 ( 0 ) . Results are meanof 2 independent experiments for each nickel compound. Bars are SD.

chromosomes.Theinactivatedsenescencegenewasnotfoundwithin heterochromatin of the X chromosome but rather was believed to be on the short arm of the X chromosome. Thus, this senescence gene was not deleted from within heterochromatin but was inactivated by DNA methylation as evidenced (4,22). The mechanism by which nickel from our experiments with azacytidine

could stimulate DNA methylation was puzzling. However, in studies using a transgeniccell

line (G12) wheretheendogenous

hpgrt gene had been

permanently inactivated and a bacterialgpt gene under the controldf an SV 40 promoter was inserted in the heterochromatic region of chromosome 1 (G12), nickel produced a very high incidence of 6-thioguanine (6-TG) (23). In contrast, placing the gene in another cell line, G10, on chromosome 6 far away from heterochromatin rendered cells unresponsive to nickel(23). Figure 4 illustrates the responsesof G12, G10 and wild typeV79 cells to carcinogenic crystalline NiS or NiO(23).

66 W.

Costa

MODEL FOR HOW NICKEL EXERTS ITS

EFFECTS ON

TRANSCRIPTION OF GENES NEAR HETEROCHROMATIN Figure 5 is a model for how nickel inactivates the transcription of genes. Nickel selectively binds to heterochromatin producing caged oxygen radicals at that site. It binds to histone H-1 substituting for magnesium and increases chromatin condensation.These

interactions cause the extension of

This can be modeled by aspool heterochromatin into neighboring euchromatin.

(heterochromatin) with thread (euchromatin) where the spool will pull in more thread. T h i s process is activated by Ni*+ binding to heterochromatin. These effects would possibly be transient, except that

DNA methylase enzymes

recognize that the new thread of DNA on the spool is not as heavily methylated

is, therefore, a as is the restof the DNAon the spool. The newly condensed DNA

Other CriticalGenes that

DNA Methyl

Cytosine Methylation

Figure 5

Gene Inactivation by Ni-Induced DNA Hypermethylation

67

substrate for theDNA methylase enzyme. DNA methylation causes the pulled in thread

to remain on the spool for subsequent generations since methylation

patterns are inherited. Thus by interacting with heterochromatin and inspooling moreDNA,nickelcaneffectivelyinactivateexpressedgenes,

such as the

senescence gene and other critical genes by hypermethylation leading to a genetically inactive state for subsequent generations.

ACKNOWLEDGMENTS I would liketo thank Dr. Catherine Klein for discussions and editorial assistance and Jane Galvin for secretarial help. This work was supported by NIEHS grant numbers ES 00260, ES 04895, ES 04715, ES 05512 and CA 16087 from the National Cancer Institute.

REFERENCES 1.

N.T.Christie, D.M. Tummolo, C.B. Klein,and T.G. Rossman, The role of Ni(I1) in mutation, in Nickel and Human Health: Current Perspectives (E. Nieboer, Ed.),JohnWiley, 1990.

2.

M. Costa, Annu. Rev. Phamcol. Toxicol. 31:321-337 (1991).

3.

M. Costa and H.H. Mollenhauer, Science 209515-517 (1980).

4.

C.B. Klein, K. Conway, X.W. Wang, RK. Bhamra, X. Lin, M.D. Cohen, L. Annab, J.C.Barrett, and M. Costa,Science 251:796-799 (1991).

5.

F.W. Sunderman, Jr., Carcinogeniaty of nickel compoundsin the animals, in Nickel in the Human Environment, IARC Monogr. Ser. (F.W. Sunderman, Jr., Ed.), IARCSci. F'ubl., Lyon, France,1984,53127-142.

6.

M.P. Abbacchio, J.D. Heck, R.M. Caprioli,

and M. Costa, Chemosphere

10897-908 (1981). 7.

M.P. Abbacchio, J.D. Heck,and M. Costa,Carcinogenesis 3175-180 (1982).

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8.

M. Costa and J.D.Heck, Cancer Res. 435652-5656

9.

J.D. Heckand M. Costa,Cancer Left. 1519-26

10.

P. Sen, K. Conway, andM.Costa, Cancer Res. 472142-2147

11.

P. Senand M.Costa, Toxicof.Appf. Phurmucof. 29:606-613

12.

N. Borochov, J. Ausio, and H. Eisenberg, Nucfeic Acih Res. l230893096

(1983).

(1982). (1987). (1986).

(1984). 13.

X.W. Wang,R J. Imbra, and M. Costa, CancerRes.486850-6854

14.

S.R Patiemo andM.Costa, Chem.-Biof.Interucf.55:75-91

15.

S.R. Patiemo, M. Sugiyama, J.B. Basilion, and M. Costa, Cancer Res. 455785-5794

16.

(1985).

(1985).

K.S. Kaspnak, M.P. Waalkes, and L.A. Pokier, Toxicol. Appl. Phurmucol. 82336-343

17.

(1988).

(1986).

KXonway, X-W. Wang,L.S.Xu, and M. Costa, Carcinogenesis81115-1121 (1987).

18.

C.B. Klein, K. Frenkel, and M. Costa, C h . Res. Toxicof.4592-604

19.

X. Huang, K. Frenkel, C.B. Klein, and M. Costa, Toxicof.Appf. Phurmucof. 12029-36

(1991).

(1993).

20.

K.S. Kasprzak, Chem. Res. Toxicof.4604615 (1991).

21.

D.W. Magerum and S.L. Anliker, Nickel(II1) chemistry and propertiesof thepeptide complexesof Ni(II) and Ni(III), in The Bioinorgunic Chemistryof Nickef (J.R. Lancaster, Ed.), VCH Publishers, New York, 1988,pp. 29-551.

22.

X.W. Wang, X. Lin, C.B. Klein, RK. Bhamra, Y.W., Lee, and M. Costa, Carcinogenesis 13555-561

(1992).

23.

B. Kargadn, C.B. Klein, and M.Costa, Mutut. Res. 300:63-72

24.

Y.W. Lee, C.B. Klein, B. Kargacin, K. Salnikow, N.T. Christie, and M. Costa, Mol. Ceff.Bwf. (submitted, 1994).

(1993).

5 Oxidative Mechanisms of Nickel(II) and Cobalt(II) Genotoxicity Kazimien S. Kaspnak Department of Human Health, Frederick Cancer Research & Development,Building 538, Room 205E,Frederick,MD217021201

I. INTRODUCTION There is experimental evidence that following in vitro and in vivo exposures, transition metals bind to cell nuclei[l-51. Therefore, current hypotheses suggest that such binding may play a direct [6,7l. role in the mechanisms of metal-induced carcinogenesis Infact,thebinding is oftenaccompaniedbygenedamaging effects,suchasconformationalchanges in DNA andnuclear proteins, strand breakage and depurination ofDNA the molecule, DNA base cross-linking chromatin of components, and modification.Thoseeffectsmaylead to mutationsdueto erroneous repair or replication of the damaged DNA template. Metal binding to enzymes which control DNA replication and 69

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Kasprzak

repair may further contribute to increased error frequency. And, competitionbetweentoxicandessentialmetalsinnuclear regulatory proteins, e.g., proteins containing "zinc fingers" [2,4], may cause abnormal gene expression. More details onthe above are presented in reviews by Sunderman [5-71 andCosta [8]. But, the essentialquestionremains:Isdirecttoxic metal binding (DNA-metal"adduct"formation)aloneresponsibleforthe observed genotoxic effects? It is likely that depurination, conformational alterations, and certain types of cross-links in chromatin are a direct result of "metaladduct"formation.However, the DNAbasedamage found in vitro [S] and in vivo [lo-131 cannot be explained in this way since the damaged bases are not metal adducts but oxidation products. A common explanation may be that the DNA damaging action of chromatin-bound metal is, at least in part, redox catalyticin nature. This assumptionis consistent with both the experimental thechemistryof the metalsinquestionand evidence in cell-free and in vivo systems, reviewed recently by several authors [14-181.

II. OXIDATIVEDNADAMAGE Carcinogenicmetals,suchasnickelandcobalt,aretransition elements and thus have rich coordination and redox chemistries. These factors, among others, allow carcinogenic transition metals to activate oxygen species. The most important reactions include 'Abbreviations and Formulae: 02,ambient oxygen;H202,hydrogen peroxide; O;', superoxide anion radical;'OH,hydroxylradical;A,adenine;G, guanine; C, cytosine; 5-me-C; 5-methylcytosine; T, thymine; His, L-histidine; NTA, nitrilotriacetate; GSH, glutathione; 8-oxodG, 8-oxo-2'deoxyguanosine; 8-oxo-dGTP, 8-oxo-2"deoxyguanosine triphosphate; intraperitoneal; iv, intravenous; GC-MS/SIM, gas chromatography-mass spectrometry with selected ion monitoring. For abbreviations of thedamaged DNA bases see Figure 1.

Oxidative Mechanisms of Nickel(ll1andCobalt(ll1 Genotoxicity

71

0; conversion to superoxide (0;')and/or H202in autoxidation H202conversiontohydroxylradicals ('OH) reactions,and through Fenton/Haber-Weiss chemistry [14,151. Formation of otherpotentoxidantssuchasmetal-oxoandmetal-peroxo [15,19]. Infact,increased complexeshasalsobeenobserved levels of oxygen activation products, which may damage chromatin, have been found in cells cultured in the presence of metals [20-241. Besides catalyzing the generation of reactive oxygen species, transitionmetalsmayassistintheattackofthosespecieson chromatinbyinhibitingcellularantioxidantandDNArepair systems [25-291and by directing oxidative attack to specific sites on DNA [30]. It is not surprising, therefore, that both in vitro and in vivo exposures of cells to carcinogenic transition metals result in production of modified DNA bases, strand breaks, and avarietyofcross-linksthat are alsocharacteristicforDNA damage caused by 'OH and other oxidants generated by ionizing radiation [14,15,311. Let us look at some examples from our own work. A. Nickel

Experiments were conducted under conditions already proven to initiaterenalcarcinogenesis in rats [10,32]. PregnantFischer rats were injected ip with Ni(II) acetate at the end of gestation [ll]. Chromatin was isolated from kidneys and livers of both 1 or 2 daysafterthelastinjectionand mothersandfetuses l), analyzedfor 11 oxidativelydamagedDNAbases(Figure usingthegaschromatography-massspectrometry/selectedion monitoring(GC-MS/SIM)techniquedevelopedbyDizdaroglu [33]. The results are presented in Figure 2. As can be seen in thisfigure,DNA in thekidney(atargetorganfornickel carcinogenesis) contained significantly elevated levels of promutagenic 8-oxo-Gua and Thy glycol. In contrast, DNA in liver (a non-target organ) did not contain increased levels of any establishedpromutagenicDNAbaseproducts.Thisfinding

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Kasprzak

supports our former suggestions of a possible role of 8-oxo-dG (DNA lesion the same as 8-oxo-Gua) in Ni(II)-induced carcinogenesis [10,34,35]. To confirm in animals our in vitro findings that L-histidine (His) enhances Nio-mediated oxidation of 2’-deoxyguanosine [35,36], male Fischer rats were injectedwith a single iv dose of the Ni(His), complex [l31 or, for a comparison,with equivalent doses of Ni(II) acetate, sodium acetate, or His. In addition to measuring the extent of DNA base damage, as above, the effect of Ni(His), on DNA-protein cross-linking was evaluated the using alkaline elution technique [13]. Asin the in vitro experiments, chelation with His was found to enhance Ni(II)-induced oxidative

Figure 1. Structures of active oxygen-generatedproductsof DNA bases, as identified by the use of the GC-MS/SIM technique according to Dizdaroglu 1331.

OxidativeMechanisms of Nickel(ll1andCobalt(ll1Genotoxicity

73

Figure 2. Levels of damaged DNA basesin kidneys and liversof pregnant rats and their fetuses following systemic exposure to Ni@) [ll]. The rats were injected with single ip dosesof 45 pm01 N i o acetatekg body wt each on days 16 and 18 of gestation and killedon day 19 of gestation. Control rats received equivalent doses of sodium acetate. A l l levels shown as > 100% wereincreasedsignificantly (P < 0.05 or better). See Figure 1 for abbreviations.

DNA base damage in the rat kidney (Figure 3). The extent of DNA-protein cross-linking was increased as well (Figure 4). At the dose levels applied, treatments other than with Ni(His), did not produce any significant increase in the base damage above control. To verify in vivo our in vitro findings that lipid peroxidesmay play a role in metal-mediated genotoxic effects [37,38], N i o -

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Kasprzak

1

1 Pyrimidinederived bases

Purinederived bases

Figure 3. Levels of damaged DNA basesinkidneys of rats following systeniic exposure to the Ni(His), complex [13]. The rats were injected with a single iv dose of 20 pm01 Ni(His)Jkgbody wt and killed 18 hr later. Control rats received an equivalent doseof sodium acetate. All levels shown as > 100% were increased significantly (P -C 0.05 or better). See Figure 1 for abbreviations.

induced DNA base oxidation was correlated with Nio-induced lipid peroxidation in the kidneys of mice [39,40] and rats [41]. In those studies, BALB/c, C57BL, B6C3F1, and C3H male mice and male Fischer rats received single ip injections of Ni(II) acetate. The levels of lipid peroxides and 8-oxo-dG in kidneys were determined 3 - 48 hr post-injection in mice (Figure 5 ) and 1 - 72 hr in rats (Figure 6), using the HPLC technique with electrochemical detection [lo]. A significant increase in 8-oxodG level was observed only in BALB/c mice, i.e., animals which were also very susceptible to Ni(II)-induced lipid peroxidation. In mice, both effects were temporary, indicating efficient repair of the damage. In rats,however, a transient increase in lipid

Oxidative Mechanisms of Nickel(ll1andCobalt(ll1

75

Genotoxicity

wan o.* Nws,

0..

0.10 0

6

S

12

0

3

6

9

12

Figure 4. Alkaline elution curves for renal DNA of rats killed 18 hr after a single iv injection of compounds specified in the legend. Closed symbols, samplesirradiatedwith 400 rads of y-radiationpriortotheelution. Open symbols, nonirradiated samples. A, without proteinase K digestion. B, after protein& K digestion [13].

peroxidation was followed by a persistent elevation of the8-oxodG level. In bothmiceand rats, the maximum levels of lipid peroxidation preceded those of 8-oxo-dG by at least12 hr. This finding casts some doubts on the possibility that lipid peroxides participate directly in the inducticn of DNA base damage in vivo. The observed species- and strain-related differences in response to Ni(I1)-mediated DNA base damage may, however, be a good indication of corresponding differences in susceptibility to renal carcinogenesis.

B. Cobalt Spectra of damaged DNA bases, similar to those described for nickel, have also been established in rats for cobalt 1121. Male and female Fischer rats were injected ip with different dosesof CO(@ acetate and killed 2 or 10dayslater.Renal,

76

Kaspnak BALWC

C57BL

BbC3F1

C3H

T

.

24 48

mln (h0

Figure 5. Lipid peroxide GPO) and 8-oxodG levels in kidneys

of mice

injected with a single ip dose of 170 pm01 of N i O acetatekg body wt and killed 3 48 hr later.Thecontrolmicereceived 340 pm01 of sodium acetatekg body wt. Asterisks indicate statistically significant differencesvs. the control levels, with P < 0.05 or better.

-

hepatic, and pulmonary chromatin from those rats was analyzed by the GC-MS/SIM techniqueaccordingtoDizdaroglu [33]. Some of the results are shown in Figure 7 [12]. No significant differences in the response were found between male and female rats. The damagedepended on CO@) doseandtargettissue, withkidneybeingthemostaffectedorgan. The renaland hepatic, but not pulmonary, DNA base damage tended increase to with time, implying possible inhibition CO@) by of DNA repair.

Oxidative Mechanisms of Nickel(ll) andCobalt(l1) Genotoxicity

77

the baseproductsfound are promutagenic the resultssuggestapossibilityofinitiationof carcinogenesis by cobalt in the investigated tissues. A bioassay to test this prediction is under way in our laboratory. The carcinogenic potential of cobalt is lower than that of nickel [43] which may be reflectedin relatively lower levels of damaged DNA bases observed in tissues of cobalt-treated rats. However, in vitro cobalt appears to be a much more potent catalyst DNA of base oxidationthan nickel [g]. Those differences are most likely due to substantial differencesin bioavailability of both metals at target sites.

Sincesomeof [14,15,42],

d

-

1

0

3

24

72

0

3

24

72

Time (hr) Figure 6. Lipidperoxide &PO) and 8-oxodG levelsinkidneys of F344/NCr rats injected with a single ip dose of 105 pmol of N i o acetatekg body wt and killed 3 - 72 hr later. The control rats received 200 pm01 of sodium acetatekg body wt. Asterisks indicate statistically differences vs. the control levels with P < 0.05 or better.

significant

l

Pyrimidine-derived bases

1

-

Punne-denved bases

Figure 7. Levels of damaged DNA bases in kidneys, livers, and lungs of rats following systemic exposure to C o o [12]. The rats were injected with a single ip dose of 100 pm01 C o o acetatekg body wt and killed 10 days later. Control rats received equivalent doses of sodium acetate. *, the control level in this case was below the detection limit. All levels shown as > 100 % were increased significantly (P < 0.05 or better). See Figure 1 for abbreviations. 78

Oxidative Mechanisms of Nickel(ll1and CobaltW Genotoxicity

79

III. DISCUSSION The significance of the above effects to carcinogenesis relieson evidence that at least some types of oxidative DNA damage are mutagenic. As shown above, the variety of DNA base damage in vitro and in vivo has been established for carcinogenic Ni(II) and C o o [g-121, without, however, verification of the resulting the otherhand,mutational spectra havebeen mutations.On determined for other metals, such as Fe(II), C u o , and Cu(II), with reversion and forward mutation assays using single stranded DNA templates exposed to the metal salts under air [44-461, but the underlying types ofDNAlesionswerenotresolved.The mutationswerepredominantlysinglebasesubstitutionswhich clusteredatdistinctgenepositionscharacteristicallyforeach metal. The most frequent mutations produced by Fe@) were G + C transversions followed by C + T transitions and G -* T Cue, the mutationswere transversions[45].WithCu(1)and predominantly C + T transitions followed by G +T transversions [46]. The G -* T transversions are characteristic for base mispairing by 8-oxo-dG (8-oxo-Gua) lesion [47,48]. A significant increase in 8-oxo-dG production has been found in renal DNA of male of carcinogenic Wistarratsgivenasingleparenteraldose in ratstreated with noncarcinogenic Fe(III)NTA, but not Na(I)NTA orFe(III)chloride[49].Interestingly,ratrenal mesenchymaltumorsinducedwithanothercarcinogen,nickel K-ras oncogene subsulfide,havebeenshowntocontaina activatedatcodon12exclusivelyby the G -* Ttransversion mutation [50]. Most recently, the 8-oxo-dG lesion hasalso been associated with altered methylation of adjacent cytosines, which, in turn, may affect proper gene expression [51] and lead to cell transformation and cancer. The origins of the remaining types ofpointmutations, mentioned above, are not clear. The mutations may be produced by various DNA lesions, including the damaged bases shown in Figure 1. For example, the C + T transitions are likely to be

80

Kasprzak

caused by mispairing on 5-OH-Cyt lesion [52], or through metalin enhancedoxidativedeaminationofCand5-me-Cresidues DNA [53]. Deamination of other bases may, perhaps, contribute Thy glycoland tothosemutationsaswell.Mutagenicityof 5-0HMe-Ura, arising in DNA of Ni(I1)-, or CO@)-treated rats, respectively [ll,121, is still debatable [15]. ReidandLoeb [54] andTkeshelashvili et al. 1551 have established that tandem double CC + TT mutations, known to occurvia W damagetoDNA, can alsobeproducedby treatmentsgeneratingactiveoxygenspecies,e.g.,by NiO, F e O , Cu(I),and C u O plus O2 and/or H202. The authors speculate that the mutations occur because of base mispairing at sites of cytosine dimers (intrastrand cross-linking), known to be produced by 'OH. The significance and specificity of cross-links, e.g., the DNAproteincross-linksobservedinrenalchromatinofNi(Hisbtreated rats [13], to certain types of mutation and carcinogenesis remain to be defined. It is believed that persistent cross-links may impair functions of the nuclear matrix, such as replication and transcription, and thus introduce genetic and/or epigenetic alterations into the affected cells [56]. Other metal-induced effects, such as DNA depurination and strand scissions,arepromutagenicevents [45,57], but they cannot be ascribed solelyto the catalytic modeof metal action on needs DNA.Theircontributiontometal-relatedmutagenesis further elucidation. Besidesmediatinggeneticdamagedirectlyatchromatin binding sites, metals may also affect genetic material remotely through promotion of lipid peroxidation, inhibition of cellular oxygenhandlingsystems,and/orinhibitionofDNArepair [14,15]. Lipid peroxidation products were found to mediatethe formationof8-oxo-dG [58] andstrandbreaks [59] inDNA. Lipid peroxidation by N i O in mice was found to be high in certainstrains(e.g.,BALB/c)that are lowinGSHandGSH B6C3F1 or C3H) peroxidasecomparedtootherstrains(e.g., [29,39,40]. Catalase and GSH peroxidase, which protect cells

Oxidative Mechanisms of Nickel(l1)andCobalt(l1) Genotoxicity

81

against metabolic peroxides, are inhibitedby N i O 1271. Carcinogenicmetalcations,including N i O and C o o , also have the ability to bind to cellular antioxidants such as ascorbate, cysteine, histidine, GSH, and others and to modify their reactions with oxygen species to produce free radicals [14,15]. Oxidative DNA damage can be prevented and/or repaired in living cells by various mechanisms [60-651. The cellular protection against 8-oxo-dG mutagenicity includes several enzymesactingagainst the 8-oxo-dGlesion.Dataonthose enzymes have been recently reviewed by Grollman and Moriya 1631. It is noteworthy that the activity of at least two enzymes the engaged in the repair depends on essential metals: that of 8-oxo-dGTP triphosphatase (MutT protein), which removes this damaged nucleotide fromthe nucleotide pool, depends on Mg(II) [66], while that of formamidopyrimidine DNA glycosylase (Fpg protein),whichremoves8-oxo-dGandsomeotherdamaged purinesfromDNA [67l, depends on Z n o (Fpg isa."zincfinger"protein [68]). Therefore, we maysuspectthatboth to inhibitionbyotherdivalent enzymesshouldbesensitive cations. In conclusion, among the various pathogenic effects produced by nickel, cobalt, and other transition metals, the mediation of oxidative damage appears to have a primary role in metal-induced carcinogenesis. REFERENCES 1. 2. 3.

S.E. Bryan, Heavy metals in the cell's nucleus, in Metul Zons in Genetic Information Trunsfer (G.L. Eichhornand L.G Marzilli, Eds.), Elsevier, New York, 1981, pp. 87-101. J.M. Berg, Science 232: 485-487(1986). A. Leonard,Chromosomedamageinindividuals exposed to heavy metals, in Metul Zons in Bwibgicul Systems Sigel, Ed.), Vol. 20, Marcel Dekker, Inc., New York, 1986, pp. 229-258.

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M. Dudaroglu, Free Radic. Biol. Med. 10: 225-242, (1991). K.S.Kasprzakand L. Hernandez, Cancer Res. 49: 5964-5968

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6 The Antimutagenic Effects of MetallothioneinMayInvolveFree Radical Scavenging E. I. Goncharova and T. G. Rossman

NelsonInstitute of Environmental Medicine, NYU Medical Center, Long Meadow Road, Twtedo, NY 10967

I. INTRODUCTION Metallothionein (MT) is a metal-binding sulfhydryl rich protein in which cysteine residues makeup one-third of the amino acid residues. MT's occur in vertebrates, invertebrates, plants, eukaryotic microorganismsandsome prokaryotes [l]. It has been well-documented that cadmium, zinc and copper salts, among others, induceMT expression [2]. Since its recognition ithas been suggested that MT plays a crucial role in detoxification of heavy metals, in the storage of metal ions and in the regulation of cellular Zn(I1)andCu(I1) metabolism [l]. MT has also been reported to play a protective rolein oxidative stress by scavenging free radicals [3,4]. V79 cells) The transgenic cell line G12 (derived from Chinese Hamster contains a single copy of the E. coli gpt gene as a target for mutagenesis [S]. The gpt target is sensitive to both genetic and epigenetic events [6] and is superior to the endogenoushprf gene for detecting mutagenesis byW, X-rays and agents causing oxidative damage [71. G12 cells have a very low level of endogenous MT. expression. A number of G12 derived MT.-over-producinga ll lines have been isolated after transfection with a vector containing the mouse MT-I gene under control of its own promoter region [81. We recently reported that M T - I transfectants have lower spontaneous mutation frequencies compared with the G12 parental cell line, and that the spontaneous mutation frequencyis by the inverselyrelatedtothelevel of MT expression[8].Mutagenesis alkylating agents N-methyl-N'-nitro-nitrosoguanidineandethylmethane 07

88

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sulfonate were not altered by MT expression. In this study, we examine the effects of MT on mutagenesis by the chemotherapeuticdrug amsacrine and the synthetic vitamin K analogue menadione. We demonstrate that MT can block "A-induced oxidative stress. In addition, we reporta protective role forMT in the survival of cells exposed to arsenite. Each of these agents is reported to produce free radicals by variousmechanisms.Ourresultssupportthe hypothesis that a protective role of MT against mutagenicity and toxicity of a number of cytotoxic agents is basedon its ability to act as a free radical scavenger.

It. MATERIALS AND METHODS A. CellCulture: The cell lines used were line G12, a gpt transfectant of Chinese hamster V79 cells [5], and MTl-2 and MT1-2A, MT transfectants of G12 cells[8]. Cells are grown in Hams mediumF-12,supplementedwith 5% fetal bovine serum (Gibco, Grand Island, NY) and penicillin-streptomycin (Gibco, Grand Island, N Y ) at 37O C in an atmosphere of 5% C02 and 95%

air.

B. Toxicity assay:

For clonal toxicityassay, cells wereseeded at a density of 300 cells/60 mm dish in normal medium. Test agents were added to the cells following attachment, and remained on the cells for the time chosen. The cultures were incubated in C02 for five to seven days, during which time clones developed to macroscopic size. Clones were fixed with methanol, stained with Giemsa (Gibco, Grand Island, NY) and counted. Agents used: mAMSA was a gift from Dr. C. Klein, NYU, menadionewaspurchasefromSigma(St.Louis, MA), andsodium (Fair Lawn, NJ). arsenite was purchased from Fisher Scientific C. Mutagenesis assay: The mutagenesis assayused has been previouslydescribed [5].

D. Gene Amplification: Gene amplification assays were performed according to the method Ottoof er a2 [91. The selective agent PALA(N-(phosphonoacety1)-L-aspartate)was added to the medium at sufficient concentration to give 10 x LD50. &D50 is the dose required to reduce clonal survival by 50%). The medium was changed weekly. After 4 weeks, PALA-resistant clones were fixed with methanol, stained with

89

Effects of Metallothionein and Free Radical Scavenging

Giemsa and counted. PALA was kindly provided Institute.

by the National Cancer

E. Fluorescent measurementof intracellular oxidants producedby TPA Cells were treated with 500 ngml TPA (phorbol 12-myristate 13-acetate) 50 PM (Sigma, St. Louis, MO) for 6 hoursandthenincubatedwith dichlorofluorescin diacetate (DCF-dAC)(Kodak,Rochester,NY)foran additional 30 min. Fluorescence was measuredas previously described forCH0 cells [lo]. Briefly, cells were washed twice with ice cold PBS, scraped from the late, and resuspended at lo6 cells/ml for fluorescencemeasurement. fluorescence was analyzed using a Fluorescent Spectrophotometer (Perkin Elmer, Norwalk, CT), using 502 nm excitation and 522nm emission.

f

3

m. RESULTS AND DISCUSSION A.

Protection by MT against mutagenesis and killing

' ' chemotherapeutic agent amsacrine (mAMSA). '

by the

Resistance of tumors and cellsin vitro to some anticancer agentswas found to correlate with elevated levelsof M T. Tumor cell lines selected for resistance to cis-platinum over-expressed MT and were cross-resistant to the alkylating agents chlorambucil and melphalan. Induction of MT by Cd(II) in these cells also conferred resistance to the same agents [ll]. Mouse C127 cells transfected with the MT IIa gene and showing high levels of its expression were resistant to melphalan and chlorambucil but showed no increased resistanceto bleomycin, doxorubicin, 5-fluorouracil or vincristine [l l]. DNA topoisomerases are unique enzymes thatcan break and rejoin the phosphodiester backbone ofDNA and thus change the topology of DNA. Topoisomerase I1 is localized at the baseof chromatin loops [12], where there aresequencescontainingconsensustopoisomerase I1 binding sites [13]. Topoisomerase I1may be involved in DNA replication, transcription, and recombination. DNA topoisomerase 11is a target of antineoplastic drug therapy [ 141. Several topoisomerase inhibitors including mAMSA, adriamycin, [ 151. ellipticines, and epipodophylotoxins are potent antineoplastic agents Type 11 DNA topoisomerases promote the passage of one double An intermediate step in topoisomerase I1 strand ofDNAthroughanother. reactions involves cleavage of DNA and covalent linkage of the enzyme to a DNA phosphate group. The binding is reversible and is normally followed by a subsequent rejoiningof the DNA ends after the strand passage [16]. mAMSA is a 9 anilinoacridine derivative developed as an antileukemicagent[17]. Originally it was thought that the DNA cleavage produced by mAMSA was

90

Gonchatova and Rossman

caused by free radicals. However, a number of lines of evidence now point toa connection between the cytotoxic actionof mAMSA and its ability to inhibit topoisomerase I1 [13,14,18]. Normally, topoisomeraseII catalyzes the breakage and rejoining of DNA strands. In the presence of mAMSA, both strands of DNA are broken and covalently attached to topoisomerase I1 [ 191. This is called stabilization of a “cleavable complex” between DNA and topoisomerase 11. Upon replication of the complex, DNA strand breaks occur [16]. Broken DNA ends are potentially recombinogenic, and may result in deletions, amplifications and translocations. The mutagenicity of mAMSA was first demonstrated inS. typhimirium, where frameshift mutations were induced [20]. mAMSAwas found to have weak but significant mutagenic activity at the hprt, but not at the Na/K ATPase locus in Chinese hamster V79 cells [21,22], and at the thymidine kinase locus of mouse lymphoma cells [23]. Cytogenetic analysis of these mutants showed that mAMSA is a potent clastogen [24], an effect also seen at the S-l (cell surface antigen) locus in a human-hamster hybrid cell line, where 92% of all mutants induced by mAMSA had megabase pair deletions [25]. The authors suggest that mqMSA-induced deletions may involve a series ofmany topoisomerase 11-DNA loop complexes, because it is unlikely that such large deletions are mediatedby the loss ofa single replicon. The toxicity and mutagenicity of mAMSA were compared in G12 cells and in MT1-2A cells, a G12 derivative which produces high levels of MT. MTl-2A cells are more resistant to the cytotoxicity of mAMSA compared with the parental G12 cells. mAMSA causes a dose-dependent increase in 6TG resistantvariantsinG12cells.However,thismutageniceffect is almost abolished in MT1-2A cells (Figure 1). To c o n f m that the MT content affects the mutagenic response to mAMSA, the mutagenicityof mAMSA was examined in MT transfectants with different levelsof MT expression (Figure 2). The cell line MTl-2, which has a lower MT content than MT1-2A, exhibited a higher mutant fraction after MT contentwasestimated by sensitivity to mAMSAadministration.The of mutant accumulation. Parental G12 cells witha cadmium chloride at the time low basal level of MT expression had the highest mutant fraction induced by mAMSA. The mechanismsby which MT reduce the toxicityand mutagenicity of mAMSA are not clear. One might speculate that its protective effect could related to its ability to scavenge free radicals. Besides acting as a topoisomerase inhibitor, mAMSA is also reported to generatefree radicals upon oxidation [26] A second possibility is that MT may act in some way to stabilize the cleavable complex, thereby preventing DNA strand breaks.

91

Effects of Metallothionein and Free Radical Scavenging

+c12 mwivsl "T1-2A Mwival +G12 mutagenesis -p"T1-2A mutagenesis

120

A

100

2

-> 0

5

n

80

F

i

(D

z

0

3

S

L

60

S

h

W

'E

-

3 -.

0

40

Y

0

c

U

e

r

E

20

I r 0

mAMSA (nglml)

Figure 1 Effects of

MT

expressionontoxicityandmutagenicity

of m A M S A

92

Goncharova and Rossman

MTl-2

-: e

0 2

MTl-2A

c

Cd" LDSo:

Figure 2

38pM

50pM

73pM

Mutagenesisinduced by mAMSA in cells expressing different levels

B. protection by MT against menadione(MD) toxicity and mutagenicity. Menadione (2-methyl-1.4 napthoquinone)is asynthetic analogueof vitamin K1. MD is capable of redox cycling and subsequently generating toxic oxygen species [27]. The cytotoxicity of MD probably results from hydroxyl radicals, since single strand DNA breaks are induced in MD-treated cells [28]. MD is also reported to cause depletion of glutathione pools [29] and oxidation of sulfhydryl groups in cytoskeletal proteins [30]. All of these effects point toan involvement of hydroxyl radical production byMD. MD has also been shown to induce MT synthesis [3l]. At low concentrations,MD reduces mutagenesis in G12 cells almost to the level seen in MTl-2A (Figure 3). MT1-2A cells are more resistant to the cytotoxic action of MD compared withG12 cells, especially at higher concentrations. MD is mutagenic to G12 cells at a concentration which allows approximately 35% survival. At the same concentration, MTl-2A cells show

Effects of Metallothionein and Free Radical Scavenging

Figure 3 Effects

of

M" expressionontoxicityandmutagenicity

93

of

menadion

little mutagenesis. Thus, MT protects cells fromthe cytotoxicity and Since the major mechanism of MD toxicity is related to mutagenicity of the production of reactiveoxygen species such as hydroxylradical, the protective roleof MT against MD-induced cytotoxicityand mutagenicity might be mediated by its free-radical scavenging ability. Low concentrations of MD are antimutagenic perhaps because MD can act as a free radical scavenger as well as a free radical generator.

MD.

C. MT Protects Against the Cytotoxicity of Arsenite Although the evidenceof a role for M" in metal resistance comes mainlyfrom studies on Cd(II), C u m , and Zn@) [32], a protective role for MT was also

1

W

0

Arsenite (pM)

metallot~ionein e x p r e ~ i o non a r ~ n i t ecytoto~icit

age in r ~ e via n f~ o ~ a of~ the o d~ i m e t h y ~ ~ n i c ies [36,37]. The i n d ~ ~ of ~on

binds arsenite in! vitro, ~senite

were grown in the 0 dose, which was th eoulsy sin^ clones resis ifica~onin GI2 and MTl-2A cells

450

4500

330.6

450

500

320.

96

Rossman

and

Goncharova

by which M" decrease spontaneous mutagenesis has no bearing on this other aspect of genetic stability.

E. Metallothionein Blocks Spontaneous and TPA-induced OxidativeStress There is much evidence that TPA acts as a tumor promoter at least in part by causing the formation of reactiveoxygen species [45, 463. Levels of intracellular oxidants were measured G12 in and MT1-2A cells grown in normal medium and after treatment for 6 hours with TPA (Figure 5). Basal levels of oxidants are lower in MTl-2A cells compared withG12 cells. Treatment with TPA leads toa two fold increase of the level of oxidants in G12 cells butonly a small increase in MT1-2A cells. Even after treatment with TPA, the level of oxidants in MT1-2A cells was lower than the basal level in G12 cells. These

6-

-s . 1

.

a

L

-

4-

e -

!!-

g- 3:$

E (D

8

9

(D

E

-

.

: 2.

u.. v . 0

.

1.

0-

GG 1 21+2

MT1-2A

TPA

Figure 5

MTl-2A+ TPA

Metallothioneinprotectsagainstspontaneous andTPA-inducedoxidativestress

97

Effects of Metallothionein and Free Radical Scavenging

mults provide further evidence for the protective roleMT ofagainst reactive oxygen species.

IV.Conclusion The agents used in this study (mAMSA, menadione, arsenite and "PA) are all one feature in common. They am all different in structure and function, but have able to produce reactive oxygen species which may as serve a key point for their MT toxicity. The data presented in this paper clearly demonstrate that high expression correlated withincreasedresistanceto the mutagenic and/or cytotoxic action of these agents. We hypothesize that a major mechanism for the increased resistance to these agents is the ability of MT to act as a free radical scavenger. The radical scavenging capability ofMT in tissues and cells was recently reviewed [47]. Data presented here provide additional support for this important physiological role h4T. of ACKNOWLEDGMENTS This workwas supported by NCI grants CA57352 and CA61319 and is part of NYU Institute of Environmental Medicine Centerprograms supported by grant CA13343 from the National Cancer Institute and grant ES00260 from the National Institute of Environmental Health Sciences. We thank Ms.Eleanor in document preparation. Cordisco for her expert help REFERENCES

1. D.H. Hamer, Ann. Rev. Biochem. 52913-951 (1986). 2.

M. Karin, Cell 41:9-10 (1985).

3. P.T. Thomalley, B.A. Teicher, M.P. Hacker, D.M. Hamer, and

K.V&,

Biochem. Biophys.Acta 82236-66 (1985).

4.

A. Bakka, A.B.S. Johnsen, I. Endersen, andU.E. Rugstad, Expenentia 28381-383 (1982).

5. C. Klein, andT. Rossman, Environ. Mol. Mutagen. 161-12 (1990). 6. C.B. Klein, Environ. Mol. Mutagen. 23(Suppl. 23):31 (1994). 7. C.B. Klein, L. Su, T.G. Rossman, and E.T. Snow,Mutat. Res. 304:217-228 (1994).

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E.I. Goncharova and T.G. Rossman, CancerRes., (1994) (In Press).

9. E.Otto, S. McCord, and T.D. Tlsty,J. Biol. Chem. 264:3390-3396 (1989). 10. X.Huang, K. Frenkel, C.B. Klein, and M. Costa, Toxicol.Appl. Phurmucol. 12029-36 (1993). 11. S.L. Kelley, A. Basu, B.A. Teicher, MP. Hacker, D.M. Hamer, and J.S. Lazo,Science 241:1813-1815 (1988). 12. W.G. Nelson, andD.S. Coffey, NCIMonographs 423-29 (1987). 13. W.C. Earnshaw, andM.M.S. Heck,J. Cell Biol. 100:1716-1725 (1985). 14. L.A. Zwelling, E. Estey, M. Bakic, Monographs 4:79-82 (1987).

L. Silberman, and D. Chan, NCI

15. L.F. Ziu, Ann. Rev. Biochem.58:351-375 (1989). 16. T.-S. Hsieh, in DNA Topology and its Biological Effects. Cold Spring Habor Laboratory Press, Cold SpringHarbor, W, 1990, pp. 243-262. 17. B.F. Cain, andG.J. Atwell, Eur. J. Cancer 10537-549 (1974). 18. L.A. Zwelling, MJ. Mitchell, P. Satipanway-cha,J. Mayer, E. Altshuler, M. Hinds, and B. Bugully, Cancer Res. 52:209-217 (1992).

19. T.C. Rowe, C.L. Chen, and Y.H.Hsiang, CancerRes. 46:2021-2026 (1986). 20. L.R. Ferguson, and W.A. Denny, J . Med. Chem. 22:251-255 (1979). 21. W.R. Wilson, N.M. Harris, and LR.Ferguson, Cancer Res. 44:4420-4431 (1984).

22. M.M.

Moore, D. Clive, J.C. Hozier, B.E.Howard,A.G. Turner, and J. Sawyer, Mutat. Res. 151:161-174 (1985).

Batson, N.T.

23. D. DeMarini, C.L. Doerr, M.K., Meyer, K.H. Brock, J. Hozier, and M.M. Moore, Mutagenesis5349-355 (1987). 24.

L.L. Deaven, M.S. Oh,and R.A. Tobey, J . Natl. Cancer Inst. 6 0 11551161 (1978).

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Effects of Metallothionein and Free Radical Scavenging

25. NL. Shibuya, A.M. Meno, D.B. Vannais, P.A. Craven, and C.A. Waldren, Cancer Res. 541092-1097 (1994). 26. J.L. Jurlina, A. Lindsay, J.E. Packer, B.C. Baguley, and W.A. Denny, Jr.,

Med. Chem.30473-480 (1987). 27. H. Thor, M.T. Smith, P. Hartzell, G. Belommo, S.A. Jewell,and S. Orrenius, J . Biol. Chem. 25212419-12425 (1982). 28. E.O. Ngo, T.-P. Sun, J.-Y. Chang, C.-C., Wang, K.-H.

Chi, A.-L. Cheng, and L.M. Nutter, Biochem. Pharmacol.42:1%1-1968 (1991).

29. D. DiMonte, D. Ross, G.Bellomo,

L. Eklow, and S. Orrenius, Arch.

Biochem. Biophys. 232334-342 (1984).

30. F. Mirabelli, A. Salis, M. Perotti, F. Taddei, G. Bellomo, and S. Orrerius, Biochem. Pharmacol.37:3423-3427 (1988). 31. K . 4 . Min, Y. Terano, S. Onosaka,and Phatmacol. 11374-79 (1992).

K. Tanaka, Toxicol. APPl.

32. L.D.H. Petering, and B.A. Fowler, Environ. HeallthPerspect. 65:217-224 (1986). 33. J. Liu, W.C. Kershaw, and C.D. Klaassen, Toxicol. Appl. Pharmacal. 10227-34 (1991). 34.

J. Liu, W.C. Kershaw,and D. Klaassen, J. Toxicol.Environ. Health.35:5162 (1992).

35. T.G. Rossman, in Handbook of Experimental Pharmacology, Toxicology of Metals -Biochemical Aspects (R.A. Goyerand M.G. Cherian, Eds.) Springer-Verlag, New York, 1994. 36. K. Yamanaka, M. Hoshino, M. Okamoto, R. Sawamura, A. Hasegawa, and S . Okada, Biochem. Biophys.Res. Commun.16858-64 (1990). 37. K. Yamanaka, A. Hasegawa, R. Sawamura, and S. Okada, Toxicol. Appl. Pharmacal. 108205-213 (1991). 38. Keyse, S.M., and Tyrrell, R.M. Proc. Natl. Acad. Sci USA 85:99-103 (1989).

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39. A. Albores, J. Koropatnick, M.G. Cherian, and A.J. Zelazowski, Biol. Interactions 85:127-140 (1992).

Chem.-

40. T. Maitani, N. Saito, M. Abe, S. Uchiyama, and Y. Saito, Toxicol.Le#. 3963-70 (1987). 41. M. Kreppel, J.W. Bauman, J. Liu, J.M. MC-, Appl. Toxicol. 20:184-189 (1993).

and C.D. Klassen, Fund.

42. 2. Wang, Y. Shore, and T.G.Rossman, A cadmium-sensitive Chinese hamster cell line with low constitutive level of metallothionein gene expression. WC.First International Symposium onMetals and Genetics, Toronto, 1994, p. 2 6 . 43. A. Albores, J. Koropatnick, M.G. Cherian and A. Zelazowksi, Chem.Bio1. Interactions85:127-140 (1992).

44. G.R. Stark, M. Debatisse, E. Giulletto, and G. Wahl, Cell 52901-908 (1989). 45. Wei, H.and K.Frenkel, Carcinogenesis 14(6):1195-1201 (1993).

46. R.A. Floyd, FASEB J. 4~2587-2597 (1990). 47. M. Sato, and I. Bremner, Free Rad. Biol. Medicine 14:325-337(1993).

7 Protection from Metal-Induced DNA DamagebyMetallothioneininan in Vitro System Lu Cai Department of Pathology,University of WesternOntario,London, Ontario N6A 5C1, Canada Jim Koropatnick and M.George Cherian Department of Oncology, London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario N6A 4L6, Canada

I. INTRODUCTION Chemicalalterationsin DNA resulting From theactionofoxygen free radicals formed during oxidant stress incells can result in mutagenic lesions. Although free radicals are formed during normal cellular metabolism, are most detoxified by physiological antioxidant systems [ 1-41. Most of the toxicity of in vivo is thought to arise from transition metal oxygen and hydrogen peroxide ion-catalyzed production of highly reactive hydroxyl radicals (.OH) by the Fenton reaction[2,5-81. For example, iron (a participant in the Fenton reaction) can promote the formationof reactive radicals that initiate oxidative damage to nucleic acids, proteins, and lipids [6-141. Cu(II) ions can also participate in 101

Cai et al.

102

formation of *OHby the Fenton reaction, and appear to be potentially more reactive in mediating oxygen radical-induced cytotoxicity and genotoxicity than Fe ions 115-191. may also enhance the effects of other oxidizing agents, including ionizing [20,21] and UV [22] radiation. Therefore, elucidating the mechanism of iron-or copper-induced oxidative damage to biological molecules is important in understanding the initiation and progress of several diseases, including cancer. Oxidant stress and DNA damage havebeen suggested to be critical in aging, mutagenesis, and carcinogenesis [23-251. Itis clear that severalmetal ions can bind with DNA or chromatin in vivo, or oxidative stress could liberate them from intracellular storage sites with subsequent binding to DNA [8-121. DNA could, in fact, be damaged in the presence of reagents capable of generating reactive oxygen radicals: hydrogen peroxide in the presence of iron [4,13-151 or copper [lS-191 is an example of such a situation. Although oxidizing radicals are produced at the cellular level,organisms can counter their deleterious effects through enzyme systems (catalase, superoxide dismutases and glutathione peroxidase) and antioxidants (vitamin C,vitamin E and thiolcontaining molecules) that inhibit cell damage, and can utilize DNA repair enzymes to correct DNA damage [25-271. Metallothioneins (MTs) are metal-binding proteins with a high (approximately 30 %) cysteine content: it has been suggested that they play a role in both essential metal homeostasis and resistance to heavy metal toxicity [28]. They may also play an antioxidant role [29-321. Similar to glutathione,MT maybindandinactivateavarietyof radicals, including hydroxyl and organic radicals induced by metals [33,34], radicals induced by ionizing radiation [35,36], and radicals induced by other chemical reactions in vivo [37]. Direct addition ofMT can inhibit hydroxyl radical-induced DNA damage in an aqueous in vim reaction system using Fe-EDTA [38]. On the other hand, Cd/Zn-MT has been shown to induce damage to supercoiled plasmid DNA through the generation of radicals [39]. The effect of MT on the ability of radicals to damageDNA in intactcellsis,therefore, an openquestion. However,thecellularcompartmentalization of MTwithinnucleiduring development [40,41] and in certain human tumours [42], and the importance of metals in chromatin structure and gene regulation [43-46] suggest that an

Cum

Protection from DNA

Damage by Metallothionein

103

intimate association of MT and DNA is of biological significance. We explored the possibility thatMT may protect DNA from radical-mediated damage using a free radical-generating system in which 'DNA was cleaved in vitro in the presenceofactivatedoxygen. These freeradicalsweregeneratedfrom hydrogen peroxidein the presence of ascorbic acid by a Fenton reaction, which is critically dependent on metals.

II. COMPARISON

OF DNADAMAGEINDUCED COPPER COMPOUNDS

BY R O N AND

When calf thymus DNA (66 pglml) was incubated with hydrogen peroxide (2 mM H,OJ and ascorbic acid (2 mM) in the presence of iron (50 PM)or copper (50 pM) salts at 30" C for 30 min in Chelex-treated 20 mM phosphate buffer @H 7.0), several degrees ofDNA damage were observed (Fig. 1). The EDTA to a concentration of10 mM reaction was terminated by the addition of and the intact DNA content was measured by fluorescence in the presence of l mM ethidiumbromide(EB)(excitationat 510 nm andemission at 590 nm)[47]. Enhanced fluorescence following interaction of EB and DNA is a measure ofthe integrity ofDNA a solution containing all reagents exceptKO, was assumed to have 100%fluorescence and0% DNA damage. DNA damage was measured as a loss in fluorescence[48]. In control experimentsEDTA was added before the addition ofcopper or iron and ascorbate to prevent the action of metal ions. Zero fluorescence wasassessed in a solution identical to control except that DNA was absent. 50 p M Cu reduced EB/DNA fluorescencebymorethan 95% while equimolarconcentrationsofironresultedinonly 30% damage. Thus, the ability of copper to damageDNA was much higher than that of iron under our experimental conditions (Fig. 1). A. Iron

Fifty p M Ferric ammonium sulfate reNH,(SO,)J induced only a 30% loss of fluorescence (Table1) compared to a95%! loss induced by equimolar Cum

104

Cai et al.

(Fig. 1) in the presence of 2 mh4 H202. Maximum damage was 67%.and was achievable only by increasing theH202 concentration to 6 mh4 and iron to 100

x 100 -

6

F 75E

a

50-

4

n 250(v

* * 0 0 c/)

3

0

v, 6,

LL

(v

0 6, LL

n

v

m n m

0 v, W

0

Z

W

a

G

0 a, LL

v I Z Q) LL

figure I Cu(I1) induces DNA damage in the presence of H202 and ascorbate more effectively than Fe(II) and Fe(I1I). Addition of 50 pM Cu(II), Fe(1I) or Fe(III) in the presence of 2 mM H202 and 2 mM Na-ascorbate caused loss of fluorescence of the ethidium bromide @B)-DNA complex. DNA damage was assessed as the loss of fluorescence. 100% damage results in complete loss of fluorescence.

Protection from DNA

105

Damage by Metallothionein

Table 1 DNA damage induced by FeNH,(SO,), in the presence and 6.0 mM H,O, and 2 mM ascorbic acid.

0 0.1 1.o 5.0 10 20 50 100 200

0

0

3.4 f 1.5 14.6 f 5.4 16.7 f 5.2 13.7 f 2.3 15.6 f 2.2 15.3 f 1.2 21.6 f 0.8 27.5 f 2.4

10.1 15.6 22.6 24.5 28.4 29.1 29.9 42.4

f 2.3 f 2.7 f 5.9 f 0.7 f 5.6 f 0.9 f 0.3 f 0.9

of 0.8, 2.0

0 6.9 f 0.1 11.7 f 3.1 26.2 f 6.6 30.5 f 0.9 28.0 f 1.7 41.4 f 6.8 67.5 f 1.5 65.4 f 2.0

also mediated a significant increase in oxygen radical-induced

DNA

damage that was dependent upon both hydrogen peroxide (Table2) and metal concentration (Table3). Relatively high levelsof F e 0 (125 pM) or hydrogen peroxide (4 mM) were required to induce a 50% loss in fluorescence.

B. Copper

Cum

was amoreeffectivemediator than of DNA damagecausedby hydrogen peroxide. It caused a 50% loss in fluorescence at less than 5 pM Cum in the presence of only 2 mM hydrogen peroxide (Table 4). Maximum loss of fluorescence in 2 mM hydrogen peroxide occurred at 10 pM copper chloride; in 0.2 mM hydrogen peroxide maximumDNA damage was induced

per chloride. Thus, Cu(I1) and Fe(I1) had gen peroxide to generate free radicals, w1 effective of the two.

t effwts on

0,on F ~ I I ) - ~ DNA d u ~damage"

1

DNA damage (95)

0

0

0.2 1.0 2.0

6.6 f 0.1

.o

A

12.4 j, 0.5 33.1 j, 0.5 49.52 f 0.02

ascorbic acid and 50 pM Fe(II).

ffwt of Fe(I1) on DNA damage in the presence of 2

F

DNA damage (%)

0 0.5 1.25

0

16.5 f 3.2 15.8 f 2.3 17 f 1 31 j , 4 47 I: 2

2

mci

Effect of Cu(I1) concentration on DNA damage in the 0.2 or 2.0 DIM H202

NA damage ((270)

2.0

0 0.1 1 .o 5.0 10.0 20.0 40.0 60.0 100.0 200.0

0

3.0 f 0.1 6.1 f 0.1 9.2 f 0.5 23.6 f 1.6 1.8 f 0.1 59 f 1 70 f 3 63.4 f 0.6 68 f 2

0 2

0 17 f 1 31 f 13 56 f 5 90 f 1 93.6 f 0.7 not done 98.2 f 0.7 98 f 2 not done

108

Cal et ai. 100.

L

i

*

95 -

90 -

*+ 85

~

0

300

900 600

1200

1500

Zn-MT (ug/ml>

100

50

I

0.0

0.5

1.0

I

1.5

2.0

H202

figure 2

Zn-MT protectsDNAfromdamageinducedby

&(IQ inthe

presence o f H202 andNa-ascorbate.(A)DNAdamageinducedby 50 p M Cu(I1) in the presence of 2 mM H202 and 2 mM Na-ascorbate'and increasing amounts of Zn-MT; (B) DNA damage induced by50 pM &Ci2 in the presence of increasing amounts of H202, (0) in the absence of Zn-MT, (A) with 4.12 pg Zn-MTlml; (0) with 41.2 pg Zn-MTlml.

109

Protection from DNA Damage by Metallothionein

III.

DNA DAMAGE INDUCED BY CU(Il)-MEDIAmD OXYGEN RADICAL IS INHIBITED BY METALLOTEXONEIN

Zinc-bound MT (Zn-MT) inhibited the ability ofCum to mediate radicalinduced DNA damage in the presence of hydrogen peroxide(2 mM) and Naascorbate (2 mM). The data are presented in Figure 2 (A,B). The inhibitory effect of Zn-MT in DNA damage was dosedependent in .the presence of hydrogen peroxide and ascorbate (Fig. 2A).As little as 40 pg MT/ml exerted a significant protective effect(Fig 2B). At a concentration of 500 pg/ml, ZnMT effectively protected 80% or more of DNA at risk for oxygen radicalinduceddamageatcopperconcentrations as high as 200 pM (Fig. 3). Compared to the marked protective effect of 100 p g / d MT. other proteins such as calf thymus histone (100 pglml) did not protect Cu(II)-induced DNA damage, and bovine albumin (100 pg/ml) exerted little protection (Fig. 4).

x

6

m a E

l

o

t

o

-

75

x

M1

a U

< 7

(3

0

@we

20

40 60 80 100120140160

1

180 200

3 Zn-MT (500 pg/rnl) protects DNA fromdamageinducedby increasing concentrations ofCu(I1) in the presence of 0.2mM H202 and 2 mM Na-ascorbate.

110

Cai et al.

100

Cu(I1) alone

+ Cu(I1)

+ MT (100 clg/ml)

7 5 +- Cu(I1) + His (100 clg/ml)

+ Cu(I1)

+

AL (100 clg/ml)

50

25

0l

I

0

10

20

figure 4 Calf thymus DNA damage induced by Cu(I1) is markedly inhibited by Zn-MT. Bovine albumin exerted little protection, and calf thymus histone did not protect. All reactions were camed out in the presence of 2 LUMH202 and 2 mh4 Na-ascorbate. DNA damage was assessed by loss of fluorescence loss of the DNA-EB complex. AL: albumin; His: histone.

111

Protection from DNA Damage by Metallothionein

W . FORMATION OF CU(I) FROM CU(n) AND ITS INHIBITION BY METALLOTHIONEIN

Cum

generated from Cu(I1)may be the active ion mediating free radical from Cu(II) in thepresenceof production[16,17]. Generation of hydrogen peroxide and ascorbate was determined using bathocuproinedisulfonic u O and acid (BCS)and the method of Li and Trush [ 161. Concentrations of C BCS (pH7.0) were varied to achieve a final BCS concentration of 0.3' mM in a total volume of 2 ml at 25" C The concentration of stable BCS-Cu(I) complex formed wasmeasured by absorption at 480 I"The amount ofmetal available for Cu(I) formation (and not hydrogen peroxide concentration) was

Cum

.

rate-limiting, with Cu(1) formation reaching a plateau at approximately 75 pM CuCl, (data not shown). The addition of Zn-MT (500 pg/ml) to a copper chloridehydrogen peroxidelascorbate system inhibited production of active from Cu(I1) (Fig. 5).

Cum

z

.75

,,P

C

0

aJ

d-

v

C 0

CUCl* (MM) Figure 5 Cu(1) production from Cu(I1) in the absence of MT (0) or presence of 500 p g / d h - M T (A). 2 mM H202+ 2 mM Na-ascorbate were included in reaction mixtures. Cu(1) formation was assessed by absorption at 480 m.

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V. DISCUSSION Superoxide radicals (O;-) are formed in all aerobic cells. Most damaging effects of systems capable of generating qhave been attributed to the metaldependent formation of more reactive species, such as hydroperoxyl radicals (Hop),singlet Oz, and hydroxyl radicals(-0H)[2,5-8]. Highly reactive *OH radicals can be produced by a reaction between Fe@) and H,O,: the process is termed the Fenton reaction and proceeds as shown in (1): Fe@)

+ H202+ Fe@) + -OH + OW

(1)

Many metal ions and their complexes in lower oxidation states (Cum, C@), and C oo,for example) can participate in this reaction to produce radicals capable of inducing oxidative DNA damage [5]. The present study confirms the previous reports on both Fe and Cu induced DNA damage in the presence of H202and ascorbate (Fig. 1). The hydroxyl radicals could also be generated from 0,- and H202in a Haber-Weiss reaction as shown in (2) and (3):

Fe@)

+ H,O,

+

Fe@)

+ *OH + OH-

(3)

Reactions (l), (2) and (3) are interrelated in that the Fenton reaction is the second step of the Haber-Weiss reaction. In reaction (2), 4"[which reduces Fe@) to Fe@)] can be replaced by alternate reducing agents- glutathione or ascorbic acid, for example [49-521. We reportthe differences in Fe andCu induced DNA damage in vitro in the presence of HzOz and ascorbic acid. The phenomenon displays two different features: (1) C u O induces much more extensive DNA damage compared to iron, confirming data from previous studies[48,53-551, and (2) DNA damage induced by Cum is markedly dependent on metal and H202 concentration. DNA darnage in thepresenceof iron, on theotherhand, was minor at equimolar metal and hydrogen peroxide levels, and became significant only at (6 mh4) and iron (125 PM). high concentrations of H202

Protection from DNA Damage by Metallothionein

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Muiras et al. [l41 demonstratedthatthedose-responsecurveforFe@)At afixed inducedDNAdamageinthepresenceofHzOziscomplex. concentration of Fe(I1) the maximum extent of DNA damage occurred at low HzOz concentrations (10-30 PM). Higher hydrogen peroxide concentrations markedly suppressed DNA damage: it was suggested that high levels of HzOz scavengedhydroxylradicalsandinhibitedthedamagingeffect[14,57,58]. Furthermore, their results also indicated that the dose-response curve Fe@)- for induced DNA damagein the presence of HzOz critically depended on the nature of the reaction buffer, ionic strength, temperature and pH [14]. However, the lack of suppressionof Cu(II)-induced DNA damage in the presence of ascorbic acid and high HzOz concentrations had previously been reported [48]: at the highest levels of peroxide applied mM), (10 almost complete DNA degradation occurred within 8 min. DNA degradation induced by Hz02 in the presence of catalyzing iron was about50 times slower than with copper (DNA damage was much less extensivethanthatobservedwithcopperunderthesame experimental conditions). They suggested that the phenomenon was due to differenca in the binding of Cum and Fe@) to DNA: scavengers of .OH radicalsprotectedagainstirondependent,butnotcopperdependent,DNA damage. It is well-establishedthatFeions[4,13-151 or Cu ions[15-19], can participate in the formation of *OH from Oz and HzOz, both in vitro and in vivo: the pattern of radical-mediated base modification in the presence of these metals is similar to that produced by ionizing radiation. However, the formation has been disputed. of .OH in reactions involvingCu(II) ions and HzOz [56,57] It has been reported that nitrilotriacetic acid inhibited DNA damage in systems containing Cu(II), butincreasedthereactivity of Fe [13]. In addition,the extensiveDNAdamageproduced by Cu(II)/HzOz/ascorbic acid is not significantlyinhibited by superoxidedismutase or by the .OH scavenger mannitol.Theinabilityofhydroxylradicalscavengerstoprotectagainst damage in several systems has been the basis of arguments that those radicals are not responsible forDNA damage [57,58]. Subsequent to those arguments, it was suggested that the production of modified DNA in systems containing ions and Hz02and/or 0,- or ascorbic acid is mediated by .OH [i.e., Cu(II) ions bound to DNA could react with HzOzand ascorbic acid to generate

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hydroxyl radicals, which then immediately attack and damage DNA bases]. Thus, in terms of its ability to promote damage to DNA, Cu(II) is an extremely active metal, much more than Fe(III) and FNII).Cu(I1) is also a very effective in promoting oxidation of some lipids [7]. The high activity of Cum in increasing hydroxyldependent damagetoDNAand tootherbiomolecules suggests that the intranuclear availability of Cum ions in vivo is likely to be carefblly controlled. The protection of DNA from Cu-induced oxidative damage may be important in limiting mutagenesis and consequent carcinogenesis. There is increasing evidence that MT can act as a free radical scavenger. Rat liver Cu-MT, for example, enhanced dismutation of superoxide radicals (W) in vifro [59]. MT has also been shown to decrease the toxic effects of both hydroxyl and superoxide radicals produced by the xanthinexanthine oxidase and whole animal reaction in virro [31]. In cultured cell [29,30,36] [35,37,60,61] experiments, a role for MT in scavenging free radicals generated from a varietyof chemical and radiation-induced sourceshas been implicated. This suggests that MT is ofimportance in cellular defense mechanisms directed against free radicals. In addition to its ability to protect, there is controversy with regard to the ability of MT, under some circumstances, to potentiate cellular damage. Cu,ZnMT actually stimulated microsomal lipid peroxidation initiated by xanthinexanthine oxidase in virro [62]. DNA damage could, potentially, be caused by unidentifiedradicalspeciesformedbyZn/Cd-MT[39]. Reports existthat overexpression of foreign MT genes in transfectedCH0 cells did not increase cellular resistance to subsequent oxidative challenges [63-66]. Here, however, it isclear thatMT did not increase Fe-or Cu-induced DNA damage,butrathermarkedlyreducedit. This supportsthereportthat FeinducedDNAdamagecould be inhibitedby MT [38],andextendsthe suggestion thatMT could directly protect calf thymus DNA from Cu-inducing damage in vitro. At the level of whole cells, induction ofMT expression may protect against subsequentfree radical-induced damage: zinc-induced Chinese hamster V79 cells exhibit both increased MT content (without increased GSH levels).and reduced susceptibility to HzOz-inducing DNA damage, while cells transfectedwithantisense MT-1RNAexpressionvectors aresensitiveto oxidant stress [60].

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DNA Damage by Metallothionein

115

The mechanismbywhichMTprotectsDNAfromfreeradical-induced in vitro ionizingradiation damagemay be bybindingthosefreeradicals: studies have shown that Zn,Cd-MT can effectively protect against radiationinduced damage to the tagadphosphate DNA backbone [67]. However, the mechanism by which MT inactivates free radicals is still unclear. Although MT does not normally bindFe in vivo, it appears to preventfree radical formation [68]. It shouldbe mentioned that anFe-MT complex can in the Fenton reaction be formed in vitro underanaerobicconditions [as]. WereportthatDNA damage caused byCu(I) generated fromCu(II) was inhibited by addition of MT (Fig. 5). These results suggest sequestration of Cu(II) by MT and thereby inhibition of the availabilityCu of m for reduction toCuo.The higher affinity of MT for copper than for zinc suggests that Zn-MT found under uninduced cellular conditionsin vivo would be available for such sequestration. In addition to a role for MT in preventing formation of damaging radical species by interaction with metals, it is also possible that MT could bind with DNA to reduce the possibility ofDNAlCuO interactions (considered tobe a key step in Cu-induced DNA damage). The sitespecific DNA damage induced in the presence of Cuhydrogen peroxide suggests that copper binds to specific DNA sites and forms singlet oxygenor copper-peroxide radicals [53]. In our studies, DNA in the presence of Zn-MT formed a precipitate in the presence of 50 pM C u m and 2 mM YO,: thatprecipitatecontainedbothDNA ( a s d by ethidium bromide fluorescence and agarose gel electrophoresis) and MT(assessedbyWesternblotanalysis). It is possiblethat Zn-MT complexed with DNA can sequester metals and thus reduce copper-induced damage to DNA. Recent studies, however, show that copper-MT can increase lipid peroxidationin vitru and can act as a pmxidant [70]. Thus the specific metals bound to MT may influence the properties of MT in oxidant stress. In summary, we report that mammalian Zn-MT has the capacity to inhibit DNA damage induced by free radicals generated from reactive oxygen donors in the presence of copper. It may do so by direct interaction with copper to prevent its participation in redox reactions. The localization of MT in cell nuclei under certain circumstances (for example, during embryonic and postnatal mammalian development, and in certain human tumours) may suggest a role for MT in protecting against DNA damage induced by free radicals.

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W. ACKNOWLEDGMENTS This research wassupportedby Research Council of Canada.

grants to MGCand

JK bytheMedical

REFERENCES l. I. Fridovich, Arch. Biochem. Biophys. 247, 1-1 1 (1986). 2. K. Brawnand I. Fridovich, Arch. Biochem.Biophys. 206, 414-419 (1981). 3. D.A.RowleyandB.Halliwell,Biochim.Biophys.Acta 761, 86-93 (1983). 4. 0.1. Aruoma, B. Halliwelland M. Dizdaroglu, J. Biol.Chem. 264: 13024-13028 (1989). 5. S. Goldstein, D. Meyerstein and G. Czapski. Free Radic. Biol. Med. 15, 435-445 (1993). 6. S.D. Aust, C.F. Chignell, T.M. Bray, B. Kalyanaraman and R.P. Mason, Toxicol. Appl. Pharmacal. 120, 168-178 (1993). 7. M.K.Eberhardt,C.SantosandM.A. Soto, BBAGEN 1157, 102-106 (1993). 8. S.D. Aust, L.A. Morehowse, C.E. Thomas, J. Free Radic. Biol. Med. 1: 3-25 (1985). 9. D.W. Feif, Free Radic. Biol. Med. 12,417-427 (1992). 10. R.F. Castilho, A.R. Meinicke, A.M. Almeida, M. Hermes-Lima and A.E. Vercesi, Arch. Biochem. Biophys., 308, 158-163 (1994). 11. E. Kukielka and A.I. Cederbaum, Arch. Biochem. Biophys., 308,70-77 (1994). 12. E.S.Driomina, V.S. Sharovand Y.A. Vladimirov,FreeRadic.Biol. Med., 15, 239-247 (1993). 13. 0.1. Aruoma, B. Halliwell, E. Gjewskiand M. Dizdaroglu, J. Biol. Chem., 264, 20509-20512 (1989). 14. M.L. Muiras, P.U. Giacomoni and P. Tachon, Mutation Res. 295,47-54 (1993).

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15. M. Dizdaroglu, G . Rao,B. Halliwell and E. Gajewski, Arch. Biochem. Biophys. 285, 317-324 (1991). 16. Y.B. Li and M.A. Trush, Arch. Biochem. Biophys. 300, 346-355 (1993). 17. Y.B. Li and M.A. Trush, Carcinogenesis, 14, 1303-1311 (1993). 18. 0.1. Aruoma, B. Halliwell, E. Gajewski and M. Dizdarogl, Biochem. J. 173, 601-604 (1991). 19. L. Miline,P.Nicotera, S. Orrenius andM.J. Burkitt, Arch. Biochem. Biophys. 304, 102-109 (1993). 20. A. Samuni, M. Chevion andG. Czpaski, Radiat. Res. 99,562-572 (1984). 21. M.A. George, S.A. Sabovljev, L.E. Hart, W.A. Cramp, G. Harris andS. H O ~ S ~Brit. Y , J. Cancer, 55 (Suppl. VIII), 141-144 (1987). 22. R.E.Lloyd,R.A. Larson, T.L. AdairandR.W.Tuveson,Photochem. Photobiol. 57, 1011-1017 (1993). 23. R.M. Rose, Evolutionary Biology of Aging, OxfordUniversity Press, 1991. 24. D.I. Feig, M.M. Reid, L.A. Loeb, Cancer Res., 54,11890~-1898~(1994). 25. M.G. Simic, Cancer Res., 54, 1918s-1923s (1994). 26. T. Miura, S. Muraoka andT. Ogiso, Pharmacology &Toxicology, 74,8994 (1994). 27. A. Meister, Cancer Res., 54, 1969s-1975s (1994). 28. D.H. Hamer, Annu. Rev. Biochem., 55, 913-951 (1986). 29. D.M. Templeton and M.G. Cherian, Meth. Enzymol. 205, 11-24 (1991). 30. N. Imura, M. Satoh and A. Naganuma, inMetalbthionein in Biobgy and Medicine (C.D. Klaaassen, andK.T. Suzuki, W), CRCpress,Boca . Raton,BostonandLondon, 1991, pp.375-382. 31. P.J. Thornalley and M.Vasak, Biochim. Biophys. Acta827,364(1985). 32. M. Sato and I. Bremner, Free Radic. Biol. Med. 14, 325-337 (1993). 33. H.M. Chan, R. Tabarrok, Y. TamuraandM.G. Cherian, Chem.Biol. Interactions 84, 113-124 (1992). 34. M.G. Cherian and M. Nordberg, Toxicol. 28, 1-15 (1983). 35. A. Bakka, A.S. Johnsea,L. Endresen and H.E. Rugstad, Experientia, 38,

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(1982).

36. M. Satoh, A.Naganumaand N. Imura, Cancer Chemother. Pharmacol., 21, 176-178 (1988).

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37. J.P. Thomas, G.L.Bachowski andA.W. Girotti, Biochem. Biophys. Acta, 884, 448-461 (1986). 38. J. Abel and N. deRuiter, Toxicol. Lett., 47. 1991-1996 (1989). 39. T. Muller, R. Schuckelt andL. Jaenicke, Arch. Toxicol. 65,20-26 (1991). 40. M.Panemangalore,D.Banerjee, S. OnosakaandM.G.Cherian,Dev. Biol., 97: 95-102 (1983). 41. N.O.Nartey, D. BanerjeeandM.G.Cherian,Pathology 19:233-238 (1987). 42. N.O. Nartey, M.G. Cherian and D. Banerjee, Am. J. Pathol., 129:177182 (1987). 43. C.E. Castro, Annu. Rev. Nutr., 7: 407421 (1987). 44. J.K. Chesters, Nutr.Rev., 50: 64-71 (1992). 45. T.V. O’Halloran, Science 261: 715-725 (1993). 46. C.O. Pabo and R.T. Sauer, AM. Rev. Biochem., 61: 1053-1059 (1992). 47. G.R. Buettner, J. Biochem. Biophys. Methods 16, 27-40 (1988). 48. R. Stoewe and W.A. Prutz, Free Radic. Biol. Med. 3, 97-105 (1987). 49. B. Halliwell and J.M.C. Gutteridge, Arch. Biochem. Biophys. 246, 501514 (1986). 50. D. Jamieson, Free Radical Biol. Med., 7: 87-108 (1989). 51. I. Karuzina and A. Archakov, Free Radical Biol. Med., 16, 73-97 (1994). 52. C.J.Reedand K.T. Douglas,Biochem.Biophys.Res.Commun. 162, 1111-1117 (1989). 53. K. Yamamoto, S. Inoue and S. Kawanishi, Carcinogenesis. 14, 1397-1401 (1993). 54. P. Techon, 1989, Free Radical Res. Commmun, 7, 1-10 (1989). 55. J.A. M a y and S. LW, Science 240, 1302-1309 (1988). 56. J.A. M a y , S.M. Chin and S. Linn, Science, 240, 640-642 (1988); 57. G.R.A. Johnson, N.B. Nazhat and R.A. Saadalla-Nazhat, J. Chem. Soc. Chem. Commun., 404408 (1988). 58. H.C. Sutton andC.C.Winterbourn,FreeRadicalBiol.Med. 6, 53-60 (1989). 59. M. Shiraishi, K. Utsumi, S. Morimoto, I. Joja, S. Iida, Y. Takeda and K. Ano, Physical. Chem. Phys.. 14, 533-537 (1982). 60. L.S. Chubatsu and R. Meneghini, Biochem. J. 291, 193-198 (1993).

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61. T.P. Coogan, R.M. Bare and M.P. Waalkes, Toxicol. Appl. Pharmawl. 113, 227-233 (1992). 62. J.R. Arthur, I. Bremner, P.C. Morrice and C.F. Mills, Free Radic. Res. C O ~, 4,. 15-20 (1987). 63. B. Kaina, H. Lohrer, M.KarinandP.Herrlich,Proc.NatlAcad.Sci. USA., 87, 2710-2714 (1990). 64. H. Lohrer and T. Robson, Carcinogenesis 10, 2279-2284 (1989). 65. M. Miura and T. Sasaki, Radiat. Res., 123,171-175(1990). 66. J. Koropatnick and J. Pearson, Mol. Pharmacol., 44, 44-50(1993). 67. C.L. Greenstock, C.P. Jinot, R.P. WhitehouseandM.D.Sargent, Free Radic. Res. &mm. 2, 233-239 (1987). 68. A.C. Mello-Filhoand R. Meneghini, Biochim. Biophys. Acta, 847, 82-89 (1985). 69. W.A. Prutz, J. ButlerandE.J.Land,Int. J. Radiat.Biol., 58,215-234 (1990). 70. G.F.Stephenson,H.M.ChanandM.G.Cherian,Toxicol.Applied Phannacol., 125, 90-96 (1994).

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DNA Strand Breakage and Lipid Peroxidation as Possible Mechanisms of Selenium Toxicity J. Kitahara, Y. Seko, and N. ImuraSchool of PharmaceuticalSciences, Kitasato University, 5-9-1, Shirokane, Minato-h, Tokyo 108, Japan H. Utsumi and A. HamadaSchool University, Tokyo, Japan

of PharmaceuticalSciences,Showa

I. INTRODUCTION Selenium isknown as an essential trace element constitutingthe active site of glutathione peroxidase which handles the active oxygen suchas hydrogen peroxide and organic hydroperoxides to protectanimalsfromoxidativestresses.Ontheotherhand, selenium has long been recognized as a toxic element which causes alkaline disease andblind stagger disease in farm animals and human poisoning in some areas of high selenium content in the soil such as Enshi district in China. Further, it was reported recently that a health food supplement having unusually high selenium content caused human intoxication [l]. Although the molecular basis of its desirable functions has recently been well documented, the mechanism of selenium toxicity has not clearly beenelucidated yet. Cytotoxicity of selenite was reported to be enhanced by glutathione (GSH) [2-4]. Further, the addition of selenite to rat hepatocyte cultures caused lipid peroxidation [5]. A few papers 121

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have suggested superoxide anion (m-)generation by the reaction of selenitewithsulfhydrylcompounds in vitro [6-81.These results seem to indicate a possible involvement of active oxygen species in selenite toxicity. However, we recently found that the experimental procedures used for proving superoxide anion formation with cytochrome c or acetyl-cytochrome c were inadequate and the results could not be an evidence for active oxygen formation in the reaction of selenite with sulfhydryl compounds [9]. These facts prompted us to re-examine a possibility o f active oxygen generation in the reaction of selenite with sulfhydryl compounds in vitro and to study its rolein selenite toxicity exertedin vitro and in vivo.

11.

DNA STRANDBREAKAGEANDLIPID PEROXIDATION IN ORGANS OF MICE ADMINISTEREDSELENITE

ICR mice (female, 6-weeks) were intraperitoneally administered 60 pmol/kg of sodium selenite dissolvedin saline. DNA strand break was examined athr2 after the selenite administration by the method of Sina etal. [lo]. Thiobarbituric acid-reactive substances (TBA-RS) were determined fluorometrically according to the method of Ohkawa et al. [1l] at 24 hr after the administration using 5% homogenate of various organs of mice. Dose dependent increases inTBA-RS and DNA single strand break were observed in various organsof mice treated with selenite, demonstrating that in vivo. selenite exerted oxidative stress

III. CYTOTOXICITY OF SELENITE Rat hepatocytes (2x106 cells/ml) obtained by the method of Hogberg and Kristoferson [l21 were incubated with or without selenite and/orthe other chemicals at37°C in a rotating tube under 95% 0 2 and 5% C02 atmosphere.Analiquot o f thecell suspensionwasremovedandusedformeasuringoxygen consumption rate. Another aliquot was centrifuged at10,000 x g for 15 sec. The supernatant was used for detenninig TBA-RS and extracellular lactate dehydrogenase(LDH) activity. Cellular GSH was determined fluorometrically by the method of McNeil et al.

DNA Breakage,LipidPeroxidation,andSeleniumToxicity

E

F

h

E

F

123

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[13]. LDH activity was assayed bythe method of Wroblewskiet al. [14]. Oxygen content in rat hepatocyte suspension was decreased (Fig. la) by the exposure of the cells to seleniteas well as GSH level (Fig. lb) and followed by the increasesin TBA-RS (Fig. IC) and LDH (Fig. Id) released from the cells. These changesin the indicators for celllesionweresignificantlyreduced by desferrioxamine-manganese(DFMn), an SOD mimic, suggesting a roleof active oxygen speciesin the cytotoxic action of selenite ~51.

IV. DNA STRAND BREAKAGE BY SELENITE AND GSH IN VITRO pUCl18 plasmid DNA (2 pg/ml) was incubated with 50 pM sodium selenite and2 mM GSH in phosphate buffer (pH 7.4) at 37°Cfor 4 min. The DNAwasextractedandanalyzedby agarose-gel electrophoresis [16]. DNA single strand breakage wasoccurredbythereactionwithseleniteandGSH.The presence of oxygen was shown tobe essential for the breakage. The breakage was not inhibited by the addition of Cu, Zn-SOD or catalase. The SOD appeared to enhance the DNA cleavage to some extent. Desferrioxamine and DETAPAC, iron chelators, failed in depressing the DNA cleavage. While mannitol and DMSO, hydroxyl radical scavengers, efficiently inhibited the DNA strand break. These results indicate a role of hydroxyl radical in the DNA strand break caused by selenite in the presence of GSH.

V. HYDROXYLATION OF SALICYLATE BY SELENITE AND GSH Sodium salicylate (2 mM) was subjectedto the reaction with50 pM selenite and2 mM GSHat 37°C for20 min in the presenceor absence of various scavengers or an iron chelatorin phosphate buffer (pH 7.4). The reaction was terminated by the addition of N-ethylmaleimide (final 9.1 mM) and TCA (final 5.2%). The mixture was centrifuged at 12,000 x g for 10 min and 2,5dihydroxybenzoic acid in the supernatant was determined by HPLCusingShodexODSpakcolumn(4.6x150mm).

DNA Breakage,LipidPeroxidation,andSeleniumToxicity

125

Ammonium acetate (0.2%, pH 5.5)/methanol(95:5) was used as the elution buffer (0.6mvmin). Fluorescence (excitation 3at 2Onm and emission at 460 nm) was monitored. It was confirmed that 2,5-dihydroxybenzoicacid,amajoroxidationproduct of salicylate, was formed by selenite and GSH. The amount of2,5dihydroxybenzoic acid formed from salicylate was decreased by the addition of Mn-SOD (25 units/ml) to50% of the control, but of the enzyme. not completely inhibited even with excess amount Heat-denatured Mn-SOD did not affect the hydroxylation. Cu, Zn-SOD (50 units/ml),ontheotherhand,enhancedthe hydroxylation by40%. Ironchelatorcoulddepressthe hydroxylation only partly. However, when xanthinexanthineoxidase system was usedin place of selenite and GSH, both Mn-SOD and Cu, Zn-SOD diminished the hydroxylation almostcompletelyanddesferalalsoinhibitedthereaction markedly.

VI. DEOXYRIBOSE DECOMPOSITION BY SELENITE AND GSH Deoxyribose (0.98mM)was incubated at 37°C for 20 min with 50 pM sodium selenite and 2 mM GSH in phosphate buffer @H 7.4) in the presence or absenceof scavengers or an iron chelator. The reaction was terminated by the addition of Nethylmaleimide (final 9.1 mM). The reaction mixture(0.55 ml)was added with 0.5 ml of 2.8% TCA and 0.5 m1 of 1% TBA and incubated at 100°C for 10min before determination of TBA-RS as decomposed products from deoxyribose[17]. The inhibition profile of the formation of TBA-RS from deoxyribose by the scavengers or desferrioxamine was similar to that obtainedin the hydroxylation of salicylate.

VII. ELECTRON SPIN RESONANCE (ESR) SPECTRUM OF THE MIXTURE OF SELENITE AND GSH The ESR spectrum of the mixture of selenite and GSH was measured by Electron Spin Resonance Spectrometer (JEOL, JESRElX)using 5,5-dimethyl-l-pyrrolineN-oxide @ W O ) as a spin

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trapping agent. The signals corresponding to those of hydroxyl radicaladductofDMPO(DMPO-OH)wereobtained.The addition of ethanol to the mixture gave the signals obviously corresponding to these of a-ethylhydroxy radical adduct of DMPO. The intensity of theESR signals of DMPO-OHwas not affected by Cu,Zn-SODandcatalaseandwasreducedto approximately half of the control by mannitol and DMSO, which are hydroxyl radical scavengers.

VIII. CONCLUSION As shown in Table 1, Cu,Zn-SODappears to substantially stimulate these reactions induced by hydroxyl radical. Mn-SOD partly inhibits the reactions, but not completely. Desfenioxamine, anironchelator,alsopartlydepressthedeoxyribose

Table 1 Summary :Effects of radical scavengers and iron chelator onH00 formation by selenite and GSH

.

j

n

DNA Breakage, Lipid Peroxidation, and Selenium Toxicity

................ )....................... ...:...... ... ! ..v e

.. .... ... ..

.......... U

m

.m Y

Q

127

128

Kitahara et al.

decompositionandhydroxylationofsalicylate.Mnnitoland DMSO which are known as hydroxyl radical scavengers almost completely inhibitthese reactions. The presence of oxygen seems to be essential. Further, it was confirmed in separate experiments that hydrogen selenide caused the same reactions in the presence of oxygen as those induced by selenite and GSH. In addition to the above mentioned data obtained by selenite, selenocystine, a selenoamino acid, also induced DNA strand break and lipid peroxidationinorgans ofmice. In in vitro reaction of selenocystine with GSH also caused DNA strand break and deoxyribosedecomposition.However,thesereactionswere effectively depressed by Cu, Zn-SOD and iron chelator. These results may suggest that superoxide radicalis a major species of active oxygen initially generated in the reaction of selenocystine with GSH. Finally we would like to suggest that selenide formed by the reduction of selenite with GSH or through the metabolism of as a final molecular selenoamino acid generates hydroxyl radical species of active oxygen by reducing oxygen molecule. A part of hydroxylradical is formedfromsuperoxideradicalthrough hydrogen peroxide and Fenton-tyte reaction in the presence of iron.However,majorpartofthehydroxylradicalmaybe generated through another unknown processin the presence of selenide as shown in Fig. 2. REFERENCES 1. R. Jensen, W.Closson and R. Rothenberg, Bobid. Morta1,Weekly Rep. 33,157-158 (1984) 2. J.H. Ray and L.C.. Altenburg, Mutat. Res. 54:343-354 (1978) Cancer Res. 3. G. Batist, A.G. Katki, R.W. Klecker Jr. and C.E. Myers, 465482-5485 (1986) 4. R.D. Snyder, Cancer Lett. 3473-81 (1987) 5. N.H. Stacey and C.D. Klaassen, J . Toxicol. Environ. Health 7:139-147 (1981) 6. P.Garberg, A. S a , M. Warholm and J. H6gberg, Biochem. Phamcol. 37:3401-3406 (1988) 7. G. F. Kramer and B.N.Ames, Mutat. Res. 201,169-180 (1988) 8. Y. Seko, Y. Saito, J. Kitahara and N. Imura, Active oxygen generation by the reactionof selenite with reduced glutathione in vitro in Selenium in Biology and Medicine (Wendel A, Ed) , Springer-Verlag, Berlin Heidelberg New York, 1989, pp. 70-73. 9. Y. Seko,J. Kitahara, H. Utsumi, A. Hamada and N. Imura,Reexamination of the proposed mechanismof active oxygen generationby the reaction of selenite with reduced glutathione (GSH), Fifth

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International SymposiumonSelenium in Biology and Medicine, Vandrbilt University School of Medicine, Nashville, 1992, p.107 10. F. Sina, C.L. Bean, G.R.Dysart, V.I. Taylor and M.O. Bradley, Mutat. Res. 113,357-391 (1983) 11. H. Ohkawa, N.Ohisi and K. Yagi,Anaf. Biochem. 95,351-358 (1979) 12. J. Htigberg andA. Kristoferson, Eur. J . Biochem. 74,77-82 (1977) 13. T.L. McNeil and L.V. Beck ,Anal. Biochem. 22,431-441 (1968) 14. F. Wr6blewski and J.S. LaDue, Proc. Soc. Exp. Biof. Med. 90, 210213 (1955) 15. J. Kitahara,Y. Seko andN.Imura, Arch. Toxicol. 67,497-501 (1983) 16. S. Toyokuni and J.-L. Sagripanti, J. Inorg. Biochem.47,241-248 (1992) 17. J.M.C. Guttenridge, FEBS Lett. 128,343-346 (1981)

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Role of Metal in Oxidative DNA Damage by Non-mutagenic Carcinogen Shosuke Kawanishi, Shinji Oikawa, and Sumiko InoueDepartmentof Public Health, Faculty of Medicine, Kyoto University, Kyoto 606, Japan

Free radicals and other active oxygen species are constantly formed in the human body. Many of themserveuseful physiological h c t i o n s , but they can be toxic when generated in excess and this toxicity is often aggravated by the presence of ions of transition metals [l].DNA damage by active oxygen species has drawn much interest in relation to carcinogenesis. Active oxygen species may be involved in initiation, promotion and conversion of multistage carcinogenesis. The active oxygen species in metal-induced DNA damage play important roles in the metal carcinogenesis and the unknown carcinogenic mechanism of some organic carcinogens. Metal ionsreact with superoxide anion radicals (02-)and hydrogen peroxide m02)to produce highly reactive species such as hydroxyl freeradicals (*OH)and metal-oxygen complexes in biological systems (Figure 1).

Figure 1. A mechanism of metal-mediated oxidative DNA damage. 131

132

Kawanishl et al.

The Fenton reaction of Fe(I1) with KZ02 is the well-known mechanism for the generation of *OHand/or the ferry1 ion 121. For the first time, Kawanishi et al. reported that carcinogenic Cr(V1) reacts with H- to produce *OHand singlet oxygen (102) which of oxygen free cause DNA damage,andemphasizedtherole radicals in metal carcinogenesis 133. Other carcinogenic metal compounds such as Fe(II1) nitrilotriacetate [41, Ni(I1) [5, 61 and Co(I1) 171produce various types of active oxygen species from H202. Cu(I1) plus H202caused damage to isolated DNA. The main active species causing the DNA damage are more likely copper-oxygen complexes with similar reactivity to Q 2 and/or *OH,rather than *OHitself [SI. These active oxygen species were suggested to give different kinds of site specific DNA damage (l'hble 1). Since H202 reaches the nucleuswhen it survives in significant concentrations, and can be produced even in nucleus [g, 103, these DNA damages mayoccur in cells. Dizdaroglualso observed oxidative DNA modifications in chromatin of cultured mammalian cells treated with H202 and in chromatin of organs of animals treated with carcinogenic metal salts C113. Table 1. Active oxygen species formation and site specificities of DNA damage induced by metal compounds in the presence of hydrogen peroxide M e a cardnoaenldtv

&(VI) ++

Ni(ll)

++

Fe(lll)-NTA

+

Co(l1)

Cu(ll)

Wl)

+

7

7

G>T-C>A

T-G20>A

.OH @T&A -R

PI

[5,61

0-T-C-A [41

m

PI

T of S-GTC

I

++; sufticient evidence of carcinogenicity in humans and animal experiments + ;evidence o f carcinogenicity in animal experiments On the other hand, Cu(I1) and Mn(I1) have ability of mediating both Hzoz formationand oxidative DNA damage by certain carcinogenswhichhave no or weak mutagenicity. Benzene, ophenylphenol (OPP) and caffeic acid, pentachlorophenol (PCP), tryptophan metabolites have not been proved to be mutagenic i n bacterial test systems, either. Our previous work showed that in

Metal and DNA Damage by Non-mutagenic Carcinogen

133

the presence of Cu(II), those compounds or their metabolites caused damage to isolated DNA through H202 formation (Figures 1 and 2). It is of interest that Ames-test negative "non-mutagenic" carcinogens or theirmetaboliteshave beenshown t o cause oxidative DNA damage in the presence of transition metal ion. I n this chapter, we reviewed the role of metal in carcinogenesis of non-mutagenic carcinogens such as benzene [12],OPP [13],caffeic acid [14],PCP [l51and tryptophanmetabolites. benzene metabolite

tryptophan acid caffeic metabolites OH

OH

OH

tm

I I

3-hydroxyanthranllic acid

CH

1,2,4-benzenetrlol

COOH

OPP metabolite

PCP metabolite

I

OH

2,s-dlhydroxyblphenyl

tm 3-hydroxykynureine

ai tetrachlorohydroquinone

Figure 2. Chemical structures of non-mutagenic carcinogens and theirmetabolites; they are derivatives of dihydroxybenzene or hydroxyaminobenzene. 11. Oxidative DNA Damage

& B e n z e n eMetaboIit.4~

Benzene, widely used in the chemical industry, hasbeen shown to cause serious hematological disorders and carcinogenic effects on humansandanimals. Extensive epidemiological evidence has beenpublished on the highincidence of leukemias inmen occupationally exposed to benzene [16, 171. The administration of

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Kawanishi et al.

benzene to animalsproducedleukemia [181, lymphomaand carcinomas of the Zymbal gland, mammary gland and liver C16, 191. Previous studies showed that benzene induced sister chromatid exchanges in mouse bone marrow [16,20,211. However, benzene has not been shown to be mutagenic in bacterial test systems.

OH I

0-

OH hydroqumone

f

benzene

0"

l,2,4-benzenetriol

catechol

Figure 3. Benzene and itsmetabolite. We previously investigated reactivitiesof benzene metabolites (phenol, hydroquinone, catechol, 1,2,4-benzenetriol)(Figure 3)with DNA by a DNA sequencing technique using =P 5I-end-labeledDNA fragments C123. Among benzenemetabolites, 1,2,4.-benzenetriol caused strong DNA damage, and hydroquinone caused slight DNA damage.Benzene,phenol,catechol and resorcinol showed no effect. lkace amounts of Cu(I1) were shown to be necessary for the induction of DNA damage by 1,2,4-benzenetriol. The cleavage sites induced by 1,2,4-benzenetriol were determined by utilizing the Maxam-Gilbert procedure C221. The result showed that cleavages at the positions of guanine and adjacent thymine weremore frequent thanthose of other bases. The cleavagewithout piperidine treatmentindicatedthebreakage of deoxyribose-phosphate backbone.Theincrease of oligonucleotideformationwith piperidine treatment suggests that the base alteration(s1 and/or liberation(s) are induced by 1,2,4-benzenetriol plusCu(I1) and subsequently the cleavages at those bases occurred. We examined the effects of superoxidedismutase (SOD), catalase, and *OH

Metal and DNA Damage by Non-mutagenic Carcinogen

135

scavengers on 1,2,4-benzenetriol-inducedDNA damage. SOD and catalase almost completely inhibited DNA damage, suggesting the involvement of 02- and H202. Methional significantly inhibited DNA damage, whereas sodium formate did not inhibit it. Since buffers and reagents areknown to be invariably contaminated with trace amounts of metal ions [23], and the addition of Cu(I1) is reported to accelerate drug-induced DNA damage [24], the effects of chelating agents and metal ions on 1,2,4-benzenetriol-induced DNA damage were examined. 1,2,4-Benzenetriol-inducedDNA damage was inhibited by the addition of a Cu(1)-specific chelating agent,bathocuproine,andwasaccelerated by theaddition of Cu(I1). On the other hand, the addition of Fe(II1) did not accelerate the DNA damage induced by 1,2,4-benzenetriol. ESR studies using spin traps demonstrated that addition of Fe(II1) increased *OHproduction during the autoxidation of 1,2,4benzenetriol,whereastheaddition of Cu(I1)did not. Several papers suggested that DNA damage was caused by H202 through Fenton reaction in vivo [2, 251. Therefore, there still remains the possibility that Fe(I1) participates in =OH production from H202 and in DNA damage. Recently, measurements of 8-hydroxy-2deoxyguanosine (8-OH-dG) have been shown to be useful to clarify the participationof oxygen radical in DNA damage. We measured benzene 8-OH-dG content in calf thymus DNA treatedwith metabolites in the presence of Cu(I1) or Fe(II1) by using a n electrochemical detector (ECD) coupled to a HPLC (HPLC-ECD). Formation of 8-OH-dGby 1,2,4-benzenetriol in the presence of Cu(I1) or Fe(II1) increased with the increasing concentration of 1,2,4-benzenetriol. The 1,2,4-benzenetriol plusFe(II1)-induced 8 OH-dG formationwassignificantlyinhibited by typical *OH scavenger,ethanol,whereasethanol did notinhibitthe 1,2,4benzenetriol plus Cu(I1)-induced 8-OH-dG formation (Figure 4). Therefore, it is considered that thespecies causing DNA damage i n the case of Cu(I1) are active oxygen species other than *OH. The inhibitory effect of catalase on DNA damage indicated that H202 plays an important role in producing active oxygen species causing DNA damage. Inthe presence of Cu(I1) or Fe(III), 1,2,4benzenetriol caused DNA damage more efficiently than H202. It can be speculated that 1,2,4-benzenetriolenhances DNA damage by H202 in the presence of metal ions, probably by promoting the conversion of Cu(I1) to Cu(1) or Fe(II1) to Fe(I1) and/ or inducing the change of DNA conformation.

Kawanishi et al.

cu

r

Fe

ij.

P'

I 0 o

b

,

0 Figure 4. The 8-OH-dG formation in DNA induced by 1,2,4-benzenetriolin the presence of Cu (11) or Fe (111) and theeffects of scavengers. Calf thymus DNA (140pM per base) was incubatedwith 50 pM 1,2,4-benzenetriol in the presence of10 p M metal in buffer(pH 7.9 ) at 37 "Cfor 10 min. The treated DNA was analyzed by a HPLCECD.

Our idea that the metal-mediated DNA damage through H202 is relevant for the expression of the carcinogenicity of benzene has been supported by Kolachana et al.'s observations regarding with the inductionof oxidative DNA damage by benzene metabolites in HL60 cells in uitmand in thebone marrow of mice in vivo[26].

m. OxidativeDNA Damagebyehenylphenol o-Phenylphenol (OPP) and its sodium salt have beenused a s fimgicides for citrus fruits [273. It has been reported that long-term administration of OPP and its sodium salt a t high dose induces carcinoma of the urinarybladder in rats [28], although OPP and its sodium salt have not been proved to be mutagenic in bacterial test systems [29]. Morimoto et al. reported that DNA damage was induced in urinary bladder epithelium of male rats treated with

Metal and DNA Damage by Non-mutagenic Carcinogen

137

OPP metabolite, 2-phenyl-1,4-benzoquinone (PBQ) [30]. However, the mechanismsof DNA damage remain to be clarified. Reactivities of OPP and its metabolites (2,5-dihydroxybiphenyl (Di-OH-BP), PBQ) with DNA wereinvestigated by a DNA sequencing technique, and the reaction mechanism was studied by UV-visible and ESR spectroscopies [131. In the presence of Cu(II), PBQ CU(I1)

NADH

- - - + + + + + -- -+ ++ -- +- -+ ++ ++

incubation time 20 20 20 20 20 20 5

20 (min)

Figure 5. Autoradiogram of =P-labeled DNA fragments incubated with OPP metabolite in the presence of NADH. .. . The reaction mixture contained the=P 5' end-labeled 337basepairfragment (PstI 234!j-AuaI* 2681)obtained from human c-Ha-ras-l protooncogene, 1@M per base of sonicated calf thymus DNA, 60 W PBB, 250 W NADH and 20 p.MCuCh in 200 p1 of 10 mM phosphate buffer at pH 7.9 containing 5 @l DTPA. After theincubation at 37."C, themixture was heated with lM piperidine at 90"Cfor 20min. and theDNA fragments were electrophoresed on an 8 % polyacrylamide/8 M urea gel and the autoradiogram was obtainedby exposing X-ray film ta the gel.

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Kawanishi et al.

Di-OH-BP caused DNA damage even without piperidine treatment. Catalase, methionine and methional inhibited the DNA damage completely, whereas -OH scavenger (mannitol, sodium formate, ethanol, tert-butyl alcohol) and SOD did not. Di-OH-BP plus Cu(I1) induced piperidine-labile site frequently a t thymine and guanine residues. The addition of Fe(II1) did not induce DNA damage with Di-OH-BP. ESR-spin trapping experiments showed that the addition of Fe(II1) produced *OH during the autoxidation of Di-OH-BP, whereas the addition of Cu(I1) hardly did so. The results suggest that DNA damage by Di-OH-BP plus Cu(I1) is due to active species other than *OH. We also studied DNA damage by PBQ plus NADH. PBQ alone did not induce DNA damage in thepresence of Cu(111, but addition of NADH induced the DNA cleavage even in theabsence of NADHFMN oxidoreductase (Figure 5). These results demonstrated that semiquinone radical produced by the reduction of PBQ by NADH reacts with0 2 to produce 02-and subsequently H202, which may be activated by transition metalsto cause DNA damage.

DbIA damage

H24

4

Figure 6. A possible mechanism of DNA damage induced by OPP metabolites.

Metal and DNA Damage by Non-mutagenic Carcinogen

139

UV-visible spectroscopicstudies showed that the autoxidationof Di-OH-BP was accelerated by Cu(I1) and the additionof catalase did not inhibit the accelerating effect of Cu(I1). ESR studies showed thattheinitialrate of semiquinoneradicalproductionwas increased by the addition of Cu(I1) and the increaseby Cu(I1) was not affected by catalase. On the other hand, DNA damage by DiOH-BP plus Cu(I1) was inhibited by catalase. SOD facilitated the autoxidation of Di-OH-BP and the production of semiquinone radical, whereas SOD itself did not induce DNA damage with DiOH-BP. These results suggest that neither semiquinone radical 2 'is the reactant to DNA.On the basis of these data, we nor 0 proposed a possible mechanism of DNA damage induced by OPP metabolites as shown in Figure 6.

IV. Oxidatim DNA Damage by Caffeic Acid Caffeic acid is a phenolic compound widely distributed in plants [31]. Caffeic acid was reported to induce forestomach squamous cell carcinoma of rats 1321. However,caffeic acid has not been shown to be mutagenic in bacterial testsystems [33,341. Previous studiesdemonstratedthat caffeicacidinducedchromosomal aberrations in Chinese hamster ovary cells L351 and DNA double strand breaks in cultured rat fetal lung cells and HeLa cells [36]. However, the mechanism of DNA damage induced by caffeic acid remains to be clarified. Our previous experiments with the isolated DNA showed that caffeic acid caused extensive DNA damage in the presence of Cu(I1) but not in thepresence of Mn(I1) [14]. The inhibitory effects of bathocuproine and catalase on the DNA damage suggest that Cu(1) and H202 have important roles in the production of active species causing DNA damage. Caffeic acid plus Cu(I1) caused cleavage frequently a t thymine residues especially of the 5'-GTC-3' sequence and 5'-CTG-3' sequence. The site specificity cannot be explained by *OH,since it is generally considered that *OHcauses DNA cleavage at every nucleotide with little marked site specificity [37]. Moreover, typical *OH scavengers (mannitol, sodium formate, ethanol, tert-butyl alcohol) did not inhibit caffeic acid plus Cu(I1)induced DNA damage,whereasmethionalandmethionine completely inhibited it. Therefore, it can be speculated that Cu(1)

140

Kawanishi et al.

and H z 0 2 produce a complex such as Cu(I)OOH, other than fiee hydroxyl radical, and that the complex participates in the DNA damage. In recent years, pulsed field gel electrophoresis (PFGE) has emerged as a powerful tool for the study of high-molecular weight DNA. PFGE is generally used for detection of cellular DNA doublestrand breaks. CafFeic acid was shown to produce strand breaksi n DNA of the cells treatedwith Mn(I1). We havedesigned a n experimental protocol which allows detection of DNA single-strand breaks andalkali-labile sites by PFGE. With this procedure, caffeic acid was shown to produce single-strand breaks and alkali-labile sites in DNA of the cells treated with Mn(I1). The enhancing effects of 3-aminotriazol(acatalaseinhibitor)andbuthionine sulphoximine (a GSH synthesis inhibitor) and the inhibitory effect of catalaseon caffeicacidplus Mn(I1)-induced DNA damage indicate the participation of H202. Theinhibitory effect of ophenanthroline indicates that endogenous transition metals such as copper and iron participate theoxidative DNA damage. Thus, it

I

.OH, metal-oxygen complex

t

DNA damage Figure 7. A possible mechanism of DNA damageinduced by caffeic acid in the cell treated with Mn (11).

Metal and DNA Damage by Non-mutagenic Carcinogen

141

is considered that through Mn(I1)-catalyzed autoxidation caffeic acid produces H202, which is activated by endogenous transition metals to cause damage to cellular DNA. As for the isolated DNA, The Cu(I1)-mediated DNA damage was enhanced by preincubation of caffeic acid with Mn(I1). The rate of H202 formation by Mn(I1) was greater than that by Cu(I1). Theseresultssupportthe mechanism that caffeic acid causes cellular DNA damage by the Mn(I1)-catalyzed formation of H202 and metal-activation of H202 (Figure 7).

V. Osidative~DNA Damam byPentacMomphen01 Pentachlorophenol (PCP) is a wide-spectrum biocide. PCP has let to a substantial environmental contamination and accumulated i n normal human population in addition to PCP-exposed workers 1381. PCP has shown to be carcinogenic for mice [391, although it does not seem to be mutagenic in bacterial test systems. A significant increase in chromosome-type abberrations has been observedin the lymphocytes of PCP-exposed workers 1383. M&onnel et al. showed that PCP is carcinogenic for mice 1391. However, PCP has not been shown to be mutagenic in bacterial test systems, whereas weak mutagenicity has been reported in other systems [38]. Studies on the metabolism of PCP in vivoand in vitrorevealed that tetrachlorohydroquinone (TCHQ) is a major metabolite of PCP in mice and rats' [401. Oxidation of TCHQ to tetrachloro-p-benzoquinone(TCBQ) can occur enzymatically [41]. Regarding PCP metabolite-induced DNA damage, two possible mechanisms have been proposed. Witte et d . reported that TCHQ covalently boundto calf thymus DNA [42]. Covalent binding of PCP to protein and DNA was observed in vitro by incubation with a metabolic activation system [41]. On the other hand, TCHQ can produce reactive oxygen species which may cause DNA damage [43]. Part of the strandbreak formation by TCHQ in human cells is supposed to be due to the action of *OH[44]. In this study, we examined DNA damage by TCHQ in thepresence of metal ions. We also analyzed the8-OHdG formation in calf thymus DNA by using a HPLC-ECD, and investigated the reaction mechanism by UVvisible and ESR spectroscopies.

142

Kawanishi et al.

NADH

N A D +

M OH tetrachiorohydroquinone

c'

0 2

oi tetrachiorobenzoquinone

+metal

Figure 8. A possible mechanism of DNA damage induced by PCP metabolites.

TCHQ caused DNA damage in thepresence of Cu(I1) but not i n thepresence of either Mn(I1) or Fe(II1).TCHQ plusCu(I1) induced piperidine-labile sites frequentlya t thymine residuea and guanineresidues.Themostpreferredsites were thethymine residues of the 5'-GTC-3' sequence. TCHQ increased 8-OH-dG i n calf thymus DNA inthe presence of Cu(I1). Typical *OH scavengers showed no inhibitory effectsonTCHQ plus Cu(I1)induced DNA damage. Bathocuproine and catalase inhibited the DNA damage, suggesting that Cu(1) and H202 have important roles in the production of active species causing DNA damage. We also studied DNA damage by TCBQ plus NADH. TCBQ alone did not induce DNA damage in the presence of Cu(II), but additionof NADH induced the DNA cleavage even in the absence of NADHFMN oxidoreductase. UV-visible and ESR spectroscopies revealed that TCHQ was rapidly autoxidized into semiquinone even in the absence of metal ions, indicating thatsemiquinone radical itself is not the main active species inducing DNA damage. These results suggest that semiquinone radical produced by the autoxidation of TCHQ and/or the reduction of TCBQ by NADH reacts withdioxygen

Metal and DNA Damage by Non-mutagenic Carcinogen

143

to form 02- and subsequentlyH202,which is activated by transition metals to cause DNA damage (Figure 8).

Boyland and Watson reported that 3-hydroxyanthranilic acid (3HAA) and 3-hydroxykynureine (3-HKyn). tryptophan metabolites ( Figure g), were carcinogenic in the bladders of mice 145,461. Implantation of cholesterol pellets containing 3-HAA or 3-HKyn into bladders in mice induced a significantly greater number of bladder cancers than cholesterol pellets alone 1471. 3-HAA and 3HKyn weredemonstrated t o inducechromatidbreakageand chromatid translocations in mammalian cells 1481. 3-HAA showed promotional effect on X-ray-initiated transformation of BALB/3T3 cells [49]. However, the mechanism of DNA damage induced by 3HAA and 3-HKyn remains to be clarified.

Figure 9. nyptophan metabolites.

In order to clarify the mechanism of the DNA damage, we examined the induction of DNA strand breaks in human cultured cells treated with 3-HAA and 3-HKyn in thepresence of metal ions. Pulsed field gel electrophoresis showed that in the presence of Mn(II), 3-HAA and 3-HKyn induced DNA strandbreaks i n cultured human cells. Enhancing effect of catalase inhibitor and inhibitoryeffect of o-phenanthroline on thestrand breakage indicated the involvement of H 2 0 2 and endogenous transition metal ion. As for the isolatedDNA,we examined DNA damage by 3-HAA and 3-HKyn in the presence of metal ions. 3-HAA and 3-HKyn induced piperidine-labile sites frequentlyat thymine and guanine residues in thepresence of Cu(I1). The inhibitory effects of catalase and bathocuproine on Cu(I1)-mediated DNA damage suggest that

144

Kawanishi et al.

Hzoz and Cu(D produce adive species causing DNA damage. The Cu(II>mediated DNA damage was enhanced by preincubation of 3-

HAA with Mn(I1). UV-visible spectroscopy showed that Mn(I1) and Cu(I1) enhanced the autoxidationof 3-HAA in differentway.

60-

" H A A e 3 - H K y n

0

25

50

75

100

125

Concentration of tryptophan metabolites

Figure 10. The 8-OH-dG formation in DNA incubated with tryptophan metabolites in the presence of Cu(I1). Calf thymus DNA(140 pMper base)was incubated with3-HAA or concentrations inthe presence of 2opM CuC12 in 4 0 0 ~ o1 f 4 mM phosphate buffer at pH 7.9 containing 5 pM DTPA at 37"c for 60 min. Treated DNA was subjected l.~ enzyme digestion and analyzed by a HPW-ECD. 3-HKyn ofthe indicated

By using a HPLC-ECD, we measured 8-OH-dG content in calf thymus DNA treated with 3-HAA or 3-HKyn in the presence of metal ions (Figure 10). The 8-OHdG formation increased with the concentration of 3-HAA in the presence of Cu(I1). On the other hand, concentration dependence was not clearly observed with 3HKyn. At a low concentration, 3-HKyn caused Cu(I1)-dependent formation of 8-OH-dG more efficiently than .3 " 3-HAA plus Cu(I1) induced 8-OH-dG more efficiently than H 2 0 2 plus Cu(I1). Neither H202 alone, 3-HM alone, 3-HKyn alone, nor Cu(I1) alone increased 8-OH-dG content.These results suggest that in the presence of Mn(I1) or Cu(II), these tryptophanmetabolites produce

Metal and

DNA Damage by Non-mutagenic Carcinogen

145

Hzoz, which is activated by transition metalion to cause damage to DNA both in the case of isolated DNA and culturedcells.

VII.

Oxidative DNA Damage byNon-mutagenicCarcinogen

Certain carcinogensmay induce both H202formation andoxidative DNA damage in the presence of endogenous metal ions [12-14,50541. The benzene metabolite 1121, the OPP metabolite [131, caffeic acid [14], the PCP metabolite and tryptophan metabolites caused oxidative damage to isolated DNA through 02- formation, although quinone type metabolites required NADH. Mn(I1) has ability of mediating oxidativedamage of cellular and isolated DNA by certain carcinogens [14,541. Figure U. shows a possible mechanism for DNA damage due to Cu(I1)- or Mn(I1)-mediated formation of Hz02 which reacts with endogenous metal ions such as Fe(I1) and Cu(1) bound to or close to the DNA to produce active oxygen species. Similarly, the active oxygen species were shown to participate in

I caffeicacid I metabolite +benzene

YO2 +peroxisome(fatty acid B-oxidation)

clofibrate phthalate esters

*OH, metal-oxygencomplex

t

DNA damage Figure 11. A possible mechanism of active oxygen formation and DNA damage by certain non-mutagenic carcinogens.

146

Kawanishi et al.

Cu(n)-dependent DNA damage by hydrazine, hydroxylamine and their derivatives, which are no or weakly mutagenic carcinogens [50-553.

Certain peroxisome proliferators have carcinogenicity and may cause DNA damage through Hz02 formation. A major role of the peroxisomes is the breakdown of long-chain fatty acids. A wide range compounds, including clofibrate, di(2-ethylhexyl)phthalate, trichloroethylene and 2,4-dichlophenoxyacetic acid increase in the number of hepatic peroxisomes. Thus, the activity of peroxisome system (including acyl CoA oxidase) for the P-oxidation of fatty acids to produce Hz02 often increases more than that of catalase. Manyperoxisomeproliferators havebeenshown to induce hepatocellular tumours when administered at high dose levels to rats and mice for long periods [561. These peroxisome proliferators have not been proved to be mutagenic in bacterial systems. Several mechanismshavebeen proposed to explain the induction of tumours. One is based on increased production of active .oxygen species due to imbalanced production of peroxisomal enzymes; it has been proposed that these active oxygen species cause indirect oxidative DNA damage with subsequent tumour formation [57]. Endogeneous transition metal ions should participate in these oxidative DNA damage.

1973

1976

1987-

90%

60%

Mutagen Carcinogen

I

Concordance 40-60%

Figure 12. Historical change in concordance between mutagenicity and carcinogenicity. Short-term tests for genotoxic chemicals were developed to assess the potentialgenetic hazard of chemicals to humans. Ames

Metal and DNA Damage by Non-mutagenic Carcinogen

147

et al. developed the Salmonella mutagenicity test and reported that 90 % of the carcinogens tested were mutagens and 90 % of the

noncarcinogens were nonmutagens [58].However, concordance between carcinogenicity and mutagenicity decreased from 90 % to 60 96 according to subsequent reportst59-611 (Figure 12). There is a need for a short-term test to detect those carcinogen that missed by the Ames test. On the basisof our data and reported data, we classified the non-mutagenic carcinogens into genotoxic type and non-genotoxic type as shown in Table 2. Hormone and Table 2. Classification of non-mutagenic carcinogen. nickel sulfides,cobalt oxide, Metal iron nitrilotriacetate Genotoxic Benzene (oxidative DNA derivatives damage) Peroxisome proliferators

benzene, OPP, caffeic acid, PCP,

tryptophan metabolites,

+ diethylstilbestrol, hydroquinone clofibrate, phthalate esters, 2,4dichlophenoxyaceticacid, WV-14.643

"

Nongenotoxic

Hormone

estradiol-l7/3, estriol, estron, ethinylestradiol, mestranol

Polychlorinated

polychlorinated biphenyl,

aromatic hydrocarbon

2,3,7,8-tetrachlorodibenzo-pdioxin

"

Unclassified

dioxin, chloroform, arsenic, thioacetamide

halogenated aromatic hydrocarbons are truly non-mutagenic or non-genotoxic carcinogens.Halogenatedaromatichydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxincan recognize specific intracellular proteins and appearto mimic the action of hormones and growth factors and perturb signal transduction pathways, resultingincarcinogenesis C621. On theotherhand, nonmutagenic carcinogens such as metals, benzene derivatives and peroxisome proliferators .can participate inactive oxygen formation and oxidative DNA damage. Shibutani et al. reported that the formation of 8-OHdG, one of the oxidative DNA products, caused misreplication of DNA that might leadto mutation or cancer [63]. Thus, it seems reasonable to conclude that the metal-mediated oxidative DNA damage through H 2 0 2 formation is relevant for the

148

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expression of the carcinogenicity of certainnon-mutagenic carcinogens.

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10 Sequence-Selective Cleavage of DNA by Cationic Metalloporphyrins Genevihe Pratviel, Pascal Bigey, Jean Bernadou, and Bernard Meunier LaboratoiredeChimiedeCoordination,CentreNationaldelaRecherche Scientifique, 205, route de Narbonne, 31077 Toulouse cedex, France

I. INTRODUCTION Transition metal complexes endowed with redox properties and DNA affinity have been developed as “chemical nucleases”. Reagents capableof efficient DNA cleavage would have potential application in medicineas antitumoral or antiviral agents and in molecular biology as footprinting reagents or artificial restriction enzymes. Well known examples of such compounds are FeEDTA [1,2], Cu(oP), [3], metalloporphyrins [4] and COor Ru 153

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complexes [5]. Their mechanismof DNA cleavage is an oxidative degradation of DNA that can be classified in two types: (i) generation of superoxide anion from molecular oxygen or/and formation of hydroxyl radicals from hydrogen peroxide (Fenton reagent) catalyzedby metal ion salts and (ii) direct oxidationof DNA by high-valent transition metal-oxo complexes. Unlike natural enzymes, theydo not hydrolyze phosphodiester bonds of DNA,but can cleave nucleic acids by oxidation of deoxyribose units. The non-specificityof the diffusible hydroxyl radical attack and the usual destruction of sugar ring by chemical nucleases is a driving force to develop the design of hydrolytic reagents for producing DNA fragments that canbe religated and cloned[6-91. Up to now, these compounds are far less efficient than natural enzymes or oxidative chemical nucleases. We will describe here the oxidativeDNA cleavage by a highvalent metal-oxo species of Mn cationic porphyrin. Oxidative damage to sugar in this case (mprecisely hydroxylationof the S’-carbon of an intrastrand sugar) can be reverted by a mild reduction,theequivalent of a ‘‘pseudo-hydrofysis”of a phosphodiester linkage.This reagent combinesthe high reactivity of a metal-oxo species with no destruction of DNA sugarphosphate backbone. For the development of a chemical nuclease, an efficient reactivity is necessary but another important point is the sequence specificity of DNA cleavage. It can be improved by tethering a DNA recognition moiety to the active metal complex and should lead to a family of synthetic cleaving reagents with tailored specificity [lo-141. We will also present some results specific on DNA cleavage by cationic metalloporphyrins covalently linked to oligonucleotides.

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XI. Mn-TMPyP:OXIDATIVEDNACLEAVAGE Synthetic metalloporphyrins are able to mimic heme-enzymesmediated oxygenation and oxidation reactions 141. Beside this mode ofmtivity,iron or manganese porphyrins with peripheral positivecharges, e.g. Fe- or Mn-mso-tetra(4-N-methylpyridiniumy1)porphyrins(Fe- or Mn-TMPyP, Figure 1; for crystallographic data onMn-TMPyP see [15]), exhibit a strong interaction with DNA that brings into the vicinity of the targeta powerful oxidizing species. The active oxidative speciesin the case of DNA cleavage is probably the same as that involved in catalytic oxygenation and oxidation reactions described for metalloporphyrins in general, namely, a high-valent metal-oxo porphyrin complex able to hydroxylate a C-H bond or to epoxidize an olefin [4]. This active speciesis generated in the c

Figure 1 Structure of Mn-TMPyP(eachaxialposition occupied bya water molecule).

is

et

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al.

p s e n c e of oxygen atom donor compounds like iodosylbenzene [1Q ,hydmgen peroxide[171, potassium monopersulfate[17- 191 or magnesium monoperphthalate [20]. Another way to generate an oxo-metalloporphyrin species is to use molecular oxygen associated with an electron source, as does cytochromeP450 in vivo [16, 21,221. Among all these different methods, the most efficient for performing oxidative cleavage of DNA is the oxidation of cationic Mn-porphyrins by KHS05 [17, 20, 231. Data on DNA cleavage by Mnm-TMPyP/KHS05 suggest that diffusible radical species are not involvedin the reaction, because the breaks arc well-defined, and also because HzOz is at least three orders of magnitude less efficientin generating the active species leading to cleavage [17]. A catalytic activatiodreaction cycle analogousto cytochrome P-450is possible : TMPyp-Mnm+ KHSOS

? TMPyP-Mnm + ROH

+

TMPyP- mv=0 + RH

e

TMPyP- "v-OH

3.

+R.

The high-valent m P " n v = O species responsiblefor DNA cleavage is too reactive to be characterized. Turnovers of the catalyst can be observed only whenprotected fkom self-oxidation (leading to the chmmophm bleaching) by strong interaction with its target. For example when the DNA/Mn-porphyrin ratio is high (75 pM bp/5 nM), up to 5 SSBs per Mn-TMPyP molecule were observed 1173. Mn-TMqrP binds in the minor groove of AT-rich regions of DNA [23-331. The Mn-porphyrin frameworkis devoid of any Hbonding donor/acceptor capacity and intercalation is precluded by the presence of an axial ligand on manganese [H]. Thus the

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157

ability of Mn-TMPyP to select AT-rich regions of DNA is apparently electmstatic/stericin origin. It has been proposed that this cationic metalloporphyrinis attracted by the high negative potential at the surface of the minor groove [34] of AT-rich sequences. A close contactwith the minor grooveis necessary to improve the binding interaction and the cleaving efficiency of the metalloporphyrin, as deduced fromstudies on variations in total charge andin charge distribution at the macrocycle periphery [2527,35361. At high ionic strength, the reagentis unable to bind and/or cleave DNA[17,371. Considering the size of MnTMPyP, it could span over5 to 6 base-pairs in the minor groove of B-form DNA, butthe preferred cleaving site consistsof three consecutive AT base-pairs creating a suitable “box” for highly selective DNA cleavage [la, 19, 28, 29, 381. At this site MnTMPyP is strictly mediating C5’ oxidation on nucleosides on both 3’-sides of the “AT box”[39]: one single-strandbreak (SSB) on each strand leads to double-strand cleavage with a 4 base-pair shift to the 3’-end of the opposite DNA strand (Figure 2). Double-strand cleavage is possible on the same binding site because the metal-oxo entity can be generated on either side of the symmetric porphyrin ring.So far, thereis no definite proof that such a cleavage pattern is the result of two SSBs by the same metalloporphyrin activated twice inside the minor groove, or whether it is due to two different activated metalloparphyrins. On large DNA substrates, SSBs prevail [l71 and on oligonucleotides substrates (with one or two “AT boxes”) DSBs are easily obtained 1391. Besides cleavage at (AV3 cleaving sites, some secondary reaction sequences are also noted. They consist of one base-pair changein the (AV3 site (one GC base-pair outof three, no matter what the position of the GC bp)

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[25]. This secondary reactivity is particularly noticeable when drastic cleaving reactionsare performed r39-401.The reactivity at these secondary sites is one order of magnitude less than for (AV3sites.

I

Figure 2 Interaction of high-valent metal-oxo TMPyP-Mnv=O in the minor groove -of (An3sequences. Four basepair 3’ stagger of oxidative attack(4).

The mechanism of DNA cleavage is shown in Figure 3. The activated cationic metalloporphyrin initiates the reaction by CS’ hydroxylation of the deoxyribose giving the 5’-OH derivative a. Spontaneous cleavagefollows, with formation of a 3’-phosphate

Sequence-Selective Cleavage by Cationic Metalloporphyrins

a

b

C

159

d

Figure 3 Mechanism of DNA cleavage.

end and a 5”aldehyde ending derivativeb (direct break of the DNA backbone). Further transformation (first p-elimination) produces a second break on DNA the backbone with release of a S-phosphate end andthe a,p-unsaturated aldehyde compoundc. Finally a second p-elimination gives rise to freebase and furfural d as sugar degradation product. Sometimes, depending on the extent of cleavage and on the sequence at the 3’-side of (An3 site, a second oxidative reactionof activated Mn-TMPyPon the same site leadsto 5’-COOH ending fragments[40-41]. This selectiveC S hydroxylation can take place on both 3’ sides of the (An3 double-strand cleavage site of the Mn-TMPyP/ KHS05 system. This leadsto direct strandbreaks with a4 basepair 3”stagger of the cleaved residues, which conf‘iis that oxidative attackof the activated metalloporphyrin occurs from the minor groove of DNA (Figure 2). The two DNA hgments are bearing 3”protruding single-stranded termini overlapping on three base-pairs that are reminiscent of restriction enzymes cleavage sites. Both strand nicks are identical and consist of 3’phosphate termini facing a 5’-aldehyde residue. By treatment

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with NaBH4, this terminus canbe readily convertedto a 5’-OH end. Thus the oxidative cleavage followed by a reduction stepis equivalentto the hydrolysis of the phosphodiester bond: hydroxylation step+ reduction step= “pseudo-hydrolysis”.

reduction

{NIB&

S’TGCGG~TT~ 5a%ACGACI’G3’ ” A C G C C ~ ~~AMCTGCMACS

Figure 4 Pseudo-hydrolysis of a DNA phosphodiester linkage. In order to develop such chemical toolsfor gene engineering,it wastempting to check .ifthese chemically cleaved DNA fragments couldbe ligated to construct a covalently linked new strand of DNA. For that purpose, natural ligases could not be used because fragments to b e , joined ..were not carrying 5’phosphate and 3”OH ends, the usual termini, but the situation was the opposite. Fortunately, chemical ligation methods described in the literature seemed especially appropriate since the 5’-OH/3’-phosphate configuration is the most favorable for alcohol nucleophilic attack ontoan activated phosphate [42-441. We chose to use the “BrCN method‘’ described by Shabarova et al. 1421on short double-strandedDNA sequences containing one (An3site.

Sequence-Selective Cleavage

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161

R ' I check thata chemically cleaved and religated DNA shows no chemicalbasemodificationsandpresents a re-formed of the cut,we tested whetherthe phosphodiester bond at the site religated duplex could be a substrate for a restriction enzyme. We used the 35-mer duplex that is bearing a restriction site forBgl I enzyme [41]. A s shown on Figure 5, Mn-TMPyPIKHSOd NaBH4 and BgZ I are cleaving the same phosphodiester bonds but leave the phosphate at the site of the cut at the 3' or the 5'end, respectively. The 35-mer duplex was chemically cleaved (route 1, Figure 5), and religated (route2, Figure 5). It was then tested asa substrate forBgl I (route 3, Figure 5). The enzymatic hydrolysis was complete. Furthermore, alkaline phosphatase removed the S-phosphate group from the Bgl I generated fragment, The S-OH 1 6 " single-stranded frasment generated by Bgl I + alkaline phosphatase had the same electrophoretic migration as the S-OH 16-mer generated by the Mn-TIWyP/ KHSO#NaBH4 system.In a control experimentBgl I digestion of control duplex showed the same cleavage pattern. One problem remaining for the development of the cationic manganese porphyrinas a chemical DNA restriction toolis that the (AV3 cleaver affinity sequence is too often encountered on random double-stranded DNA.Oneway to improvethe sequence selectivity of this chemical nuclease would be to covalently link such cleaver with a sequence-recognition vector like an oligonucleotide.

m. CATIONIC

PORPHYRIN-OLIGONUCLEOTIDES

The manganeseporphyrinconjugateMn-trisMPyP-5'" I T m G G G G G T was synthesized [13,45] (see Figure

162 Pratviel et al.

z

d 0L

\o

m

82

0

ru

Sequence-Selective Cleavage by Cationic Metalloporphyrins

r,

X

Ti

u, ru 0

U

0

Y 0

ru

3

C

4 X

0

Y

Q

a

Q)

L

em kl

.I

163

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Pratvlel et al.

6 for structure). The tether has 17 bonds and the 16-mer vectoris complementary to the 29-mer target sequence 5'AGCTAKCCCCAAAAGAAAAGTAGAX(X. In vitro assays of the nuclease activityof the vectorized manganese porphyrin were performed by usingKHS05,to generate activated manganese-oxo porphyrin complexes (such metal-oxo species can be generated inside of a cell by molecular oxygen and an electron source a c d n g to a mechanism similar to thatof P450 enzymes and activated bleamycin).In the experimental conditions each DNA cleavage reaction(Figure 7) contained 7 n M of the 5'labeled 29-mer, from0.1 n M to 1 pMof Mn or Fe-trisMPyP-16mer, and 400 p M in nucleotides of double-stranded salmon testes DNA (2000 equivalents with respectto the 29-mer)in a solution of 125 mM NaCl and 50 mM TrisHC1buffer (pH8). Annealing of the free metallo-trisMPyP-16-mer with the 29-mer was obtained by heating at 90"C for 3 min and followedby a cooling to 37 "C within 4 h and stored overnightat 4 "C. All assays (total volume = 16 pL) were performed at 4 'C. For reactions without conjugate, the free 16-mer was hybridized with the 29-mer and then pre-incubated with 100 nM and 1 p M of Mn-"yP, the parent DNA cleaver without vector, for 15 min at 4 "C before addition of KHS05. DNA cleavage reactions were initiatedby addition of 1 mM KHSO, for Mn-or 1 mM ascorbate for Feporphyrins and maintained at 4 "C for 1 h. Reactions were then quenched by'40 mM HEPES (pH 8) and heated at 90 "C for 30 min (all concentrations listed are final concentrations). After cooling at 4 "C samples were dilutedwith 1 p.L of yeast tRNA (10 mg/ml) and 100 pL of 0.3 M sodium acetate (pH 5.2) and precipitated with300 pL of ethanol and finallyMsed with 75% ethanol and lyophylized. Fragments of DNA were analyzed by

165

Sequence-SelectiveCleavage by Cationic Metalloporphyrins

metal number of bonds G CTAG

45

“n 17 6-10

24 11-15

1 30 16-20

,

Fe

17 2 1-25

C T

.“#l

* G21

TA20

TA C G 16 TA 15 TA TA TA GC G C io GC

GC GC GC TA T C A

GI Figure 7 Analysis of the cleavageof a 5’ labeled 29-mer singlestranded DNA by oligo”n or Fe-porphyrin conjugates(* is the location of the metalloporphyrin reagent).

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20% polyacrylamide gel electrophoresis under denaturing conditions (7 M urea). The manganese porphyrin conjugate Mn-trisMPyP-l 6-meris able to cleave the single-stranded 29-mer target at a very low concentration, 100 nM,with only 14 equivalents of the vectorized DNA cleaver with respectto thetarget.Such is obtained in the presenceof a large remarkable nuclease activity excess of a random double-stranded DNA of salmon testes.From previous studies, free Mn-TMPyP was expected to only cleave the duplexf m e d by the 29-mer and the free 16mer vector at the vicinal nucleotides on the3' side of each AT base-pair triplet, 4 and G21, A20, A15 andG16. This is effectively the case (lanes 5 ) for the non-vectorized manganese porphyrin, but at higher concentration (1 PM). Almost no cleavage was detected at 100 nM concentration (lane 4). The presence of an intense smear (lanes 9 and 10) near the junction of single and double-stranded region of the duplex suggests that the mechanism of the DNA is not restricted cleavage by the vectorized manganese porphyrin to thespecific 5' mechanismobserved forthefree metalloporphyrin Mn-TMPyP. We suspected that the porphyrin was not free enough tooptimise its interaction within the minor groove of double-strandedDNA due to a too short linker.. The length of the tether was then increased from 17 to 30 bonds in order to improve the cleavage efficiency of the metalloporphyrin and to mediate non dispersed lesions. In lanes 14 and 15, the cleavage pattern was cleaner and located on the same bases T22, G21, A20 ofthe junction between single and doubleregions. In lanes 19 and 20 smears were observed in the single-stranded part of the 29-mer and they span over more bases as the lengthof the A20 cleavage sites tether rised30 bonds butthe classical G21 and

Sequence-Selective Cleavage by Cationic Metalloporphyrins

167

persist. As the lengh of the tether increased the specificity of cleavage of thereagentresembledthat of thefree metalloporphyrin (Mn-TMPyP): atG21 and A20 sites. If the arm is too long, the rea&ive moiety was reaching new remote sites. Interestingly, theFe derivative could be activated with molecular oxygen in the presence of a reductant, confirming the possibility of in vivo activationof these metalloporphyrin compounds (lanes 21 to 25). The cleavage sites were unexpectedly located on the single-stranded region and were remarkably restricted to two sites G21 and "22. Withoutquestion,the attachement of thecleaver motif tris(methylpyridiniumyl)porphyrinato-manganese(III) to an oligonucleotide vectorallowed selective recognition and cleavage of the complementary target, even in the presence of alarge excess of random DNA. Site selectivity of cleavage within the selected sequence is more challenging in order to obtain artificialDNA restriction tools. Among allpossible DNA cleaversto be attached to oligonucleotides to prepare active antisense oligonucleotides, the motif tris(methylpyridiniumy1)porphyrinatomang) (MntrisMPyP) has some significant advantages:(i) (Mn-TMPyP) is an efficient DNA cleaver; (ii) manganese is not removed from n& (iii) these cationic manganese synthetic porphyrins in vivo a porphyrins exhibit a non-negligible anti-HIV activity [4q. Inhibition of the expression of the Hnr transactivation factorTAT or other viral proteins likeREV or ENV has been obtainedwith antisense oligonucleotides[47-48]. These data provide support for further investigations with nuclease-resistant metalloporphyrin conjugates to target RNA or DNA via triple-helix approach at cellular level [49].

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ACKNOWLEDGMENTS. Thefinancial support of the CNRS, the ‘Association pour la Recherche contre le Cancer’ (ARC, Villejuif), the ‘Agence Nationale de Recherches sur le Sida’ ( A N R S , Paris), the ‘Region Midi-PyrMes’ and Genset (Paris) is gratefully acknowledged.

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26. B. Ward, A. Skorobogaty, J. C. Dabrowiak, Biochemistry 25,7827-7833 (1986). 27. G. Raner, B. Ward, J. C. Dabrowiak, J. Coord Chem 19, 17-23 (1988). 28. G. Raner, J. Goodisman, J. C. Dabrowiak, in Metal-DNA Chemistry (Ed.:T.D. Tullius), ACS SymposiumSeries 402, 1989, pp. 74-89. 29. R. F. Pasternack, E. J. Gibbs, in Metal-DNA Chemistry (Ed.:T D. Tullius), ACS Symposium Series 402, 1989, pp. 59-73. 30. R. F. Pasternack, E. J. Gibbs, J.J. Villafranca, Biochemistry 22,5409-5417 (1983). 3 1. J. A. Strickland, L. G. Marzilli, K.M. Gay, W. D. Wilson, ibid 27, 8870-8878 (1988). 32. R.J. Fiel, J. Biomol. Struct. & Dynamics 6, 1259-1273 (1989). 33. L. G. Marzdli, New J. Chem 14,409-420 (1990). 34. a) X. Hui, N. Gresh, B. Pullman, Nucleic Acids Res. 18, 1109-1114 (1990); b) P.K. Weiner, R. Langeridge, J. M. Blaney, R. Schaefer, P. A. Kollman, Proc. Nutl. A c d Sci. USA 79,3754-3758(1982). 35. L. G. Marzilli, G. Pethti, M. Lin, M. S. Kim, D.W. Dixon, J. A m Chem Soc. 114,7575-7577 (1992). 36. a) M. A. Sari, J. P. Battioni, D. Dupd, D. Mansuy, J.B. Le Pecq, Biochemistry 29,4205-4215 (1990); b) U. Sehlstedt, S. K. Kim, P. Carter, J. Goodisman, J. F. Vollano,B. Norden, J. C. Dabrowiak, ibid 33,417-426 (1994). Atta, J. Bernadou, B. Meunier, S. M. Hecht, 37. R.B.Van Biochemistry 29,4783-4789 (1990). 38. J. C. Dabrowiak, B. Ward, J. Goodisman, Biochemistry

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by Cationic Metalloporphyrins

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28,3314-3322 (1989).

39. M. PitiC, G. Pratviel, J. Bernadou, B. Meunier, Proc. Natl. Acad Sci. USA 89,3967-3971(1992). 40. M.PitiC, G. Pratviel, J. Bernadou,B.Meunier in The

Activation of DioxygenandHomogeneousCatalytic Oxidation (Eds.:D.H. R. Barton, A. E. Martell, D. T. Sawyer), Plenum, New-York, 1993, pp. 333-346. 41. G. Pratviel, V. Duarte, J. Bernadou, B. Meunier, J. Am. Chem. Soc. 115,7939-7943 (1993). 42. N. I. Sokolova, D. T. Ashirbekova, N. G. Dolinnaya and Z.A. Shabarova, FEBSLetters 232,153-155(1988). 43. K. J. Luebke and P.B. Dervan, J. Am. Chem. Soc. 113, 7447-7448 (1991).

44. N. Dolinnaya, N. I. Sokolova, D. T. Ashribekova and 2.A. Shabarova, Nucl. Acids Res. 19,3067-3072 (1991). 45. C. Casas, C. J. Lacey and B. Meunier, Bioconjugate Chem. 46.

4,366-371 (1993).

L.Ding, J. Balzarini, D. Schols, B. Meunier, E. De Clercq,

Biochem Phurmucol. 44, 1675-1679 (1992). 47. E ' Shimada. H. Fugii, B. Maier, S. Hayashi, H. Mitsuya, S. Broder, A.W. Nienhuis, Antiviral Chem Chemotherapy , 2, 133 (1991). 48. a) G. Sczakiel, M. Pawlita, J. Wrol. 65,468 (1991); b) S. T. Cload, A. Shepartz, J. Am.Chem.Soc. 116, 437-442 (1994); c) C.HClbne, J.J. ToulmC, Biochem Biophys. E. Uhlmann, A. Peyman, Acta 1049,99-125(1990); Chem. Rev. 90,543-584 (1990). 49. a) H. Han, P. B. Dervan, Proc. Natl. Acad. Sci. 90,38063810 (1993); b) C. H6li?ne, N. T. Thuong, Angew. Chem. Int. E d Engl. 32,666-690 (1993).

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l1 Lanthanidemu Complexes as SyntheticNucleases:Hydroxyalkyl Group Participation in Catalysis Janet R Morrow, K. 0.Aileen Chin, and Kelly Aures Chemistry Department, Acheson Hall, State University of New York, Buffalo, NY 14214

I. INTRODUCTION It has been known for many years that metal ions efficiently promote substitutionreactions o f phosphateestersandphosphoricanhydrides. Thereiscurrentlyarenewedinterest in thedesign of metalion goal of producing complexcatalysts for thesereactionswiththe catalysts for the specific cleavage of RNA (l), DNA (2) or the cap structure of RNA (3). Here we present new lanthanide@) complexes of macrocycles the 1,4,7,10-tetrakis(2-hydmxyethyl)-1,4,7,10tetraazacyclododecane (THED) and lS, 4S, 7S, lOS-tetrakis(2hydroxypropyl)-tetraazacyclododecane (S-THP) thatmay be useful as synthetic nucleases. The mechanism o f transesterification of RNA and phosphateestersbytheselanthanide(1II)complexes is discussed. In addition, the lanthanide(1II) complexes of THED promote an unusual substitution reaction o f a phosphate diester whereby a bound alkoxide group of themacrocycleactsasanucleophiletowardthephosphate diester to form a covalent adduct. This type o f reaction is reminiscent 173

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of the covalent adducts formed in the hydrolysis of phosphate esters by metalloenzymes such as alkaline phosphatase.

r H0

H0

L~I(THED)~+

L~(s-THP)~+

Ln = La, Eu

Ln = La, Eu, Lu

Figure 1 Lanthanide(II1)HydroxyalkylMacrocyclicComplexes.

II.

LANTHANIDE@I) HYDROXYALKYL MACROCYCLIC COMPLEXES

Although lanthanide ion salts promote rapid RNA cleavage, o f common ligands such as the lanthanide(II1) complexes as cleavingagents (4). Ligands polyaminocarboxylatesareinactive must be designed such that a positive overall charge is maintained on the macrocycliccomplex. In addition,theligandmustbindstrongly all availableexchangelabile to thelanthanideionwithoutblocking coordination sites. We have prepared lanthanide(III) macrocyclic complexes that catalyzeRNA cleavage by transesterification (5, 6). An importantclassofligands for the lanthanide(II1)ionscontains the 1,4,7,1O-tetraazacyclododecanemacrocycle with four additional pendent donor groups that are neutral (6-9).

LanthanideWl) Complexes as Synthetic Nucleases

175

The Eu(n1)and La(III) complexes of THED and the La(III), Eu(III) and Lu(III) complexes of S-THP have been prepared (Figure 1) as their trifluoromethylsulfonate salts (7, 8).Solution'H, 13C and ? L a N M R studies and solid-state structural data support octadentate coordination of the THEDand S-THP ligands to thelanthanideions.Withthe exception of the La(THED)(CF,SO,), complex, all of the lanthanide(II1) hydroxyalkyl macrocyclic complexes are extremely inert to lanthanide ion release in water at 37 "C,at pH 6.0 or pH 7.4. In the presence of excess Cu2+ as a trapping agent, half-lives for the dissociation of the are 0.87 and 11 days, THED complexes of La(II1) and Eu(III) respectively, and the half-livesfor dissociation of the S-THP complexes of La(III), Eu(II1) and Lu(III) are 73, 100 and 53 days respectively. The ionic radii of the trivalent lanthanide ions decrease by approximately 15% upon traversing the series from La(III) to Lu(III). This contraction has two importanteffects from the standpointof catalysis. First, the coordination number will be greater for the lighter lanthanide ions than for the heavier lanthanide ions. If the THED and S-THPmacrocyclesremainoctadentate,thenonewouldpredictthat Eu(I1I)andLu(II1) therewill be fewersites for catalysisforthe complexes than for the La(III) complexes. Solid state studies bear this out. The La(III) complex of an octadentate macrocyclic ligand that is structurally similar to THED has a ten coordinate La(II1) cation that contains two sites for smallmoleculebinding(6).Thesolidstate structure of Eu(S-THP)(H,O)(CF,SO,), featuresa nine-coordinate Eu(II1) cationwithasinglecoordinationsite for bindingwater(8)and is 2. Fluorescencelife-timestudiesof the Eu(II1) showninFigure complexes of S-THP or THED in aqueous solution indicate that 1.3 f 0.5 water molecules are bound to the Eu(1II) cation in these complexes (10). Second, the decrease in ionic radius across the lanthanide series has the effect of increasing the Lewis acidity of the later lanthanide ions. One would anticipate that this would increase the potency of the lanthanideion as atransesterification or hydrolysiscatalyst.The anticipated Lewis acidity order is observed with the 1anthanideaI) STHPcomplexes(9). AS determinedbypotentiometrictitrations,pK, values for a lanthanide-bound water or lanthanide-bound hydroxyalkyl groupdecreaseacrosstheseries:La(S-THP)(CF,SO,),,8.40(kO.05); Eu(S-THP)(CF,SOJ,, 7.80 (kO.1) and Lu(S-THP)(CF,SO~)~, 6.40 (fo.1) and9.30 (kO.1).

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Figure 2

IIL

The structure of the [Eu(TtP)(H,O)I3+ cation. Reproduced with permission from ref. 8.

RNA CLEAVAGE

The S-THP complexes of L a m ) and Eu(III) and the Eu(III) complex of THED promote cleavage of the oligomers of adenylic acid A&,, at37 "C,pH7.60 (11). Productsinclude2',3'-cyclicadenosine monophosphate, consistent with cleavage by transesterification of RNA. Pseudo-first-orderrateconstants for thecleavage of &-A,, in the presence of 2.00 x 104 M complex are 7.1 (fo.8) x l@'S", 4.2 (B.5) x 10'' S" and1.9 (fo.3) x lo4 S" forLa(S-THP)(CF3S0d3, Eu(STHP)(cF3so3)3and Eu(THED)(CF,SO,), respectively.Incomparison, a hexadentate Schiff-base macrocyclic complex of Eu(III) has a pseudo-

177

Lanthanide(lll1 Complexes Synthetic as Nucleases

first-order rate constant of 4.2 x lo4 S" under similar conditions (5). Cleavage of A,-A,, by Eu(THED)(CF3S03),is first order in complex for a complex concentration ranging from 1.00 to 2.00 x lo4 M, and 0.95 (M.15) M"s" isobtained. In asecondorderrateconstantof contrast, the Lu(II1) S-THP complex does not promote substantialRNA cleavage under similar conditions.

7.0

Figure 3

7.5

8.0

8.5

9.0

The pHdependence of thesecondorderrateconstant for the transesterification of 1 by La(S-THP)(CF3SOJ, at 37°C.

The pHdependence(1 1) of thepseudo-first-orderrateconstant for cleavage of adenylic acid oligomers by the Eu(III) THED complex and

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for transesterificationofthemodel RNA compound, 1 U = 4nitrophenylphosphate ester of propylene glycol), suggests that a lanthanide(II1)-bound hydroxide or a lanthanide(II1)-bound alkoxide is the activecatalyticform ofthecomplex.ThesigmoidalpH-rate profiles are fit to equation (1) below with K, the apparent kinetic K, second-order the and constant. rate Values of k, for transesterification of 1 byEu(THED)(CF,SO,),, La(S-TW)(CF3S03),, and Eu(”IED)(CF,SO,), at 37°C ‘are respectively: 7.2 x lo2,6.3 x 10, M1s” with K, values of 1.0 x lo’, 7.9 x lo9and 10’and1.4x 4.0 x lo4. K, and k, values for the cleavage of A,,-A,, oligomers by EuWD)(CF,SOJ, at 37°C are 7.9 x lo-’ and 1.1 M”s”, respectively. An example of the pH dependence of the rate constants for transesterification of 1 by the La(1II) complex of S-THP is shown in Figure 3.

The most striking aspect of the kinetic data for RNA cleavage by the various complexes is that the most Lewis acidic complex as indicated bypK,values(Lu(S-THP)(CF,SOJ,) is theleasteffectivepromoter. In addition, the La(1II) S-THP complex is a better promoter than the Eu(II1) analog although pK, values indicate that the reverse should be true. In fact, the order that is followed for the S-THP lanthanide(II1) complexes correlates to that of the basicity of the bound hydroxide or alkoxide group which presumably participatesin general base catalyzed RNA cleavage.Alternately,anotherfactorwhichwouldproduce the observedtrend is thedecrease in coordinationnumber for trivalent lanthanide ions on traversing the lanthanide series from lanthanum(II1) to lutetium(I1I).Withanencapsulating ligandsuch as S-THP or THED,there are relativelyfewsitesopen for bindingandcatalysis. As discussed in section 11, solid state and solution studies indicate that S-THP and THED have at least one the Eu(II1) complexes of coordinationsite for smallmoleculebindingwhereas the La(I1I) two sites.Therearenosolid-state complexesarelikelytohave structural studies of analogousLu(II1) macrocyclic complexes. Solution N M R data of similar macrocyclic complexes indicate that the= is one small molecule binding site for complexes of the heavier lanthanide ions such as Lu(II1) (12). l is J catalytic in Transesterification of RNA and the phosphate ester ( lanthanide(III) complex. Several turnovers are observed in the transesterificationof a dinucleotide by a Eu(III) hexadentate Schiff-base

179

Lanthanide(lll1 Complexes Synthetic as Nucleases

complex. The Eu(III) complex THED of appears to be a tiansesterificationcatalyst as well. In thepresence of atwenty-fold catalytic turnovers are observed for the excess o f 1, several transesterification of 1 by Eu(THED)(CF3S03), at 3TC, pH 7.40. The sole phosphorus product of the reaction of 1 with the Eu(III) THED catalyst even after several days at 37°C as observed by use of 31PNMR is the cyclic phosphate ester (Figure 4).

-

4 OH

0

I

-o-p=o I

+

4-N02PhU

+ I"+

4-NO2Ph 0

1 Figure 4

W.

Productsfromthetransesterificationofthephosphate ester 0.

SUBSTITUTION REACTIONS'OF PHOSPHATE ESTERS

There are manyexamplesofmetalionpromotedhydrolysisof phosphate diesters (13-15) and phosphoric anhydrides (16-21). For the are thought to very efficient metal complex catalysts, these reactions proceed by metal ion binding to the phosphate ester followed by attack of ametal-boundhydroxide or metal-boundwatermoleculeatthe (22) phosphoruscenter.Elegantstudies bySargesonandcoworkers demonstrated this important pathway. An ''0 label in a water molecule Co(II1) complex was incorporated into the product bound to a phosphate. Therearerelativelyfewstudies ofphosphate ester substitution by metal-boundnucleophilesother than hydroxide or water.Examples include phosphate ester transesterification by a Co(II1)-amido complex

180

Morrow et al.

(23), and more recently phosphate ester substitution reactions promoted An interestingearly bylanthanide(III)peroxidecomplexes(24-25). example is the nucleophilic attack of a Zn(II)-carbaldoxime anion on aphosphorylimidazole (26). Thelanthanide(III)complexesdiscussed hereprovide an additionalexample.Productanalysisandkinetic studies indicate that a bound hydroxyalkyl group acts as a nucleophile toward a phosphate diester. Treatment ofbis(4-nitropheny1)phosphate (BNPP) with eitherthe L a m ) or Eu(III) complex of THED at 37 "C, pH 7.40 results in therapid production of 4-nitrophenolate. Pseudo-first-order rate constants for the production of 4-nitrophenolate (0.1M NaCl and 0.01 M Hepes buffer) are 2.7 (fo.2) x W S-' and1.0 (fo.05) x 10' S" in the presenceof 1.00 x lo-, M La(I1I) or Eu(III) THED complexes, respectively. Rate constants for Eu(THED)(CF,SO,), are slightly higher in the absence of NaCl (1.9 f 0.2 x lo4 S"). The production of 4-nitrophenolate is first order in Eu(THED)(CF,SOJ,. ThepH-rateprofile is sigmoidaland computer-assisted fitting of the data to equation (1) gives an apparent pK, of theactivecatalyst(7.38)that is close to that measuredby potentiometric titration (7.50) and a k, of 0.19 M's". In contrast, the rate constant for production of nitrophenolate from treatment of BNPP (1.8 f 0.2 x l o 6 S") withEu(S-THP)(CF3S03), is fifty-fold less than This isin contrastto the five-fold that for Eu(THED)(CF,SOJ,. difference in rate constants for RNA cleavage by the two complexes.

In contrast to the catalytic nature of phosphate ester transesterification by Eu(THED)(CF,SO,),, the production of 4-nitrophenolate from BNPP in the presence of the Eu(III) complex is stoichiometric in nature. An equivalent of 4-nitrophenolateis produced under conditions where there is atwenty-fold excess o f BNPP to Eu(III) THED complex. Product o f this inhibitionbynitrophenylphosphate(NPP) is notthecause behavior.Additionofanequivalentof NPP didnotinfluencethe initial rate of 4-nitrophenolate production. The reaction products were examined by use of 31PNMR. A solution of 0.01 M La(THED)(CF,SOJ, or Eu(THED>(CF,SOJ, was added to a 0.01 M solution of BNPP and the pH was adjusted to 7.6. A new NMR resonance was observed in the Eu(THED)(CF,SOJ, reaction (-5.2 ppmversus Hp0.J andtwonewresonanceswereobserved for the La(THED)(CF,SOJ, reaction (-4.7 ppmand -5.0 ppm). These ,'P resonances are assigned to a covalent adduct of the macrocycle with 4nitrophenylphosphate. 'H N M R data of the La(I1I) complex adduct is

Lanthanide(ll1) Complexes Synthetic Nucleases as

181

supportive of this assignment. The two 31PN M R resonances observed with the La(III) complex are attributed to an adduct with the La@) ion bound or to an adduct of the free macrocycle. This is consistent with the faster dissociation rate of the La(III) complex of THED. The adducts are readily isolated as solids from treatment o f solutions of the of potassium Ln(THED)(CF,SO,),complexeswithoneequivalent hydroxide and BNPP in ethanol. Elemental analysis of the products are consistent with covalent adducts of the formula [Ln(THED-H+)(P03(4NO,C,H,O))][CF,SO,],where thephosphateester is bound tothe hydroxyethyl group of the THED ligand as shown in Figure 5. HPLC analysisand 31P studiesindicatethat NPP is produced in small amounts. Studies are underway to determine whether NPP is produced by the hydrolysis of the covalent adduct.

OR I

0 Figure 5

Production of a covalent adduct from attack boundalkoxidegroupoftheTHEDligand phosphatediester. R is 4-nitrophenyl.

of a

on a

preliminary studies as monitored by N M R suggestthatsimilar substitutionreactionspromotedbyLn(THED)(CF,SO,),occur for phosphate esters with good leaving groups. Cyclic phosphate estexs do not react measurably with the Eu(III) complex of THED after 24 hours at 37°C. In contrast phosphoric anhydrides are rapidly hydrolyzed by both theLa(III)andEu(I1I)complexesofTHED.Thenucleotide product ofATP treatment with La(THED)(CF,SOJ, is exclusively ADP.

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Other 31PN M R resonances indicate that inorganic phosphate is produced as well as a new species with a chemical shift of 0.58 ppm. This resonance is tentativelyassignedtoacovalentadductofthe macrocycliccomplexwithphosphatebasedon its appearancewhen La(THED)(CF,SO,), is treated with other phosphorylating agents such as NPP. In addition, over time the 31PNMR resonance for inorganic phosphate increases at the expense of the resonance for the covalent adduct. The proposed pathway for production of 4-nitrophenol from BNPP and Ln(THED)(CF,SOJ, is shown in Figure 5. This pathway is congruent with the fact that theEu(THED)(CF,SO,), complex has a single site for binding small molecules (10) and with the stoichiometric nature of the reaction. In addition, thelesserreactivity of the Eu(II1) S-THP complex toward BNPPis consistent with the rate determining formation of an adductbynucleophilicattackofadeprotonatedhydroxyalkyl group.Nucleophilicattackonthephosphatebythemorebulky be anticipatedtoproceed deprotonatedhydroxypropylgroupwould more slowly than attack by a deprotonated hydroxyethyl group. It is ofinterestthatmetalloenzymessuch as alkalinephosphatase catalyze the hydrolysis of phosphate monoesters through the formation of a similar covalent adduct (27-29). Hydrolysis is thought to proceed in two steps. First, a serine group attacks the phosphate ester to form a covalent adduct and it is thought that one of the two Zn(I1) ions may facilitate this step by lowering the pK, of the serine group. This step is analogous to the pathwayobservedherewiththelanthanide(1II) complexesof THED. Finally, the adjacentzinc(II)-boundhydroxide hydrolyzes the covalent adduct to complete the hydrolysis reaction.

In summary,hydroxyalkylmacrocycles are good ligands for the trivalentlanthanides.Thependenthydroxyalkylgroupsparticipate in general base catalyzed transesterification of RNA and phosphate esters and participate as nucleophiles in stoichiometric substitution reactions of phosphate esters.

ACKNOWLEDGMENTS

the NationalInstitutesofHealth (GM46539)andby the donorsof the PetroleumResearchFund, administered by the American Chemical Society.

This work was supported by

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as Synthetic Nucleases

183

REFERENCES 1.

2. 3.

4. 5. 6. 7.

8. 9. 10. 11. 12.

13. 14.

15. 16. 17.

18.

For a review see: J. R. Momw, Artificial Ribonucleases, in Advances in Inorganic Biochemistry (G.L. Eichhorn and L. G. Mamilli, Eds.), prentice Hall, EnglewoodCliffs, N.J., 1994, vol. 9, ch. 2. L. A. Basile, A. L. Raphael, and J. K. Barton, J. Am. Chem.

SOC. 109: 7550-7551(1987). B. F. Baker, J. Am. Chem. Soc. 115: 3378-3379(1993). J. R. Morrow and V. M. Shelton, New Journal of Chemistry 18: 371-375(1994). J.R. Morrow, L. A. Buttrey, V. M. Shelton, K. A. Berback, J. Am. Chem. Soc., 114: 1903-1905 (1992). S. A. Amin, J. R. Morrow, C. H. Lake, M. R. Churchill, Angew. Chem. Int. Ed. Engl., 33: 773-775 (1994). J. R. Morrow,and K. 0. A. Chin, Inorg.Chem. 32: 3357-3361(1993). K. 0. A. Chin, J.R. Morrow, C. H. Lake, M. R. Churchill, M. R., Inorg. Chem. 33: 656-664 (1994). J. R. Morrow, S. A. Amn i , C. H. Lake, M. R. Churchill, Inorg. Chem. 32: 4566-4572 (1993). J. R. Morrow, S. A. Amin, D. A. Voss, Jr, C. H. Lake, M. R. Churchill and W. Dew. H o m k s , Jr., in

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Chin,and J. R. Morrow, submitted. Bryden, C. N. Reilley and J. F. Desreux, Anal. Chem. 53: 1418-1425(1981). See ref. 1 for references through 1992. J. H. Kim and J. Chin, J . Am. Chem. Soc. 114: 97929795(1992). J. N. Burstyn and K. A. Deal, Inorg. Chem. 32: 35853586 (1993). H. Sigel and P. E. Amsler, J . Am. Chem. Soc. 98: 73907400(1976). H. Sigel, F. Hofstetter, R. B. Martin, R. M. Milburn, V. Scheller-Krattiger and K. H. Scheller, J. Am. Chem. Soc. 106: 7935-7946(1984). R.M. Milburn, M. Gautam-Basak, R. Tribolet and H. Sigel, J. Am. Chem. Soc. IO7 3315-3321 (1985). C.C.

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J.Visscherand A. W.Schwartz, NucleicAcids Res. 20(21): 5749-5752(1992). R. N. Bose, R. D. CorneliusandR. E. Viola, Inorg. Chem. 24: 3989-3996 (1985). G.P. Haight, Jr., Coord. Chem.Rev. 79:293-319 (1987). D. R. Jones, L. F. Lindoy and A. M. Sargeson, J . Am. Chem. Soc. 105: 7327-7336 (1983). J. MacB Harrowfield, D. R. Jones, L. F. Lindoy and A. M. Sargeson, J. Am. Chem. Soc.102: 7733-7741 (1980). B. K. TakasakiandJ.Chin, J. Am.Chem.Soc. 115: 9337-9338(1993). B. K. Takasakiand J. Chin, J. Am. Chem. Soc. 116: 1121-1 122 (1994). G. L. LloydandB. S. Cooperman, J. Am. Chem. Soc. 93: 4883-4893(1971). E. E. Kim and H. W. Wyckoff, J. Mol. Biol. 218: 449464(1991). J. E. Butler-Ransohoff, S. E. Rokita, D. A. Kendall, J. A. Banzon, K. S. Carano, E. T. Kaiserand A. R Matlin, J. Org. Chem. 5 7 142-145(1992). P. Gettins and J. E. Coleman, J. Biol. Chem 258: 408416(1983).

12 Initiation of DNA Strand Cleavage by Iron Bleomycin: Key Role of DNA in DeterminingthePathway of Reaction David H. Petering,Patricia F’ulmer, Wenbao Li, andQunkaiMao Departmentof Chemistry, Universityof Wisconsin-Milwaukee, Milwaukee, WI 53201 William E. Antholine Medical College of Wisconsin, Milwaukee, WI 53226

I.

INTRODUCTION

Metallodrugs are rare agents in the modem apothecary. Next to the multitude of organic c~mpw~lds, coapuatively few metal complexes, metal bmding ligands, and other structures that interact with intracellular metals are used to treat human disease. For example, among theapproximately 30 drugs c o d y employed in cancer chemotherapy, onlythree or four require metals, cisdiamminedichlom Ptand its cyclobutanedicarboxylatederivative, bleomycin, md possiblyadriamycin [l-31. Amongthese,only the second generation platinum complexresulted from deliberate inorganicpharmacological research. Considering the remarkable development of inorganic chemistry over the past several decades and the rmccess of the drugs mentioned above,it is surprising that relatively little attention has been devoted to metal complexes and related compounds as sources of drugs. 185

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Petering et al.

An importaut site of reaction of chemdhenpevtic agents is DNA. Among the types of damageresultingfrom such d o n s are oxidative alterations in St~cture initiated bythe attack of .ctivoted forms of oxygea m the bases or backbone of the polymer. Metal complexes that include redox active metal ions are particularly adeptat catalyzing the reductionof dioxygen in d l s to species such as hydroxyl radical, which readily attack organic molecules in their vicinitysuch as DNA [4]. Bleomycin is the model for a number of inorganicnucleaseswhichhave been designed during the past decade [5]. Shown in Figure 1, its structureand that hypothesizedfor Fe bleomycin display the essentialfeatures of all of thesecomp0undS"thepresence of a metal coordination site where dioxygen activation occurs, a domain that has affinity for DNA, and a linker between them. In this type of structure, a dox-active metal ion becomes associated with the polymer and so generates an activated species of oxygen in the vicinity of the target DNA. Reaction of Iron Bleomycin with Dioxygen

A.

Bleomycin is isolated froma streptomycete as a partial copper complex [6]. Considering its strong biological activity in mammalian systemsand even greater potency against microrganisms such as Euglena gracilis, it probably tames as an antibiotic in nature which has been designed to cause lethal DNA damage [7]. As described below, Blmacts against tumor cells as an iron not a copper complex. In an elegantset of papers by Peisach, Horwitz, and coworkers [8-10], it has been shown that FeBlm can be converted to an activated form, probably HO2-Fe(III)Blrn, in three general ways(Figure 2). Fe(II)Blm c811 seme bothas site of activation and reductant, Fe(III)Blm can m t with 02 and two electrons from another source, or Fe(IIQBlm and H2% can react directly to form the activated intermediate. Once formed in the presence of DNA, H02-Fe(III)Blm reacts with theC4'H of deoxyribose to initiate strand scission or base release in a reaction equivalent to hydrogen abstraction by an hydroxyl radical [l 1,121. Inits absence, H02-Fe(III)Blm~dergoesan ill-defined self-inactivation mction which renders the drug much less capable of causing further DNA damage when reacted again with reductant, 0,.and DNA [13,14]. The reaction of HO2-Fe(IIr)Blm with DNA Ic+ds either to outright single strand scission as a 3carboo base propad from deoxyribose is formed W h base releaseleaving alkaline labilesites (Figure 3) [15]. The first PJhWay requiresdioxygen and another electron. As writtea, starting with H02'

'Abbreviations:bathophenanthroline disulfonate, BPS;bleomycin,Blm; major congeners of Blm, Blm A2 andB.& redox-inactivated bleomycin, RIBlm.

Initiation of DNA Strand Cleavage by Iron Bleomycin METALBINDING DOMAIN BINDING DOMAIN DNA I

CO-TERMINAL AMlNE

Figu~? 1 Structures of bleomycins. (a) Metal-free ligand and (b) metallobleomycin.

187

Petering et

188 ~

al.

SELF INACTIVATION

Figun 2 Routes of formation and reaction of activated FeBlm. Fe(III)Blm, the reaction produces one hydroxyl did,which is not deteded by spin trapping: H02-Fe(IQBlm

+ DNA + O2 + e' ->F.(m)Blm + cleavage products

+ 'OH

(1)

The other pathway can occur under anaerobicconditiws and effectively d u c e s the peroxide ligand to two water m o l e c u l e s U 8 base is released from DNA leaving a modified alkaline labile sugar: H02-Fe(III)Blm

+

+ DNA -> -k

Fe@I)Blm free base modified DNA 2H20

+

(2)

This paper examinesthe m e c h a n s im ofcellular DNA damage caused by bleomycin, focusing attentionon the chemistrywhich occurs when metallodrug and DNA interact to initiate DNA damage and considering bow the drug may cause double strand cleavage.

B.

Cellular DNA Strand Cleavage by Bleomycirrs

Both single and double straad damage is ddected in cdls. It hs beat shown that the formation of double strrnd damage follows the same Blm concentration dependace as inhibition of cell proliferatioa [16,17]. Furthermore, damage is prognssive over tims unless hlted by the dditim of 1, IO-phenmthroline [18]. Once inhibited, angoing DNA np.ir is observed in

which a significant fraction of double but not single s t z d piof DNA remains fragmented. Thus,8 strong relrtionshipbetwaen double strand scimion and cytotoxicityhas beenestrblished. Importantly, under conditionsleuiing the

0

I

0

a

.

1 I = 0-a-o

189

l90

Petering et al.

cellular DNA damage, the concentration of drug in the nucleus is less than 1 molecule per 16base pairs [16]. Chemicalstudiesdescribedabovedemonstratethat under suitable conditions FeBlmcan cleave the DNA backbone in the pl.lesence of O2 [8-lo]. Other reports indicatethat a'+, Blm,and 0, also produceDNAstrand scission [19]. Blm, FeBlm, and CuBlm CIUI also inhibit cell proliferation and cause DNA damage [16,20]. However, in HL-60 and Euglena gracilis cells which were made iron deficient by reducing iron available to them in their growthmedia,onlyFeBlmretained full DNA strandscissionactivity[2]. Similarly, in preparations of nuclei, Blm was unable to causemore than 1% of the DNA double strand breakage resulting from exposure to Fe(I1I)Blm [21]. Thus, it is evident in cells that Blm requires iron for its activities and that the comparable activity ofBlm, CuBlm and FeBlm is due to the facile conversion of the first two forms into FeBlm.

II.

REACTIONS OF METALLOBLEOMYCINS WITH DNA

The DNA damaging action of Blm is striking because it occurs when the base pair to drug ratio in the nucleus is on the orderof 16:1 and because it results in both single and double strand damage at all concentrations of drug as discussed below.To determine which of the pathways of activation of FeBlm might take place under physiological conditions and how double strand scission might occur despite the presence of only onesite of activationof 0, in the drug, studies of the reactionsof DNA-bound CO- and FeBlm havebeen undertaken.

A.

Redox Reactions of Co(IQBlm with 0,: Influence of DNA and Structure of Produds

The following set of reactions characterizethe oxidation of Co(II)Blm by 0, [22]: Co(II)Blm O2 e O2400Blm Ft BlmCo(II)~~-Co(lI)Blm + 02 202-Co(lI)Blm BlmCo(II)-02-Co(II)Blm + H+ "> H02-Co(III)Blm co(III)Blm (Form I) (Form II)

+

+

(3) (4) (5)

A dioxygenated species of Fe(II)Blm and H02-Fe(III)Blm are thought to form during the corresponding reaction of F@)Blm withO2 so that reactions 3-5 appear to be an excellent model for the reaction of Fe(I1)Blm with OT A similar set of reactionsoccurs when Co(I1)Blmreacts with O2 in the presence of DNA [23]. Nevertheless, there are significant differences. First, the rate of conversion of 02-CoBlmto Form Iand 11 is dramatically dependent

Initiation of DNA Strand Cleavage

by Iron Bleomycin

191

upon the base pair to drug ratio. Given a binding site si= of 2-3 bum pairs, OSKC this Wio 6-8: 1 reactions4 and 5 almost leaving st.ble %Co(II)BlmDNA. Apparently, once the ratio of base pairs to CoBlm molecules iacrersesbeyond the size of the bindingsite of 2-3base pairs and adjwxntdrug molecules can not make direct contact, reaction 4 can not take place without raorgaaizrtian'of drug molecule to establish conditionsfor dimerization. The dioxygmted adductassociatedwithoriented DNA f i h was examined by ESR spectroscopy to determine whether the presence of DNA ccmstrriwd the orientation of the paramagnetic center [24]. It was found that the dioxygen species was fixed in a plane nearly perpendicular to the double helical axis (Figure h). This finding provides strong evidence that the metal coordination site 1s well as the DNA binding domain interacts with DNA. Combined with the i n d i m evidence that Blm binds in the minor groove of DNA, these dah arc .Is0 consistent with a model in which the oxygen+xygen bond of O+cQI)Blm a n be approximately colinear with the C4"H bond on either strlad that is the initialsite of attack in the cleavage reaction (Figure 4b). NMR structures of Form I and I1 have been completed,revealing conformntid complexity not reported for the structures of ZnBlm andOCFe(II)Blm [B-271. As seen in Figure 5, the bithimle is folded back upon the pyrimidine in Form I to constitute a compact structure with a central pocket containing the peroxy ligand to co(m). Form I binds strongly to DNA and provides a model for how both themetal and DNA domains mayjointly interact with DNA.

I

0

I

O;Jgt;.....&0:j

2'

3'

a

4' H

I'

......... f 1'

0

4' t0 i )

0-

/

\o

b

Figure 4 Structure of O,-CoBlm bound to DNA. (a)Orientation of the oxygensxygen bond with respect to the DNA helix axis. (b) Hypothetical orientation of dioxygen in the minor groove of DNA. The dot represents the double helix axis.

I

Bithiazole Peroxide ligand ("L"in cleft) COW) (Hexagon in cleft)

+I

Charged

I Pyrimidine Figure 5 Energy minimized structure of H02..Co(III)Blm NMR analysis.

4 determined by

B. Redox Reaction of Fe@)Blm with 0,: Influence of DNA The oxidation of Fe(II)Blm by 0, occurs rapidly in the absence of DNA. As with CoBlm, the rate of d o n slows in the presence of DNA. Recent studies illustrated in Figure 6 support a pathway of reaction in which 0, rapidly bmds to Fe(I1)BlmDNA (step 1, reaction 6) [28]. This is followed by a slower releaseof dioxygea into solution that occurs with kineticsthat are seoond order in 02-FeOBlmDNA (step 11, reaction 7 and 8).

+

F@)BlmDNA 02 it OyFe(II)BlmDNA (6) 202-Fe(lI)BlmDNA P [DNABlmF@)-O2-Fe(II)BlmDNA] +0 2 (7) [DNABlmFe(II)-02-Fe0BlmDNAI+H+ -> H02-FQ)BlmDNA F m B l m D N A (8)

+

Although a dimer intermediate may form in reaction 7, that is uncertain. The important finding is that the secondader rate constant for step II decreases dramaticallyfrom 1180 M"s" to 160 as theDNA base pair to drug ratio increases from 511 to 8/1. Thus, as in the oxidatim of Co(lI)Blm bound to DNA,theseparation of FeBlmmolecules-alongtheDNAdoublehelix effectively inhibitsthe biomolecular reaction described in reactions 7 and 8. As the base pair to FeBlm ratio increases to 20 to 1, large amounts of 02-

Initiation of DNA Strand Cleavage by Iron Bleomycin

193

100

90

z

P l-

a LL 2

5cn

80

0"

ml

60 0

IO

20 30

TIME (MINUTES)

F'igure 6 Reaction of 0, with Fe(I1)BlmDNA. (IUptake ) of O2 a h Fe2+ is added to BlmDNA. Ratio of base pairs to F e o B l m is 1 0 1. (II) Release of 0, from 02-Fe(II)BlmDNAduring redox reaction. (m) Release of O2 from 02-Fe(II)BlmDNA after reaction of the in the d u c t with bathophenanthroline disulfonate.

Fe(II)BImDNA can be detected by addition of the F e 0 chelating ageat, htbophenanthroline disulfonate to the reaction mixture, which liberates s t o i c h i d c amounts of Fe@) and 0, (step III of Figure 6 ) from bound dioxygearted drug. These results show clearly thatat large base pair to drug ratiossuch m are found in cells treated with Blm, 02-FeBlm willbe stabilized on DNA and will require the input of electrons from sources other than Fe(II)Blmto cany out the activation and DNA cleavage reactions (Figure 2). Examination of the properties of dioxygenated metallobleomycinshas revded that it is difficult to remove 0, from O 2 ~ B l m D N A and 0,Fe(II)BlmDNA.

Petering et al.

194

The rate constant for dissociationof 0, From the latter is 0.003 S-', which is remarkably small in comparison with that for the loss of 0, from the a chain of hemoglobin,28 s-l[28]. This fmding suggeststhat the irondioxygen adduct l i e that of 02€o(II)BlmDNA exists in a constrained site that restricts the rate of dissociation of 0,. Anothermodel for 02-Fe(II)BlmDNA is ON-Fe(II)BlmDNA. A comparison of the interaction of nitric oxide with Fe(II)Blmin the absence or presence of DNA by electron spin resonance spectroscopy has shown that the ESR spectrum of theNO adductis altered when the drugis bound to DNA [29]. As with O 2 ~ ) B l m D N Ait, appears that ON-Fe coodination site 6 ~ l s e sthe presence of the polymer, consistent with a folded conformationof the molecule and perhaps the association of metal both and DNA binding domains with DNA.

m.

REDOX-INACTIVATED IRON BLEOMYCIN

Figure 2 indicates that once the drug is rcciv.tsd to HO2-Fe(IIl)Blm, it is competent to initiate DNA damage. A l t d v e l y , in the abseace of DNA it undergoes a suicide-like reaction thatmodifies its own structure such that it is W longer effective in DNA strand scission chemistry. h l i e r work hd demonstrated that this material, called redox-inrctivated bleomycin (RIBlm), was fully capableof activating 0, to H~-Fe(III)RIBlmin the pnsenceof Fe2+ 1151. However,therewasevidence that tbe bithiamle chromophore in the molecule had been modified [30]. Returning to the structure of H02-Co(III)Blm 4 as a model for the corresponding iron species,theperoxideligand is positioned in close proximity to the bithiazola moietyd, thus, might be ex ted to react with this portion of the molecule. In addition, unpublished ll&lm Nh4R experiments show that the '13Cd resol~ancesfor the Blm 4 aud 4 complexes differby several ppm (Figure7a). This result shows that the metal center senses the positively charged tails ofthe two, othewise identical molecules, as might be expected in flexiblefolded conbmations for such structures. Indeed, in an ongoing NMR analysis ofRIBlm 4,it has bees found that the sole differences in the modified structure are the destruction of the native bithiamle group and the loss of one of the methyl groups from the dimethyl sulfonium group in the tail of the molecule (Figure 7b). All other rotons in the molecule are accounted for sccording to detailed 2dimeasional NMR analysis of m l m A2 From these combined results, it appears that RIBlm 4 fails to cleave the DNA backbone because neither of the DNA binding elementsof Blm 4remains intact. As a consequence, RIBlm A2does not strongly associate with DNA and like &methylBlm A2, which also lacks a methyl group in the dimethyl sulfonium moiety, has little DNA cleavage activity [15].

L

.

Initiation of DNA Strand Cleavage by Iron Bleomycin

-3

1 3

t c

l$

195

1

196

Petering et al.

N.

MECHANISM OF DOUBLE STRAND SCISSION BASIC CONSIDERATIONS

Cellular studies point to a relationship between double straad DNA cleavage and cytotoxicity [16,17]). While past studies have ducidatcd many aspects of the alternative pathwaysof DNA damage, single strand scissiaa and base release (Figure 3), much less is known about double strand cleavage. Nevertheless,thefollowingpropertieswill need to be inoorporrted into a mechanism for this process. First, it has been shown that after reaction of Fe(n1)Blm and ascorbate with supercoiled plasmid DNA (Figure 8), double strand cleavageis observed (band III) dong with single strand brealrage (band II) at every concentration of FeBlm. In contrast, d o n of the plasmid with Fe2+ first convertsthesupercoiled structure (band I) to the single strand scission product, relaxed circularDNA, and then further cleaves it to double strand linear mol~ules,probably as a result of producing enough proximate singlestrandbreaks on opposite strands to get double strand breakage. Therefore, as in cells single and double strand p d u c t s are produced at all effective concentrations of FeBlm, suggesting that the two types of damage are mechanistically related. The kinetics of formation of single and double strand bmaks am also similar (Figure 9). As such, they are not consistentwith independent, sequential formation of singleandthendoublestrandbreaks. I n s t e a d , they support reaction to c a w single or double strand breaks by single FeBlm molecules at particularsites. Indeed, because the base pair to dmg ratio is large, one hypothesizes that dissociation of FeBlmfrom the site of doublestrand cleavage does not occur prior to complete double strand brenkage; othewise random rebinding of the drug would prevent further reaction at the original site. n d e d DNA Finally, double strand damage to produce largely blunt e breaks or ones offset by one nucleotide on the two strands occurs at the same sites where base releasetakes place [3 1.321. Pmbbly, single strand damage is also confined to these same sites (321. The presenoeof these site-spccific alternative reaction productssuggests that reaction of activatedFeBlm is kinetically partitioned among three pathways. A d i n g to a racent modd. H%-Fe(III)Blm, reacting at a site, initidly auses either bre Felersa or the introduction of a single strand break at a sitaspscific G-pyrimidine [32]. If the former, no further &ion occurs on the other straad. If tbe latter, no reaction may occur, another break might be formed on the second otrpnd leading to double strand cleavage,or thc second stranddso may be damaged by base release. A constraint on the mechanism foreach pathway is that little free radical production is observed in these DNA damage reactions [33]. To 8ccoullt for resction on both strands, the same model wggeskd a collcerted mechanism for reactivation of FeBlm following the initiationof single strand breakageat the primary site of attack that involves reactionof Fe(III)Blm

Figure 8 DNA strand scission of pBR322plasmidby species of F$+. Reaction of 15 FM plasmid base pairs, 25°C in phosphatebuffer, pH 7.4, with specified concentrations of (left) Fe2+ added as Fe(NHd2(S0d2 and (right) Fe(II1)Blm plus 100 mM ascorbate. Reaction times: left, 15 min and right, 30 min.

W

2

5 W

U

10

30 40 TIME, MINUTES

20

50

60

Figure 9 Kinetics of single and double strand scission of pBR322 plasmid by Fe(In)Blm andascorbate.Form I, nativeplasmid;form XI, r e l a x e d circular DNA with single strand breaks; form 111, linear DNA with double strand breaks. 15 p M base pairs was reacted with 0.1 CM Ft(l1I)Blm in phosphate (pH 7.4) buffer at FWN)$.

25".

Reactions were stopped at each time point with 0.2 mM

Petering et al.

198

with the hydroperoxide intermediatein the strand scission pathway in analogy to its reaction with hydrogen peroxide (Figures 2 and 3). In this model, the hydroperoxide provides the hydroxyl radical equivalent to abstract the C4"H from a deoxyribose on the second strand. The problem with this hypothesis is that in the process the first strand is left without the necessary hydroperoxide intermediate to proceed to cleavage products. Two other possiblemechanisms are consistentwith much of the information about double strand scission. First, a um~rtedreaction might occur as follows to initiate cleavage at C4"H and C4"-H on both strands:

+ C4'-H-> (oOH)Fe(III)BlmDNA + C4'. + H20(10) (oOH)Fe(nI)BlmDNA + C4"-H->Fe(III)BlmDNA + C4"' + Hz0 (11)

HOZ-Fe(III)BlmDNA

In this mechanism, the drug%ound peroxide homolytidy provides two hydroxyl radicalsto abstract thetwo hydrogen atom. Like the base-release path (reaction 2), which also converts the peroxide ligand to two water molecules, reactions 10 and 11 do not produce residual hydroxyl radicals. h the dbet mechanism*~OH)F~(III)B~~DNA must lmd~rgoa secund cycle of activation to HO2-Fe(II1)BlmDNA, which thm initiatcs cleavage011 the other strand. Two hydroxyl radicals arc produced sequmtially in this mechanism which m not deteded, making it a less attractive alternative to reactions 10 and 11 [M].However, the first mechanism does not provide an obvious to achieve a mixture of siugle and double strand cleavage, whereas in the second mechanism a mixture of these damaged products could result if reactivation occufi at a rate comparable to the rate of dissociation of Fe(I1I)Blmfrom the single stranddamaged site. Underthatcondition, the dissociation event would effectively limit DNA damage at the site to single strand cleavage. V.

CONCLUSIONS

The bindmg interactions within andbetween various forms of Fe- or CoBlm and DNA play critical roles in determining the chemical reactivity of these species with respect to DNA. The particular stoichiometryof base pairs to Fe@)- or Co(1I)Blm alone specifies whether adduct formation or oxidationreduction in the presence of0, will occur. Adduct formation has consequences for structure and reaction. Binding of 0, to CoBlmDNA is accompanied by rigorous orientation of02 with reqxct to the DNA structure. Thennodynamic andkineticstabilizationofdioxygen species result.Dioxygenbinding also decreasestheligandsubstitutionreactivity of Fe(1I)BlmDNA [28]. Taken together thw results support the hypothesis that both metal and DNA binding

Initiation of DNA Strand Cleavage

by Iron Bleomycin

199

domains interact intimately with the DNA structure. The perturbgtim of the ESR signal of ON-Fe(I1)Blm by DNA also supports this hypothesis. Indeed, the findingthat H02€oBlm 4 existsin a foldedstructurenowprovides a structural model in which bothmetal and DNA binding domainsof the drug may jointly interact with the minor groove. It is expected that the mechanism of initiation of single and double strand damage will depend on these binding interactions between drug and DNA which are now emerging.

ACKNOWGEMENTS The authors thank the United States National Institutes of Health National Cancer Institute, and theAmerican Cancer Society fortheir support of thisresearch through grants NIH-CA-22184and American Cancer Society -DHP 3 1C.

REFERENCES l. 2. 3.

4. 5. 6.

7.

8. 9. 10.

11.

K. Rsdtke, R. Bymes, F. brnitzo,W. E. Antholine, and D. H. Petering, J. Inorg. Chem. 43: 456 (1991). K. Radtke, F. A. brnitzo,R. Bymes, W. E. Antholine, and D. H. u b d i lo n (1994). Petering, Bidem. J., acceptd for p D. H. Petering, R. W. Bymes, .ad W. E. Antholine. Transitim metpl requirements for the nction of antibiotics, in Handbook on Metal-Ligand Interactions in Biological Fluidr, Vol. 2 (G. Berthon. Ed.), Marcel Mer,in p m , 1994. R. W. Bymes, M. M o b , W. E. Antholine, R. X. Xu, d D. H. Petering, Biochemistry 29: 7046-7053 (1990). D. S. Sigman, Biochemistry 29: 9097-9105 (1990). H. Umezawa, Y. Suhara, T.Takita, .ad K. Maeda, J. Antibioz., Sa.A 19: 210-215 (1966). S. Lyman. P. Taylor. F. brnitzo,A. Weir, D. Stoat, W. E. Antholine, and D.H. Petering, Biofhcn Phannaml. 3& 42734282 (1989). E.A. Sausville, J. Pcisach, awl S. B. Horwitz, Bi17: 2740-2745 (1978). E. A. Sausville, R. W. Stein, J. Pcisach, .ad S. B. Honrvitz, Biochemistry 17: 2746-2754 (1978). R. M. Burger, J. Peisach, .ad S. B. Horwitz, J. Biol. Chan. 256 11636-1 1 6 4 4 (1981). J. C. Wu, J. Kozarich, and J. Stubbe. J. Biol. Chem. 257: 3372-3375 (1982).

200 12. 13. 14. 15. 16. 17. 18. 19.

U). 21.

22. 23. 24. 25. 26.

27.

28.

29.

30. 31. 32. 33. 34.

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J. C. Wu, J. Kozarich, and J. Stubbe, J. Biol. chem 258: 4694-4697 (1983). R. M. Burger, J. Peisach, and S. B. Honrvitz, J. Biol. chem.257: 3372-3375 (1982). J. Templin, L. Berry, S. Lyman, R. W. Byrnes, W. E. Antholiue, and D. H. Petering, B i o h Phunna~url.43: 615-623 (1992). J. Stubbe, and J. Kozarich, c h m r . Rw. 87: 1107-1136 (1987). R. W. Bymes, J. Templin, D. Sem, S. Lyman, and D. H. Petering, Cancer Res. 5& 5275-5286 (1990). R. W. Bymes and D. H. Petering, Rud. Res. 237: 162-170 (1994). R. W. Byrnes and D. H. Petering, Bioahem. Pharmaml. 42: 1241-1248 (1991). 0. M.Ehrenfeld, J. B.-Shipley, D. C. Heimbrodr, H. Sugiyanm, E. C. Long, J. H. van Boom, G. A. van der Mad, N. J. oppenheimer, and S. M. Hecht, Bioahanisrry 26: 931-942 (1987). E. A. Rao, L. A. Suyan, W. E. Antholine, and D. H. Petaing, J. Med. ChRm. 23: 1310-1318(1980). R. W. Byrnes .ad D. H. Petering, Bh&m l%urmumL, in press (19w. R. X. Xu, W. E. Antholine, and D. H. petering, J. BM. chcm 267: 944-949 (1992). R. X. Xu, W. E. Antholine, d D. H. Petering, X BM. chcm 267: 950-955 (1992). M. Chikira, W. E. Antholine, urd D. H. Pehing, J. Bwl. than, 264: 21478-21480 (1989). R. X. Xu, D.Nettesheim, J. D. Otvos, and D. H. Petering, BioCJlcmhtry 33: 907-916 (1994). M. A. J. Merman, C. A. G. Haasaod, U. K. Pandit, & C . W. Hilbers, Eur. J. Bioahem. 273 211-225 (1988). M. A. J. Akkeman, W. J. F. Neijman, S. S. Wijmenga. C. W. Hilbers, and W. Vermel, J. Am. chsn.Soc. 112: 7462-7474 (1990). P. Fulmer and D. H. Petering, Biochemistry 3 3 5319-5327 (1994). W. E. Antholine and D. H. Petering, BioQhan, Biophys. Rcr. C o m m ~ n .91: 528-533 (1979). M. Nakamura and J. Peisach, J. Antibiot. 42: 638-647 (1988). L. F. Povirk, Y.-H. Han, and R. J. Steighaer, Biodtemistly 28: 5808-5814 (1989). R. J. Steighner and L. F. Povirk, Proc. Natl. A d Sci. USA 87: 8350-8354 (1990). L. 0. Rodriguez and S. M. Hecht, Biochem. Biophys. Res. Cbmmun. 104. 1470-1478 (1982).

D. H. Petering, R. W. Bymes, and W. E. Antholine, them.-Biol. I~U~TUCL 73: 133-182 (1990).

13 NickelComplexesinModification NucleicAcids

of

Steven E. Rokita, Ping Zheng, Ning Tang, Chien-Chung Cheng, Ren-Hwa Yeh, James G. Muller, and Cynthia J. Burrows Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY 11794

1. lNTRODUCTlON Issues of nickel metaholism, toxicity, and carcinogenicity inevitably relate back to the umrdination and oxidation chemistry of this metal. The ligand environment strictly controls the efficiencyand diversity of nickel-dependent reaction. For example, certain nickel(I1)-peptide complexes undergo spontaneous oxidation in the presence of the dioxygen [l], whereas free nickel salts such as Ni(l1) chloride are quite inert to oxidation. The laboratories of Burrows and Rokita have combined their respective expertise in nickel-based catalysis and conformation-dependent modification of DNA to explorethe intrinsic reactivity of nucleicacids with biomimetic and macrocycliccomplexes of nickel. A series of square planar complexes were found to promote efficient and selective modification of guanine residues that were highly accessible to solvent. Theorigin of this specificity likely derives from direct interaction hetween guanine N7and nickel and concomitant delivery o f an associated peracid. Most recently, additional nickel complexes have been developed to promote nucleohase arylationand strand scission alternatively. 201

202

Roklta et al.

11. SELECTIVITY OF NICKEL-PROMOTED OXIDATION The initial goal of our collaboration wasto define the fundamental specificity and reaction determinants of macrocyclic nickel complexes with nucleic acids. NiCR (shown below) was one of the first species examined since it appeared to exhibit many properties known to promote effective catalysis also of olefin epoxidation 12,3]. Most subsequentinvestigationshave continued to focus on this example.The initial targets of modification were synthetic oligodeoxynucleotidesthat provide well defined systems for rapid and unequivocal characterization of nucleic acid specificity [4,5].

A. Modification is Specific for GuanineResidues Allguanineresidues (G) of single-strandedoligodeoxynucleotidesare subject to modification in the presence of NiCR and a water soluble oxidant such as KHSO, (Figure l). Typically, equimolar concentrationsOtM) of NiCR and a DNA strand are incubated for minutes in the presence of excess peracid. These conditions induce an alkaline lability that leads to strand scission at the effected guanines as a result of subsequent treatment with hot piperidine (lane 1, Figure 1B) [ 6 ] . The final pattern of fragmentation is equivalent to that generated by theMaxam-Gilbertsequencing reaction for G (alkylation by dimethylsulfate, lane 2, Figure 1B). In contrast, no spontaneous strand scission is evident directly after the metalmediated oxidation reaction (lane 3, Figure 1B). DNA modification based on nickel required the participationof both the peracid and nickel complcx, and accordingly no strand scission was observed after incubating DNA with either the metal or oxidant alone and assaying with piperidine. In addition, nickel chloride could not substitute forNiCRnor could an oxidant such as H,O, substitute for the peracid. Further mechanisticcharactcrization is discussed Collowing thesections below on the conformational specificity of guanine oxidation.

B. Only Solvent Accessible Guanines are Oxidized in the Presence of NiCR Synthetic oligodeoxynucleotides were further used to examine the relationship between DNA secondary structure and modification by NiCR. Guanine residues remained the only target of oxidation throughout our investigations. Most interestingly, the extent of reaction was very dependent on

z" v

m

3

Nickel Complexes

d

U I

In

in Modification Nucleic ofAcids

C

e

u u

h

0

m

W

n

E3

rrl

W

C

CI)

U

v)

.E

2

m

203

204

Rokita et al.

the local environmentsurroundingguanine. Modification wasseverely limited or not detectable for G residues that were paired to their complementary base, C, within the interior of a standard Watson-Crick duplex. In contrast, most non-canonical arrangements of G were readily oxidized by NiCR [7]. While all of the G residues in single-stranded A were susceptible to modification by NiCR, none reacted under equivalent conditions in the presence of the complementary strand A' (Figure 2). The only G-C pairs that exhibited an apparent reactivity were those at a helical terminus, and eventhen modification could have been due to base pair fraying. For example, the 3' terminal G in (he radiolabeled strand B was not protected from reaction in the presence o f the fully complementary strand B'. Other guanine residues accessibleto NiCR werc either mispaired (C) or unpaired as in an extrahelical bulge (A+[A'-C]) or loop (D and E) (Figure 2).

5 ' -

3'

3-T

lG-0 m

+ L

I

A 1

H T-A

-b&---k

B B '

L

4-4 T-3

A

A-T

m;"$c

D

\-I. 3-T

7-T l-A c c

Figure 2 Conformationspecificmodification deoxynucleotides using NiCR.

-

J ~P+G-T+T-@\

4J-A"LLly E t f of

["PI-labeled oligo-

Nickel Complexes in Modification of Nucleic Acids

205

Thisconformationalspecificity is not generally mimicked by other metal or non-metal reagents. The G residues in the loop formed by D were clearly distinguished by NiCR but not by other metal complexes based on iron, copper or manganese [S]. The most commonly applied reagent for G modification, dimethylsulfate, exhibits little or no sensitivity to secondary structure 191. The unique specificity of NiCR then likely derives from the distinctcoordination and redox chemistry of the metal center. Taillander's laboratory had previously demonstrated that Ni(I1) salts selectively coordinate to G N7 [lo], and this might also provide a basis for nucleic acid recognition by NiCR. Our attention next focused on modification of yeast tRNAph"in order to establish a precise correlationbetween reactivity and solvent accessibility of a single functional domain within the guanine nucleotides. This target was quite valuable sinceits surface properties have heen extensively characterized by experimental and theoretical techniques[ 11-13]. NiCR readily modified 12 out ol the 23 residues of guanine and i t s natural derivatives when the RNA was partially denatured by the absence ofMg" [ 14). In the native conformation, only 4 residues were reactive (Figure3) and these residues corresponded exactlyto the guanines with the greatest accessibility and surfacepotential at theirN7 positions 113). Thisrelationship has greatly enhanced the value of NiCR as a probe for nucleic acid conformation. Now, guanine modification induced by this complex can be used to indicate the structural environment of G N7 specifically. 4

G-18

G-l9 G -20

Figure 3 Selective reaction oftRNA*' in thepresence of NiCR (adapted from ref. 15). G-34

64

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Rokita et al.

C. NiCR has Utility as a Probe for Nucleic Acid Folding The structure of the self-splicing ribozymeof Temzlrymena was next examined with NiCR to test the more general applicability of this reagent as a probe for nucleic acid folding. The polynucleotide target in this case contained a total of 409 nucleotides and formed a compact and stahle conformation in solution. Only 11 G residues in the native structure were able to react with NiCR. Most importantly, those results could be anticipated from the exposure of G N7 suggested by a model of the ribozyme structure [16]. Although NiCR is large enough to express a high degree of conformational specificity, it is still small enough to react with a G residue within the ribozyme active site. This residue had not previously been shown to be accessible by the much larger probe, RNase TI, which may he prohibited from entering the active site. The length of the ribozyme represented a maximum for direct analysis of a 5'-I3'P] labeled polynucleotide. An alternative method for characterizing reaction of long sequences relies on a primer extension assay. In these cases, sites of modification are detected by their ahility to terminate chain elongation as catalyzed by reverse transcriptase. This second procedure wasrecently used to reinvestigatethe reaction of the Tefrahymerra ribozyme with NiCR (Figure 4) [ 171, and it essentially confirmed the earlier results. Hydroxyl radical protection studies of the Cech laboratory 1181 have also been summarized in Figure 4 to illustrate the complementary nature of this modification technique. Hydroxyl radical reaction measures the solvent cxposure of the ribose backbone which is clearly independent of the exposure of G N7. Interpreting the results generated by a single modifying reagent can be extremely difficult in the absence of supporting data. Chemical modification reflects the most reactive and not necessarily the most abundant conformation. Both RNA targets described above folded into a unique conformation, and therefore thepattern of reactive sitescould be used to diagnose secondary and tertiary structure directly. For sequences that equilibrate between two or more structures, individual species are not easily distinguished in the ensemble of d a h . Our laboratories attemptedto characterize a model RNA pseudoknot with NiCR but were unable to prevent rapid interconversion between alternative conformations (Figure5). NiCR failed to detect the dominant pseudoknot even after reducing the reaction time to one min and temperature to 4 'C. Analysis of this system was originally attempted because the laboratory of Tinoco had previously characterized

Nickel Complexes in Modification of Nucleic Acids 163

?

207

169 U

d

Q

C A A

S'

226 G A G G G4

-

288

Figure 4 Reaction of the fefrdynenaintron (L-21Sca RNA) with NiCR and hydroxyl radical. Sequences that wereprotected from hydroxyl radical are indicated by shading [ 181. Residues that were modified by NiCR and detected by primer extension are indicated by arrows. A solid arrowhead represents reaction in the presence and absence of Mg"; an open circle, in the ahsence of Mg" only; and an open arrowhead, in the presence of Me" only.

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pseudoknot J

A CU G U c u C-G A-U G C G-C G U-A AUUUC-G 3

3' hairpin

5' hairpin

*

NiCR inducedcleavage not determined

Figwe 5 (A)Conlhrrnationalequilibrationand

(B) reaction of a model

pscudoknot. the structural equilibrium of this RNA quite successfully hy other chemical and physical methods [19, 201.

Nickel Complexes in

Modification of Nucleic Acids

209

Ill. LIGAND CONTROL OF NICKEL ACTIVITY A. Selective Oxidation of Guanine is Not a General Characteristic of Most Metal Complexes A variety of metal complexes related to NiCR were surveyed during the early phase of this project to highlight the major requirementsfor selective modification of G residues. Only positively charged, square planar Ni(I1) complexes represented by NiCR and Ni(cyclam) were active[ h ] . Although the nickel complex of glycylglycylhistidine is square planar and known to promote oxidative reactions [21,22], i t s net negative charge appeared to prevent interaction with the polyanionic DNA in this application. Octahedral species such as Ni(cyclen) and Ni(tren) provided no vacant coordination sites and were unreactive. Square planar copper derivatives, CuCR and Cu(cyclam), with a minimal tendency to gain two axial ligands were also unable to effect modification of DNA in the presence o f 21 peracid.

B. Ligand Donor Strength Regulates the Activity of the Macrocyclic Nickel Complexes The initial studies used to compare coordination geometry and reactivity couldnot easily distinguish hetween the relative contributions of ligand field strength and Ni(III/II)potential. This required synthesis and examination of complexes that expressed independent variation of electrochemical potential and field strength. Fifteen compounds derived fromNiCR, Ni(cylam) and Ni(Me,cyclam) were chosen, and their reactivity was tested with the hairpin-forming oligodeoxynucleotide D 1231. Selectivity for the unpaired guanines remained constant throughout the comparative investigation. Onlytheextent of modification varied. The order of increasing reactivity, 1 c 2 c 3 c 4 c 5, correlated closely to an increase in ligand donorstrength(Figure 6). In contrast, the trend in E,, values(ranging from 0.78 V for 4 to 1.25 V for 1) did not coincide with the nickel reactivity. This suggest.. that the high in-plane ligand field provided by a tetraazamacrocycle is repsonsihle for the appropriate ligand exchangerates necessary for reaction.

210 v)

b

v)

b

0

m

0

U)

cu

v)

v)

cu

(U

NOIlVaIXO VNa %

0

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Rokita et al.

Nickel Complexes

in Modification Nucleic of Acids

21 1

C. Nucleic Acid Recognition and Oxidation May Occur Through Direct Association of the Nickel Complex, Oxidant, and Guanine Thecorrelation between macrocyclic ligand and metal-promoted DNA oxidation suggested that the optimum geometry of the initial Ni(I1)complexis four-coordinate, square planar. Although this would preclude direct interaction between the Ni(I1) species and DNA, it would provide axial coordination sites that were thermodynamically and kinetically accessible once the metal center was oxidized from Ni(I1) to Ni(II1). Accordingly, a macrocyclic complex of Ni(Il1) was expected to bind DNA more strongly then its Ni(l1) derivative. This was confirmed using an electrochemical technique first developed by the laboratory of Bard [24]. For Ni(cyclam), the ratio of binding constants, KNi[111)/KNi(1,), was approximately 30:l (Figure 7) 1231. Preliminary analysis further suggested that this ratio increased to greaterthan 200: 1 when calf thymus DNA substituted for GMP. In contrast,AMP and CMP didnot greatly affecttheelectrochemistry of Ni(cyclam). Spectroscopic investigations arenow in progress to define the nickel-DNA interactions more fully.

l

with 50mM GMP

E(V0LT)

Figure 7 The effect of GMP on the electrochemistry of Ni(cyclam).

Rokita et al.

212

A variety of mechanisms can be proposed for selective oxidation of guanine through intermediate formation of Ni(lI1) complexes. Of the nucleohases, guanine is most susceptible to electron abstraction. Consequently, a pathway involving electron transfer to regenerate theNi(I1) complex and form the radical cation of guanine was consideredhut later found unsatisfactory.Theoxidantdependence of G modificationsupports an alternative process that requires a two electron oxidant and may involve direct oxygen atom transfer. One electron oxidants including K&O, and NazIrCI, were unable to replace KHSO, for modification of DNA. Furthermore, Ni(I1I)cyclam did not induce guanine oxidation alone or in the presence of these one electron oxidants [S]. Theactiveperacids, KHSO, and magnesium monoperoxyphthalate (MMPP),additionally provided theopportunity for coordination to the Ni(II1) intermediate. A simplc peracid such as peracetic acid functioned poorly in this application in accord with its lack of ready ligation to the nickel complex 161. A ternary complex of Ni(IIl), guanine N7 and oxidant might then assemble during reaction to achieve the target specificity and oxidant dependence described here (Figure S). The cis orientation of the oxidant and G is shown in order to indicate that direct oxygen transfer could be possible.

2+ A -

A

S.. ..

.iL'w..,&

&&ne i n d u c e d strand scission at guanine

n

S"0H

__*

residueswith tigWy accessible N7 positions

Figure 8 Proposed mechanism for selective modification of guanine by NiCR.

D. Oxidation Products are under Investigation A common product of deoxyguanosine oxidation, %oxodeoxyguanosine, might also be generated during the nickel-dependent modification of DNA. This guanine derivative could account for the alkaline lahility at sites of reaction [ E ] but , it.. formation has not yet been confirmed. Control studies revealed that 8-oxodeoxyguanosine was degraded by KHSO, (or "PP) alone more rapidly than dG was oxidized by the combined presence of NiCRand KHSO, (MMPP). If the S-oxo derivativedoes indeed form

213

Nickel Complexes in Modification of Nucleic Acids

during the DNA reaction, then it too would likely he oxidized further. The search for possible secondary derivatives is ongoing. The ultimate strand fragmentation induced by piperidine treatment yielded both 3' and 5' terminal phosphate groups(Figure 9). Theseproducts are consistent with a variety of processes that include hase oxidation and deglycosylation.

A

B

5' A

3' T A

T ( A ' T

T

C

A G"

A T

-

i

2'" -=

A T

C

T A

:\ C

T

A T 32PA

c

C

T A

0 G

yr*

*

/

-phosplloglycolate

@@ma

A C T A

32pT

Figure 9 Electrophoretic analysis identified the formation of phosphate termini in (A)5'-13'P1 and (B) 3'-[32P] labeled oligodeoxynucleotides.

E. Alternative Ligands Promote Distinct Reactions of DNA Macrocyclic ligands play a dominant role in the activation and control of nickel-dependent reactions. As descrihed ahove, CR, cyclam and many of their derivatives stahilizea characteristic coordination geometry and oxida-

TMAPES

Rokita et al.

214

tion chemistry of nickel that allows for selective oxidation of guanine. Active nickel complexes formed by an alternative ligand might then be expected to induce nucleic acid modification that is consistent with the distinct ligand chemistry. In addition to the macrocyles described above, salen derivatives also form square planar complexes with Ni(I1) and stimulate the metal redox chemistry. However, unlike CR, salen has the added ability toform ligand-derived radicals. Since Ni(salen)is not water soluble, our laboratories synthesized a cationic salen derivative (NiTMAPES) [26]. Consistent with the unique chemistry of salen, NiTMAPES induced arylation rather than oxidation of DNA in the presence of KHSO,. This modification was indicated by the formation of products with apparent molecular weights greater than the target oligodeoxynucleotides 1261. Only a small fraction of the adducts induced strand scission after piperidine treatment,and consequently, a primer extension assay was uscd to identify the complete set of modification sites. Reaction targets were once again limited to solvent accessible guanine residues of DNA and RNA [17,26].

H

NiCR

A final ligand system used tocontrolnucleic

Ni(N,HF)

acid reactivity in this report was originally developedby the laboratory of Kimura [27] for nickel activation of molecular oxygen. Under these conditions, electron transfer between Ni(1I) and oxygen was facilitated by metal coordinationto a pentadentate macrocycle. A related set of ligands have now been prepared and their complexes tested in a preliminary manner with plasmid DNA. The nickel complex Ni(N,HF) was found to promote spontaneous strand scission in the presence o f 0, without need of a subsequent alkaline treatment 1281. This process apieared independent of hydroxyl radical since standard trapping agents such as mannitol (50 mM) and ethanol (1.7 M) did not inhibit the scission reaction. The sequence and conformation de-

Nickel Complexes

in Modification Nucleic of Acids

215

pendence of modification is now under investigation and not expected to mimic the selectivity of NiCR. As currently designed, Ni(N,HF) should have little affinity for DNA. The overall charge of this complex is neutral, and the nickel would become coordinatively saturated when bound with oxygen. Logical derivatives of this and other ligands are now being synthesized for selective recognition and reaction in vitrQ and in vivo.

ACKNOWLEDGMENTS We greatly appreciate the dedication, enthusiasm and support of our coworkers and collahcmtors. In addition, we thank Professor Woodson for the ferral1ytnerta intron RNAand ProfessorTinoco for thepseudoknot RNA. Research support was provided by the American Cancer Society, National Institutes of Health, National Science Foundation and the Stony Brook Center for Biotechnology sponsoredby the New York State Science and Technology Foundation.

REFERENCES Bossu, E. B. Paniago, D. W. Margerum, S. T. Kirksey, and J. L. Kurlz, Inorg. Cltern. 17: 1034-1042 (1 978). 2. J. F. Kinneary, J. S. Alhert, and C. J. Burrows, J. Am. Cltern. Soc. 110: 6124-6129 (1 988). 3. H.Yoon, T. R. Wagler, K. J. O’Connor, and C. J. Burrows, J. Am. Cltern. SOC.112: 4569-4570 (1990). 4. S. E. Rokita and L. Romero-Fredes, Nucleic Acids Res. 20: 3069-3072 ( 1 992). 5. U. Hiinsler, and S. E. Rokita, J. Am. Cltern. Soc. 11.5: 85544557 (1993). 6. X. Chen, S. E. Rokita, and C. J. Burrows, J . Am. Clrem. Soc. 113: 58x45886 (1 991). 7. X. Chen,C. J. Burrows, and S. E. Rokita, J . Am. Clrem. Soc. 114: 322325 (l 992). 8. J. G. Muller, X. Chen, A. C. Dadiz, S. E. Rokita, and C. J. Burrows, Pure R.Appl. Cltern. 6.5: 545-550 (1993). 9. P. E. Nielsen, J. Mol. Recop?. 3:1-25 (1990). 10. J. A. Taboury, P. Boutayre, J. Liquier, and E. Taillaadier, Nucleic Acids Res. 12: 4247-4257 ( 1 984). 11. S. H.Kim, F. L. Suddath, G.J. Quigley, A. McPherson, J. L. Sussman, A. H. J. Wang, N. C. Seenlatl, and A. Rich, Science 18s: 435-440 ( 1 974). 12. J. D.Rohertus, J. E. Ladner, J. T. Finch, D. Rhodes, R. S. Brown, B. F. 1. F.P.

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C. Clark, and A. Klug, Nature 2.50: 546-551 (1974). 13. R. hvery, and A. Pullman, Bioplrysicnl Clrem. 19: 171-181 (1984). 14. X. Chen, S. A. WOodSo11,C. J. Burrows, and S. E. Rokita, Biochemistry 32: 76 10-76 16 ( 1993). 15. S. R. Holbrook, J. L. Susslllan, R. W. Warrant, and S. H. Kim, J . Mol. Biol. 123: 63 1-660 (1 978). 16. F. Michel, and E. Westhof, J.Mol. Biol. 216: 585-610 (1990). 17. S . A. Woodson, J. G. Muller,C. J. Burrows, and S. E. Rokita, Nr.lcleic Acids Res. 21: 5524-5525 (1993). 18. J. A. htha111, a11d T. R. Cech S C ~ ~ I 24.5: ~ C L 276-282 . (1989). 19. J. D.Puglisi, J. R. Wyatt, and 1. Tinoco, J. Mol. Biol. 214: 437-453 (1990). 20. J. R. Wyatt, J. D. Puglisi, and 1. Tinoco, J. Mol. Biol. 214: 455-470 (1990). 21. T. Sakurai, and A. Nakahara, Inorg. Chim. Actn 34: L243-244 (1979). 22. D.P.Mack, and P. B. Dervan, Bioclremisrry 31: 9399-9405 (1992). 23. J. G. Muller, X. Chcn, A. C. Dadiz, S. E. Rokita, and C. J. Burrows, J. Am. Clrem. Soc. I I4: 6407-641 1 (1992). 24. M. T. Cqrter, M. Rodriguez, and A.J. Bard,J. Am. Clrem. Soc. I l l : 8901891 1 ( 1 989). 25. M. Kouchakdjiaa, V. Bodepudi, S. Shibutani, M. Eisenberg, F. Johnson, R. P. Grollman, and D.G. Patel, Biochemistry 30: 1403-1412 (1991). 26. J. G. Muller, S. J . Paikoff, S. E. Rokita, and C. J. Burrows, J . Inorg. Bioclrem. 54: 199-206 ( 1994). 27. E. Kimura, R. Machida, and M. Kodama, J . Am. Clrem. Soc. 106: 54975498 (1984). 28. C. C. Cheng, S. E. Rokita, and C. J. Burrows,Angew. Clrem. Int. Ed. Ertgl. 32: 277-278 ( I 993).

14 NewMethodsforDeterminingthe Structure of DNA and DNA-Protein Complexes Based on the Chemistry of Iron(1I) EDTA Thomas D. Tullius Department of Chemistry, The Johns Hopkins University, 3400 North CharlesStreet,Baltimore, MD 21218

I.

DNA-PROTEIN COMPLEXES

DNA must be associated with protein in order to function in a biological system. The study of DNA structure inits own right has provide many surprises and new insightsrecently, with the structural characterization of left-handed Z-DNA [l],DNA triplexes [2], four-stranded (telomeric) DNA [3, 41, and the Holliday junction recombination intermediate [5]. But while DNA structural polymorphismis now a well-precedented pheof these unusual structures, let alone nomenon, the biological exploitation simple B-form DNA, invariably requires the agency of DNA-binding proteins. Even RNA, which has been conclusively demonstrated to possess its own [6],most often performs its the capacity for catalytic activity on biological functions in association with protein. So, to understand how nucleic acids participatein the life of the cell it is necessary tostudy their interactions with proteins. By the late 1970's only a single eukaryotic transcription factor had been isolated (the zinc finger protein Transcription Factor mA, about 217

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Tullius

which morewill be said later this in chapter), and only a small handful of prokaryotic DNA binding proteins had been crystallized.But in parallel with the characterization during the last decade of the unusual DNA structures mentionedabove, therehas been aflood of DNA binding proteins discovered. Beyond the cloning and isolation of both eukaryoticand prokaryotic DNA binding proteins, several have been co-crystallized with their DNA binding sites,and so high-resolution three-dimensional [A.N M R has also becomean important structures are available for many technique for structure determination [S] of both DNA binding proteins and in some cases complexesof protein with DNA 191. With this large amount of new structuralinformation has come the recognition of several distinct families of DNA-binding proteins, each using a particular protein structural motif (the zinc finger, the helix-turn-helix, and so on) to bind to DNA [lo]. While it might be thought thatwith the recent successof the crystalstructure deterlographers and N M R spectroscopists, other methods for mination have become less important, this is not the case. Perhaps the most important reason this for is that complexesof protein and DNA that are often highlycomare competentto functional in a biological system plicated, with several proteins bound to a long segment'of DNA. Molecular biologists have been able to reconstitute functional transcription, replication, and recombination systems using purified components, but if not impossible these very large macromolecular assemblies are difficult subjects of study for crystallographyor NMR.

A. Chemical Probe Methods This need to study large assembliesof DNA and protein has resulted in the development of a strategy called "chemical probing" [U, 12,131. A key advantageof this experimental approachis that very small quantities of protein and DNA are required, so that the functional assemblages produced by in vitro reconstitution may easily be studied. Chemical probe DNA sequencexperiments makeuse of the high resolution afforded by ing gel electrophoresisto monitor the reactivityof each DNA nucleotide toward a chemical reagent. A variety of small-molecule DNA cleavage reagents is now available, each having particular recognition properties for the structural features of a DNA-protein complex 1121. Some, like potassiumpermanganate,reactmorereadilywithsingle-stranded regions of DNA. Others, like dimethyl sulfate, alkylate particular positions in the DNA bases, and can be used to demonstrate whether these positions are exposed or hidden in a DNA-protein complex. While the atomic resolution of crystallography or NMR is obviously not available

New Methods of Determining Structure

of DNA

219

from a “map” of the chemical reactivity of the DNA in a DNA-protein complex, the ability to study complicated systems as easily as simple this ones, andthe relative easeof chemical probe experiments, have made experimental strategy widely used.

B. Hydroxyl Radical Footprinting In my laboratory we have been engaged in developing a particular chaical reagent, the hydroxyl radical, for use in structure determination of DNA-proteincomplexesinsolution. We producethehighlyreactive hydroxyl radical by a convenient inorganic reaction, the Fenton reaction [14]:

[Fe(EDTA)]’-

+ H202 + [Fe(EDTA)]l- + OH- + .OH

(1)

We use theEDTA complex ofi r o n 0 as a sourceof electrons to reduce hydrogen peroxide, because the negative charge of this metal complex reducesitsaffinitytowardthepolyanionic DNAmolecule[15].The chemical probe in our system, then, is the neutral hydroxyl radical and not the metal complex. This is unlike the many other DNA chemical probe reagents that are based on inorganic complexes [l61 in which the properties of the metal complex itself (its shape or reactivity, for example) are exploited. In our method we make useof the ability of the hydroxyl radical to cleave the DNA backbone by initial hydrogen atom abstraction from a deoxyribose [lq.The result of this homolytic reaction is a deoxyribosebased radical, which by subsequent chemistry decomposes, leaving a single-nucleoside ”gap”in the DNA strand. Because the hydroxyl radicalso reactive it is quite non-selective in its reactions with the DNA backbone, so that each nucleotide in a normalDNA molecule is cleaved to nearly the same extent. However, if a protein is bound to a specific site in the DNA the deoxyribosesthat are covered by the protein become inaccessible to the hydroxyl radical and no cleavage is seen at the protein binding site [18]. Becausethe hydroxyl radicalis so small, the “footprints”it produces of bound protein are the highest-resolution available. We have used this chemistry to make images of a wide variety of DNA-protein complexes. Hydroxyl radical footprinting is now a widely-used method. Since there is an extensive literature on this technique, covering both applications and experimental methodology[19], I will not discuss hydroxyl radical footprinting further. A newer method that makes use of hydroxyl is aimed at radical chemistry, the Missing Nucleoside Experiment, which

220

Tullius

Obtaininginformationontheenergetically-importantcontactsmade between a protein and its DNA binding site,is the subject ofthe next section.

C. The Missing Nucleoside Experiment The footprint shows where a protein physically covers DNAa molecule. as a direct measure of the The hydroxyl radical footprint can be regarded solvent accessible surface area of the DNA in a protein-DNA complex [19].While the footprint gives a picture of the overall shapeof the complex, the question of how the protein recognizes this particular DNA sequence among all others is not addressed. This question gets at the - what are the problem of molecular recognitionin a DNA-protein complex energetically-importantcontactsmadebyaproteinwith DNA? To answer this question we developed a new experimental strategy, the ”Missing Nucleoside Experiment”[20]. The method is based on the experimental approach called ”chemical interference” [U].It has a more direct antecedent in the missing contact method developed by Brunelle and Schleif[21].In previously-developed interference methods [22]the effect on protein bindingof alkylation of a is monitored. If, for example,the N-7 posinucleotide base or phosphate tion of a particular guanineis methylated, and the proteinis then unable to bind to that modified DNA molecule, it is concluded that the guanine makes an important contact with the protein, likely a hydrogen bond with theN-7 nitrogen. Several studies have found that phosphates that are implicated in proare invariably found tein binding by ethylation interference experiments [B].Recently Verdine to be sites of protein contact in co-crystal structures and coworkers [24]have developed a seriesof modified nucleotides that can be incorporated by automated synthesis into DNA oligomers. This collection of syntheticmodifiednucleotidessigrufrcantlyextendsthe kinds of protein-DNA contacts that may be studied by interference methods. One difficulty with previously developed interference methods is that different chemistry must be used for each of the fourDNA basesin order to detennine which nucleotidesin a DNA molecule make contactwith a in one protein. We sought to develop a method that would identify, experiment, all of the nucleotidesin a DNA molecule that make energetically-importantcontactswithaDNA-bindingprotein.TheMissing Nucleoside Experiment relies on the ability of the hydroxyl radical to generate a family of DNA molecules eachhaving a single-nucleoside gap in one of its strands. Itis the absence of a chemical p u p in theDNA (the

New Methods of Determining Structure

of DNA

221

missing nucleoside)that we monitor for its effect on protein binding. In contrast, in conventional interference experiments it is the addition of a chemical group by alkylation, and thus the added steric bulk or elimination of a hydrogen bond donor or acceptor, that gives information on con tacts of protein withDNA. A diagram of the Missing Nucleoside Experiment is shown in Figure 1. Because the hydroxyl radical is so nonselective in its reactivity with duplex DNA, a uniform population of gapped DNA molecules can be produced by useof the Fenton reaction on a sampleof DNA containing Protein is then added the binding site for the protein of interest (Step0). If a nucleoside thatis importo the mixtureof gapped DNA’s (Step 0). is missing in a particularDNA, the proteinwill be unable tant to binding to bind (left).If, however, the missing nucleosidein a DNA molecule is outside the protein binding site (or, in fact, it is in the binding site but is still able to bind to the does not make an important contact), the protein gapped DNA (right). DNA’s The gapped DNA molecules boundto protein, and the gapped are unable to bind, encode the information concerning which nucleosides necessary for formation of the protein-DNA complex. To recover this information the mixtureof protein-bound and unbound gappedDNA is ). separated by electrophoresis on a native polyacrylamide gel (Step 0 The bands containing boundand unbound DNA are excised from the gel and the DNA is denatured and electrophoresed in separate lanes of a Missing nucleosidesthat interfered with DNA sequencing gel (Step protein binding show up as prominent bands in the lane containing unbound DNA (right). A complementarypattern,inwhichmissing is seen in the lane bands correspond to the important nucleoside contacts, containing boundDNA (left). We first applied the Missing Nucleoside Experiment to the complex of ORl operator site [20].This the bacteriophage lambda repressor with the protein had been studied extensively in many other laboratories. In particular, the X-rayco-crystal structure had been determined [ E ] ,and exhaustivemutagenesisexperiments had establishedtheenergetic importance for protein binding of each base pair in the operator sequence

m).

[261.

The results of the Missing Nucleoside Experiment on lambda repressor are shownin Figue 2 along with a diagramof the contacts made by repressor with DNA from the co-crystal structure [25]. There is a great deal of information in the Missing Nucleoside pattern, which for lack of space cannot be discussed in detail here. To summarize the results, we found that essentially every signal the in Missing Nucleoside Experiment could be explained by a hydrogen bonding or hydrophobic contact that

222

Tullius

* -

l+

4

LIlr Unbound

G9 Sequencing Gel

Native Gel

-

I

Important Contacts

Figure 1 Diagram of the Missing Nucleoside Experiment. was found inthe co-crystal structure. Conversely, nucleosides for which Missing Nucleoside signals were not observed were not assigned in the X-ray structure as contacts. A very interesting feature of the correspondence betweenthe crystal structureand the Missing Nucleoside Experiment is the stmad-specific nature of the contacts between protein and that contacts DNA. That is, for base pairs 1-3the crystal structure shows are made by proteinside chains with the bases on thebottom strand (as drmm in Figure2). For base pairs 4-8 the contacts switch to top thestrand. 'I"& switching of contacts from bottom to top strandis also clearly seen in the Missing Nucleoside pattern. Furthermore, our resultswereinexcellentaccordwithpublished mutagenesis experiments [26]. The correspondence with mutagenesis is

223

New Methods of Determining Structure of DNA

Missing Nucleoside

11111111

TTTACCTCTGGCGGTGAZA IIII I I I

1' 2' 3 ' 4 ' 5' 6' l' 8'

8 l 6 5 4 3 2 l

0,l

X-Ray Crystallography GlyM

---

PhWh* " . . ) Hydrophobic

ATACCACTGGCGGTGATAT 1'2'3' 4 * 5 ' 6 ' 7 ' d

g

8 l 6 5 4 3 1 l

fI

TATGGTGACCGCCACTATA lle54

"\\

011144)

Gin33

OLl

Figure 2 Comparison of the MissingNucleoside pattern and the contacts derived by X-ray crystallography for the lambda repressorDNA complex. Missing nucleoside data are from Hayes & Tullius [20]. The length of a vertical bar is proportional to the effectof that missing nucleoside on repressor affinity. Horizontal bars mark nucleosides that can be lost without effect on repressor binding. Crystallographicallyderived contacts are from the report of the CO-crystal structure [25]. For clarity, crystallographic contacts for only one half-siteare shown.

224

Tullius

worth considering in more detail. Mutagenesis by necessity involves changing both halves of a base pair simultaneously. One particularly interesting aspectof the Missing Nucleoside Experimentis that, in some cases, one half of a base pair gives a strong signal, suggesting thatit is essential to binding, while the other base in the base pair can be remove without effect on complex formation. An example of this phenomenon can be seenat base pair 2in Figure 2, in which the adenine gives a strong signal, but the base-paired thymidine does not. The Missing Nucleoside Experiment, therefore, in contrast to mutagenesis, permitseach base of a base pair to be studied individuallyitsfor contacts with protein.

D. Structure of the TFIIIA-DNA Complex Having established the experimental validity of the Missing Nucleoside Experiment we went on touse it to determine the structureof the comIIIA with the 55RNA plex of the zinc finger protein Transcription Factor gene, a DNA-protein complex for which no X-ray structure has yet been solved. Transcription Factor LUA is the prototypical zinc finger protein [27l. TFIIIA binds to a 50-bp long segment of DNA internal tothe 5s ribosomal RNAgeneof Xenopus, and regulates transcription of this gene. Since the fist suggestion, based on the internal sequence homology of TFIIIA, that small domains of the protein might fold around a zinc ion and form nine ”fingers”, the zinc finger protein structural motif has been found tooccur hundreds of times in the genomesof higher organisms. Berg’s prediction of the three-dimensional structureof the zinc finger [28] was subsequently confirmed by solutionof the structures of single N M R methods [29,30]. Since natuzinc fingers using multi-dimensional ral zinc finger proteins invariably contain multiple tandem repeatstheof zinc finger sequence motif, it was of interest to determine how arrays of zinc fingers bind DNA. to Pavletich& Pabo [31] solvedthe first co-crystal structure of a zinc finger protein, Zif268, which contains three zinc finrn of the DNA gers. The protein was found to wrap around almosttuone helix, following the major groove. Each finger interacts with three base pairs of DNA and the three fingers are related by almost exact threefold screw symmetry. How a protein like TFIIIA, with its nine zinc fingers, might interact with DNA was still an open question. Would it resemble Zif268 taken three times, wrapping for nearlythree complete turns around the major groove of its DNA binding site?Or is there another modeof zinc fingeran X-ray structure? Our earlier DNA interactionnotyetfoundin and a series of delehydroxyl radical footprinting experiments TFIIIA on Zif3 model was tion mutantsof the protein[32]suggested that the simple

New Methods of Determining Structure of DNA

225

unlikely to be correct, because the footprints were more complicated than that simple model might predict. We recently proposed a detailed three-dimensional model for TFIthe ITA-DNA complex based on the results of Missing Nucleoside analysis [33]. This work represents arguably the most complicated protein-DNA complex for which only solution chemical probe methods were usedin proposing the structure.It will be interesting to see how close we have of the TFIlIA-DNA comcome in our prediction when a crystal structure plex is solved. .The results of the Missing Nucleoside experiment on the TFIJ”5S DNA complex are shown in Figure 3. It is immediately apparent that there are three separate regions of the DNA that make energeticallyimportantcontactswiththeprotein.Thesethreeregionscorrespond almost exactly tothree “boxes” determined by mutagenesis experiments to be necessary for regulation of transcription by TFIIIA. Each of these regions of interaction covers slightly more than one helical turn of DNA. One might imagine a model for the complex that involves three of subset of DNA. the nine zinc fingers of TFIIIA binding to these segments

Figure 3Missing Nucleosidepattern for the complex of TFIIIA with the Internal ControlRegion of the 5s ribosomal RNA gene of Xenopus.The length of a vertical baris made to be proportional tothe effect of that missing nucleoside onTFIIIA affinity. Taken from Hayes & Tullius [33].

226

Tullius

Our final, detailed, model for the structure was based on a consideration of the symmetryof the Missing Nucleoside pattern in Figure 3. The two envelopes of contacts at the two ends of the DNA binding site have pair DNA - that is, the peaks in the near mirror symmetry across a base of set of contacts on the two strands occu at the same base pair.We conthis sort of symmecluded that a protein-DNA complex that would give try in its contacts would resemble theZif complex, which consists of an of DNA and object (the three fingers of Zif) that follows the major groove acts almost like tahird DNA strand. The envelope of contacts in the tenter of the site have a different strand symmetry. The peaks in the contact pattern are offset from one strand to the otherby 5 base pairs in the 5' direction. We observed an identical offset in the lambda repressorMissing Nucleoside pattern (see Figure 2). Lambda repressor binds to DNA through an alpha helix that is inserted in the major groove. Lambda on one strandof the DNA at the repressor makes contacts with the bases end of the site, and then switches strands to make contacts farther within the site.

IE

C 87

I

77

I

A m

I

57

I

COOH

Figure 4 Model of the TFIIIA-DNA complex, based on the Missing

Nucleoside Experiment. Fingers 1-3and 7-9 were modeled based on the [31]. Thesefingers are foreshortenedto indicate that theygo into and out of the planeof the paper.A, IE, C:the approximate positionsof the three "boxes" defined by mutagenesis as importantfortranscriptionalactivation. C, coding;NC,noncoding strands. Taken from Hayes& T a u s [33].

Zif268 co-crystalstructure

Our model for the TFIIIA-DNA complexis shown in Figure 4. Fingers 1-3and 7-9 were placed on the DNA in the same relative orientationas the three fingers of Zif268, and are shownas wrapping aroundthe major groove. Fingers 4-6 are depicted as making a much different interaction with the DNA, extending head-to-tail along one side of the DNA. This set

of three fingers resembles lambda repressor, which also binds to one side

New Methods of Determining Structure

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227

of DNA parallel to the helix axis. Finger 5 is the only oneof the three that contacts the major groove. Fingers 4 and 6 cross the minor groove and serve mainly tolink finger 5 with thetwo sets of three fingersat the ends 4 6 make withDNA is of the complex. Whilethe interaction that fingers unprecedentedforzincfingers,footprintingand Missing Nucleoside experiments are wholly consistent with the model. In addition, after we had submitted o m paper two other groups [34,35] used different experiTFImental results to propose similar (but less detailed) models for the IIA-DNA complex, givingus confidence that our analysis is correct.

11. "UNUSUAL" DNA STRUCTURES

In the last decade much evidence has accumulated that the structure of DNA is important to its ability to function in a biological system. Perhap the most ubiquitous example of an "unusual" DNA structure that is essential to biologyis curved (or bent)DNA. It has of course long been known that DNA must bend to associate with the histone octamer to form the nucleosome, the first level of compaction of DNA in the eukaryof DNA bending otic nucleus. Itis only recently that the wide occurrence associated with protein binding has been appreciated[36]. Indeed, it is now standard practice to measure the degree of bending of DNA that is caused by any new DNA-binding protein that is discovered. The assay used for this measurement, the additional retardation in gel mobility caused by the protein being bound at the center compared to being bound at the ends of the DNA fragment [37l, gives information on the overall shape of the DNA molecule when it is bound by protein. Less information is available from this experiment on the local structure of DNA that is associated with bending, curving, or kinking, whether protein-induced or the result of the nucleotide sequence of the DNA itself. Another unusual DNA structure that is inarguably associated with biological functionis the fourstranded Holliday junction, a key intermehasbeenmade diatein DNA recombination.Whilemuchprogress recently in defining the shapeof the Holliday junction and the topology of its strands [5], no atomic resolution structural data are yet available for a DNA junction. Several other non-duplex DNA structures have been the subject of study, but are less clearly implicated in biological processes. The lefthanded &form of DNA, while known for more than a decade, has yet to DNA, and the related be proven essential to a biological process. Triplex H-form structure, is of wide experimental interest [38], but aside from possible use as a drug for inactivatingDNA inside a cell, the biological

228

Tullius

function of triple-stranded DNA remains enigmatic. Similarly although the q u a r t e t structures adoptedby the guanine-richends of the chromo[3,4], their relevance some (the telomere) have been studied extensively is not yet completely understood. to chromosome structure and dynamics What is beginning to emerge from the workof many laboratories is that nature makes use of the structural repertoireof DNA, but in ways that have yet to be defined in many systems and for many of the possible "unusual" structures of DNA. Indeed, perhaps the most unusual DNA structure is the one we besthow, the uniform, symmetrical, straightR form duplexDNA structure that came from the original X-ray fiber diffraction experiments on DNA a half-century ago. A challenge beforeus is to detect the occurrence of unusual DNA structures during biological function, to define in detail the nature of these structures, and to manipulate these structures to affect the biological process.

A. Chemical and Enzymatic Probe Methods forDNA Structure Determinationin Solution To address these structural questions, chemical and enzymatic probe experiments have been found to be useful to work out the structuresof nucleic acids in solution [13]. The major advantage of these methods is that they may be applied to DNA molecules that are much larger than could be studied by crystallography or M.As a consequence, structural features of DNA as it is involved in a biological process are now accessible to determinationand study. Some of the reagents that have been developed recently as probes of [39], osmium tetroxide DNA structure include potassium permanganate [40], theuranylion [41], bis(o-phenanthrolinecopperfl)[Q], 5-phenylphenanthrolinecopper(1) 143, 441, and derivatives of tris(o-phenan[16]. throline) complexesof rhodium, ruthenium and cobalt h general, each of these reagents is sensitive to a different aspectof DNA structure. For example, potassium permanganate or osmium tetroxide are thought toreact preferentially with unstacked DNA bases, which might occur at sites ofDNA kinking [39]. The phenanthroline-based reagents developed by Barton are designed to interact shape-selectively with DNA, and thus to detect the presence of a stretch of the A-form or the left-handed Z-form in a DNA molecule [16]. 5-phenylphenanthrolinecopper(1) seems to react withDNA that is "distorted" as the result of protein binding[M]. These reagentsare most often used by allowing the DNA molecule of interest to react with the probe compound, and then observing of the site

New Methods of Determining Structure of DNA

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reactivity (andthus the segmentof DNA with theunusual structure) by inducing DNA strand cleavage at the placeof reaction. The key to analysis of the experiment is the ability to separate DNA molecules differing in length by a single nucleotide,via electrophoresis of the reaction products on a denaturing polyacrylamide gel. By this means information on the occurrence of the unusual structure can be obtained rapidly for a very small quantity (a few femtomoles) of DNA, at single-nucleotide resolution. Furthermore, quantitative analysisof the gel allows accurate determination of the fractionof DNA moleculeshaving the structure. My laboratory has been engaged in developing the hydroxyl radical as a chemical probe of DNA structure. We have adopted an experimental strategy that is in some ways opposite to those discussed above. Since we generate the hydroxyl radical by the Fenton reaction of a negativelycharged transition metal complex, [Fe(EDTA)]*-, with hydrogen peroxide (see Equationl),the metal complex has a negative charge, andit thereif any affinity for the polyanionic DNA molecule [15]. fore will have little Rather than the metal complex, then, the probe of structure inour system is the hydroxyl radical, perhaps the smallest imaginable chemical probe. The hydroxyl radical reacts to nearly the same extent with each nucleotide in a normalB-form DNA duplex.If the DNA is in an unusualconformation,however,thepatternisdifferent. We usethesecleavage of unusual DNA molecules. patterns to make “images”

B. The Structure of Tn&, Tracts Despite much effortin many laboratories, the detailed naturein solution of the unusual structure (called B-DNA)that is adopted by shortruns of this structure leads toDNA bending, adenines (adenine tracts), and how have yet to be definitively determined. While isolated, phased A-tracts to [37,45,46], the dependence are wellknown to impart curvature DNA is also of importance. of the structureof an A-tract onits sequence context This is not a trivial point, since an important benefit of understanding of natural DNA DNA bending wouldbe the ability to predict the shapes sequences (suchas promoters or originsof replication) for which curvature is suspected to be necessary for optimal biological function.If the sequence context of an A-tract affectsits structure, and therefore the degree of curvature, then simply considering the occurrence and phase of only of a naturalDNA. the A-tractsis not enough to predict the shape An experimentbyHagerman [47]graphicallydemonstrated this point. He discovered that the sequence d(GAAAA’I??TC), ran anomalouslyslowlyon an electrophoresisgel,thehallmark of bent DNA. Remarkably, the simple sequence isomer d(GTTTTAAAAC), ran with

Tullius

normal mobility., leading to theconclusionthat this sequencewas straight. These results were explained[48] by assuming that in the bent A4T4sequence small local bends added in phase, giving theDNA molecule an overall curvature, while these same bends out were of phase with the helical repeat in theT4A4 sequence, making this molecule appear to be straight. The key assumption in this analysis was thatthe structure of the A-tracts in both sequence isomers was the same. We decided to test this assumption by performing hydroxyl radical cleavage experiments on these two sequences [49]. We previously had shown [50] that the adenine tracts in a highly natural bent DNA sequence fromatrypanosomeparasitehadavery unusual and characteristic of cleavhydroxyl radical cleavage pattern. We had found that the extent to 3’ along a age by the hydroxyl radical decreased monotonically5’from Ashort A-tract, and then increased in the mixed-sequence DNA between tracts. The thymine-rich strand showed a similar cleavage pattern, but shifted 2 or 3 nucleotides in the 3’ direction. Our conclusion from these observations was that the structure of the A-tract is not uniform, but B form at the 5’ end of an A-tract into changes gradually from the normal at the 3’ end of a short the unusual B’ form, whichis most fully expressed run of adenines. Becauseof the shift in the phase of the cleavage pattern between the two strands, we also concluded thatkey a characteristicof the unusual structure of an adenine tractis a narrow minor groove,that becomes progressively more narrow from 5’3’toalong the A-tract.This prediction was borneout in subsequentX-ray structures of A-tract-conin whichbothanarrowminor tainingoligonucleotides [51,52,531, groove, and its progressive narrowing from 5’ to 3,were seen. Subsequently, other experiments [X, 551 also have noted the same 5’ to 3’ “polarity” of the structure of adenine tracts that we first observed by hydroxyl radical cleavage. Our experiments with the “Hagerman sequences” [49] showed that 4T4, the bent sequence,had an unusual hydroxyl radical cleavage pattern that was very similar to that of the bent trypanosome sequence, while the straight sequence T 4 4 had a normal cleavage pattern. We concluded that the reason why theT4A4 sequence wasnot bent was thatits structure was close to normal B-form, and not that its small local bends were out of phase. We thus provided evidence for a different explanation funof Hagerman’s data, namely that the two sequence isomers adopted damentally different structures. We ascribed this difference in structure to the presence of a 5’-T-A-3’ step in the straight sequence. Others had asserted that T-A steps would interfere with the abilityof base pairs to propellertwist [56]. Aremarkablecharacteristic of theA-tractsthat emerged from the X-ray structures [51,52] was the extreme of prodegree

New Methods of Determining Structure of DNA

23 1

peller twist in the A-T base pairs of the A-tract. It had previously been noted that high propeller twist was associated with narrowing of the minor groove [57. We reasoned that the presence of a T-A step might cause the minor groove to remain wide because of its damping effect on propeller twist, thus leading to the inability of the T4A4 sequence to adopt the unusual structure of A-tracts in bent DNA. Haran and Crothers [58] later showed by gel mobility experiments if the valueof that some sequences of the form TnAn were indeed curved, n were large enough. The sequence ( T ~ A T N for ~ ) ~example, , wasfound to be quite curved. Haranandcrothers explained their data in termsof the model that all A-tracts have the same structure, and that overall curvature depends on the phasing of small local bends.We, however, decided to investigate the hydroxyl radical cleavage patterns of the bent and straight TnAn sequences studied by Haran and Crothers, to detennine whether the local structu~s of all indeed were the same [59]. The cleavage patterns of the T7A7N7 sequence, and for reference the highly bent A& sequence, are shown in Figure 5. We found that the hydroxyl radical cleavage patternof the bent T7A7N7 sequence has the characteristic featuresof the cleavage pattern of other bentDNA's, that is, cleavage decreases monotonically along A-tracts, and increases in the mixed sequence DNA separating the A-tracts. However, we also found that the T-A step in theT7A7N7 sequence, and one or two base pairsto either side,reach a local maximum in cleavage.This observation is consistent with our previous reasoningT-A thatsteps interfere with propeller twisting, and therefore sequences with T-A steps lack the ability to form the narrow minor groove thatis associated with curvature. The observation that the cleavage pattern of T7A7N7is not uniform (in contrast to the T4A4 sequence [49]) shows that the T-A step influences the structure only locally (i.e., over4-6base pairs),and not globally. We used theseand other data to propose a new model for the structure of TnA,, sequences. The model asserts that in aTnA,, sequence, the T-A step and 2 to 3 base pairs to either side are in the normal B-form conformation. However, if the T tract or the A tract are longer than3 or 4 base from the T-A step. pairs, theunusual B structure still can form away base pairsof mixed We tested this model by substituting either 4 6or sequence DNA for the TzAz or T3A3 core sequence of T7A7N7 Wefound that these new DNA molecules had hydroxyl radical cleavage patterns very similar to the patternof T7A7N7, showing that the mixed sequence DNA and the T2A2 or T3A3 sequences adopt the same (presumably R form) structure. Moreover, the new sequences had gel mobilities nearly identical to thatof the ( T T A ~ Nsequence, ~)~ demonstrating that substitution of mixed sequence DNA forA/T DNA had no effect on the curva-

232

Tullius '

"C

5 Hydroxyl radical cleavage patterns of cloned oligonucleotidescontaining four repeats of A5N5 (left), and two repeats of T7A7N7(right). The A$J5 sequence is known to impart substantial curvatureto DNA. The smoothly varying hydroxyl radical cleavage pattern of this synthetic sequence is nearly identical to the pattern of anatural,highlycurvedsequencefromtrypanosome parasites. T7A7N7 has a similar modulated cleavage pattern, and it also is curved[58]. The T-A steps in the T7A7N7 pattern thatare discussed in the text are indicated by arrows.

Figure

ture of T7A7Np

We thus showed [59] that the hydroxyl radical cleavage pattern provides sufficiently detailed structural information DNA on that structural principles can be inferred from the data, andDNA that molecules can be structure but a differentsequence compared to designed that have the same another DNA.

C. Concluding remarks I have describedtwo examples of how metal-based chemistry can be used as a high-resolution structural tool study to DNA and DNA-protein complexes. A key characteristicof the chemistry used,the Fenton reactionof iron@) EDTA with hydrogen peroxide to generate the hydroxyl is radical, the simple and inexpensive natureof the reagents required.This feature its wide use by molecular biologists as of the method has contributed to are interested in nucleic acid structure. well as chemists who The principle behind the experiment, thatan exceedingly non-specific reaction can provide detailed structural information, is different from the This princiidea behind most other metal-based chemical probe methods. is now subple might profitably be used in other systems; indeed, there stantial experimental activity in the use of iron@) EDTA-based hydroxyl radical chemistry for the structural analysis of proteins. A further goal of our work is to use hydroxyl radical chemistry to study the structure of DNA and DNA-protein complexes inside living

New Methods of Determining Structure

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cells. My group has already made substantial recent progress toward this end.

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D. M. J. Lilley, Current Opinion Cell Biol.2: 464-467 (1990). T. R Cech, Ann. Rev. Biochem 59 543-568 (1990). C. 0.Pabo and R. M. Sauer, Ann. Rev. Biochem 62: 1053-1095 (1992). G. M. Clore andA. Gronenborn, Annu. Rev. Biophys. Biophys. Chem 20

29-63 (1991). 9. G. Otting, Y. Q. Qian, M.Billeter,M.Muller,M. Molter, W. J. Gehring, andK. Wuthrich, EMBO I. 9: 3085-3092 (1990). 10. S. C. Harrison, Nature 353: 715-719 (1991). 11. T. D. Tullius, Ann. Rev. Biophys. Biophys. Chem.2 8 213-237 (1989). 12. P.E. Nielsen, J. Mol. Recognition 3: 1-25 (1990). 13. T. D. Tullius, Current Opinion Struct.Biol. 2: 428-434 (1991). 14. C. Walling, Acc. C h . Res. 8 125-131 (1975).

15. T.G. Wensel, C. F. Meares, V. Vlachy, and J. B. Matthew, P m . Natl.

Acad Sci. (USA) 83: 3267-3271 (1986). 16. A. M. Pyle and J. K.Barton, Progress Inorg. C h . 38 477-516 (1990). 17. R.P.Hertzberg andP. B. Dervan, Biochemistry 23: 3934-3945 (1984). 18. T.D. Tullius and B. A. Dombroski, Proc. Nufl. Acud.Sci. (USA) 83: 5469-5473 (1986). 19. W. J.Dixon, J. J. Hayes, J. R, Levin, M. F. Weidner, B. A. Dombroski, and T. D. Tullius, Meth. Enzymol.208 380413 (1991). 20. J.J. Hayes andT. D. Tullius, Biochemistry 28 9521-9527 (1989). 21. A. Brunelle and R F. W e i f , Proc. Nufl.Acad. Sci. USA 84: 6673-6676 (1987). 22. U. Siebenlist and W. Gilbert, P m . Nufl. A d . Sci. USA 77: 122-126 (1980). 23. F.D. Bushman,J. E. Anderson, S. C. Harrison, and M. Ptashne, Nuhrre 326: 651-653 (1985). 24. K. C. Hayashibara and G.L.Verdine, Biochemistry 32: 11265-11273 (1992). 25. S. R.Jordan andC.0.Pabo, Science 242: 893-899 (1988).

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26. A. Sarai and Y. Takeda, Proc. Nutl. Acud. Sc.i (USA) 86 6513-6517 (1989). 27. J. Miller, A. D. McLachlan, and A. Klug, EMBO J. 4 1609-1614 (1985). 28. J. M. Berg, P ~ o c N . d . Acud. Sci. (USA) 85 99-102 (1988). 29. G. Parraga, S. J. Horvath, A. Eisen, W. E. Taylor, L. Hood,E. T. Young, and R.E. Klevit, Science 242: 1489-1492 (1988). 30. M. S. Lee,G.P. Gippert, K. V. Soman, D. A. Case, and P. E. Wright, Science 245 635-637 (1989). 31. N. P.Pavletich andC. 0.Pabo, Science 252: 809-817(1991). 32. K.E. Vrana, M. E. A. Churchill, T. D. Tullius, and D. D. Brown, Mol. Ce2f. Biol. 8 1684-1696 (1988). 33. J. J. Hayes andT. D. Tullius, J. Mol. Biol. 227: 407- 417 (1992). 3 4 . K. R Clemens, X. Liao, V. Wolf, P. E. Wright, and J. M. Gottesfeld, Pmc. Natl. Sci.(USA) 89: 10822-10826 (1992). 35. L. Fairall andD.Rhodes, Nucleic Acids Res. 20: 4727-4731 (1992). 36. A. A.Travers, Current OpinionStruct. Biol. 2: 114-122(1991). 37..H-M Wu and D. M. Crothers, Nature 308 509-513 (1984). 38. H. Htun and J. E. Dahlberg, Science 243: 1571-1576 (1989). 39. J. A. Bomwiec, L. Zhang, S. Sasse-Dwight, and J. D. Gralla, I. Mol. B i d . 196: 101-111 (1987). 40. D. M. J. Lilley and E. Palecek, EMBO J. 3: 1187-1192 (1984). 41. P. E. Nielsen, C. Jeppesen, and 0.Buchardt, FEBS Lett. 235 122-124 (1988). 42. C. Yoon, M. D. Kuwabara, R Law, R Wall, and D. S. Sigman, J. Biol. Chem 263: 8458-8463 (1988). 43. T. Thederahn, A. Spassky, M. Kuwabara, and D. S. Sigman, Biochem. Biophys. Res. Comm. 168 756-762(1990). 44. B. Frantz andT. V. OHalloran, Biochemistry 29 47474751 (1990). 45. J. C. Marini, S. D. Levene, D. M. Crothers, and P. T. Englund, Pmc. Natl. Acud. Sci. (USA) 79 7664-7668 (1982). 46. P.J. Hagerman, Biochemistry 24 7033-7037 (1985). 47. P. J. Hagerman, Nature 321: 449450 (1986). 48. L.E. Ulanovsky andE. N. Tiifonov,Nature 326: 720-722 (1987). 49. A; M. B~khoffand T. D.Tullius, Nature 331: 455457(1988). 50. A. M. Burkhoff and T. D. Tullius, Cell 48 935943 (1987). 51. H.C. M. Nelson, J. T. Finch, B. F. Luisi, and A. Klug, Nature 330 221226(1987). 52. M. Coll, C. A. Frederick, A. H. -J. Wang, and A. Rich, Pmc. Natl. Acad Sci. (USA) 84 8385-8389(1987). 53. A. D. DiGabriele, M. R. Sanderson, andT. A. Steitz, Pmc. Natl. Acad Sci. (USA) 86: 1816-1820 (1989). 5 4 . J. G. Nadeau andD. M. Crothers, Proc. Nutl. Auzd. Sci.(USA) 86 2622-

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2626 (1989). 55. V. Lyadtichev, NucZeic Acids Res. 2 9 4491-4496 (1991). 56. C. R Cayadine, J. Mol. Bioi.262: 343-352 (1982). 57. A. V. Fratini, M. L. Kopka, H. R Drew, and R. E. Dickerson, J. Bioi. Chem 257: 14686-14707 (1982). 58. T.E. Haran and D. M.Crothers, Biochemistry 28 2763-2767 (1989). 59. M.A. Price and T.D. T a m , Biochemistry 32: 127-136 (1993).

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15 DNA Recognition by Steroid HormoneReceptorZincFingers: Effects of Metal Replacement and Protein-ProteinDimerization Interface Bibudhendra Sarkar BiochemistryResearch, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1x8, Canada

I . INTRODUCTION The steroid hormone receptor superfamily is a group of cytoplasmic receptors which act as transcriptional enhancer proteins. These receptors bind specificallyto short DNA sequences and control the transcription of a number of genes (1). Sequence comparisons of regions of varying degrees of conservation revealed that a number are shared by almost all the receptors (Figure 1). The A/B domain, which is the most variable and differs considerably in size from one receptor to another is known to contain promoter- and cell-specific trans-activation function. The C domain isthe most highly conserved of the regions and encodes the DNA binding domain. This region is connected to the regionof next highest conservation, the E region, by the hingeor D region. The E region constitutes the 237

a

230

DNA Recognition by SteroidHormoneReceptorZincFingers

239

hormone binding domain. It also contains transactivating and dimerization functions. The DNA binding domainC of these receptors are highly related and similarly stabilized by two zinc atoms each coordinated to 4 cysteine residues. The two fingers present in the DNA binding domain are not equivalent. The first finger (P box) is responsible for sequence specific DNA recognition while the second finger (D box) is involved in protein-protein cooperative interactionin the dimerization process. The hexameric sequence of the consensus response elements, their directionality and spacing dictate their specificity for receptor binding. Although zinc is generally presumedbetothe endogenous metal ion within the zinc fingers, few studies have actually demonstrated in some this to be the case. In vitro studies have demonstrated that are capable of functioning in zinc instances, metals other than zinc fingers (2-8). Consequently the studies of interaction of metals other than zinc with zinc finger domains have become focus a major in our laboratory. Protein-protein interactions betweenDNA binding domains of these proteins in the dimerization interface is responsible for mediating the recognition of base pair spacing and orientation of response element halfsites (Figure 2). The recognition of specific half site sequence is mediated by direct protein-DNA contacts of individual domains. Two estrogen receptor (ER) DNA-binding domain (DBD) polypeptides bind cooperatively as a homodimerto the half site of 2 estrogen response elements (EREs) consisting of inverted repeatsof the consensus sequence AGGTCA. On the other hand retinoic acid receptor (RAR) and retinoid-X receptor (RXR) DNA-binding polypeptides bind cooperativelyas a heterodimer to two retinoic acid response elements (RAREs) consisting of direct repeats of the AGGTCA consensus sequence. Dimerization interfaces required for homodimerization of steroid receptors bound to symmetric hormone response etements (HREs) have been identified within both the ligand - and DNA-binding domains (911). The DBD interfaces in steroid hormone receptors, through head to head interactions, mediate recognition of half-site orientation and spacing as cooperative binding of DBD polypeptidesto DNA occurs only with proper half-site positioning(12,13).Onthe contrary, the asymmetric nature of direct repeat RAREs suggests that non-reciprocal headto tail DBD dimerization interfaces in and RXR are likely to be required for efficient interactions between each heterodimeric partners. Thus RAR and RXR should bind n preferentially to either the up- or down- stream half site core imotif order to achieve specific and cooperative dimer interaction.

U

G C

C3U

ou

f 240

t3u t3u 4E.c

c!Ju t3u

DNA Recognition by SteroidHormoneReceptorZinc

11.

Fingers

241

EFFECT OF METAL REPLACEMENT

A purified, bacterially expressed polypeptide encompassing the ER DBD was used in all our studies (2). When apopolypeptide was dialyzed against buffer without metal no specific binding could be detected as measured by a mobility shift assay with a consensus ERE hexamer-containing oligonucleotide. However, specific DNA binding was restored by dialysis against buffer containing zinc, as cadmium or cobalt but not with buffer containing copper or nickel showninFigure 3, althoughthesemetalsdoappeartointeract Cd

CO

Cu

W

I

.

Ni

Zn

-bound

c free

Figure 3 Mobility shift assay of cadmium-, cobalt-, copper-, nickel and zincreconstituted polypeptidesas indicated (2).

directly with the polypeptide (2,4). Dissociation constantsfor DNA binding domain polypeptide and zinc, cadmium and cobalt reconstituted apopolypeptide with the ERE consensus hexanucleotide-containingoligonucleotide were determined from mobility shift results using the double-reciprocal binding plot. The native DNA-binding domain polypeptide and zinc- and cadmiumreconstituted polypeptide all had very similar affinity for the ERE hexamer whereas cobalt-reconstituted polypeptide had a decreased affinity compared to the native polypeptide (Table 1).

Table 1 Dissociation constants of polypeptide bindingto ERE hexamer sequence as determined by double reciprocal plot analysis of mobility shift assay results(2) Polypeptide form

Kd nM

Native Zinc Cadmium Cobalt

48

66 48 720

242

Sarkar

Methylation interference experiments showed that native, zinc-, cadmium-, and cobalt- reconstituted forms of the polypeptide interact with theAGGTCA (read TGACCT on the other strand) half site in a qualitatively similar manner (Figure 4). The reduced intensity of the guanine band within the TGACCT half-site indicates that in each case, the polypeptideis interacting specifically with the half-site sequence.

T Q+ 4

I

C

C T

G C

Figure 4

A

Methylationinterferenceassays of native,zinc-, cadmium- and cobalt-reconstituted polypeptides with ERE hexamer containing oligonucleotide. In eachcase, bound (B) and free 0DNA lanes are indicated and the ERE hexameric sequenceis denoted inbold. The specific guanine residue in the ERE hexamer which is "required" for specific binding is indicated by arrow(2).

The ability of zinc, cadmium and cobalt to reconstitute theDNAbinding propertiesof the native polypeptide are consistent with the expected structural contributionof these metals. They are knownto coordinate with tetrahedral geometry and are all capable of binding to cysteine sulfhydryls, which are the zinc ligands. The inability of aporeceptor DNA-bindingto be restoredby copper or nickel is also not unexpected. Square planar geometries are more common for nickel. Nickel-binding may result in a distorted finger from the in a conformationally normally tetrahedral metal site resulting

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243

changed polypeptide incapableof specifically interacting with the DNA. Copper on the other hand in the form of copper(1) hasa high affinity for sulfhydryl ligands but has less stringent geometric requirements than zinc. Thus copper may not determine the proper folding of the polypeptide for DNA-binding. These results suggest that misincorporation of copper, for example, would lead to a transcriptionally inactive receptor affecting transcriptional regulation. In fact, if copper binding causes wrong folding, the receptor may be rapidly degraded within the cell. In certain conditions intracellular concentration of copper may become significant. This could occur in Wilson and Menkes diseases in which certain cells and tissue retain elevated levels of copper. Furthermore, in a zinc-deficient state, copper may be able to compete effectively with the decreased cellular concentration of zinc. It is possible that some symptoms of zinc deficiency may directly attributable to misfolding of steroid receptors(14). It has now been shown that iron can replace zinc in the zinc finger motif of ER and still retain the DNA-binding activity (15). Furthermore it has been demonstrated that iron can generate free radicals, specifically hydroxyl radicals, while coordinated to the four cysteines of zinc finger. Hydroxyl radical is known to damage DNA, which may lead to mutagenesis and/or carcinogenesis.

111. PROTEIN-PROTEIN COOPERATIVE INTERACTION WITH INVERTED REPEAT RESPONSEELEMENTS A. Homodimerization of ER It was possible to resolve both the monomeric and dimeric bound forms of the ERDBD upon binding to an actual ERE and to quantitate the extentof cooperativity (3). A model forERE binding is presented in Figure5. According to this model ERDBD bindsto "K'. either of the two hexameric half sites with identical affinity

244

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sk A

l

l .t+

rk

B

Figure 5 /“el for estrogen receptor DNA-binding domain interaction with theERE. Free polypeptide (hatched box) binds to either half site with an association constant “ K ’ . The complex formed is the with either siteA or site B occupied monomeric complex, while the complex formed with both sitesA and B occupiedis the dimeric complex (3).

Binding of the second polypeptide occurs with affinity an equal to oK,where K is the polypeptide association constant for a half site and o is the cooperativity parameter. Thus, the binding equation canbe derived from the equilibria:

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where P=free protein,fAfg=free DNA, bAfg=protein bound to site A only, fAbB=protein boundto site B only,bAbg= protein bound to both sites A and B. Cooperativity values were determined by mathematical best fit of data over a rangeof o values using a computer program developed by us. A cooperativity parameter of >l indicates positive of cooperativity, a valueof 1 indicates no cooperativity and a value
Table 2

Cooperativity values for interaction with an ERE for various metal substituted formsof the estrogen receptor DNA-binding domain polypeptide(3) Polypeptide form

0

Native Cadmium Cobalt

109

85 516

to an ERE. But cobalt substituted ERDBD shows an approximately 5-fold increased cooperativity. It appears that cobalt substitution has the effectof decreasing the affinity of hexamer recognition but at the same time increasing cooperative protein- protein interaction. It is possible that the increased cooperativity may be related to a conformational change that the polypeptide seems to undergo upon binding, as happens to the glucocorticoid receptor DNA binding domain (13). It is likely that the distortion may be more energetically favorable with cobalt than with zinc or cadmium polypeptide. We have recently found that the protein-protein interactions mediating this cooperativity have the surprising capacityto direct high affinity specific recognition of a glucocorticoid response element (GRE) hexamers (AGAACA) when it is oriented and spaced appropriately relative to an ERE hexamer (AGGTCA) (16). This induced binding to a sequence usually uniquely associated with another DNA-binding protein is facilitated by protein-protein interaction. The ability of protein-protein interactions to direct recognition of such a non-cognate sequence is of relevance to other

246

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DNA-binding proteins as well. In particular, it suggests that protein-protein interactions between some transcription factors may similarly direct recognition of factors to binding sites usually associated with the other protein.

IV.

PROTEIN-PROTEIN COOPERATIVE INTERACTION WITH DIRECT REPEAT RESPONSEELEMENTS

Retinoic acid, a pleotropicregulator of developmentand homeostasis, controls the expressionof specific gene networksvia direct interactions with nuclear receptors. RAR and RXR bind as heterodimers on theDNA recognition sites known as retinoic acid of a direct response elements(RAREs) that are generally composed repeat of the half-site core motif AGGTCA spaced by 2 (DR-2) or 5 (DR-5) base pairs. To investigate the potential interactions between RAR and RXR DBDs in RARE recognition, peptides encompassing the zinc finger region of RAR and RXR (referred to as AD and XD (Figure 6) respectively) were expressed in a bacterial system. Bacterially expressed DBD peptides were first assayed for DNA binding activity using a mobility shift assay. A probe was synthesized consisting of a consensus RARE having two identical AGGTCA half sites with a spacing of 5 bp (DR-5 RARE). When assayed individually, AD andXD DBD peptides bind to the synthetic DR-5 RARE predominantly as monomers at the concentrations employed. However, when equal volumes of AD and XD containing extracts are mixed, the resulting dimer band is more intense than can be accounted for by additivity alone. Enhanced dimer formation is indicative of cooperative interaction. The level of cooperativity appears to be low, raising the possibility that some but not all the cooperativity determinants are presentin the zinc finger regionof RAR and RXR DBDs(17). By analogy to the steroid hormone receptors (9, 12, 13), the cooperativity observed with the DBD peptides likely arises from dimerization interaction between the DNA bound proteins. To help visualize possible elements of the putative RARRXR DBDs dimerizationinterface, molecular modeling techniques were employed using an Evans and Sutherland PS 390 graphics terminal and the available GRDBD-DNA cocrystal structure (13). We modeled possible interactions of RAR and RXR DBD boundto half-

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" I

247

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sites spaced by 2 or 5 bp. These studies indicated that the carboxyterminal regionof the RAR DBD, when boundto the down stream half-site motif, would be placed in close proximity to the RXR DBD upon binding to both DR-2andDR-5RAREs. Therefore, a carboxy terminal extended version of AD, termed ADe,was constructed. To study if the corresponding region in RXR also plays a role in DR-2 and DR-5 RARE recognition, a carboxyterminal extended versionof XD, named XDe wasalso constructed. When assayed for DNA binding activity, cooperativity between ADe RARE binding. and XDe peptides was clearly enhanced upon DR-5 RARE To test whether the DBD polypeptides also recognize a DR-2 and display response element specificity, ADeandXDe were incubated with a seriesof naturally occurring direct repeat response elements with half-site spacing ranging from 15tobp and assayed for DNA binding activity. As expected ADe and XDe peptides readily form dimer complexes when natural DR-5 and DR-2 RAREs are used as probes. However, the DR-1 RXRE, the DR-3 vitamin RE fail to show significant dimer D3RE and DR-4 thyroid hormone formation (17). To individually test the role of RAR and RXR extended carboxy terminal sequences on RAREs binding, different combinations of AD + XDe and ADe + XD were used in mobility shift assays on both RAREs. Significant dimer formation is not observed on either DR-2 or DR-5 RAREs in the absence of the extended carboxy terminal regionof the RAR. In contrast cooperativity is observed on the DR-2RARE with the XD+ ADe combination in which only the RAR extended carboxy terminal region is present. However, significant dimer formation is not observed when the XD + ADe peptide combination is assayed with the DR-5 RARE. Taken all these results together it appears that the RAR carboxy terminal extended sequence, whichwe term "H-box" (Figure 6) is essential for DR-2 and DR-5 RARE binding. However, the RXRDBD carboxy-terminal extended sequence, that includes the previously defined T-box (17), appears to be requiredfor DR-5 binding. In order to explore the possibilities that RAR and RXR may show a preference for either of the two half-site core motif in DR-2 and DR-5 RAREs we designed experiments in which the DNA binding specificity of the RAR is changed to the DNA binding 7). This was specificity of the glucocorticoid receptor (GR) (Figure achieved by changing three amino acid residues within the RAR DBD "P-box" (9, 19, 20) to the residues present in the GR DBD. The mutagenized FUR (RARg), which now possesses the ability to bind the half-site core motif AGAACA, should only recognize

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I n

a

2 a

n

m

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hybrid response elements in which the AGAACA and AGGTCA half-site core motifs are properly positioned. As expected RARg/RXR heterodimer fails to bind with high affinity consensus DR-2 or DR-5 RAREs. When a seriesof hybrid response elements DR-2 hybrid response element with was tested for binding, only the the AGAACA half-site in the down-stream position is bound with high affinity. With theDR-5 series, the same relative specificity is observed for a down-stream GRE half-site, however the affinityof this interaction is lower. To confirm that similar interactions occurin vivo, each hybrid response elementwas linked to the reporter gene thymidine kinase luciferase and the ability of the RARg mutant to induce luciferase activity was assayed by transient co-transfection in COS-7 cells. Only the DR-2 and DR-5 hybrid response elements with the AGAACA half-site in the downstream position confers the ability of RARg to increase luciferase activity following retinoic acid treatment (17).

Our results show that RAR/RXR heterodimeric partners bind DR-2 and DR-5 RAREs in an ordered but non-equivalent configuration(Figure 8) andprovideevidencethattheH-box,a

Figure 8

RAR and RXR heterodimericpartners differentially use cooperativity determinantsto recognize DR-2 and DR-5 response elements(17).

novel functional domain encompassing a region conserved among RARs, mediates these interactions. A consequence of ordered but non-equivalent binding of RAlURXR partners to DR-2 and DR-5 RAREs is that RARE-dependent heterodimer configurations may result in the presentationof distinct setsof transactivation domains

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in the native receptors. Non-equivaTent heterodimeric complexes could then be expected to display different transactivation potential. Evidence of such a proposal is provided by the recent finding that RAR and RXR isoforms exhibit RARE- and promoter context(21). dependent transcription activation abilities

V.

CONCLUSIONS

Studies collectively demonstrate that metal requirement in the zinc finger is not highly stringent, consistent with the role in the structural stabilizationof the domain. Results show that copper and nickel are able to bind "zinc finger'' residues but do so nonproductively. On the other hand, the ability of cadmium, cobalt and iron to substitute for zinc in the zinc finger suggests a structural "flexibility" in the DNA-binding domain. The ability of metals to substitute for zinc in theDNA binding domain suggests that metal in vivo may be of relevance to the substitution in these 'zinc fingers' toxicityand/orcarcinogenicity of some of thesemetals. Involvement of protein-protein interaction inDNA binding is well documented. We have presented the evidence that these cooperative interactions are capable of mediating the recognition of ERE sequence degeneracy. In broader context, the ability of proteinprotein interactions to mediate recognition of non-cognate DNA sequencesalsohas potentially significantimplicationsfor ERE are transcription factors in general. While the half-sites in an RARE are oriented as oriented as inverted repeats, the half-sites in direct repeats. Similar cooperative interactions occur betweenthe DNA binding domains of RAR and RXR but the cooperative interactions reside outside of the zinc fingers region, a situation ER and GR DNA binding domains. distinct from that seen with the Results also reveal an additional level of complexity in nuclear receptor action and suggest that formation of ordered but nonequivalent RARE dependent heterodimerRAR/RXR complexes may be an important determinant of the pleiotropic natureof the retinoid signal.

ACKNOWLEDGEMENTS The research was supported by grants from the National Cancer Institute of Canada and the Medical Research Council of Canada.

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REFERENCES 1. R.M.Evans, Science 240:889-895 (1988). 2. P.F.Predki and B.Sarkar, J.Biol.Chem. 2675842-5846 (1992). 3. P.F.Predki and B.Sarkar, Env.Hlth.Persp. 102: 195-198 (1994). 4. H-J.Thiesen and C.Bach, Biochem.Biophy.Res.Comm. 1761551-557(1991).

5. L.P.Freedman, B.F.Luisi, Z.R.Korszun,R.Basavappa, P.B.Sigler and K.R.Yamamoto,Nature 334543-546 (1988). 6. A.D.Franke1, D.S.Bredt and C.O.Pabo, Science, 240:70-73 (1988).

7. J.Kuwahara and J.E.Coleman, Biochemistry 29:8627-863 1 (1 990).

8. J.G.Omichinski, C.Trainor, T.Evans, A.M.Gronenbom, G.M. Clore and G.Felsenfeld,Proc.Natl.Acad.Sci.USA 90: 16761680 ( 1993). 9. K.Umesono and R.M.Evans, Cell 57:1139-l146 (1989). 10. S.E.Fawel1, J.A.Lee, R.White and M.G.Parka, Cell 60:953962 (1990). 11. B.M.Forman and H.H.Samuels, MoLEndocrinol. 4:1293-1301 ( 1990). 12. K.Dahlman-Wright, H.Siltala-Roos, J.Carlstedt-Duke and J.Gustafsson, J.Biol.Chem. 265: 14030-14035 (1990). 13. B.F.Luisi, W.X.Xu, Z.Otwinowski, L.P.Freedman, K.R.Yamamoto and P.B.Sigler, Nature 352:497-505 (1991). 14. G.E.Bunce and M.Vessa1, J.Steroid Biochem. 26:303-308 (1987).

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15. D.Conte and B.Sarkar, Proceedingsof The First International Symposium on Metals and Genetics p.13 (1994) 16. P.F.Predki and B.Sarkar, Bi0chem.J. In Press (1994). 17. P.F.Predki, D.Zamble, B.Sarkar and V.Gigu&re, Mol.Endocrino1. 8:3 1-39 (1994). 18. T.E.Wilson, R.E.Paulson, K.A.Padgett and J.Milbrandt, Science 256: 107-110 (1992). 19. M.Danielsen, L.Hinck and G.M.Ringold, Cell 57:1131-l138 (1989).

20. S.Mader, V.Kumar, H.deVerneui1 and P.Chambon, Nature 3381271-274 (1989). 21. S.Nagpa1, M.Saunders, P.Kastner, B.Durant, H.Nakshatri and P.Chambon, Cell 70:1007-1019 (1992).

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16 HIV-l Tat Protein Forms a Zinc-Finger-like Structure Jean-Pierre Laussac Laboratoire de Chimie de Coordination,C N R S , 205, route de Narbonne, 3 1077 Toulouse cedex, France Honor6 Mazargail andDanide Prom6 Institut de Pharmacologie et de Biologie Structurale, CNRS, 205, route de Narbonne, 31077 Toulouse cedex, France MoniqueErard Institut deBiologieCellulaire et deGMtique, route de Narbonne, 31077 Toulouse &ex, France

CNRS,

Manh-Thong Cnng Laboratoire de Chimie Physique Macromolkulaire, Nancy, France

205,

CNRS,

I. INTRODUCTION Among essential trace elements, zincis just behind iron in terms of the quantity found in the living system. The importanceof this element in human health and diseasesis becoming increasingly recognized and its biological importance is now beginning to be understood. Zinc is an essential component for normal growth and developmentis known and to be an important cofactor fora variety of metalloenzymes[l]. Zinc also [2]. Zinc-finger domainsare known to be of occurs in zinc-finger proteins great importance in number of proteins thatare involved in nucleic acid binding and transcriptional control [3,4]. It is now clear that the zincfinger family includes several distinct classes of structural motifs including the transcription factor IIIA-like fingers [5], retroviral nucleic acid binding proteins [6], steroidreceptors [7], and several fungal transcriptionregulatory factors [8]. Eachmemberof this family 255

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coordinates oneor more zinc atoms via cysteine or histidine side chains, an event required for proper protein folding. More recently zinc has been discovered to playa functional role in the human immunodeficiency virus, type 1 (HIV-l) Tat protein [g]. The HIV encodes several regulatory proteins that are not found in other retroviruses. One of these regulatory proteins, the Tat protein, mactivates genes that are expressed from the HIV long terminal repeat (Lm) [lo] and is essential for viral replication[11-14]. The mechanism of action of Tat is complex and poorly understood [15]. The target sequence for Tat transactivation is a 59-nucleotide RNA stem-loop [l61 that functions structure called transactivation response element (TAR) in the form of nascent RNA [17]. Purified recombinant Tat protein is able to bind TAR RNA specifically in vitro and formsa one-to-one complex [18,19]. However, binding of Tat to TAR RNA is not in itself sufficient for function. Indeed, it has been observed thatm-activation probably depends upon the presence of both Tat and additional cellular factors [20,21].

B t is a small protein of86 residues, which is conserved in both HTV-2 and simian immunodeficiency viruses. However, only the72 Nterminal amino acids are required for full activity [22] while, Siegelset al. report that only the first 58 residues are sufficient for transactivating activity [23]. Examination of Tatamino acid sequence reveals the presence of at least three distinct functional domains affecting localization and transactivation: a cysteine-rich region, a ‘core’ region and a basic region. The cysteine-rich cluster region, located between residues 21 and 37, containssevencysteines.Thefunctionofthecysteineresidues is unknown, but they have been proposed to be involved in binding metal ions and in protein-protein interaction [24]. The cysteine residues are crucial for Tat function. Indeed, mutational analyses of six of the seven cysteines (cysteine31 is the exception) completely abolishes Tat function [9,14,25,26]. Thus, the ability of these residues to coordinate with metal seems likely to serve an important function [g]. The ‘core’ sequence (residues 38 - 47) plays a role inTAR recognition and contributes to Tat binding specificity by orientating the basic region within the major groove [27,28]. The third functional domain encompasses the group of positively charged amino acids, which contains eight lysines and arginines between residues 48 and 57. This basic regionis important for nuclear localization [9,29,30] and mediates specific binding to TAR RNA [31,32]. It has been proposed that the cysteine-rich domain has the potential to form a zinc-finger [33] common to several other

257

HIV-1 Tat Protein Forms Zinc-Fingerdike a Structure

transcriptional regulatory proteins [34]. Frankel et al. have proposed an alternative structure that is distinct from a zinc-binding finger and is involved in metal-linked dimer formation [24]. However, this finding is in contrast with other recent data which show that Tat is a monomer when expressed in vivoin mammalian cells[35] and in vitroin the wheat germ cell-free translation system [26]. Similarly, Tat protein, expressed and purified fromEscherichia coli,which has been refolded in the presence of Zn2+ appears to be monomeric [l81 while, Slice et al. have found no evidence that the Tat protein exists as a dimer in solution [36]. Because of these contradictions,a structural characterizationof the zinc-coordinated cysteine-rich region of HIV-1 the Tat protein should help clarifying the role played by zinc in the biological activity of this protein. To this end, we report the synthesis of a21-57 peptide fragment that contains the three distinct functional domains of the Tat protein (Figure 1) and the secondarystructureofthecorrespondingZn2+ complex by Mass Spectrometry(MS), Circular Dichroism (CD)as well as by one- and two-dimensional Nuclear Magnetic Resonance (NMR) spectroscopy.

25

30

35

45

40

50

55

ACTNCYCKKCCFHCQVCFITKALGISYGRKKRRQRRR cysteine-rich region 'core' region basic region Figure 1. Amino acid sequenceof the W - 1 Tat protein fragment corresponding to thethree distinct functional domains.

All these data and those observed with Cd2+ and CO2+ complexes indicate the formation of1:la monomer Zn2+ complex witha zinc-finger like structure and not a clustering dimeras proposed elsewhere ~41.

II. MATERIALS AND METHODS A. SynthesisandPurification

of HIV-1 Tat-37Peptide

l&-37 peptide was synthesized by solid-phase using Fmoc chemistry on an Applied Biosystems model 430A automated peptide synthesizer. A

258

st al.

Fmoc-Arg (Pmc)-HMP (4-hydroxymethyl phenoxymethyl polystyrene resin) support (0.1 mmole) was used. The subsequent Fmoc amino acids (1 mmole, i.e. 10 eq. resin) were incorporated using the DCC/HOBt method (dicyclohexylcarbodiimide and 1-hydroxy-benzotriazole reagents). Protecting groups were 2,3,5,7,8-~entamethylchroman-6sulfonyl (Pmc)for Arg, t-butyloxycarbonyl (Boc) for Lys, trityl (Trt) for Gln, Asn, His, and Cys, and t-butyl (tBu) for Ser, Thr, and Tyr. After the 20th coupling step, half of the resin was removed both to reduce the peptide-resin volume and to increase the amount of Fmoc amino acid. Moreover, the standard program of the synthesizer was modified to progressively increase the coupling time as a function of the size of the growing peptide. Deprotection of the Fmoc groups was obtained by two successive treatments of 3 min and 15 min with 20% piperidine in Nmethyl pyrrolidone. At the end of the synthesis, the peptide (I") resin (100 mg) was treated for two hours with 10 ml of solution containing trifluoroacetic acid ("FA) (82.5%), phenol (5%), 1,2 ethanedithiol (2.5%), thioanisole (5%) and H20 (5%) to remove the protecting groups and to cleave the peptide from the resin. At the end of the reaction, the TFA solution was filtered and concentrated under vacuum. The peptide was then precipitated with 10 mlof cold t-butyl methylether and centrifuged for 10 minutes at 4°C. The supernatant containing the scavengerswasremoved, the precipitate resuspended in t-butyl methyl ether and centrifuged again. The precipitate was then solubilized in acetic acid 20% and lyophilized. The peptide was purified by reverse-phase high-performance liquid chromatography onanAppliedBiosystems151A apparatus equipped with a diodearray as detector anda Brownlee AquaporeW300 C8 column 2.1 x100 mm (7 pm,300 A) and Brownlee Aquapore RP300 C8 column 10 x 220 mm (20 pm, 300 A) respectively. The pure fractions were pooled, lyophilized and characterized by mass spectrometry (vide infra). All manipulationsof the reduced peptide were performed under anaerobic conditions, and all solvents were degassedwith N2 prior to use.

B. Samplepreparation The Zn2+ complex, noted here as. Zn(Tat-37), was prepared by adding one equivalent ofZn2+ ions (1.l-fold molar excess) to a 8.5 m M (NMR) or 0.8 mh4 (CD) aqueous solution of the peptide. After adjusting the pH withmicromolar aliquots ofNaOD, the solution was lyophylized,

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dissolved in either 99.996% D20 or 90% H20/10% D 2 0 and transferred to anN M R tube or CD cell. Because the peptide solutions were sensitive to air oxydation of the thiols, care was taken to exclude oxygen. All solvents were degassed with nitrogen prior to use, nitrogen was bubbled into the solution during pH adjustement and the N M R tubes were flushed with nitrogen before and after sample addition and then were sealed.

C. Mass spectroscopy An electrospray-dedicated TRIO-2000 instrument was used (FISONS Biotech, Manchester,UK). The solvent, either 50% aqueous methanol or 50% aqueous acetonitrile at 10 pVmin. About 500 ng sample in 10 p1 solvent was injected for each experiment. The cave-to-skimmer voltage was set to 40 volts. Averaged mass-to-charge spectra were transformed into time mass scale spectra using the MaxEnt program.

D. Circulardichroism Circular dichroism (CD) spectra were recorded at 20°C with a Jobin-Yvon dichrograph VI connected to a compatible PC microcomputer. A50 pm optical path length cell was usedto record spectra of the free or Zn2+complexed peptide in the far ultraviolet region (260-185 nm) at a concentration of 2 mg/ml peptidic material inH20,pH 6.4. The results are presented as molar ellipticity values based on the amino acid mean residue mass of110Da.

E. NMR Spectroscopy Wo-dimensional NMR spectra were obtained on a Bruker AMX 500 spectrometer, equipped with an inverse probehead, at 298 K and 278 K. The spectra were acquired in the phase-sensitive mode using the time proportional phase incrementation method [37] and the water resonance was suppressed by gated irradiation during the relaxation delays. The TOCSY experiments [38] were acquired with spin-lock times of 23 ms and 69 ms. The NOESY experiments [39] were performed with five mixing times(20,100,200,300 and 500 ms). Spectra were acquired with 32 scans per t1value and 512 1 t values. The 2D maps result from 512 x 2048 data matrices and the spectra width was 6002 Hz in both dimensions. All spectra were Fourier transformed using a 45' shifted squared sine bell filtering in the t2 and t1dimensions and a polynomial

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baseline correctionwas applied. All theN M R data were processed on a 4D35 TG+ SiliconGraphics IRIS workstation using theFELIX software (Biosym, Hare Research).

111. RESULTS

A. Mass Spectrometry Electrospray ionization mass spectrometry was used to determine the stoichiometry of Zn2+ binding with Tat-37. The free peptide, withoutany addition of Zn salts, gave a series of multiple charged peaks from which the averaged molecular weight was found at 4430. 1 (* 0.15 standard deviation). This measurement was in agreement with the theoreticalvalue 4430.4. The zinc complex was studied in two different solvents. In methanol solution, two different componentswere detected. The major one (60%)corresponded to the free peptide while, the minor onewas due to a 1:l monomeric zinc complex with a molecular weight of 4493.2 corresponding to [(Tat-37)+ Zn2+ - 2H+]. In the raw mass spectra data, these species were desorbed as multiprotonated ions and the above mentionned compositions were those of the corresponding neutral components as deduced from molecular mass calculations. The presence of free peptide was detectedin the mass spectrum, suggesting that some dissociation of the complex occured in this acidic solvent [40]. Figure 2 shows the electrospray mass spectrum of zinc complex with a one-to-onestoichiometryinaqueousacetonitrilesolution (50%/50%). In this more basic solvent, three peaks are detected and two zinc-containing ions appeared. The major one was the 1:l adduct as depicted above (51%). The second one (25%) at mass 4554.5 was attributed toa 1:2 complex, corresponding to[(Tat-37) + 2Zn2+ - 4H+]. The last peak is due to the excess of free peptide. Interestingly, from Figure 2, no dimeric formsof Tat-37 with Zn2+ could be detected. In the same solvent conditions (50% aqueous acetonitrile), the well characterized complexof the HN-2 nucleocapsid protein (NC-39) whichcontainstwozinc-fingerdomainsexhibitedtwospecies, a monomeric 1:l complex (59%) and a 1:2 complex (41%). The presence of a 1:1 complex shows that a partial loss of zinc atoms occurs under electrospray conditionswhich may be related to the lower affinity of the second finger for the metal ion in NC-39 (to be published). In any case, no dimeric forms can be detected. This disagreewith

HIV-1 Tat Protein Forms

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26 1

a previous [41] published experiment describing the Tat 21-38 complexes with zinc and cadmium cations. These authors used plasma desorption mass spectrometry for the characterization of the molecular weight of the complexes. In contrastto electrospray where ionization takes place from solution, plasma desorption is a solid phase ionization technique from material adsorbed on a nitrocellulose filter.

4000

5000

9000

mass

Figure 2. Electrospraymassspectrum of Tat-37-Zn2+complexin aqueous acetonitrile solution(50%/50%). The molecular weights of two zinc species are indicated.

Thus, in these two methods, molecular interactions are very different within the condensed phase from which ionization occurs. It seems that relatively weak molecular interactions, such as non-covalent bonds, may survive during the electrospray ionization process. Proteinligand non covalent complexes [42] as well as ternary complexes between the dimeric HIV-1 protease and a substrat-based inhibitor have been reported [43]. Even though a true correlation between electrospray generated gas phase complexesandsolution-phasenoncovalent

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intreactions have not been fully established, this technique appears the most reliable mass spectrometry method for the visualization of solution phase complexes. B. Circulardichroismanalysis

Circular dichroism has been extensively used for the exploration of peptide conformation in solution and diagnostic bands specific of various secondary structure elements have been characterized. As CD has a very fast time scale (10-15S) one cansee bands for virtually all species present. Thus, the presence of significant populations of &turn conformers in solution canbe quite adequately detected by CD.

Mol. E : l i p . x l E 3 -1.000..

190.00

200.00

210.00

220.00

nm

230.00

240.00

250.00

Flgure 3. Circular dichroism spectra of apo-Tat-37 (curve 1) and &(Tat-37) complex (curve 2) in aqueous solution. Results are expressed in molar ellipticity.

The CD spectraof the Tat-37 peptide in the absence (curve 1) or 2) of Zn 2+ (Figure 3) are fully consistent with the in the presence (curve presence of type I B-turns as reviewed by Smith and Pease [44]. In particular these so-called ‘helix-like’ spectra differ from random coil with regard to the intensityof the minimumat 200 nm and to the absence aof positive bandat 220 nm. According toa previously established correlation

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between a low absolute value of negative ellipticityat 200 nm anda high B-turn content of a peptide chain [45], it can be deduced that the addition of Zn2+ stabilizes the B-turn structure of the Tat-37 peptide. As stated above, this peptide comprises three regions which are functionally independent. It is likely that they are also distinct at a structural level. Taking into account this assumption, we can deduce that the cysteine-rich region (residues 21-37) which has been known to bind 2n2+ [41] has a tendency to form a B-turn structure enhanced by Zn2+ addition. However one cannot totally rule out the possibility of a concomitant stabilizationof the basic region (residues48-57) which has &turns [46]. also been reported to contain potential This CD result leads us to suggest that at least one part of the cysteine-rich region might form a zinc-finger like structure similar to the one already described for several retroviral nucleocapsid proteins [47,48]. C. 'H NMR 1. One-dimensional 'H NMR studies

One-dimensional 1HN M R studies were performedin order to determine the influence of Zn2+, pH and temperature on the H N M R spectral properties. The addition of a zinc salta to solution of Tat-37 peptide H20 in at pH = 6.3 produces substantial changes in the NMR 'H spectrum of the Zn(Tat-37) complex (Figure 4). These modifications are confined to the in the frequenciesat which downfield region and appear to reflect changes the amide protons resonate and also around 2.8 ppm where all the CysCBH2 appear. In fact, the chemical shift perturbations, inducedby zinc complexation, may be due to the direct influence of changes in electron density and/or may result indirectly from an altered conformation of the peptide with, as a consequence, an important dispersion of the NMR signals, particularly the NH signals. Recent results according to study in our laboratory, on the retroviral-type zinc-finger domain of the W - 2 nucleocapsid protein [47,48], provide evidence that the chemical shift dispersion of signalswithin the 'H NMR spectrumclearly demonstrates the presence of a well defined folded compact structure. Curiously, theZn2+ complexation produces no chemical shift dispersion of the NH signals but rather a marked tendency for these resonances to get closer togetheras well as a notable broadening. A similar behavior

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&(Tot-37)

278K

PPm Figure 4. One-dimensional 1H NMR spectra of the amide and aromatic K (upper region of Tat-37 in H20 in the absence of Zn2+at 298 of equimolar Zn2+(1 .l-fold spectrum) and in the presence molar excess) at 298K and 278K (lower spectrum).

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has been observed with Zn 2+ complexed thymulin, a nonapeptide hormone produced by thymic epithelial cells [49]. The titration resultsof the Zn2+-bound form of Tat-37 peptide show that this peptide binds one equivalent of metal stoichiometrically. The addition of an excess of zinc, up to a 1:l ratio, did not change the original N M R spectrum. From pH titration studies, it has been observed that this complex startsto form around pH 4.20 while up to pH 10.20 the formation of zinc hydroxyde is not observed. These observations relate closely with those encountered with the first zinc-finger domain of the W - 2 nucleocapsid protein [47]. Finally, witha few exceptions, the resonances do not exhibit notable changes in chemical shifts and linewiths at 278K (Figure 4).

-

2. Two-dimensional'H NMR studies Sequence-specific resonance assignments were achieved following the conventional method proposed by Wuthrich [50] using NOESY and HOHAHA spectra measured consecutively under the same conditions. The fingerprint region of the NOESY spectra containing connectivities between amide protons and amide and aliphatic protons are shown in Figure 5. Curiously, rather fewer NOE enhancements are present in these NOESY spectra than might be expected fora protein of this molecular weight. A similar observation has been made in the spectra of rabbit liver of cysteines [S11 andin the metallothionein which contains also a cluster spectra of thymulin [49]. It is striking to note that no NH(i)/NH(i+l) NOE sequential connectivities are observedin these two maps. A study conducted at different mixing-time values from 20 msto 500 ms, gives the same results. Consequently, it is clear that all spin systems expected from Tat-37 and Zn(Tat-37) could not be observed because some of them present ambiguities arisingfrom chemical shift degeneracy or experience broadening through the influence of complexation. The degeneracy problem is particularly crucial for the Tat complex where of most the NH signals get closer together upon binding. Nevertheless, for the apo-peptide, a lot of relayed cross peaks are observed in the 69 ms HOHAHA spectrum of Tat-37, while together the CaH(i)/NH(i+l) NOE sequential connectivities and the intra-residue CaH(i)/NH(i) through-bondcorrelationconnectivitiescreatethree continuous patterns of sequential connectivities: C( 22) to T(23), C(34) to R(5 2) and Q( 54) to R( 5 5). Nevertheless, distinction among the remaining spinsystems,fourcysteines (c(25,27,30,31)), three arginines

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(R(53,56,57))

and two lysines (K(28,29)), was not possible at this early stage of the analysis. The assignmentof the N-terminal regionproved to be significantly more difficult than that of the center region of the sequence. This could be explained by a substantial local mobility of the residues or conformational disorder in this region. In addition, upon complexation, an important broadening of the cysteine resonances occurs which significantly complicates the assignment of the N-terminal region. Similarly, the assignmentof the C-terminal region suffered fromproblems comparable to those of the N-terminal region. Indeed, although very strong cross-peaksare observed for the arginine and lysine residuesin all the TOCSYspectra, their NOE signals are barely observed in the NOESY spectra. Onewould expect this phenomenon to be temperature-dependent and experimental NOESY conditions-dependent. Unfortunately, these experiments failed to yield any new informations.

3. Complexes with Cd2+ and C$+ Cadmium(I1) and cobalt(1I) can substitute for Zn(I1) in a number of metalloproteins and are useful as a spectroscopic probeof Zn(II) metal binding sites [52]. Interestingly, the additionof cadmium and cobalt salt to a solution of Tat-37 in H20 at pH 6.3, produces the same perturbations than those observed with zinc salt(Figure 6).These changesare confined at 2.8 ppm where all the Cys CBH, resonate and to the downfield region.Fromthesedata,itappearsthatZn(II),Cd(II)andCo(I1) probably induce a similar conformational changeof the Tat-37 fragment upon complexation. Furthermore, electronic spectroscopy of the bluegreen CO@) complexstrongly suggests a tetrahedral metal-binding site consistent with a four cysteinatecoordination environment.

-

IV. DISCUSSION Since it was established that Zn2+ is required for the binding of Tat to nuclear matrix [53], much effort has been devoted to the structure understanding of the corresponding complex (es) and what specific functional role it might play. Indeed, the knowledge of the Tat structure represents the first step towards understanding the role of Tat in mactivation of viral gene expression. Examination of the amino acid sequence of the Tat reveals the presenceof four potential zinc-binding sites, each encoded by the amino acid sequence Cys-X-X-Cys. The spacing of the cysteine residues is similar to that found in analogous

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PP" Figure 6. One-dimensional1HNMRspectra,corresponding to the

aliphatic region,of Zn(Tat-37) (8.5 mM, pH 6.30), Cd (Tat-37) (4.3 mM, pH 6.50) and CO(Tat-37) (3.9 mM,pH 6.30) in90% H20at 298 K

regions of known metal-binding regulatory proteins [3] and is thought to form a metal-binding finger [33]. To date, the informations about the structure of Tat remain unclear and conflicting results have been reported [ 18,24,26,35, 361. In fact Zn 2+ has a d' electronic configuration with, as a consequence, the lack of suitable spectroscopic properties for identifying com lexes formed in solution. Furthermore, the coordination geometry of Zn!+ is easily distorted by the ligandsin the coordination

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sphere, leading toan important flexibility and adaptability of Zn 2+ which mayinfluenceitscoordinationmode.Thesepropertiescombineto demonstrate the difficulty in studying zinc-proteins in solution. However from this study, all our observations converge to the conclusion that the Tat-37 peptide fragment forms a complex with Zn2+, Cd2+ and CO2+.The stoichiometry of Zn2+ binding, as determined by mass spectroscopy and confirmed by 'H NMR, is one Zn2+ ion per peptide fragment(complex 1:l). Parallely, we found that theHIV-1 Tat-37 forms a monomercomplexwithZn2+asevidenced by themass spectroscopy technique. On the other hand, we have found no evidence that the Tat fragment exists under the form of a dimer in solution as reported elsewhere[24]. It has been observed that upon addition of Zn2+ to Tat-37, the linewidthsof most of the resonances (particularly those corresponding to the Cys residues) became visibly broader in the Zn complex than in the apo-form. These observations are indicative of a significant structural changeof the peptideas well as a tightZn binding. Unfortunately, the presenceof this broadening prevents the determination of the 3D solution structure of this metalloprotein by NMR technique. It is reasonable to assume that the zinc ions arein equilibrium with several binding sites since the total number of thiolate ligands is higher than requiredfortheformation of a tetrahedralZnS4environment. Fluxionability, similar to that encountered in the Cd metallothionein complex [54] may explain the broadening. An other alternative is the formation of intermolecular disulfide bonds between cysteines which are not coordinated toZn2+. However, the N M R spectra of Tat-37, with and without metal bound, indicate that metal binding exerts its effect only on the conformation of Tat around the cysteine-rich region and does not affect the global foldingof the entire molecule. Indeed, our NMR-data suggest that the C-terminal basic region must be exposed to solventand it is likely to have a major functional importance. It be cananticipated that binding toa specific ligandwould stabilize this part of the molecule, as found by CD studies where this region becomes partially or fully structured when bound to TAR [55]. In conclusion, we have shownby MS,CD and N M R that the37residue peptide fragment representing the 21-57 domain of the HIV-1 Tat proteinforms a stable 1:l monomercomplexuponadditionof equimolecular amountsof zinc, cadmium and cobalt ions. Nevertheless, the hypothesis of the presence of a (Zn),l%t-37 (2:l) complex, as observed from the MS spectra, cannot be ruled out. This hypothesis is currently under investigation. The structure of the 1:1 complex shows the

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presence of a type I S-folded domains stabilized by a tetrahedral coordination of four cysteine residues to Zn2+. At this stage of the analysis, we proposed a model in which the C(22), c ( 2 5 ) , C( 34) and C(3 7 ) are coordinated to the metal ion,as depicted in Figure 7, a structure similar to the one proposed by Patarca and Haseltime [33]. Further

K

C

K

C

C

F

Y

H

C

C

N

\

Q

,S'"

,Tnz, NH2-A

v

C

C

22

31

FITKALGISYGRKKRRQRRR-COO-

Figure 7. A possible schematic representation of a 1:l Zn (Tat-37) complex. In this representation Zn2+ is coordinated to C(22), C@), C(=) and C(37) residues.A tetrahedral coordination geometry is assumed. This structure is analogous to that proposed by Haseltime et al. [33].

analysis of the cysteine region, in particular the ability of the mutant cysteine substitution derivatives to form metal complexes, is in progress. In light of our results,we propose that theZn(Tat-37) complex might be structurally related to the zinc-finger like structure, common to several retroviral nucleic acid binding proteins, which are well known to bind to ssDNA and RNA [3]. We remark that a strongly basic region is present in the Tat protein near the proposed metal binding finger, a feature also common to many retroviral metal-binding proteins [3]. More detailed analyses will be necessary to fully understand the details of the Tat 3D zinc complex structure.

REFERENCES 1. B.L. Vallee, and D.S. Auld, Acc. Chem. Res. 26: 543-55 1 (1993). 2. A. Klug, and D. Rhodes, TZBS 12: 464-469 (1987).

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27 1

3. J.M. Berg, Science 232: 485-487 (1986). 4. J.E. Coleman, Annu. Rev. Biochem. 61: 897-946 (1992). 5. J. Miller, A.D. McLachlan, and A. Klug, EMBO J. 4:1609-1614 (1985). 6. L. Green, and J.M. Berg, Proc. Nutl. Acad. Sci. USA 86: 4047-4051 (1989). 7. J.M. Berg, Cell 57: 1065-1068 (1989). 8. M. Johnston, Nature 328: 353-355 (1987). 9. S. Ruben, A. Perkins, R. Purcell, K. Joung, R. Sia, R. Burghoff, W.A. Haseltine, and C.A. Rosen, J. Virol. 63: 1-8 (1989). 10. M.A.Muesing,D.H. Smith, and D.J.Capon, Cell 48:691-701 (1987). 11.S.K. Arya, G. Guo, S.F. Josephs, and F. Wong-Staal, Science 229: 69-73 (1985). 12.J.G. Sodroski, C.A. Rosen, F. Wong-Staal, S.Z.Salahuddin, M. Popovic, S.K. Arya, and R.C. Gallo, Science 227: 171-173 (1985). 13. A.I.Dayton,J.G.Sodroski,C.A.Rosen,W.C.Goh,andW.A. Haseltine, Cell 44:941-947 (1986). 14. M.B. Sadaie, T.Benter, and F. Wong-Staal, Science 239: 910-913 (1988). 15. P.A. Sharp, andR.A. Marciniak, Cell 59: 229-230 (1989). 16. S. Feng, and E.C. Holland, Nature 334: 165-167 (1988). 17. B.Berkhout,R.H.Silverman,andK.T.Jeang Cell 59:273-282 (1989). 18. C.Dingwall, I. Emberg, M.J.Gait,S.M.Green, S. Heaphy,J. Karn,A.D. Lowe, M. Singh, and M.A. Skinner, EMBO J. 9: 41454153 (1990). 19. J. Kam, C. Dingwall, J.T.Finch, S. Heaphy,andM.J.Gait, Biochimie 73: 9-16 (1991). 20. A. Gatignol, A. Kumar, A. Rabson, and K.T. Jeang, Proc. Natl. Acad. Sci. USA 86: 7828-7832 (1989). 21. C.A. Rosen, Trends Genet. 7: 9-14 (1991). 22. B.R. Cullen, Cell 46: 972-982 (1986). 23. L.J. Siegels, L. Ratner, S.F. Josephs, F. Derse,M.B.Feinberg, G.R. Reyes, S.J. O’Brien, and F. Wong-Staal, Virology 148: 226231(1986). 24. A.D.Frankel,D.S.Bredt,andC.O.Pabo, Science 240:70-73 (1988). 25. J.A.Garcia,D.Harrich,L.Pearson,R.Mitsuyasu,andR.B. Gaynor, EMBO J. 7 : 3143-3147 (1988).

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26. A.P. Rice, and F. Carlotti, J. Virol. 64:1864-1868 (1990). 27. M.J. Churcher, C. Lamont, F. Hamy,C.Dingwall,S.M.Green, J. Mol. Biol. A.D. Lowe, P.J.G. Butler, M.J. Gait, and J. Karn, 230: 90-110 (1993). 28. M.J. Gait, and J. K m , TIBS 18: 255-259 (1993). 29. J. Hauber, M.H. Malim, and B.R. Cullen, J. Virol. 68: 1181-1187 (1989). 30. H. Siomi, M. Shida, M. Maki, and M. Hatanaka, J. Virol. 6 4 : 18031807 (1990). 31. K.M. Weeks,C.Ampe,S.C.Schultz, T.A. Steitz, andD.M. Crothers, Science 249: 1281-1285 (1990). 32. B.J. Calnan, S . Biancalana, D. Hudson, and A.D. Frankel, Genes Dev. 5: 201-210 (1991). 33. R. Patarca, and W.A. Haseltime,AIDS Res. Hum. Retrovimses 3: 12 (1987). 34. J.M. Berg, Annu. Rev. Biophys. Chem. 19: 405-421 (1990). 35. A.P. Rice,.and F. Chan, Virology 185: 451-454 (1991). 36. L.W. Slice, E. Codner, D. Antelman, M. Holly, B. Wegrzynski, J. Biochemistry 31: Wang, V. Toome, M.C.Hsu,andC.M.Nalin, (1992). 12062- 12068 37. D. Marion, and K. Wiithrich, Biochem. Biophys. Res. Commun. 113: 967-974 (1983). 38. D.G.Davis,and A. Bax, J. Am.Chem. Soc. 197:2821-2822 (1985). 39. J.Jeener, B.H.Meier, P. Bachman,andR.R. Emst, J. Chem. Phys. 71:4546-4553 (1979). 40. C.L. Gatlin, and F. Turecek, Anal. Chem. 66:712-718 (1994). 41. A.D. Frankel, L. Chen, R.J. Cotter, and C.O. Pabo, Proc. Nutl. Acud. Sci. USA 85: 6297-6300 (1988). 42. B. Ganem, Y.T. Li, and J.D. Henion, J. Am. Chem. Soc. 113:62946296 (1991). 43. M. Baca, and B. Kent, J. Am. Chem. Soc. 114:3992-3994 (1992). 44.J.Smith,andL.Pease, C.R.CCrit.Rev.Biochem. 8:315-399 (1980). 45. M. Erard, F. Lakhdar-Ghazal, and F. Amalric, Eh-.J. Biochem. 191: 19-26 (1990). 46. E.P. Loret, P. Georgel, W .C. Johnson, Jr., andP.S. Ho, Proc. Nutl. Acud. Sci. USA 89: 9734-9738 (1992). 47. J.P. Laussac, G. Peyrou,H. Mazarguil, M. Erard, M. Bourdonneau, and M.T. Cung, New. J. Chem. 17: 607-612 (1993).

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48. J.P. Laussac, G.Peyrou, M. Bourdonneau, M. Erard, H. Mazarguil, and M.T. Cung, Trends Znorg. Chem. 3: 39-44 (1993). 49. M.T. Cung, and J.P. Laussac, New. J. Chem. 14: 293-299 (1990). 50. K. Wuthrich, NMR of Proteins and Nucleic Acids, John Wiley & Sons, Inc., New York, 1986. 51. N. Braun, G. Wagner,E. Worgotter, M. Vasak, J.H.R. Kagi, and K. Wiirhrich, J. Mol. Biol. 187: 125-129 (1986). 52. I. Bertini, and C. Luchinat, Adv. Znorg. Biochem. 6: 72-111(1986). 53. W.E.G. Muller, T. Okamoto, P. Reuter, D. Ugarkovic, and H.C. Schroder, J. Bid. Chem. 265: 3803-3808 (1990). 54. M.Good, R. Hollenstein, P.J. Sadler, and M. Vasak, Biochemistry 27: 7163-7168 (1988). 55. B.J. Calnan, S. Biancalana, D. Hudson, and A.D. Frankel, Genes Dm.5: 201-210 (1991).

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17 Menkes Disease: From Patients Gene

to

S. Packman DepartmentofPediatrics,DivisionofGenetics,Universityof California,Box 0748, SanFrancisco,CA94143

C. Vulpe Department of Biochemistry and Biophysics, University of California,SanFrancisco,CA94143 S. Whitney Departments of Pediatrics and Medicine, University of California,

SanFrancisco,CA94143 B. Levinson, S. Das, and J. Gitschier DepartmentofMedicineandthe Howard Hughes Medical Institute, University of California, San Francisco, CA 94143

I.

INTRODUCTION

Copper is an essential trace metal required as a co-factor for approximatelythirtyenzymes. Roteins as fundamental as cytochrome oxidase and as specialized as dopamine B-hydroxylase exploit the oxidative capacityof copper in their biological roles. However, that same oxidative potential can cause extensive cellular damage through free radical production and direct oxidation of lipids, proteins and nucleic acids. Accordingly, biologic systems must regulate copper transport to achieve a balance between specificity of function at low levels and the toxicity of copper excess [l]. Several human inherited disorders illustrate the need for this balance. The autosomal recessive disorders, Wilson disease and Indian childhood cirrhosis, reflectthe effects of copper toxicity. In Wilson disease, decreased copper export from the liver results in copper-induced chronic liver disease and contributes to pathologic [2]. changes in other tissues, especially the brain, kidney, and eye InIndianchildhoodcirrhosis,anunknowndefect in copper transport causes massive accumulation of hepatic copper and rapid hepatic injury[3]. 275

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In contrast, X-linked Menkes disease results from a systemic copper insufficiency. The deficiency results from the trapping of copper in to some tissues, most notably intestinal mucosa and kidney, leading a reduction in serum copper and ceruloplasmin-copper oxidase concentrations and a concomitant failure of copper delivery to other tissues. Patients with stereotypic Menkes disease manifest pili torti, hypopigmentation, hypothermia, growth failure, skeletal defects, arterial aneurysms, seizures, and progressive degeneration of the centralnervoussystem,withdeath in earlychildhood[4-61. Deficiencies in specificcuproenzymes --- e.g. tyrosinase (hypopigmentation), lysyl oxidase (defective collagen, elastin crosslinking), and dopamine-B-hydroxylase (catecholamine production) --- are likely tobe responsible for someof the clinical findings. The contributions of deficient cytochrome c oxidase and superoxide dismutase 1 activities to the clinical phenotype are not clear, and the enzyme involved in the etiology of pili tom is not known [2]. 11.

MENKES DISEASE AND COPPER TRANSPORT

Defective export of copper is thought to be the basic cellular defect in Menkes disease. With the exception of hepatocytes [7] and cultured chorionic villus cells [g], most tested cultured cells from Menkes patients exhibit the characteristic phenotype of copper accumulation [2]. A s demonstrated in cell culture studies, Menkes in copper efflux, with normal uptake, and cells have a specific defect with normal transport of cadmium and zinc in mutant cells[9,lo]. mRNA have Increased concentrations of metallothionein protein and been repeatedly demonstrated in Menkes cells and tissues [2, 11, 121.However,theregulation of metallothionein synthesis was shown to be normal in Menkes cells[ll-131, and recent focus has been on cellular copper transport as the possible defective step in this disorder. A number of hypothesis can be entertained regarding the cellular location ofthetransportstepdeficientinMenkesdisease. A

transport function in the plasma membrane, e.g., could well be involved in copper egress from the cell. However, detailed copper effluxandcellfractionationstudiessuggestthatthecopper accumulation of Menkes cells results from a defect in translocation across a subcellular compartment, rather than a plasma membrane defect in copper export[lo, 14, 151.

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The subcellular location of the Menkes transport step can be considered in the lightof a numberof experimental data and disease in mitochondria and related observations. Decreased copper content lysosomes has been demonstrated in mutant cells by biochemical measurements [ 161, electron microscopic cytochemistry[ 171, and cell fractionation experiments[ 141. Given the changes in lysosomal copper contentin Wilson disease[2], Indian childhood cirrhosis[3], and Bedlington terrier copper toxicosis [18], it is intriguing to speculate that there exists a complex lysosomal copper-translocating system, defective in Menkes disease and related disorders. Such a transporter would be an addition to the growing list of lysosomal transporters of small molecules [19]. The aggregate data are also consistent with an additional hypothesis, viz,., the existence of a copper-transport system in the membrane of the endoplasmic reticulum. A mutation would then result in failure of copper delivery to some cuproenzymes (e.g. lysyl oxidase), but not to others (e.g. superoxide dismutase 1) [20,21]. Indeed, it has been suggested that the small but significant amount of copper associated to transport with endoplasmic reticulum membrane may be attached proteins such as ceruloplasmin, or to unknown transport proteins needed forthe movement of copper across the cell surface [22]. The identification of the Menkes transport protein can be viewed as a major stepin the dissectionof one or more ofthe above pathways. 111.

ISOLATION OF THE GENE DISEASE

FOR MENKES

In 1993, we [23] and others [24,25] reported the identification of the gene responsiblefor Menkes disease. This gene was identified to the by a positional cloning approach, based on gene localiization Xq13 region of the X chromosome by: linkage analysis in Menkes disease families [26]; chromosomal rearrangementsin each of two patients [27,28]; and the mappingof the gene for mottled, a mouse homologue of Menkes disease, to a region of the mouse X chromosome showing homology of synteny to human Xq13 [29, 301. Particular advantage was taken of the reciprocal X:2 translocation in a female Menkes patient [27]. We constructed a physical map of the X-chromosomeregion around the site of the translocation breakpoint and identified closely flanking probes, DXS441 and PGKI. Alterations in the physicalmap of genomic DNA from the

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translocation patient suggested the translocation within lay 300 kb of PGKI. These results were consistent with mapping studies of other groups [3 1, 321. Therefore, P G K I - containing yeast artificial chromosomes (YACs) were obtained from several sources, and two were determinedto span the siteof the translocation breakpoint.

The Menkes cDNA was isolated using two approaches. In one approach, a placental cDNA library was screened with a 100-kb YAC fragment that contained the of sitethe translocation breakpoint butnot P G K I . The fragment identified a 3-kb cDNAwhich mapped -80 kb from the translocation breakpoint. Sequence analysis of the cDNA clone andof the corresponding genomic DNA suggested theentire clone represented the 3'-untranslated region of a gene, including the polyA+ tail.In the second approach, we utilized exon trapping vectors to identify coding regions in two sets of genomic h clones, one set encompassingthe breakpoint and oneset 20 kb distal. One exon-trapped clone with an open-reading frame was obtainedfrom each set ofh clones. Each of the three cDNAs detected an 8.5-kb transcript by Northern blot analysis and were used to isolate a totalof eleven different overlapping cDNAs from an unamplified fibroblast library. Together, these comprised thefulllength cDNA designatedMcl, for Menkes cDNA 1. We estimated that the Mcl gene spans roughly 100kb of genomic DNA and was likely to cross the translocation breakpoint [23].

IV.

EXPRESSION OF THE MENKES GENE

Hybridization of the MC1 cDNA to a Northern blot of polyA+RNA showed a pattern of Mcl expression consistent with the Menkes phenotype. The gene is expressed in heart, brain, lung, muscle, kidney, pancreas, and, to a lesser extent, placenta, but little or no MC1 mRNAwas detected in liver. Additional studies showed expression in fibroblasts and lymphoblasts [23]. Abnormalities in expression of the MC1 gene were found in fibroblast cell lines derived from many severely affected Menkes patients [23-251. Fibroblasts from the female Menkes translocation patient showedno detectable expressionof this transcript. In initial studies, 23 of 32 unrelated Menkes patients showed alterations in the expression of this gene, including the following: no detectable of this mRNA, a transcriptof Mcl mRNA, greatly decreased amount

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a smaller size than expected; and greatly reduced level of two hybridizing mRNA's, one of normal and another of larger size [23251. V.

MUTATIONS IN THE MENKES GENE

Four of 26 Menkes patients analyzed by Southern blot were found to have partial deletions of the Mcl gene. In one patient and his affected brother, an altered band was observed only with a 5'-most cDNA probe, whereas bands were absent with probes from the central portion of Mcl. A probe from the 3' untranslated region showed no abnormalitiesin these two patients. Together, the results indicated that there wasan interstitial deletion of the Mcl gene in the pair of brothers [23]. In a separate analysis [24], 16 of 100 patients showednon-overlappingdeletionsofthegene.Theaggregate expression data and deletion analyses provided compelling evidence that Mcl was indeed the Menkes(MNK) gene.

To begin to understand the relationship between disease severity and the underlying mutations, we studiedan additional twelve Menkes patients, ofuniformlyseverephenotype,whoexhibitedno abnormalities by Southern analysis. We screened for mutations in Menkes cDNA using RT-PCR and chemical cleavage mismatch in ten of twelve (CCM) detection [33,34]. Mutations were detected patients with severe Menkes disease, and these mutations were all different. These results clearly demonstrated that independently occurring mutations in the MNK gene are responsible for the vast majority of cases of Menkes disease. All of the mutations, including nonsense, frameshift, splice site, and non-conservative amino acid replacements,aswellasthesmalldeletions and frameshifts generated by alternative splicing, would be predicted to adversely affectexpressionoftheMenkesprotein[35].Mutationsthat resulted in splicing abnormalities, detected by RT-PCR alone, were observed in six of twelve patients and included two splice site changes,anonsensemutation,amissensemutation,asmall duplication and a small deletion. Chemical cleavage analysis of the remaining six patients revealed the presence of one nonsense mutation, two adjacent 5-bp deletions and one missense mutation. A valinefleucine polymorphism was also observed. These findings, combined with the prior observation of deletions in 1520% of Menkes patients, suggest that Southern blot hybridization and RTPCR will identify mutationsin the majority of patients[X].

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VI.

THE MENKES PROTEIN IS A COPPER TRANSPORTINGATPase

Sequence analysisof the MNK cDNA and analysisof the predicted 1500 amino-acid protein revealed that the Menkes locusencodes a cation-transporting ATPaseof the P-type. P-type ATPases form a family of integral membrane proteins which utilize an aspartyl phosphate intermediateto transport cations across a membrane. The MNK derived amino acid sequence hasall the features described in other members ofthe P-type ATPase family[36]. These include the following: a cation channel, formed by transmembrane hairpin loops; an ATP binding domain; a phosphorylation domain which contains a conserved, phosphorylated aspartic acid; an adjacent transduction domain; and a phosphatase domain, which removes the as part of the reaction cycle. phosphate from the aspartic acid The predicted MNK protein showed the greatest similarity to two Enterococcus hirue Cu++-transporting ATPases (copA andcopB, [37]). It is closely related to a subfamily of prokaryotic ATPases including cadmium and potassium transporters, and an ATPase of unknown cation specificity from Rhizobium meZiZoti [38]. At the the added feature of six nonamino-terminus, the MNK protein has in many identical copies of a heavy-metal binding motif used bacterial proteins. The motifs in the MNK protein most closely resemble thatof the Enterococcus hirae copA protein. These homologies, combined with the specificity of the Menkes cation sequestration defect for copper, support the contention that the Menkes gene encodes a copper-transporting ATPase.

VII.

CONCLUSIONS AND OVERVIEW

Several important clinical applications will directly accrue from these findings. The ability to rapidly delineate mutationsin the Menkes gene will permit accurate prenatal and canier diagnosis of Menkes disease and related disorders. The development of a correlation be between the natureof the mutation and the clinical phenotype will valuable in assessing prognosis and in making decisions about therapeutic strategies. In the longer term, an understanding of the molecular basis of disease phenotypes, the function of the gene of expression product, and the prenatal timing and tissue specificity of the Menkes gene will provide a basis for the design of rational

Menkes Disease: From Patients to Gene

28 l

and effective therapy that builds on current therapeutic approaches [42-441. The discovery that the Menkes gene encodes an integral membrane protein which likely functions as a copper-transporting ATPase has opened a new chapter in our understanding of metal transport. This of study of Menkes disease, but is an important stage in the progress the significance of this discovery does not lie only with this disorder. All cells appear to maintain copper balance by sequestration of copper in an inert form bound to specialized proteins, andby the exportingof copper from the cell. The Menkes coppertransporting ATPase is the first such heavy-metal transporting ATPase described in any eukaryoticorganism (including lower eukaryotes such as yeast) and, as such, suggests in defects in trace approaches to identify other transporters involved metal handling. At these Proceedings, e.g., we have seen that this approach has alreadyled to the description of a liver-specific gene encoding a similar cation-transporting ATPase as the basis for Wilson disease[39-411. Future linesof investigation should leadto the identification of other proteins involved in the transport of copper, as well as proteins involved in the transportof other trace metals, such as zinc, cobalt, molybdenum,andselenium. In a broader context, then, work on this rare but paradigmatic genetic disorder will have implications not only for other inherited disorders, but also for an enhanced understanding of nutritional deficiencies affecting large numbers of children. ACKNOWLEDGEMENTS The work by the authors was supported by: a grant from the March of Dimes Birth Defects Foundation: NIH grant Mol-RR01271 to the UCSF Pediatric Clinical Research Center; and NIH grant DK 47192. CV is supported by the UCSF Medical Scientist TrainingProgram (NIH NIGMS grant GM07618) and is jointly advised by JG and SP. JG is an associate investigator of the Howard Hughes Medical Institute.

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E.Hanis, Proc SOCExp Bid Med 196: 130-140 (1991). D. Danks, Disorders of Copper Transport in The Metabolic Basis of Inherited Disease (C. Scriver, A. Beaudet, W. Sly,

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and D. Valle, MS), McGraw Hill: NewYork, 1989, pp 141 1-1432. M. Adamson, B. Reiner, J. Olson, 2. Goodman, L. Plotnick, I. Bernardino, and W. Gahl. Gastroenterology 102:1771-1777(1992). J.H. Menkes, M. Alter, G.K. Steigleder, D.R., Weakley, J.H., Sung. Pediatrics 29: 764-779 (1962). R.O. Barnard, P.V. Best, M. Erdohazi. Child Neurol. 20: 586-597 (1978). H. Uno and S. Arya. Am. J . Med.Genet. 3:367-377 (1987). H.M. Darwish, J.E. Hoke and M.J. Ettinger. J . Biol. Chem. 258: 13621-13626 (1983). T. Tonneson and W. Horn. J . Inher. Metab. Dis. 12: 207214 (1989). S. Packman, C. O'Toole, D. Priceand M. Thaler. J . Inorganic Biochem.19: 203-211(1983). S. Packman and C. OToole. Pediatr. Res. 18: 1282-1286 (1984). S. Packman. Arch. Dermatol. 123: 1545-1547a, 1987. S. Packman, R.D. Palmiter, M. Karin, and C. O'Toole. J . Clin. Invest. 79: 1338-1342 (1987). A. Leone, G.N. Pavlakis, and D.H. Hamer. Cell 40:301309 (1985). S. Packman, S. Sample, and S. Whitney. Ped. Res. 21: 293 (1987). S.M. Herd, J. Camakaris, R. Christofferson, P. Wookey.and D.M. Danks. Biochem. J . 247:341-347 (1987). H. Kodama, I. Okabe, M. Yanagisawa and Y. Kodama. J . Inher. Metab.Dis. 12: 386-389 (1989). H. Kodama. J . Inher. Metab. Dis. 16: 791-799 (1993). G. Johnson, A. Morell, R. Stochert and I. Sternlieb. Hepatol. 1: 243-248 (1981). H.F.Chou, J. Vadgarnaand A. Jonus. Biochem. Med. Metab. Biol. 48: 179-193 (1992). P. Royce, J. Camakaris and D. Danks. Biochem. J . 192: 579-586 (1980). S. Packman, P. Chin, P. and C. OToole. J . Inher. Metab. Dis. 7:168-170(1984). M. Linder, Biochemistry of Copper. New York: Plenum Press (199 1).

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C. Vulpe, B. Levinson, S. Whitney, S. Packman and J. Gitschier. Nature Genet. 3: 7-12 (1993). J. Chelly, Z. Tumer, T. Tonneson, A. Petterson, Y. Ishikawa-Brush, N. Tommerup, N. Horn and A. Monaco. Nature Genet. 3: 14-19 (1993). J.F. Mercer, J. Livingston, B. Hall, J. Paynter, C. Begy, S. Chandrasekharappa, P. Lockhart, A. Grimes, M. Bhave, D. Siemieniak and T. Glover. Nature Genet. 3: 20-25 (1993). T. Tonnesen, A. Petterson, T. Kruse, A.M. Gerdes and N. Horn. Am. J . Hum Genet. 50: 1012-1017 (1992). S. Kapur, J.V. Higgin, K. Delp, K. and B. Rogers. Am. J . Med. Genet. 26: 503-510 (1987). Z.Tumer, N.Tommerup, T. Tonneson, J. Kieuder, I. Craig and N. Horn. Hum. Genet. 88: 668-672 (1992). M.T. Davisson, T.H. Roderick andD.P. Doolittle,in Genetic VariantsAnd StrainsOf The Laboratory Mouse ( 4 s Lyon, M.F. & Searle, A.G.) 432-505 (Oxford University Press, 1990). N. Brockdorff. Genomics 10:17-22(1991). V. Verga, B. Hall, S. Wang, S. Johnson, J. Higgins and T. Glover. Am. J . Hum. Genet. 48: 1133-1138 (1991). Z. Tumer, J. Chelly, N. Tommerup, Y. Ishikawa-Brush, T. Tonneson, A. Monaco and N Horn. Hum. Molec. Genet. 1: 483-489 (1992).

R.G.H. Cotton, N.R. Rodrigues, R.D. Campbell. Proc. Natl. Acad. Sc. USA. 85: 4397-4401 (1988). A.J. Montandon, P.M. Green, F. Giannelliand D.R. Bentley. Nucleic Acids Res. 17: 3347-3358 (1989). S. Das, B. Levinson, S. Whitney, C. Vulpe, S. Packman and J. Gitschier. Am.J . Hum. Genet., in press. S. Silver and M. Waldenberg. Microbiol. Rev. 56: 195-228 (1992).

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l8 Variability in Clinical Expression of an X-Linked Copper Disturbance, Menkes Disease Nina Horn, TenneTennesen,andZeynep Turner Institute, G1. Landevej 7, Glastmp 2600, Denmark

The John F. Kennedy

I. INTRODUCTION

Menkes disease is an X-linked, recessive disturbance of copper metabolism associated with abnormal hair. The disease is dominated by neurological symptoms combined with connective tissue manifestations. Most of the symptoms can be explained by the lack of important copper requiring enzymes, indicating a deficiency of an intracellular copper carrier 111. The gene defective in Menkes disease has recently been isolated and it is predicted to encode a copper transporting P-type ATPase 12-41. Though many patients with Menkes disease present a progressive clinical course with death in early childhood, variable forms of the disease can be distinguished i1.5.8- 10,lOal. In this chapter we discuss the existence of a broad clinical spectrum of Menkes disease similar to that observed in the animal model, the mottled mouse 111-121. 11. CLINICALSPECTRUM IN MENKESDISEASE

During a 20 year pre- and postnataldiagnostic service for Menkes disease, about 428 unrelated Menkes families have been referred to the JohnF. Kennedy Institute. This study is based on extensive clinical records obtained on the affected patients. Furthermore, we have included patients from the literature, who we have been able to identify unambiguously. The classification of the patients is arbitrary and the major 285

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TABLE 1. Clinical Variation in X-linked Copper disturbances Symptoms\MR

Neurologicalsymptoms: Mental retardation Motoric dysfunction Seizures Hypothermia Feeding difficulties Diarrhoea Muscle tone changes Orthostatic hypotension Ataxia Connective tissue symptoms: Tortuous vessels Skeletal changes Occipitalhorns Bladder diverticulae Cutis laxa Loose joints Additionalsymptoms: Distinctivefacies Abnormal hair Hypopigmentation Laboratoryfindings: Serumcopper 4 Serumceruloplasmin 4 Cu-64 incorporation t Age at diagnosis: Present age: Age of death: Copper therapy:

-

*) 4/7

W.

Classical Classical

181

long sum. n=6

++ ++ ++ + + ND

616 616 516 313 314 111 515 ND

ND

ND

+ +

ND

(+l

+

616 ND

+

U4

+

U3

(+l

ND

+ + +

616 414

++ ++ ++

616 515 515

6-8 mo

3d-5y 116 (7Y) 516 (7%-17~) 416

<3 Y many

U2

means 4 abnormal findings out of available information on 7;

+ = yes; - = no; ND = not described

opper

287

Variability in Clinical Expression of Menkes Disease

respond. n=4

Lateonset n=2

OHS n=21 L451

012 1/2 012 012 112 1/1 111 011 U2

+/-

618

114 314 013 313 113 314 1/2 2/3 314

v2 418 113 418 7/14 515

1/2 1/4 314 113 1/2 111

U2 011 112 1/2 U2

+ ++ + ++ + +

9/15 13/14 4/ 10

011 44

012

ND

111

+ + +

Moderate n=lS

15/15 11/11 8/15 4/7* 117 1/2 919

ND

5/13 U9 515 3m0-241tv 15/15 (7M-17~)

2/15

313 313 313 in utero-l mo 414 (7-17%~) 414

m 242

-

-

+ + + ND

+

ND

1/2 (+l 1/2 (+l 2/2 7/7 12-24 mo 18-21 mo U2 (14-l6y) 18/19 (9-5Oy) 1/19 (45Y) U2 0121

Horn et al.

criteria include mental retardation. neurological symptoms, life span, as well as start and effect of parenteral copper therapy. In addition onset of symptoms anda positive familyhistory are taken into consideration. We divided the patients into six groups and three of them have already been proposed: Classical form, late onset mild form and Occipital Horn syndrome [ 1.5.8-9. loa]. Many patients do not belong to these groups, therefore. we suggest the following additional groups: Classical form with long survival. moderate form and copper responding form. The clinical variability is summarized in Table 1.

A. Classical Form In the classical and most common formof Menkes disease the clinical picture is characterized by severe progressive psychomotoric retardation with seizures, markedconnective tissue symptoms and conspicuous hair. Patients often have a characteristic facial appearance with frontal or occipital bossing, micrognatia, and pudgy cheeks 1131. The developmental milestones are usually normal for the flrst couple of months, though hypothermia and subtle hair changes may already be present 1141. From about 2-3 months of age therapy resistant seizures start.They may either be generalized or focal, and are the most frequent cause of hospital admission. Additional initial symptoms are marked hypotonia, vomiting, diarrhoea, and feeding problems. Developmental regression becomes obvious around 5-6 months of age. Pili torti are prevalent, spontaneous movements become limited, and drowsiness and lethargy emerge. At this age Menkes disease is often diagnosed due to the peculiar hair and significantly decreased serum copper and ceruloplasmin levels. In familial casesthe X-linked mode of inheritance supportsthe clinical by accumulation of suspicion. The diagnosis is confirmed radioactive copper in cultured fibroblasts 1151. Connective tissuesymptoms arenumerous,and comprise vascular abnormalities, multiple bladder diverticulae and characteristic skeletal changes, loose joints and cutislaxa. The initial hypotonia is later replaced byspasticity, which ilnally develops into paresis. Late manifestations are blindness, subdural haematomas, and respiratory failure. The patients die

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usually, before they reach 3 years of age due to infections or vascular complications. B. Classical Form with Long Survival

Despite a severe clinical course, a number of classical patients have a much longer life span, whyfore we have grouped these patients separately (Table 1). The group consists of 6 patients. four ofwhich have been reported previously 18.16-251. Seven additional patients with long survival (6- 18 years) are known to us [ 14.2 1.27301, but presently we do not have sumcient information to include them in this survey. All thepatients have severe mentalretardation andthe neurological symptoms can be compared to the classical form with early death. The main criteria is the long life span between 7 and 16 years. In addition delayed onset of seizures were noted in three of the patients. Two patients canonly be distinguished from the classical form because of the long survival. Only one of them received copper therapy for 1%year and died at the age of 12 years 18,221. The other is 7 years old and still alive I3 11. In one patient seizures started at 2.5 years of age, though his first symptoms started at 2 months of age. The correct diagnosis was made at 6 years of age and he died 8.7 years old without any with classical initial copper therapy 1241. In anotherpatient symptoms seizures were first noted at four years of age. A 6 year copper therapy trial had started one year earlier. The patient died 13.5 years old18.17.251. In a thirdpatient mild generalized seizures started rather late, at almost 7 years of age. He was first diagnosed at 9 months of age and copper therapy was instituted until death at 7.5 years I261. Seizures were never observed in a patient receiving copper therapy since 1 month of age until death at almost 17 years of age [ 18-231. A male cousinof his developed seizures 2 months old and died 15 months old despite copper therapy from 4 months of age. The three latter patients were not grouped under copper responders because thecopper therapy has not affected the clinical course, although it probably influenced the survival.

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C. Moderate Form At present 15 patients can be placed in this group, three families comprising a total of 10 affected males and 5 singletons. All but two patients have been reportedpreviously [6,10,32-341. The main feature is a less severe mental retardation in all the patients,and at leastthree of them areable to some verbal communication [10,341.Theneurological symptomsarenot as severe as in classical Menkes patients though hypotonia and ataxia are prevalent in this group. We know that 13 patients are unable to walk, and we do not have specific information about the remaining two patients [341. 7 patients never had any seizures of age, [6,8,10,32-331, and one had seizures between 2 and 4 years which remittedspontaneouslywithouttherapy [61. Onlytwo patients have received parenteral copper, but the details of the therapy is not know to us. All the patients in this group are alive, the oldest being36 years of age 1351. Skeletal involvement does not appear to be a very prominent feature in this group. However, in an American family l341 with four affected members, bony occipital exostoses were apparent on skull radiograms of the 15 years old patient. In the two affected adults multiple joint abnormalities are present and may be the reason for their inability to walk. Pili tortiarefoundin all patients except fortwo English patients I321 and the four affected members of the American family 1341. In the latter family, fine and sparse hairwas noted in infants and the older patients had coarse hair. Distinct facies were noted in four Mexican patients 161. and in all affected members of the Americanfamily[341.The oldest Mexican patient [61 has the same fragile-X like facial appearance as the oldest patient in the English family. Serum copper was significantly reduced in all but three, at the only marginally time of diagnosis. These threepatientshad reduced serum copper levels. In an untreated patient the value increased spontaneously [81. D. Copper Responding Form

This is indeed not a true clinical subgroup of Menkes disease. In three of the patients itis obvious that the clinical course has been

Variability in Clinical ExpressionMenkes of

Disease

29 1

modified by copper therapy. In the fourth patient it is impossible to predict the clinical course without copper therapy. All four patients in this grouphave been reported previously [ 1.24.36-401. Two of thepatients (7.5 and 17.5 years old) are mentally normal, and attendschool in age matched grades 138-401. A third patient wasdiagnosed prenatally and received copper therapy since day oneafter induced delivery at 35 weeks of gestation. He is now, at 7 years of age, one year behind his peers intellectually 11,371. The fourth patient is 15 years old and attendsa special school, but hecanreadand write. His motoric problems combined with dysarthria has made the intellectual scoring dimcult (411. None of the patients have had seizures. In general, the 7.5 years old patient has normal motor development, except that we think he has a slightly clumsy gait. In the7 years old patient motoric developmentwas grossly delayed, and ataxia could be observed (301. The 15years old patient needed crutches for a period, but is now walking without support [411. The 17.5 years old patient can walk, but dueto general hypotonia combined with skeletal deformities he is essentially wheelchair bound. Ataxia is a substantial problem in three of the cases, and orthostatic hypotension has been observed in two. The 7 and 17.5 years old patients have developed skeletal deformities similar to the OccipitalHorn syndrome including occipital exostoses 130,401. I n the 15 years old patient occipital horns were palpable, but not yet confirmed by X-rays (411. It is noteworthy that oneof the patients wasonly 7 years old, when the horns were noticed. X-ray examination has not yet revealed occipital horns in the 7.5 years old patient. All the four patients were diagnosed within the first month of life, and copper therapy was startedwithin the first 3 months. The 15 years old patient 1361 had an affected maternal uncle. He was never treated with copper andhe is included in thegroup of classical form with long survival. Both the 7 and the 17.5 years old patients 137-391. had an untreated sibling who died of the classical form of Menkes disease. E. Late Onset Mild Form

In two Menkes patients the diagnosis was established first at 18 and21monthsdue to abnormal hair and delayed motor

Horn et al. development (Table 1) 15.8-9.441. This is remarkable as the diagnosuc symptoms are usually apparent before 6 months of age. We have categorized these two patients as late onset mild form. They are now 16 and 17 years old and have only slight mental retardation, if any, although they are both one year behind in school. The 16 years old American patient 18-91 has only minor motoric problems with a slightly unsteady gait. The 17 years old Australian patient has severe motoric problems with poor coordination. His ataxia is very pronounced, and he had to walk with arm sticks until about 7 years of age 1441. Neither of them have seizures, nor major skeletal changes. Recently occipital horns have been observed in the American patient 144al. In the Australian patient occipital horns were not palpable at 14 years of age 1301,but a follow-up is planned now at 17 years of age 1301. In the American patient 191. the serumcopper level was normal until he was 7 years old, whereafter it decreased significantly. At this time copper therapy was initiated. The other patient had a marginally decreased serum copper level at diagnosis, and copper therapy was started at 2 years of age. At diagnosis the hair of the American patient was described as coarse, and microscopy showed pilitorti and mild monilethrix. The Australian patient had lustreless light brown hair and pili torti were observed in about half of the hairs examined. F. Occipital Horn Syndrome (OHS)

The mildest form of Menkes disease is the Occipital Horn syndrome, originally described by Lazoff et al. 1421. At present we have knowledge about 14 families comprising 22 affected males 1451. This includes a 34 years old Japanese patientwhowas recently reported byWakai et al. 146-471. Because of severe mental retardation this patient might equally well be placed in the group of moderate Menkes. He had seizures from early childhood and he is unable to speak or stand without support. This patient patients, whom we have limited and two other Japanese information about 148-491.are not included in Table 1, The Occipital Hornsyndrome patients aremainly characterized by connective tissue symptoms. They have only slight mental

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retardation, if any, and few but significant neurological symptoms like orthostatic hypotension, and diarrhoea. Numerousskeletalabnormalitiesarepresentincluding the occipital horns, which become prominent around puberty. However, occipital exostoses have been noticed in a six years old patient, and re-examination of earlier skull X-rays revealed that horn-like protuberances were already present at 2 years of age 1431. Genu valgum is frequently observed giving thepatients a characteristic knocked-kneed gait. In onepatienttheskeletal dysplasia is so pronounced, that he is using a wheelchair [501. Other connective tissue symptoms are bladder diverticulae. joint laxity and cutis laxa. The facial appearance is often distinctive. Unusual features include long thin face, high forehead, hooked nose, long philtrum, and prominent large ears. The hair is usually not conspicuous, is described as coarse [10a,431. Serum but in some patients it copper and ceruloplasmin levels are low in most patients. but a few have normal or borderline normal values 1511. All patients except four are still alive, the oldest living patient being 50.5 years old. Two Occipital Horn syndrome patients died in car accidents [45],one died in his forties of unknown cause [45]. and one died 49 years old because of ruptured bladder [52]. 111. DISCUSSION

The clinical spectrum of Menkes disease is indeed broad (Table 1). Previously Occipital Horn syndrome and the late onset mild form were distinguished as variants of Menkes disease. Now the existence of a number of patients with a moderate clinical course is evident. Within this group some variability also exists, and it is conceivable that Menkes disease covers a clinical continuum from the severe classicalform to the very mild Occipital Horn syndrome. This is in analogy with the mottled mouse model. where at least 15 different alleles are known [ 11,121. In the mottledmousethemost severe variants are only heterozygotes, observed in the phenotypically affectedfemale because these forms are male lethal in utero. This might be the case in Menkes disease a s well, and it is probable that some mutations in the Menkes gene will be expressed in females only. X-linked condition. This is similar tothe situation seen in another

’

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Ela deficiency of the pyruvate dehydrogenase complex [531. A French Menkesgirl withnormal chromosomes and with no affected male relative could be an example of such a variant 154-551. 1.

Mental Retardation

Mental retardation between and within the groups varies considerably. It spans from the decerebrated stage in the classical form, and classical form with long survival, until the normal, or almost normal, intellect of the mild forms, good copper responders. late onsetmild form, and Occipital Horn syndrome patlents. In the moderate form, the patientsare able to interact with their a few are capable of some verbal surroundings, and communication. In some milder Menkes patients the intellectual development has been dimcult to assess dueto motoricproblems combined with dysarthria. 2. Seizures The therapy resistant seizures are prevalent in the classical form, and in the classical form with long survival. The seizures normally start in early infancy.In the group of classical formwith long survival onset was delayed in three cases. One patient from this group does not have seizures and copper therapy might have been a contributing factor. In the moderate form about half of the patients never had seizures. In four patients from this group the seizures started late [32,341 and remitted spontaneously without copper therapy in one of them. Seizures have not been reported in the copper responding form, the late onset mild form or in the Occipital Horn syndrome. 3. MotoricFunction

The motoric dysfunction also covers a wide span in severity. The patients with the classical form, and classical form with long survival are bedridden. The patients with the moderate form are able to sit in a chair. Within the group of copper responding

Variability in Clinical Expression

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patients, motoric functions vary from normal to moderate dysfunction. In the two patients with the late onsetmild form the motoric abilities are opposing. The Australian patient has pronounced motoric problems while the American patient has minor problems like the Occipital Horn syndrome patients. Two researchers (David Danks and Ilkka Kaitila) have suggested, that this patient looked rather like a severe Occipital Horn syndrome patient. Most Occipital Horn syndrome patients have only slight problems with motor co-ordination. Ataxia and orthostatic hypotension are essential problems in Menkes disease. In Occipital Horn syndrome patients ataxia is not present. On the other hand in the classical form. ataxia or orthostatic hypotension are notrecorded because the patients are too retarded to stand or walk. 4. OccfpftalHorns

Previously the occipital horns have been considered pathognomonic for the Occipital Horn syndrome. But these ossifications are now reported in several patients from the intermediate groups as well. Occipital exostoses, therefore, cannot be considered as a diagnostic marker for the Occipital Horn syndrome alone. Occipital horns areusually observed around puberty, but may be present much earlier. For example horn-like protuberances were noted in retrospect in a two years old OccipitalHorn syndrome patient, who had clearly noticeable horns at 6 years of age. In a boy with the copper responding form, occipital exostoses were found already at 7 years of age. It is still an open questionwhether untreated classical Menkes patients with long survival will develop occipital horns. 5. Dfstfnctlue Fades

Classical Menkes patients often have peculiar facies. With age the features become morepronounced and it seems that patients, living long enough, develop the distinctive facial appearance characteristic of the Occipital Hornsyndrome I6,8,14,17,25,32,341.

Horn et al. 6. Hair Abnormaftty

The peculiar hair is a valuable diagnostic marker and when seen in a male patient the diagnosis of Menkes disease should always be taken into consideration.The microscopic changes like pili torti areseenin all patients with theclassical form andin some patients with theintermediate forms. In the Occipital Horn syndrome microscopic changes are not described. 7. Copper 7'hempy

Parenteral copper therapy has been used with variable success. It is evident that it has influenced the clinical course in some cases. The majority of patients do not benefit from the therapy though a positive effect on seizures and survival are observed in some cases. However, many parents feel that copper therapy relieves the child's pain. We have grouped some patients as a copper responding form, because the effect of copper therapy is indeed striking. This is substantiated by a family history of classical Menkes disease in three of the cases. However. copper therapy cannot explain the milder clinical phenotype in patientswith the moderateform of the disease since only 2 of them have received any. The effect of copper therapy in the late onsetmild form cannot be predicted because we do not know the natural course of the disease. It is conceivable that some patients with the moderate form, and Occipital Horn syndrome would benefit from copper therapy, although theeffect on the connective tissue disturbances has not proven very promising. 8. Survival

Menkes patients with the classical form usually diebefore 3 years of age. Unusual long survival was seen in some patients with classical symptoms, and this could not be explained by copper therapy. Therefore we have grouped these patients separately. Good parentalcare could be a contributing factor to thelong survival in this group.

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In the intermediate forms the life span increases in parallel to the decrease in the severity of symptoms and the mildestform, the Occipital Horn syndrome, has an almost normal life span. I n the copper responding form the long survival can be explained by the successful copper therapy. IV. COPPER IN MENKES DISEASE

In Menkes disease the tissue distribution of copper is abnormal. In blood, copper and ceruloplasmin levels are significantly decreased in the classical form. In the milder forms the values maybewithinnormal or low normalrange. Therefore, in the milder forms these two parameters cannot be used to exclude the diagnosis of Menkes disease. It is conceivable that some of the families with Ehlers-Danlos type V 1561. which is also X-linked, belong in the Menkes group. although they have normal serum copper and ceruloplasmin levels. Incell culturethedisturbance of copper metabolism is characterized by increased copper accumulation [57-581 providing a usefuldiagnosticmarker for bothpostnatalandprenatal accumulation and retention cultured in diagnosis I151. fibroblasts is significantly increased in all the groups withdifferent clinical expression of the disease (Fig.1). It is thus by far the best diagnostic marker forMenkes disease, but the different groups cannot be distinguished on the basis of this test. V. THE MENKES GENE AND MUTATIONANALYSES

The Menkes gene has been cloned 1.5 years ago,by three different groupsincluding ours [2-41. The predicted protein is a P-type ATPase, with 6 putative metal binding sites at the N-terminus. I t is expressed in alltissues,though lower inthe liver. These findings are in accordance with themultisystemicintracellular copper transporting defect in Menkes disease. Following the isolation of the gene we started analyzing 230 unrelated Menkes patients for micromutations. Until now wehave found rearrangements in 20% of the patients, where 75% were deletions 12,591. One importantresult of this study is that deletions are of different sizes and locations, indicating that each

298

X

X

0

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of Menkes Disease

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family has a unique mutation. At the Toronto Meeting, Packman et al., reported a distinct point mutation in each of 12 unrelated patients [SO]. These findings may be used to explain the clinical variability observed among Menkes patients. The DNA analysis of all the patients with classic or other forms are not accomplished yet. However, except for the copper responding group,we found at least one patientin each subgroup, where the Menkes gene was affected 1591. This strongly supports thatthesegroupsare allelic to theclassical form of Menkes disease. The mottled gene, the mouse homolog of the Menkes gene, has recently been cloned using Menkes gene sequences [61-621. The predicted protein is a metal transporting P-type ATPase showing 80% sequence homology to its human equivalent. Analyses of the underlying mutations inmore than 15 alleles of the mottled mouse will help in understanding the broadclinical spectrum of Menkes disease. Two groups 161-621 have already found that the mottled gene was defective in Modpand Mob". two of the most severe and mildest alleles of the mottled mouse, respectively. Investigation of the defect at the protein level will give clues to how the copper dependent enzymes are affected in each subgroup and possibly the pathogenesis. Little is known about the activity of different copper dependent enzymes inthe milder forms of Menkes disease. The elucidation of the mutations in eachindividual will hence help understanding the clinical effects of the underlying genetic impairment. ACKNOWLEDGEMENT We wish to express our gratitudeto colleagues at different genetic centres, who havegenerouslysupplied us with the clinical information. Without their contributionthis survey would not have been possible. REFERENCES 1.

D.M. Danks.Disorders of copper transport, in The metabolic basis of inherited dfsease. 6th ed.,(C.R.

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2.

3.

4.

5. 6. 7.

8.

9. 10.

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11. 12. 13. 14.

Scriver, A.L. Beaudet, W.S. Sly and D. Valle. Eds.), McGraw-Hill. New York, 1989, pp. 1251-1268. J. Chelly, 2. Tumer, T. Tgnnesen, A. Petterson, Y. Ishikawa-Brush, N. Tommerup, N. Horn and A.P. Monaco, Nature Genet. 3: 14-19 (1993). C. Vulpe. B. Levinson, S. Whitney, S. Packman and J. Gitschier, Nature Genet 3: 7-12 (1993). J.F.B. Mercer. J. Livingston, B. Hall, J.A. Paynter, C. Begy,S. Chandrasekharappa. P. Lockhart. A.Grimes, M. Bhave, D. Siemieniak and T. Glover, Nature Genet. 3: 20-25 (1993). P. Procopis. J. Camakarisand D.M. Danks, J. PedIatr 98:97-99 (1981). R.H. Haas, A. Robinson, K. Evans, P.T. Lascelles and V. Dubowitz, Neurology 31: 852-859 (1981). K. Baerlocher and D. Nadal. Ergeb. Inn. Med. Kfnder heflk. 57: 79-144 (1988). A-M. Gerdes, T. Tgnnesen. E. Pergament. C. Sander, K.E. Baerlocher. R. Wartha, F. Guttler and N. Horn, Eur. J. Pedfat. 148: 132-135 (1987). J.A. Westman. D.C. Richardson, O.M. Rennert and G. Morrow, Am. J. Med. Genet. 30: 853-858 (1988). T. Tplnnesen, C. Garrett and A-M. Gerdes. J. Med. Genet. 28: 615-618 (1991). 1.1. Kaitila, L. Peltonen, H. Kuivaniemi, A. Palotie, J. El0 and K.I.Kivirikko. A Skeletal and Connective Tissue DisorderAssociatedwith Lysyl Oxidase in Deficiency and AbnormalCopperMetabolism, Skeletal Dysplasias. Progress in Clinical and Biological Research, Vol. 104 (C.J. Papadatos and C.S. Bartsocas, EDS.). Alan R. Liss, Inc., New York, 1982. pp. 307-315. D.M. Hunt. Nature 249: 850-854 (1974). M.T. Davisson. Cenornfcs 1: 213-227 (1987). N. Horn, T. Tgnnesen and Z. Tiimer, Brain Pathology 2: 35 1-362 (1992). W.R. Collie, T.J. Goka, C.M. Moore and R.R. Howell, Hair in Menkes disease: a comprehensive review, in Hair, trace elements and humanillness (A.C. Brown and R.G. Crounce, Eds.), Prayers Scientific, New York. 1980. DD. 197-209.

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S ~ p p l1: 207-214 (1989). A. Nussbaumer, E. Werder. H. Weber and K. Baerlocher, Helo. Paed. Acta 29, suppl. 32: 25-26 (1974).

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H. Wehinger, I. Witt, I. Msel, G. Denz-Seibert and C. Sander, Lancet i: 1143-1144 (19751. W.D. Grover and M.C. Scrutton, Pediatrics 86: 216-

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A.S. Dekaban. Materia Medica Polona

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220 (1975).

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R.I. Henkin and W.D. Grover, Tricholiodystrophy (TPD).New Aspects of Pathology and Treatment, in Trace ElementMetabolism fnMan and FreisinsAnimals, (M.Kirschgessner.Ed.), Weihenstephan, Germany, 1978, pp. 405-408. W.J. Dalyand H. Rabinovitch, J. Ilrol. 126: 262-264 (1981).

24.

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62 1-625 (1988).

26. 27. 28. 29. 30. 31. 32. 33.

34.

33: 228-233 (1988). A. Garnica, personal communication

W.D. Grover, personal communication. S.I. Goodman, personal communication. G. Romeo, personal communication. D.M. Danks, personal communication. G. AnnerCn, personal communication. M. Alvarez de Santos and P.M. Guerra. Rev. Invest. CZh. (M&) 36: 151-154 (1984). M. Inagaki, K. Hashimoto, K. Yoshino. K. Ohtani, I. Nonaka. M. Arima. M. Kobayashi and N. Sugiayama. NeuropedfuMcs 19: 52-55 (1988). V.K. Proud, M. Balan. S.G. Kaler and A.K. Percy, Family Study of Three Generations with a Variant of

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Menkes Syndrome and Features of X-linked Cutis Laxa (Occipital Horn Syndrome),Pedfatr. Res.33 (4) Part 2: Abstract 74A.p. 426 (1993). S.G. Kaler, L.K. Gallo, V.K. Proud, A.K. Percy, C.S. Holmes, D.S. Goldstein and WA. Gahl, A to T Transversion at the +3 Position of a Splice Donor Gene Associated with a Site in the Menkes Distinctive Phenotype. VI InternationalCongress InbornErrors of Metabolism, Milano, Abstract W10.1, p. 93, 1994. K.E. Baerlocher, B. Steinmann, V.H. Rao. R. Gitzelmann and N. Horn, J. Inher. Metab. D f s . 6 S ~ p p l 287-88 . (1983). D.M. Danks.Treatment from Birthconverts Menkes Disease into Occipital Horn Syndrome, VI International Congress Inborn Errors of Metabolism, Milano, Abstract W10.4. p. 95. 1994. G. Sherwood, B. Sarkar and A. Saas-Kortsak, J. Inher. Metab. D f s . 12 Suppl. 2: 393-396 (1989). B. Sarkar. K. Lingertat-Walsh and J.T.R. Clarke, J. Pedfat. 123: 828-830 (1993). J. Christodoulou, B. Sarkarand J.T.R. Clarke, Evolution of Classical Menkes Disease (MD) to an Occipital Horn Syndrome (OHS) Phenotype with VI International Copper-Histidinate Therapy, CongressInbornErrors of Metabolism,Milano, Abstract W10.3. p.94. 1994. K. Baerlocher. personal communication. S.G. Lazoff, J.J. Rybak. B.R. Parker and L. Luzatti, Bfrth Defects 11: 71-74 (1975). T.E. Herman, W.H. McAlister. A. Boniface and M.P. Whyte, Pedfatr. Radfol. 22: 363-365 (1992). D.M. Danks, Am. J. Med. Genet. 30: 859-864 (1988). S.G. Kaler, personal communication.

I. Kaitila. personal communication. S. Wakai, Y. Ishikawa. M. Nagaoka. R. Minami and T. Hayakawa, Rfnsyo Sfnkefgaku 31: 534-537 (1991).

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R.D. Blackston, personal communication. M. Tsukahara, K. Imaizumi. S. Kawai and T. Kajii. C l h . Genet. 45: 32-35 (1994). D. Weaver, personal communication. H.-H.M. Dahl. L.L. Hansen. R.M. Brown, D.M. Danks, J.G. Rogers and G.K. Brown, J. Inher. Metab. Dfs. 15: 835-847 (1992). A. Favier. C. Boujet and A. Joannard, J. Inher. Metab. Dls. 6 Suppl. 2:89 (1983). A.". Gerdes. T. Tmnnesen, N. Horn, T. Grisar, W. Marg. A. Muller, R. Reinsch. N. W. Baron, P. Guiraud, A. Joannard, M.J. Richard and F. Guttler, C h . Genet. 38: 452-459 (1990). P. Beighton,The Ehlers-Danlos Syndromes, in McKusick's Heritable Disorders of Connective Tissue, 5th ed. (P. Beighton. Ed.) Mosby. St. Louis. 1993. p. 223. N. Horn, Lancet I: 1156-1 158 (1976). T.J. Goka. R.E. Stevenson. P.M. Hefferan and R.R.Howel1, Proc. Nut[.Acad. ScI. USA 73: 604-606

52. 53.

54. 55.

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(1976). 59. 60.

61.

2. Tiimer. unpublished results. S. Packman et al:, Oral presentation, First International Symposium on Metals and Genetics, Toronto, 24-27 May, 1994. B. Levinson, C. Vulpe. B. Elder, C. Martin, F. Verley, S. Packman and J. Gitchier, Nature Genet. 6: 369-373 (1994).

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19 Development of Copper-Histidine Treatment for Menkes Disease Bibudhendra Sarkar Biochemistry Research, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1x8, Canada

I.

INTRODUCTION

Menkes diseaseis an X-linked genetic disorderof copper'transport which causes progressive cerebral degeneration, convulsions, growth retardation, skeletal changes and gross abnormality of elastic fibres in the arterial wall (1). Death due to severe progressive neurodegeneration almost invariably occursby the age of three (2). Danks and coworkers (3) reported the first evidence of copper deficiency in this disease. The low levels of serum copper and ceruloplasmin have been found to be the universal characteristics of the disease. There is a widespread disorder of copper transport. Because of the association of copper deficiency with hypomyelination, maldevelopment of the central nervous system may be implicated in human copper deficiency. Recently the candidate gene for Menkes disease hasbeew reported independentlyby three groups (4-6). The genecodes for a 1500 amino acid protein predictedto be a P-type cation transporting ATPase. The gene product is very similar to a bacterial coppertransporting ATPase. Additionally, it contains six putative metal binding motifs at the N-terminus. Menkes disease fibroblasts in 305

culture accumulate significant amounts of copper but the exit of copper from these cells is greatly reduced ('7, 8). Copper is mostly bound to metallothionein in Menkes disease ~broblasts. levels of copper are found in liver and brain where the activities of cop~r-dependentenzymes are decreased.

~op~r-histidine was first detected in human serum in our laboratQ~ in 1966 (9). The isolated copper-histidine complex from human serum showed a 1:2 copper : histidine stoichiometry. These results were c o n f i ~ e dby amino acid recon~titutionex~erimentsutilizin~ dialyzed and undialyzed sera (10). Besides copper-histidine, several ternary amino acid complexes of copper having one of the amino acids as histidine were also detected (9, 11).

n equilibrium analysis of copper-histidine showed the presence of one major complex species with a 1 2 s t o i c ~ o m eatt ~physiological (12). The log stability constant of this species was found to be 453. The structure of this species was elucidated by visible and spectra and proton displacement analyses (13). Th s~ructuresof the copper-histidine species at phy siolo shown in Figurel. The structure of the species was

6 Proposed structures of the physiologically impo~ant1:2 cop~r-histi~ine complex (33).

involve 0 (carboxyl), N (imidazole) and N (amino) in both histi~ines (carboxyl), N (imidazole) and N (amino) in one histi~inean^ no), N ( i ~ d a ~ o line the ) other or an equilibrium stru~tures.

er

e

The major copper transport protein in human serum is albumin. was found that when copper-histidine is allowed to react with human albumin, a ternary complex species albumin-copper-histidine is formed (14, 15). ~e proposed that the ternary complex may play a important role in the transfer of copper between histidine an n order to elucidate the mechanism of this transfer it was necessary to solve the structure of the copper-transport site of human . The structure shows copper bin to albumin at the minus of the protein involving the 2, two intervening peptide nitrogens, an imidazole ni istidine residue in the third position and the carboxyl s (16). A, synthetic peptide: representing the native copper-trans used to carry out kinetic analysis of the exchange of copper bet wee^ ine and albumin. The results revealed that the ternary lexes formed by protonation and deprotonation are both ically and thermodyn a ~ c a l l yi ~ p o ~intermediates n t in copper exchange reactions between albumin and histidine (17, 18).

e The initial studies of tissue uptake of copper influenced by amino acids were carried out utilizing rat liver slices (19, 20). facilitation of copper transport by amino acids was demonstrated wide range of copper concentrations used in these experiments. Since copper-histidine is the main copper-amino acid complex in the plasma, it was evident that histidine caused the enhanced upta copper in the tissues. Subse~uentlyseveral other labora showed histidine to transpo~copper across cellular membranes of hepatocytes, ~ ~ r o b l a s thypothala~ic s, tissues etc. whereas albumin inhibited the copper uptake (21-24). ore recently histidine has been shown to enhance the uptake of copper in human placental cells in the presence of serum, a phenomenon which is considered to be due to the release of copper that is bound to albumin demonstrating further the importance of the albu~n-copper-histidineternary complex (25). It has also been shown that histidine facilitates copper ~ p t a in ~ mam~alian e brain tissues as well (26).

Parenteral therapy of Menkes disease with various copper salts commenced either before or after neurological i m p a i ~ e nhas t never convincingly showed significant effect on the devastating

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neurodegenerative progressionof the disease (27-30). Cumulative results of our studies of copper-histidine and the mechanism of copper-transport led us to use copper-histidine in the treatmentof Menkes disease since 1976 (11,31-34).

A.

Copper-histidineformulation for subcutaneous administration

Copper-histidine solutionis prepared using full aseptic technique ina laminar air flow hood under conditions that minimize damage from light. Volumetric flasks and beakers to be used are covered in foil. The light in the laminar air flow hood must be turned off during copper-histidine preparation: Anhydrous CuC12 (0.1060g) and Lhistidine (0.24448) are weighed out. Copper-chloride is hygroscopic and should be weighed out quickly and the bottle returned to the desiccator immediately. Copper-chloride and Lhistidine powder are placed together in a beaker and dissolved in approximately 90 mL sterile 0.9% sodium chloride for injection (USP). Vigorous stirring shouldbeavoided as the product is sensitive to oxygen. A solutionof 0.2N sodium hydroxidein sterile sodium chloride 0.9% injection is used to adjust the pH of the solution to pH 7.38-7.40. The sodium hydroxide solution for pH adjustment is prepared as follows: sodium hydroxide pellets (400mg) are placed in a 1OOmL sterile, pyrogen free beaker in a laminar flow hood, dissolved in 30mL sterile sodium chloride 0.9% injection USP, transferredto a 5OmL volumetic flask, and made up to 5OmLwith sodiumchloride 0.9% injection USP. After adjustment of the pH of the copper-histidine solution,the final volume is made up to lOOmLwith sodium chloride 0.9% injection USP. The copper-histidine solution is drawn up into a sterile, disposable, plastic 20mL syringe and the desired aliquot (5 or IOmL) filtered through a 0.22 micron filter into sterile glass vials. Tests for sterility, copper and pyrogens are performed on each batch. Samples are stored refrigeratedin brown, UV-light resistant bags. An expiry of 56 days is assigned based on our stability studies. Copper-histidine is highly sensitive to light, oxygen and temperature. We have used copper-histidine in solution form, however, some institutionsmay opt for a freeze-dried form of copper-histidine. It should be noted that copper-histidineis a relatively unstable inorganic complex and freeze-drying is a very stressful process in itself. Also, addition of any potentially reactive excipients such as mannitol or other carbohydrates during the freeze-drying process must be avoided. For example, copper forms stable complexes with mannitol ( 35) which will adversely affect the copper-histidine equilibrium.

Copper-Histidine Treatment for Menkes Disease

309

B. Treatment of MenkesPatients Between 1976 and 1994, Menkes disease has been diagnosed in seven patients at The Hospital for Sick Children, Torontoand all have been treated with daily injections of copper-histidine in an effort to ameliorate their disease. Clinical information on these patients is summarized in Table 1. In every case, except one, the diagnosis was based on the presence Table 1

Clinicaloutcome of patientswithMenkesdiseasetreatedwith copper-histidine at the Hospital for Sick Children, Toronto, during 1976 to 1993 (33)

Case No.

Date of birtth Nov.12, 1976 Oct. 4, 1982 May16,1984 Sept. 29, 1986 March 17, 1988 March 9,1990 Aug. 27, 1991

Age at diagnosis Outcome 2 wk* Nearly normal

IQ 7 mo Lost follow-up to 4 m0 Died mo 14 at 1 mot Nearly normal IQ 6 m0Died at 1 112 yr of age Died at 22 mo of age 4 m0 2 m0 Profound mental retardation

*Born at 37 weeks of gestation, corrected age 39 weeks. tBorn at 35 weeks of gestation, corrected age 39 weeks.

of characteristic clinical findingsand markedly depressed plasma copper and ceruloplasmin levels. Patient 1 was born 3 1/2 years after the death of a sibling who had died with severe neurodegeneration associated with confirmed classic Menkes disease. Patient 1 was the first patient treated with injections of copper-histidine in 1976. At age 18, he has been receiving daily subcutaneous injections of copper-histidine. The case was reported in detail when he was 12 years of age (32 ). His copper and ceruloplasmin levels are shownin Figure 2. At age 18, clinically, the patient is intellectually within the normal range (WISCR, Full-scale IQ, 87) but has extensive hyperextendabilityof joints and generalized muscle weakness. However, he has never had a seizure and enjoys relatively goodgeneral health.

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Copper-Histidine Treatment Menkes for Disease

31 1

Patients 2 to 7: On the basisof our initial experience with patient

1, all the subsequent patients with Menkes disease were treated from the time of diagnosis with once daily subcutaneous injections of copper-histidine at a dosageof 50 to 15Op.g elemental copper per Kg per day.

C. Biochemical outcome In all cases plasma copper and ceruloplasmin levels returned to the normal range within 2-3 weeks of the commencement of copperhistidine injections. With the exception of our first patient the levels have remained in the normal range, on the same dose of copperhistidinethroughoutthecourse of theirdisease.Regular measurements of bilirubin, alkaline phosphatase, AST, and ALT have consistently been within normal limits. Periodic measurements of BUN and plasma creatinineand urinalysis have consistently been normal.

D. Clinical The patients fall intotwo groups on the basisof the clinical courseof their disease. Two patients have done well neurologically, though both have had major non-neurological problems attributable to Menkes disease. Both are intellectuallynormal, and neither has ever had seizures. Importantly, in both cases, the diagnosis of Menkes diseasewasmadeandcopper-histidinetherapybegunearly. However, the other five patients have done poorly who were diagnosed at 2-7 months. All five have failed to thrive and showed neurological deterioration progressivelywhich did not appear tobe influenced significantlyby the copper-histidine therapy. All had or developed intractible seizureswhich were resistant to conventional anticonvulsant therapy.

IV.

DISCUSSION

The detection of copper-histidine in normal serum (9) formed the basis of treating our first patientwith copper-histidine (33). Copperhistidine therapy appears to be effective in preventing the severe neurodegenerative problems in patientswith Menkes diseasewhen the treatmentis initiated very early in life. Two patients who have done well were born prior to 40 weeks gestation and the diagnosis was madeand treatment initiated before one month of age. Our first patient was born 3 1/2 years after the death of a sibling who died of confirmed classic Menkes disease. The treatment with copper-histidine not only raised the serum copper and ceruloplasim tonormal levels but also resulted in normal levels

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Sarkar

of various brain metabolites. We believethat this patient likelyhad a severe formof Menkes disease evidenced by the rapidly progressive course and deathof his sibling. We can attribute his relatively mild clinical courseto the effectivenessof copper-histidine therapy. Many of the clinical symptomsof Menkes disease are attributed to the lackof copper availability for copper dependent enzymes such as lysyl and amine oxidase, tyrosinase, dopamine-p-hydroxylase and cytochrome C oxidase. There are several reports giving further evidence for the efficiency of copper-histidine therapy (36-37). Copper-histidine treatment resulted in normal copper values in the cerebrospinal fluid suggesting that copper is transported into the central nervous system facilitated by histidine (36). Although there is no documented case of spontaneous regression in neurologic symptoms of Menkes disease, a recent report demonstrated that copper-histidine therapy caused a regressionin neurologic deficits along with improved activities of copper enzymesin the brain (37). It should be pointed out that one mustbe aware of the complex nature of the solution chemistry of copper-histidine which can lead to poorresults if notcontrolledproperly.Thequestion of bioavailability of copper-histidine has tobe addressed. This can be potentially critical determinant of a successful clinical outcome. Minor variationsin the formulation andhandling of copper-histidine preparation for parenteral administration may cause a significant decrease in the bioavailability of copper-histidine which may jeopardize the clinical outcome of the treatment. The solution of copper-histidine ishighly sensitive to light,oxygen and temperature. One must avoid additionsof any potentially reactive excipients such in thecopper-histidine asmannitolorothercarbohydrates preparation since mannitol forms strong complexeswith copper (35) whichwillinterferewithcopper-histidineequilibriumandits bioavailability. Menkes diseaseis characterized by impaired intestinal absorption of copper and abnormal copper concentrations in multiple organs (38-40). Although the copper deficiencyis implicated in the rapidly progressive neurologic deterioration andearly death of the affected individuals, copper therapyby the use of inorganic copper salts has not been found to alter favorably the clinical courseof the disease (27-30). The inefficiency of this form of treatment may be related to the failure of copper utilization when administered in this form. Albumin binds copper avidlyand any form of inorganic copper salt readily attaches to albumin. Albumin has been shown to inhibit copper transport in cells. However, copper-histidine appears to maintain a sufficient amount of copper-histidine in equilibrium to cross theblood-brain barrier (41) throughthe formation of a ternary complex: albumin-copper-histidine. Menkesdiseaseisacomplexheterogeneousdisease. Any genetic variability may influence the clinical response to copper-

Copper-Histidine Treatment for Menkes Disease

313

histidine treatment. Once the gene mutations are delineated and the resultinggeneproductsarecharacterizeditwillallowbetter of understanding of the disease process and perhaps prediction clinicaloutcomeofcopper-histidinetreatmentwithspecific mutations. From our experience, copper-histidine therapy when started very early in life, appears to prevent at least some of the major complicationsof Menkes disease. Thus serious consideration should be given to initiate treatment with copper-histidine as early as possible.

ACKNOWLEDGEMENTS The research was supported by grants from the Medical Research Council of Canada and the Lunenfeld Foundation.

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30. A.D.Garnica, Eur.J.Pediatr., 142:98-102(1984). 3 1. B.Sarkar in Elements in Health and Disease (Eds., R.B.Arora, S.B.Vohora and M.S.Y.Khan) Institutes of History of Medicine and Medical Research, New Delhi pp.27-41 (1984). 32. G.Sherwood, B.Sarkar and A.Sass-Kortsak J.Inher.Metalb.Dis. 2:393-396 (1989). 33. B.Sarkar, K.Lingertat-Walsh and J.T.R.Clarke,J-Pediatr., 123~828-830-(1993). 34. B.Sarkar, J.Pediatr. 125337-338 (1994). 35. J.Briggs, P.Finch, M.C.Matulewicz and H.Weige1, Carbohydr.Res. 97:181-188 (1981). 36. P.R. Kollros, R.D.Dick and GJ.Grewer, Prdiatr.NeuroZ. 7~305-307(1991). 37. J.Kreuder, A.Otten, H.Fuder, Z.Tumer, T.Tonnesen, N.Horn and D.Dralle, Eur.J.Pediatr. 152:828-830 (1993). 3 8. N.Horn, KHeydorn, E.Damsgaard, I.Tygsturp., S.Vestermark, CZinGenet. 14: 186-187 (1978). 39. J.L.Nooigen, C.J.DeGroot, C.J.A.van den Hamer, L.A.H.Monnens, J.Willemse and M.F.NiermeijerPediatr.Res. 15~284-289(1981). 40. D.L.Williams and C.L.Alkin, Arn.J.Dis.ChiZd 135375-376 (1981). 41. B.M.Katz and A.Barnea, J.Bio.Chem. 265:2017-2021 (1990).

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20 Copper-Histidine Therapy in Menkes Disease: Clinical, Biochemical, and Molecular Aspects Stephen G. Kaler Human Genetics Branch, National Instituteof Child Health and Human Development, National Institutes of Health, Building 10, Room 8C429, 9000 Rockville Pike, Bethesda, MD 20892

Menkes disease is an X-linked recessive disorder of copper transport that is usually fatal in infancy or early childhood [l]. Three fundamental issues must be addressed in configuring therapeutic strategies for this condition: (1) the block in intestinal absorption of copper must be bypassed, (2) copper must be made available to the enzymes within cells that require it as a cofactor, and (3) affected infants must be identified and treatment commenced very early in life before irreparable neurodegeneration occurs. Copper histidine, a physiologically suitable preparation [2] fulfills the first requirement when administered parenterally.

The recent cloningof the Menkes gene [3-51 established that the Menkes gene product is a member of a small family of copper-transporting ATPases. Since this molecule presumably acts to translocate copper across biological membranes, and requires several critical functional domains as well as ATP for this process, copper replacement alone would not be expectedto correct the basic defect. 317

318

Kaler

However, in the brindled mouse mutant,the genetic homologof classical Menkes disease [6,7J, parenteral copper replacementboth enhances the activity of copper-dependent enzymes cytochrome c oxidase, lysyl oxidase and tyrosinase [8-111 and restores normal viability if provided during the first week of life. Copper treatment given later(e.g., on day 12) is ineffective [12,13]. These findings suggest thatthe brindled mutation doesnot completely impede proper utilizationof copper when the block in intestinal absorption is bypassed, and that there isa critical period in murine neurodevelopment during which copper is essential. In humans, copper replacement from an earlyagehas mitigated but notcompletely eliminated the serious neurological effects of this disorder in several patients [14-17], while otherreported patients treated froman early age derived no substantive benefit[18-201. Differences between human and mouse in the timing of neurologic maturation, metallothionein induction, andthe kinetics of copper clearance could, in part, account for the different responses to copper replacement. Alternatively, the brindled defect could represent a unique mutation that is amenable to such treatment. While this mutation has not yet been defined, the size and quantity of the murine copper-transporting ATPase mRNA produced in brindled mice appears normal [7J, implying that a point mutation which does not prevent is present. Since over70% of Menkes production of a full length message patients studied at the molecular level to date show quantitative or [3,5], qualitativeabnormalities of mRNA byNorthernanalysis examples of a human mutation precisely comparable to brindled are likely to be rare. In a study designed priorto these recent advances in our understanding of the Menkes geneand gene product, we treated 19 Menkes patients with copper histidine and tracked their clinical and biochemical responses. Thepatientpopulation covered a range of clinical phenotypes, reflecting the considerable clinical variability associated with this syndrome. While the detailed results of this study will be presented elsewhere, some general trends are reviewed here. Copper histidine was prepared as a freeze-dried product with or without 5% mannitol and was approved by the U.S. Food and Drug Administration asan investigational new drug. The mannitolcontaining preparation was stable for at least 3 months at O'C and 4'C, and less than 1 month at room temperature. When slightly improved stability at all storage temperatures was documented for freeze-dried preparations without mannitol [21], this diluent was no longer added.

Copper-Histidine Therapy

in Disease Menkes

319

Table. Menkes Disease Patients Treated with Copper Histidine at National Institutes of Health, 1990-1994

Infants c 1month

5

2

Older infants (3-27 months)

11

4

3

0

Less severe clinical cases

(aged 15mos, 2 y, 13y)

Administration was by daily subcutaneous injection in the following doses: copper histidine 250 pg S.C. b.i.d. in infants under 12 months of age, and 250 pg S.C. q.d. in patients olderthan 1year. Treatment invariably restored circulating copper and ceruloplasmin levels to the normal range, and increased urinary excretion of uric acid and p2-microglobulin, a marker of renal tubular injury, but failed to enhance the activity of dopamine-p-hydroxylase (DBH), a copperdependent enzyme, as assessed by plasma and CSF neurochemical analysis, a reliable indicator ofDBH deficiency [22,23]. The above Table summarizes outcome with respect to survival for this cohort of Menkes patients, who are divided into 3 subgroups based on age when treatment began and clinical severity. While early copper treatment has been associated with better than expected neurological functionin certain Menkes patients,I believe that a patient's mutation will prove to bea more critical predictor of ultimate clinical outcome. Recent evidence indicatesthat splice junction mutations in which a small but significant amount of proper Menkes mRNA splicing is preserved are associated with less severe clinical phenotypes [24]. Splicing mutations of this type may underlie the several reported cases of improved clinical outcome following early copper replacement. In arecently reported family with adenosine deaminase deficiency, differences in biochemical and clinical severity between siblingswith the same splice junction mutation were correlated with differences in the degree of correct splicing maintained [25]. Similar intrafamilial variation in Menkes disease could confound interpretation of early copper histidine replacement in patients whose

320

Kaler

previously affected siblingsshowed the classical phenotype. Delineation of the specific mutations in such patients should help to clarify this issue. It seems conceivable that copper couldhave a rolein neurodwelopment that does not directly involve the Menkes gene product. Therefore, while restoring circulating copper levelsin Menkes patients would not overcome the basic intracellular transport defect, it could be beneficial in supporting processes thatareimpaired secondarily due to hypocupremia. S i c e n o mbrain 1 developmentis most rapidduring the first year of life, with a 12 centimeter increase in head circumference, ultimate neurological outcomeinMenkespatients with "mild" be maximized by exogenous copper replacement mutations could perhaps during this critical period. In my experience with 5 Menkes patients who came to very early attention (less than 1 month of age), the success (attenuation of neurological severity relative to the classical phenotype) or failure (no significant improvement over the classical phenotype) of parenteral copper histidinetreatment seems evident by 6 months of age. Assessment of early infant neurodevelopment criteria (e.g., head control, rolling over, smiling, visual fixation and tracking), as well as growth (particularly in head circumference) at 6 months is generally adequate to renderan opinion regardingthe benefit of treatment in such cases. In Menkes patients diagnosed after the onset of neurodegeneration, copper histidine treatment often reduces irritability and improves sleeping. The reason for these effects is not clear, but the improvements can facilitate the care of these illchildren andare frequently meaningful to their parents and families. There is no evidence that copper replacement affects longevity in such patients. In instances where parents desire it, I consider a3 month trial of copper histidine to evaluate behavioral effects reasonable.

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Copper-Histidine Therapy in Menkes Disease

REFERENCES 1. S.G. Kaler, Menkesdisease, in Admnaes in Pediatrics,

(L.A. Barness, Ed.),

2. and J. Gitscher,

3.

4. J.Chelly, Z. Tumer, T.Tonnesen, A. Petterson, Y. Ishikara-Brush, N. Tommerup, N. Horn, and A. P. Monaco, Nuture Genet.,314-19, 1993. 5. J. F. B. Mercer, J. Livingston, B.Hall,J. A. Paynter, C.Begy, S. (zhandrasekhara a, P. Lockhart,A. Grimes, M.Bhave, Glover, NutureGenet.,3:20-25,1993. D. Siemieniak, 6. B. Levinson. C.Vulm. B. Elder. C. Martin, F. Verley,S. Packman, and J.Gitscher,'Nuture %&et., 6:369-373,1994. 7., J.F.B. Mercer,A. Grimes, L. Ambrosini, P. Lockhart,J. A. Paynter,H. Dierick, and T.W. Glover, NutureGenet.,6:374-378,1994. 8. M. Philli J. Camakaris, D. M. Danks, B i o c h . I., 238:177-183,1986. 9. T. Fujii, PIto, H. Tsuda, H. Mikawa, J. Neuroch., 55:885889,1990. 10. P. M. R0 ce, J. Camakaris,J. R. Mann, and D. M. Danks, Biochm. J., 202:369-g71,1982 11. T. J. Holstein, R.Q. Fung, W. C. Quevedo, Jr., P m . Soc. Erper. Bio. Med., 162264-268,1979. 12. J. R.Mann,J. Camakaris,D. M.Danks, E. G.Wallicmk, Biochem.I., 180605612,1979. 13. G. Wenk and K.Suzuki, Biochem.I., 205:485- 487,1982. 14. D. Nadal,K Baerlocher, Eur. I. Pediat~.,147:621-625, 1988. 15. G. Sherwood, B. Sarkar, A. Sass-Kortsak,J. Inher. Metub. Dis. 12 (Suppl2): 393396,1989. 16. D. M. Danks, Disorders of copper transport,in The Metabolic Basis of Inhen'ted Disease, C R. Scriver, A.L. Beaudet, W.S. Sl ,D. Valle, Eds.), 6th Edition, McGrawL l , New York, 1989,p 1422-142g. 17. S. G. Kaler and W. A. Gahl, Pediutr. Res., &(4) Part 2:186A/1101,1992. 18. W. D. Grover and M. C. Scrutton, J. Pediutr., 86:216-220,1975. 19. P. Daish, E.M. Wheeler, P. F. Roberts,and R.D. Jones,Arch. Dis. CMd,, 53: 956-958, 1978. 20. A. D. Gamica, Eur. Pediatr., 14298102,1984. 21. M. Gautarn-Basak, {F. Gallelli, and B. Sarkar, Inorg. Biochem., 51:415,1993. 22. I. Bia ioni, D.S.Goldstein, T. Atkinson, and Robertson, NeuroI., 40:370373, B90. 23. S. G. Kaler,D. S.Goldstein, C. Holmes, J.A. Salerno,and W. A. Gahl, Ann. N~uYo~., 33~171-175, 1993. 24. S. G. Kaler,L. K. Gallo, V. K Proud, A. K.Per ,D. S.Goldstein, C. S.Holmes, and W. A. Gahl, Pediatr. Res.,35(4) Part 215%/898,1994. 25. F.X.ArredondmVe a I. Santisteban, S. Kelly, C. M. Schlossman,D.T. Umetsu, and M. S . krschfield, Am. J. Hum.Genet., W82@830,1994.

anzf.

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21 Biochemical and Clinical Benefits of Copper-HistidineTherapyinMenkes Disease J. Kreuder, A. Otten, A. Borkhardt,and

F. LampertDepartmentof Pediatrics, Children’s Hospital, Justus Liebig University, Feulgenstrasse 12, D35385 Giessen, Germany

K. BaerlocherChildren’sHospital,St.Gallen,Switzerland H.J. BohlesDepartmentofPediatrics,JohannWolfgangGoetheUniversity, D-6000 Frankfurt, Germany A. Dorries Department of Pediatrics,UniversityClinics,Wurzburg,Germany

I.

INTRODUCTION

Menkes disease is an X-linked recessivedisorder of cellular c o p r (Cu)handling.Biochemically, this disease is characterizedbylow serum, liver and brain Cu levels, whereas the Cu content in nearly all other organs is increased.Clinical features include rapidly progressive neurodegeneration,severe mental retardation, seizures,abnormalities of hair, skin andbonesanddeathinearly childhood [l, 2, 31.

323

324

Kreuder et al.

Recently,acandidate gene for Menkes disease has been identified independently by three groups [4, 5, 61. The gene product established from the nucleotidesequence is most similar to abacterialCutransporting adenosinetriphosphatase and additionally contains six putative Cu-binding motifs at theN-terminus. Initial neurological symptom can be explained by impaired activity in brain tissue of the respiratory chain enzyme cytochrome c oxidase andthedopamine-&hydroxylasewhichcatalyzestheconversionof dopamine to norepinephrine[2, 3, 7, 8, 91. Thereforecorrection of both intracellular Cu content and distribution within neural tissue may be therationale ofCusupplementation in patientswith Menkes disease.

A. Role for histidine in copper transport Detailedstudies on cellular Cu transport revealedthephysiologic importance of theCu-histidinewhich is found as part of the third plasma fraction, that bounds to amino acids [lo]. Cu-histidineat physiologicalpHformsa strong complexwith1:2 stoichiometq. Histidine facilitates Cu uptake by hepatocytes and brain tissue suggestingthat this amino acidmayform part of the complex recognizedby the specific Cu carrier [H]. Rat brain tissue slices transport Cu at a rate that critically depends on complexationwith amino acids, histidinethe most effective, whereasalbumininhibitsCuuptake.Twotransportsystems,low been affinity-hgh capacity and high affinity-low capacity, have charactizedthroughhistidine[12].Tests of effectivenessofspecific analoguesshow that L-His > D-His > Me-3-N-His by both carrier systems. Histidine at low concentrations is more effective than other amino acids; at high, the differences are less distinguishable 1131. In vitro results suggest that Cu-histidine dissociates at the level of the membrane and that the dissociated Cuis preferentially transported by a specific carrier [13-151. Therefore,Cu-histidine may be the most

BiochemicalandClinicalBenefits

of Copper-Histidine Therapy

325

probable formin which Cu is able to cross the blood-brain barrier. Long-termprevention of neurologicaldeteriorationinboyswith Menkes disease by treatment with Cu-histidine was first reported by Sarkar [l61 andBaerlocheretal. [17,18].Sarkar etal.summarizing their 17 years’ experience with subcutaneous administration of Cu-His reported a favorable outcome in 2 patients whose therapy was begun within 1 month of age [19].Recently, Kaler et demonstrated partial deficiency of dopamine-&hydroxylase in Menkes patients by catecholamine analysis of cerebrospinal fluid( 0 ; they proposed the CSF catecholamine patternto be a baseline against which the influence ofCureplacementtherapies can be judged [20]. Here, we report biochemicalandclinicalresponses toCu-histidhe treatment in 4 patients with Menkes disease.

al.

11. CLINICAL AND EXPERIMENTAL

DESIGN

A. Patients Menkes disease has been diagnosed in four boys born between 1979

and 1991;diagnosis was confirmed by an increased Cu accumulation infibroblasts [21]. The mainclinicalandbiochemicalfeatures are summarized in tables 1 and 2; data of an additionalpatientwith Menkes diseasenevertreated with Cureplacement are included. pt. 2 disclosed several boys with severe Family history in childhood, very suggestive for neurodegeneration and death in early typical severe Menkes disease 1181. In pt. 1, cytogenetic studies in fibroblasts and lymphocytes detected a rearrangement of the X-chromosome involving the insertion of the long arm segment Xq13.3-21.2 intotheshort arm [22].The same abnormal X-chromome was presentinhisphenotypically normal mother, where it was preferentially inactivated. This unique chromosomalrearrangementhas been valuableincloningofthe Menkes gene [4,231.

328

Kreuder et al.

B. Materials and Methods 1. Copper-hktidine treatment Cu-histidine supplementation was initiated at the ages of13,23 and 22 weeks in pts. 1, 3 and 4. In pt. 2, therapy with Cu-acetate (0.25-0.5 mg/dayintravenously for 5 days) andsubsequentlyCu-EDTA (0.50.75 mg/intramuscular once or twice a week) started at the age of 7 weeks. At 6 months, oral D-penicillaminewasincludedinthe treatment (250 mg/week; after his 2nd birthday, 150 mg every 2 days). Cu supplementation was switched to intramuscular Cu-histidine every 2 days at the age of18 months. Cu-histidine was prepared according to the recipe of the Pharmacological Department of the Kantonspital St.Gallen, Switzerland. 536,6 mg cupricchloride(dihydrate)and 1026 mgL-histidine (free base) were separately dissolved in sterile water at mom temperature. The solutions were mixed and the volume adjusted to 100 ml. Portions of 0,55 ml weretransferred to sterile, dark glass vialswitha filling volume of 1 ml through a 0,2 pmultipore filter. The ampules were stored at -20'~. The final cu concentrationwas2000 pg/m~. The molar ratio of Cu to histidine was 1 : 2.

2. Copper studies Serum and urine Cu were determined by conventional atomic ,absorption spectrometry. In pt. 1, measurement of CSF and tissue Cu contentwas performed .by flameless atomicabsorptionspectrometry (phifips TU 9400; GermanCancerResearchCenter,Heidelberg, Germany).Thenormalrange of Cuconcentrationin CSF was determined in 15 children at the age of 4 months to 11 years without any evidence of degenerative and inflammatory neurological disorders. For pts. 2 and 3, CSF and liver Cu content were locally measured by atomic absorption spectrometry d t i n g in higher background signals and higher normal values(see tables).

BiochemicalandClinicalBenefits

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329

OralD-penicillamineloading test was performed inpts. 1 and 2 after various times of therapy by administering 3 x 75 mg at8 h intervals [18].Cu was measuredin 24 hurinethe daybeforeand duringthedayof D-pnicillamine medication.Thecompletenessof urine collections in pt. 1was checked by measuring the total amount of excreted creatinine.

3..Catecholamines Dopamine (DA), norepinephrine (NE) and epinephrine Q concentrations in CSF were assayed after absorption onto AI203 using reversed phase high-performance-liquid-chromatography and electrochemical detection [U]. The previously reported pediatric controlvalues ofDAand NE andadultcontrol values of E were determined by radioenzymatic a method [7, 25, 261. A strong correlation has been reported between results obtained by radioenzymatic or chromatographical methods [a]. 4. Side effecis Routinemonitoringforside effects ofCu-Histherapyincluded: complete blood count, creatmne, urea, liver enzymes, alkaline tests phosphatase,ammonia,serumproteinelectrophoresis,clotting (monthly); fractional sodium and potassium excretion, tubular phosphate reabsorption, glucose, protein and amino acid excretion in urine;renalultrasound(every 2-3 months);electrocardiogramand echocardiogram (every 3-4months).

..

A. Biochemical tindings The individual courses of biochemical parameters during Cu-histidine therapy are listed in tables 3-6.

Kreuder et al.

330

Table 3: Biochemical Response to Cu-Histidine (Pt.1) duration of treatment (months)

13 wk

1.5

3

300 55

450 65

450 58

450 50

500 52

66 21

129 40

149 36

147

189 38

2.6

4.0 80 210 0.38 90 1.1

1780 300 5.9 60 Liver Cu bg/g dw) Muscle Cu (mdg dw)

-

-

6 16

12

38

4.4 40 200 0.2 200 100a 6.3b

Cu-His = copper-histidine, Cpl = ceruloplasmin, DA = dopamine; NE = norepinephriae; E= epinephrine. For nonnal values see table 2 except: a(15-45); b(2.1-5.05)

331

BiochemicalandClinicalBenefitsofCopper-HistidineTherapy

Table 4: Biochemical Response to Copper-Histidine (R. 2) 7wk

duration of treatment 3 p 6 p

4mo 12mo

-

Cu-His h d d ) 400 300 150 150 & H i s hg/kdd) 21

-

senun

15

14

21

1Oyrs

15yrs

700 28

1200 24

[Cu-EDTA][&-EDTA]

16 13

64

95 40

82 18.4

125 38.4

95

24

34

89 25

Liver Cu duglg dw) 13

12

-

8

-

-

-

cu Wdo

Cpl (mg/dl)

Cu-His = copper-histidine, Cpl = d o p l a s m i n For normal values see table 2 except:a(36-56; ref.18) Table 5: Biochemical Response to Copper-Histidine (Pt.3) 23 wk

1wk

duration of treatment 2 wk 4 wk

8 wk

500 83

500 82

500 80

70 11

6 < l

12

-

96 22

186

56

50 24

0

< 10

< loa

96

10 33 343 0.1 150

40

381 66 5.8 21

38 271 0.15 1%

91 95 0.95 125

440

0.2 293

332

Kreuder et al.

Table 6: Biochemical Response to Copper-Histidine (Pt.4) 22 wk

6 4.9

301 112 2.7 < 10

duration of treatment 3 wk 1.5 mo

3 m0

400 62

450 64

450 62

31 21.4

101 24

152 40.9

376 186 2.0 60

1. Copper and ceruloplasmin

In each patient, Cu and ceruloplasmin levels in serum returned to the normalrangewithinthe first 4 weeks of Cu-histidineinjections. of serum Cu in Higher Cu-histidine doses caused a more rapid increase pt. 3. CSF Cu concentrations roseto normal values with in 1.5 months of treatnmt in bothexaminedpatients (pt.1, pt.3). CSF Cu levels imitated thechanges of serum Cuconcentrations; this is especially demonstrated in pt. 3 by the rapid drop of serum and CSF levels after Cu dose was reduced from500 &day to 500 pg/wk during the second month of therapy. 2. Cbtecholamines in CSF In everycase, DA content wasmarkedlyincreasedwhereaslow normal or subnormal leves of NE and E wereobserved.The

ratio

BiochemicalandClinicalBenefits

of Copper-Histidine Therapy

333

between DA and NE was consistently increased indicating an impaired activity of dopamine-f3-hydroxylase. During Cu-histidine treatment, all patients displayed normal values of NE and E and an improved ratio between DA and NE. These impressive changes of the catecholamine patternin CSF appearedconcomitantlywith or evenpruceededthe increaseof CSF copper, as observedinpt.3whosecatecholamine pattern normalized within 1 week on Cu-histidine injections. The dose reduction of Cu-histidine in this patient led to the reappearance of the disturbed catecholamine levels.

3. Urine copper excretion In all patients, the urinary Cu exretion increased during Cu-histidine treatment. The D-penicillamine loading tests gave different results in the two assessedpatients. In pt. 1, none of theseloading tests augmented the daily Cu excretion or the Cdcreatinine concentration ratio. In contrast, administration of D-penicillamine induced a 2-3-fold increase of urinary Cu excretion in pt: 2 (table 7).

Table 7: 24-h Urinary Copper Excretion During D-Penicillamine Loading Tests patient 1 patient 2 S y r s 13yrs

durationoftherapy

3mo 6mo 12mo 4mo 3yrs

& H i s Cugld)

450 450450

Cu excretion Gug/day) before D-P mdet D-P

192 184

248 198

290

Wcnxthine@gimgl before D-P Under D-P

3.2 2.8

3.3 2.6

3.0 2.6

150 330

200 1000 350

1057a 439 127a 5308 1770 1W82701a 5748

-

-

-

-

-

-

334

Kreuder et al.

4. llksue copper content

After 16 months of treatment, the Cu concentration of liver tissue was twice the upper normal limit in pt. 1; Cu content of skeletal muscle was slightly increased (table 3). In pt. 2, liver tissue Cu was decreased beforethecommencementofCusupplementation; after 4 mo (CuEDTA) and 3 yrs (Cu-histidine) of therapy, liver Cu content remained in the subnormal range (table 4).

B. Clinical findings 1.Neurological symptom

Clinical outcome, the improvement of major neurological deficiencies and the neurological status are summarized in table 8. In all symptomatic patients, Cu-histidine consistently caused an increase of muscletone,vigilance,andspontaneous motor activities;feeding difficultiesimproved. Also thefrequency of hypothermiaepisodes diminishedduringthecourse ot treatment. In pt. 1, convulsions completely disappeared after 6 weeks on Cu-histidine, except during a short period of reduced mpper dosage. After 8 months of treatment, 0.45 to 0.35 mg per day. the Cu dosewasslowlyreducedfrom DespiteunchangedserumCulevels,the recurrence of seizures, particularlyinfantile spasms wasobserved within 1 month; EEG revealed typical hypsarrhythmia. Clinical as well as electroencephalographic signs of epileptic activity resolved after teadministration of the previous Cu dosage within 1 week. The 2 patients with the longest delay to Cu replacement didn't show any significant developmental progress. In pt. 1, however, a catch-up of lostdevelopmentalstepsstarting from theneonatallevel was observed during the first 6 weeks Cu supplementation. of Subsequently, developmental progress was delayedbut did not stop. At the age of 18 months, motor development corresponded to a

8

F

m

E

0

m

E

57

8

4

0

%l

I

a)

v) v)

;* * *

335

++ + +

+

+

= , + I

+

I

1

II

2 ;

g I

II

336

Kreuder et al.

developmental level of 3 months, whereas mental achievements met a developmental age of 5 - 6 months. Muscular hypotonia was reduced but persisted with the symptomsof hypotonic cerebral palsy. Pt.2 whose Cu replacement therapy started from a pre-symptomatic level showed the most favorable neurological outcome. Psychomotor and speech development was mildly retarded during childhood. Ataxia became the main neurological symptom since the second year of life; walkingwithoutcrutches is nowpossible.Due to ataxia, speech is partially inarticulate, whereas vocabulary and sentence formation are normal. He now attends a school for handicapped children and is able to read and write.

2.Growth Normal growth and weight increase were noted in both patients who were treated more than 1 year with Cu-histidine. The other 2 patients failed to thrive despite reduced feeding problems.

3.Connective t i w e manifestations Metaphysealanddiaphysealbonelesions as m e of the features of connective tissue involvement in Menkes disease continously regressed within 3 monthsin pt. 1. (fig.1).Bonematurationprogressedina nearly normal pattern, a less pronounced hypomineralization persisted [27].Similarskeletalchangesintheother affected patientsslowly improvedduringthe first yearcorresponding to thespontaneous amelioration of osseous lesions in patients withMenkes disease [3]. Abnormalhairgraduallydisappearedinthe first two patients. Bladder diverticula and lobular lung emphysema were first detected in pt.1, at the age of 13 months. Bladder abnormalities occured in pt. 3 after the discontinuationof Cu-histidine therapy.

Biochemical and Clinical Benefits of Copper-Histidine Therapy

337

Figure 1: X-rays of the left knee before (a), after 3 months (b) and 12 months (c) of Cu-histidinesupplementation.Before Cu-histidinetreatment, metaphyseal widening and spurring m well m lamellar periostal thickening in the diaphyses were observed. These typical osseous alterations of Menkes disease regressed during treatment. Bone maturation also progressed, but bone mineralizationmaineddiminished (From Ref. 27)

.

338

BiochemicalandClinicalBenefits

of Copper-Histidine Therapy

339

4. Side efects

Regularlaboratoryexaminationsshowednoabnormalities of liver, kidneyandbloodmarrow function (table 9). 4 exhibiteda persistent increase of ultrasonographic density of kidneys and liver, ultrasound density was occasionally augmented in pt. 1 and 3. Pt. 2 displayed no abnormal Cu deposition on ultrasonography.

Pt.

IV. DISCUSSION Our d t sindicate, that Cu-histidine is capable of crossing bloodbrain barrier and improving Cudependent metabolic pathways in brain tissue. For animalmodels of Menkesdisease, Kodoma described trapping of blood-borne Cu in cerbral blood vessels and astrocytes and little Cu transport to neurons [281. In contrast, we conclude that the administration of parenteralCu-histidineprovidesdosedependent transport of Cu to neuronsevenatnormal or slightlysupranormal serum levels. Persistent normal CSF Cu levels during 16 months on Cu-histidine also indicate that immaturity of the blood-brain barrier is not required for Cu-histidine to reach neuronal cells. The increased DA content and high DANE ratios in CSF before treatmentindicatethe impaired activity of dopamine-&hydroxylase. This pattern of CSF catecholamines is in accordance to the findings of Kaler et in Menkes patients without Cu supplementation [20]. The normal or slightlysubnormal NE concentrationswhichcontrast to findings in patients with congenital dopamine-&hydroxylase deficiency were also reported by Kaler et al. and suggest only partial deficiency of dopamine-&hydroxylase andor compensatory mechanisms to preserve NE levels in CSF [20,25]. The improvement and n o d i o n of DA, NE and E concentrations suggest restoring of dopamine-&hydroxylase activity in patients on Cu-histidine.The strong correlation between increased Cu levels and the catecholamine pattern in CSF as well as the increase of DA

al.

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contentand DAME ratio after Cu-histidinereduction illustrate the CSF Culevels. This improvementofdopamine-& criticalroleof hydroxylase activity is quite different from the results of Kaler et al. whodemonstratedecreasedsympatheticactivity,butnot enhanced dopamine-&hydroxylaseactivityinMenkespatientsonCu-histidine, eveninthecase of normalized CSF Culevels[29].Sincethe improvement of catecholamines was observed after intramuscular and subcutaneous injections, the mode of Cu-histidine administration doesn't explain these differences. The failure to improve non-neurological symptoms was also observed in other long-term surviving Menkes patients on Cu-histidine [19, 301. The different response to Cu-histidine between bone tissue and other connective tissues remains unclear. ExcessiveCuaccumulationandsecondaryorgandysfunctions will be one major problem of long-term Cu supplementation. Augmentation of urinary Cu excretion by D-penicillamine or other chelators hasbeen recommended as an adjunctivetherapy [18, 301. Our results of ultrasonographic monitoring suggest that the addition of Dpenicillamine may be effective to prevent chronic Cu overloading. The role of D-penicillamine and the predictive value of loading tests have to be determined in long-term Cu-histidine treatment. The inmed Cucontent of livertissueinpt. 1 illustrates therisk of hepatic overload by Cu-histidine replacement. Danks [31] therefore proposed regularliverbiopsies to monitorhepaticCuconcentration after normalization of serum Cu levels. Despitetheclearevidence of metabolicimprovement,theneurologicalbenefit of Cu-histidineinsymptomatic Menkes patients is limited. Making Cu available beyond the critical postnatal period of myelination and development of the central nervous system appears not to beeffective to reconstituteneurologicallesions. This findings confirmtheextendedexperience of Sarkaret al. and mer et describingafavorableclinical outcome only in3patientswhose treatment started early in life [19, 291. Cu-histidine, however, may be an effective agent for supportive therapy, especially for patients with poorly controlled seizures.

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1. J.H. Menkes, M. Alter, G.K. Steigleder, D.R. Weakly, J. H0 Sung, Pedhtrics, 2 9 : 764-779 (1962) 2. D.M. Danks, Disordersof copper transport, in The metubolic busk of inherited disease (C.R. scriver, A.L. Beaudet, W.S. Sly and D. Valle, Eds), McGraw Hill, New York, 1989, pp 1411-1431 3. N. Horn, T. TO” and 2. Tiher, Bmin PUthOl, 2 351-362 (1992) 4. C. Vulpe, B. Levinson,S. Whitney, S. Paclunan and J. Gitschier, Nature Genet 3: 7-13 (1993) 5. J. Chelly, 2. Timer, T. Tonnesen,A. Pelterson, Y.Ishikawa-Brush, N. Tommenrp, N. Hom and A.P. Monaco, Nutum Genet 3: 14-19 (1993) 6. J.F.B. Mercer, J. B. Hall, J.A. Paynter, C. Begy. S. Chandrasekharappa,P. Lockhart, A. Grimes, M. Bhave, D. Siemieniak and T.W. Glover, Nature Genet 3: 20-25 (1993) 7. W.D Grover, R.I. Henkin,M. Schwarz, N. Brodsky, E. Hobdell and J.M. Stolk, Ann Neuml12 263-266 (1982) 8. M. Maehara, N. Ogasawara, M. Mizutani, K. Watauabe and S. Suzuki, Bmin Dev 5: 533-540 (1983) 9. J.R. Prohaska, Physiol Rev 67: 858-901 (1987) 10. B. War and T. h&, Copper amino acid complexes in human serum, in Bwchemktry of copper (J. Peisach,P. Aism and W. Blumberg, Eds), Academic Press, New York, 1966, pp 183-1% 11. E.D. Hanis, Roc Soc Exp Biol Med 196: 130-140 (1991) 12. D.E. Hartter and A. Barnea, J Biol Chem263: 799-805 (1988) 13. B.M. Katz and A. Barnea, J Biol Chem265: 2017-2021 (1990) 14. A. Barnea and B.M. Katz, JInorg Biochem 40: 81-93 (1990) 15. A. Bmea, D.E. Hartter, G. Cho, K.R. Bhasker, B.M. Katz and M.D. Edwards, J Inorg Biochem40: 103-110 (1990) 16. B. Sarkar, Recent trends in the application of cootdination chemistry in biology and medicine, in IUPAC Cooardinution Chemktry, Vol. 20 Band=, Ed), Oxford, P-mon Press, 1980, pp 191-200 17. K.E. Baerlocher, B. Steinmann, V.H. Rao and R. Gitzelmann, J Inherit Metub D k 6 (Suppl.2): 87-88 (1983) 18. D. Nadal and K. Baerlocher, f i tJ Ped& 147: 621625 (1988)

Livingston.

(D.

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19. B. Mar, K. Lingertat-Walsh and J.T.R. Clarke, J Pediah. 123: 828-830 (1993) 20. S.G. Kaler, D.S. Goldstein, G. Holmes, J.A. salerno and W.D. Gahl, Ann NeumZ33: 171-175 (1993) 21. T. Tonnesen and N. Horn, JZnherit Metab Dis 12 (suppl.1): 207-214 (1989) 22.2. Tiher, N. Tommerup, T. T o " , J. Kreuder, I.W. Craig and N. Horn, Hwn Genet 88: 668-672 (1992) 23. 2. Tiimer, J. Chelly, N. Tommentp, Y.Wawa-Brush, T. Toonesen, A.P. Monaco and N. Hom, Hwn Mol Genet 7: 483-489 (1992) 2 4 . T. Halbriigge, T. Gerfiardt, J. Ludwig, E. Heidbreder and K.H. Graefe, Lye Sci 43: 19-26 (1988) 25. A.J. Man in't Veld, F. Boomma, P. Moleman and M.A.D.H. Schalek~mp, Lancet 1: 183-188 (1987) 26. J.D. Peuler and G.A. Garland, Lve Sci 21: 625436 (1977) 27. J. Kreuder, A. O t t e n , H.Fuder, 2. Tiimer, T. Tonnesen, N. Horn and D. Dralle, &r J Pediah. 152: 828-830 (1993) 28. H.Kodoma, JZnherit Metab D& 16: 791-799 (1993) 29. S.G. Kaler and W.D. Gahl, Am JHwn Genet 51 (suppl.1): A 170 (1992) 30. G. Sherwood, B. Sarkar and A. Sass-Kortsak, J InheritMetab Dis 12 (~~ppl.2): 393-396 (1989) 31. D.M. D&, Menkes' disease, in Inborn metabolic d&wm (J.Fmandes, Spinger, Berlin, 1990, p~ 515-521 J.M. SaudUbray and K. T A , M),

22 The Wilson Disease Gene: A CopperBinding ATPase Homologous to the Menkes Disease Gene Diane W. Cox and Gordon R. Thomas Department of Genetics, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1x8, Canada

L INTRODUCTION The transport, utilizationand elimination of copper involves a series of processes, including absorption through the intestinal epithelial cells, transfer to albuminand copper histidine for blood transport, uptake by the liver, where incorporation into ceruloplasminand excretion into the bile take place.Excess hepatic copper can be stored inthe low molecular weight, metal-inducible protein, metallothionein. Two genetic abnormalities of this transport system have been identified, as shown in Figure 1. Menkes disease isan X-linked disorder in which copper, imported into the intestinal epithelial cell, is not adequately exported [l]. The result is a widespread copper deficiency, resulting in insufficient levels of all of the copper containing enzymes, including lysyl oxidase involved in connective tissue and elastin cross linking, superoxide dismutase, cytochrome oxidase, tyrosinaseanddopamine beta hydroxylase.Thefeatures of Menkesdisease:abnormalconnective tissue, twistedhair and arteries, neurological abnormalities, developmental delay and hypopigmentation can all be explained by the absence of these enzymes. Copper histidine shows some success in treatment of this condition, which, without treatment, results in death in early childhood [21. 343

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Intestinal Absorption Plasma Albumin

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Biliary Excretion (1-4 mg124 hr)

Figure 1 Transport of Copper in Humans

chromosome 13

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Wilson disease (hepatolenticular degeneration) is a recessively inherited disorder of copper transport, with an incidence of about 1/30,000 in most populations, probably more frequent in Sardinia, Japan up to the pointof entry and China [l]. Copper is transported normally into the liver cell. However, copper is not incorporated intothe serum As a protein ceruloplasmin and excess is not excreted through the bile. result, copper accumulatesin the liver, leading to hepatocyte damage and subsequent cirrhosis or liver failure. Copper also accumulates in brain and in the rim of the cornea, the latter resulting in characteristic brownish Kaiser-Fleischer rings. Neurological damage occurs apparently from the toxic effects of copper in the brain, particularlyin the lenticular nucleus.Thevaried signs and symptoms of Wilson disease occur betweenthe ages of 8 and 30 and include cirrhosis of the liver, tremor,loss of speech, erythrocyte hemolysis, kidney abnormalities and occasionallypsychiatric disorders. Treatment of this disease is generally accomplished by the use of chelating agents, such as penicillamine [3] and triethylene tetramine [41, which remove the excess copper that has built up in the liver, allowing it to be excreted in the urine. The chelating agents sometimes produce toxic side-effects, and excess copperdepletionmust be avoided. Administration of zinc salts is also usedblock to copper absorptionand prevent buildup of the metal to toxic levels[51. Our studies have focused on finding the basic defect in this disorder.

IL CLONING THE WILSON DISEASE GENE A. Linkage Studies Wilson disease was shown to be locatedon human chromosome 13due to co-segregation of the disease state with the red cell enzyme marker, esterase D, in several large MiddleEastern kindreds [6]. Further linkage analysis with more families and a greater number of polymorphic .DNA markers in the 13q12-22 region [7-91, subsequently defined a regionclose to the retinoblastoma (RBI locus, flanked by the two random polymorphic markers D13S31 and D13S59, asthe candidate region for the Wilson disease gene. These markers defined about 2 megabases of DNA, in which the Wilson disease gene was in Figure 2. located [9]. A diagram of the linkage map is shown

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Our studies to identify the gene defectivein Wilson diseasebegan with the follow-up of a series of 25 Canadian families with Wilson disease, which had been the study of classical genetic studies more then 20 years previously [lo]. We showed that segregation in our families was consistent with the presence of a gene in the designated region, and that our families could be useful for narrowing the candidate region bythe study of recombinants [11,121. However many new markers were needed in the region.

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B. New Markers for Localizing the Gene A somatic cell hybrid panel was assembled.This consisted of a number of rodent/human cell lines containing, amongotherhuman chromosomes, only one intact chromosome 13, or one with a portion deleted. The panel was used to map new markers from a chromosome 13 flow sorted library,but this could allow the mapping of only a fewin the designated region [131. The DNA markers which had been shown to flank the disease locus, D13S31 and D13S59, were both located in a region defined by the hybridsKSF39, which lacked the region distal to q14.2, and ICD, which lackedthe region distal toq14.3. Thesecond approach was to target on this specific region of chromosome 13, using PCR to amplify DNA between Alu repetitive elements. The PCR amplification was carried out on cell line ICD, which contained only a piece of chromosome 13,in addition to hamster chromosomes, with no other human chromosomes. PCR products generated in this way were cloned, and hybridized against PCR products from cell line KSF39, selecting only those clones which did not hybridize to the latter and therefore lay within the candidate region. This method generated five new DNA markers that were located within the Wilson disease region.

A long-range restriction map (Figure 3) was then obtained using pulsed field gel electrophoresis [141. These studies indicated that four of the new probes lay between D13S31 and D13S59.Becauseof the characteristics of the fragments generated with various rare cutting restriction enzymes,it was not possible to join the two segments of the map around each of the two flanking markers. Studies of allelic association were usefulin deciding which portionof the map was most likely to contain the gene. Allelic association is the term used when the alleles of a specific marker show a different distribution on the disease chromosomes in comparison with the distribution on normal chromosomes. Since D13S31 showed evidence of allelic associations [121, we focused our efforts on the D13S31 segment of the pulsed field map.Yeastartificialchromosomes(YACs)were obtained using D13S31, D13F71S1, D13S196 and D13S59 as probes to screenthe library of YACs distributed by the Centre de Polymorphisme Humaine, Paris, France. DNA fragments from the endsof these YACs were then used as probes to obtain overlapping YAC clones. A total of. 19 YACs were placed within the map generated by pulsed field gel electrophoresis.

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(S) (C) =

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Figure 3 Pulsed Field mapof the WND region. Letters above the line represent cut sitefor restriction enzymes:C = SacII, F = SfiI, M = Mld,N = NotI, R = NruI. The centromere is located to the right of the dia m and the telomere to the left. YAC clones shown as thick lines were i s o l a t x n the first round of screening, YACs shown as thin lines were obtainedfrom subsequent screens, and dotted l i e s indicate chimeric sectionsof a YAC.

In order to further refine the linkage disequilibrium observed for D13S31, More highly polymorphicDNA markers were needed nearthe disease locus. Markers used for the constructionof the pulsed field map were used to screen a cosmid library containing DNA from chromosome 13 (obtained from the Human Genome Center, Los Alamos National Laboratory). The isolated cosmid clones that were identified were screened for stretches of repeating CA dinucleotide pairs which are oftenpolymorphic.The repeats werecloned,sequenced and PCR primers developed to amplify them. If the repeats are long enough, the number of dinucleotides varies between individuals, resulting in a highly polymorphic DNA marker. We identified three such markers near the WND gene [15], and their locations are given in Figure 4. Another marker, D135133 [16], was also identified as being located close tothe disease locus.

C Identification of the Wilson Disease Gene After a disease gene has been mapped to a specific area, precise localization has frequently been aided by small deletions (e.g. retinoblastoma, Wilms tumour) or by chromosome translocations in affected individuals (e.g. muscular dystrophy, Menkes disease). No in identifying the chromosome breaks or translocations were useful location of the WND gene. However, in January 1993, the cloning of the

S196

The Wilson Disease Gene

349

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CEN

H = 200 kb

Figure 4 Location of CA repeat markers located near the WND gene. The markers shown in bold were isolated from cosmidsas described in the text. The flanking markers D13S31 and D13S59 are shown for reference.

Menkes disease gene, using a translocation breakpoint, was reported [18-20]. The Menkes genewas expressed in a varietyof tissues, with the notable exception of the liver. With no other leads as to the type of defect in Wilson disease,and with the lack of expression of Menkes in the liver, we believedthat the WND gene could be a liver counterpart to the MNK gene. Using reverse transcriptasePCR from muscle tissue, 13 143 2-l

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Figure 5 Identificationof homology to MNKgene probes on WND region YACs. YAC 27DS was found to contain material similar to the MNK ene (Wcl) and YAG derived from the end of this clone (95C3,53C12 and 68F4Talso contained homologous region. Modified from[l71with permission.

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we obtained a Menkes disease gene transcript and from this amplified two regions of the gene which we predicted might be conserved in a homologous gene. Using oneof these clones, we identified a region of homology on three of the nineteen YACs (Figure 5). Studies of linkage disequilibrium, outlined below, indicatedthat the Wilson disease gene lay in this region of homology. Using the conditions of stringency established on the YAG, we screened the chromosome 13 cosmid library and obtained two cosmids which remained positive for Menkes gene fragment hybridization after further screening. Using the appropriate primers and probes, we were unable to obtain a relevant cDNA from several liver cDNA libraries. We therefore used a technique of direct DNA selection to obtain fragments of the WND gene, a method developed by Johanna Rommens at HSC [211, which involves fixationof a YAC or, in our case, a cosmid, on a membrane towhichamplified fragments of liver cDNA are hybridized. Several rounds of selection of those fragments which hybridize to the cosmid results in amini library of gene segments which is then cloned into a plasmid vector. The fragments obtained from our cosmids were screened with the Menkes gene fragment to identify putative WND cDNA fragments. Clones were categorized according tohybridization to each other, were sequenced as overlapping fragments, and aligned. The resulting gene was 82% identical by amino acid sequence to the Menkes disease gene. The major features of the gene, includingallfunctionaldomains (six copperbinding,nine transmembrane, transduction, channel, phosphorylation, and ATP binding regions) were conservedas shown in Figure 6. The functional 75 to 100% identical in amino acid sequence with the domains were from equivalent segmentsof the Menkes gene. A proposed modelis shown in Figure 7.

111. THE MUTATIONS OF THE WILSON DISEASE GENE A. Haplotype Studieswith Microsatellite Markers A deviation of allele frequencies on disease chromosomes from those found in the normal population occurs for markers very close to the disease gene. We therefore examined the allele frequencies in the region of the proposed gene assupport for identification of this gene as that for Wilson disease.

The Wilson Disease Gene

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Figure 6 Alignment of the human Menkes and Wilson disease genes and the rat WND homologue. Functional regions are shownabove the bar, putative transmembrane regions are shown as black boxes, and hydrophobic stretches are indicated as hatched boxes.

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(b>

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In addition to looking at the allele frequenciesof isolated markers, we examined the frequencies of haplotypes of these markers. Haplotypes are combinations of marker alleles thatare located together on the same chromosome. Thus, a patient who is homozygous for allele 1 at marker A, homozygous for allele 3 at marker B and heterozygous for alleles 3 and 6 at marker C has two haplotypes, designated 1-3-3, and 1-3-6. Haplotypes of markers that are very close (ie: less than 1 C M ) tend to remain togetherduring meiosis. This results in a smaller number of haplotypes in a population than one would expect from multiplyingthe number of alleles for eachof the markers. In disease patients, it would be expected that each mutation at the disease locus would be represented by a single haplotype of close markers which could be found at a different frequency in normal individuals. We determined that the allele frequencies of each of the close markers, D13S316,D13S133, and D13S314, are different on Wilson disease chromosomes when compared with normal chromosomes. This

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effect was moreapparent when we considered haplotypesof alleles at the three loci. These results are shown in Figure 8 and have been described 1151. We observed that a number of haplotypes occurred only on Wilson disease chromosomes,and not on the control chromosomes. Control chromosomes were the normal, non Wilson disease chromosome carried by the parents. Theoccurrence of a number of different haplotypes suggestedthat there would be a numberof Wilson disease mutations, as it is expected that each mutation occurs on a specific haplotype background. A high degree of linkage disequilibrium with nearby markers supported the idea that the homologous sequence actually represented the Wilson disease gene.

B: Identification of Mutations The finding of a mutation in the Wilson disease candidate gene in patient DNA was the ultimate proof that this is the gene responsible for the disease. We identified an indisputable mutation, a seven base pair deletion which created a prematurestop codon, in two patients of Icelandicorigin 1171. This mutation was identified by using single strand conformation polymorphism (SSCP) and heteroduplex analysis. Confirmation of the mutation was made by sequencing. In addition, four other mutations have been describedin a different setof patients [231. Using SSCP analysis, we have now identified17 additional mutations, by amplification of many of the exons from genomic DNA. These studies are ongoing as more exonsare yet to be amplified, and mutations have been identifiedin approximately half of our series of 56 patients. The mutations to date include the following. 6 deletions of one or more base pairs,2 insertions of one or more base pairs,2 nonsense mutations resulting in stop codons, 4 missense mutations occurring in conserved amino acid sequences, and 3 mutations in splice sites. Because of the large numberof mutations, most patients carry two different mutations. Although the Icelandic, 7 base pair deletion is predicted to completely disrupt the Wilson disease gene, the presentation is not hepatic disease with early onset, as might be expected, but rather neurological disease at 18 to 20 years of age. The deletion occurs in a region of the gene that is rich in transmembrane sequencesand is not present in the equivalent gene cloned by another group from a brain cDNA library 1231. Possibly the effects of the 7 base pair deletion are modified because of alternate splicing in this region.

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IV. PRACTICAL APPLICATIONS Although many of the mutations present in Wilson disease patients have yet to be determined, the information we presently have can allow us to improve diagnosis of Wilson disease. The highly polymorphic CA repeat markers, which closely flank the gene, are being used to followthe segregation of the mutantWilson disease gene. The markers allow us to follow each specific chromosome, with its normal or Wilson disease gene, through any designated family. This method of diagnosis is generally only useful where oneindividual in the family is identified clinically as a patient. Diagnosis can then be reliably carried out in the sibs. An illustration of this is shown in Figure 9.

FSD REM

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Diagnostic pedigree of family ,525. DNA markers are listed in centromeric to telomeric order. Number represent marker alleles. The proband is shown as a filled circleand the diagnostic queryas a shaded circle.

Figure 9

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355

In the example shown, the sib of the patient was expected, from biochemical studies, to bea patient. In this sib, ceruloplasmin was low, urinary copper excretion was borderline, and incorporation of copper isotope in the ceruloplasmin was reduced, as reported by Drs. G. Fell and D. Gaffney. Segregation of close markers indicates that each the two girls inherited different chromosomes from the mother, therefore the sister is not affected. In this case, the father was deceased and no DNA was available. In spite of this, the diagnosis could be made. Usually both parents are required for this type of diagnosis: if a different chromosome had been inherited from the father, assumptions would have to have beenmade about his genotype.

This family demonstrates one of the features wehave reported [24] and are consistently findingas we extend our studies of Wilson disease. A small percentage of heterozygotes show biochemical features indistinguishable from presymptomatic homozygotes, but will apparently never develop symptoms of the disease. This illustrates a need to re-test all presymptomatic sibs of patients with Wilson disease to ensure that they are indeed patients. The toxic effects of life-long treatment on chelating agents could be expected when heterozygotes are treated in error. These erroneous diagnoses canmade be in spiteof using all available methods for the study of the biochemistry of copper, and reflect unusual features of certain heterozygotes which have not generally been recognized. We have found that some haplotypes ofCA repeats are unique to Wilson disease chromosomes and are exceedingly rare or absent on normal chromosomes. The identification of these unusual haplotypes could lend support to the diagnosis of Wilson disease. However, the most reliable diagnosis will only be possible whenthe majority of the mutations for the'disease are identified and DNA from patients can be tested directly when clinical features suggest . . a diagnosis of Wilson disease.

V. THE LEC RAT: A MODEL FOR WILSON DISEASE The Long-Evans Cinnamon (LEC) rat is a mutant identified in Japan from the Long-Evans (LE) strain 1251. This rat develops hepatitis at approximately four months of age which is frequently fatal, and rats that survive later develop liver cancer. This strain has therefore been useful as a model for studies of liver cancer. The strain has been found

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to have copper biochemistryand clinical symptoms similar to features ofWilson disease. It therefore seemedalikely rodent modelfor Wilson disease with a defect in the rat homologue of the Wilson disease gene. a Clones fromthe human Wilson disease gene were used to screen rat cDNA library. Various clones were obtained and sequenced, and overlapping clones combined to obtain the complete sequence of the homologous gene in the rat. Amino acid identity betweenthe rat and human genes is 86% and all of the functional domains are highly conserved. The rat gene is shown withthe human Wilson and Menkes disease genes in Figure 6. Analysis of Southern hybridization blots on genomic DNA, and PCR techniques have indicated that the LEC rat carries a deletion of the last quarter of the gene, eliminating key functional regions, including the ATP binding fold 1251. This rat is therefore an excellent model for the study of both pathology and treatment of Wilson disease.

VI.

THEMETAL TRANSPORTING ATPases

In addition to the high degreeof identity betweenthe genes for Menkes of the Genbank sequence database and Wilson diseases, a search indicates a high degree of homology with other metal transporting ATPases. We have recently reviewed the features of these genes [141. To date, this type of gene has been found only in bacteria and in humans. This suggests that there are many more genes to be foundin other organisms. The bacterial genesare generally presenton plasmids, and replicate in the presence of high metal concentration in the surrounding environment, as provided by pollution, fungicides, or any othersituation where the metal content of the environment is increased. A particularly high degree of homology is seen with those bacteria which are resistant to either copper or cadmium, because of their efficiency in transporting these metals from the cell. A high level of amino acid identity is seen in the functional regions. The metal binding regions are compared in Figure 10. A high degree of conservation is also foundin the energy transduction, phosphorylation, and ATP binding regions. The particular configuration of cysteine residues in both the metal binding and channel domains is predicted to be important for the interaction with metal.Identicalconfigurations are seen in those

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bacteria that transport either copper or cadmium. Since cadmium is a non-essential element in humans,it is possible that the Wilson disease gene is also involved in the export of cadmium from the liver. A similar sequence is present in the nitrogen fixation geneFIX 1, although no metal binding functions haveyet been shown for this gene. Copper transporting genes have now been found in a number of different bacteria, and can be involved either in copper resistance or copper sensitivity through their roles intransporting copper across the membranes of the cell. These bacteria are discussed further in our review [221. Metal Binding Domains WC1

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Figure 10 Alignment of the metal binding domainsof several human and bacterial heavy metal ATPases. Highly conserved residues are shown as larger bold letters.

W. CONCLUSION The cloning of the Wilson disease gene has practical applications for the diagnosis of this disease, which is frequently difficult because of the extensive variation of symptoms. Treatment with chelating agents is sometimes problematic, and the cloning of the gene offers possibilities for the development of gene therapies. One of the most exciting aspectsof the cloningof both the Menkes and Wilson disease gene is the discovery of similarity with bacterial metal transporting genes.Bacteria resistant tochromium,cobalt, nickel, zinc, arsenic, silver, antimony, tellurium, mercury, thallium, lead and bismuth have been identified but genes have not yet been identified in higher organisms. This suggests that there is much to be done to increase our knowledge of metal transport. Although we now

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have a reasonable mechanism for export of copper from both intestinal and liver cells, there are still many gaps in our knowledge of how copper enters cells and how it becomes toxic. The future discovery of metal transporting pathways offers the promise of excitingnew revelations.

REFERENCES 1. D. M. Danks, Disorders of Copper Transport, in Metabolic basis of inherited disease (A. L. Beaudet, W. S. Sly, and D. Valle,Eds.), McGraw-Hill, New York, 1989, pp. 1411-1431. 2. B. Sarkar, K. Lingertat-Walsh, and J. T. R. Clarke, J Pediutr 123: 828-830 (1993). 3. J. M. Walshe, Q I Med 4 2 441 (1973). 4. J.M. Walshe, Lancet 1: 643 (1982). 5. T. U. Hoogenraad, R. Koevoet, and E. G. W. M. de Rutyer Korver, E u ~ .N e ~ 18: d 205-211 (1979). 6. M. Frydman, B. BonngTamir, L. A. Farrer, P. M. Conneally, A.

Magazanik, A. Ashbel, and Z. Goldwitch, Pmc Nutl Acad Sci USA

82: 1819-1821 (1985). 7. A. M. Bowcock, L. A. Farrer, L. L. Cavalli-Sforza, J.M. Hebert, K. K. Kidd, M. Frydman, and B. Bonn&Tamir, Am J Hum Genet 41: 2735 (1987). 8. A. M. Bowcock, L. A. Farrer, J.M. Hebert, M. Agger, I. Sternlieb, I. H. Scheinberg, C.H.C. M. Buys,H.Scheffer, M. Frydman, T. Chajek-Saul, B. Bonn6-Tamir, and L. L. Cavalli-Sforza, Am J Hum Genet 43: 664-674 (1988). 9. L. A. Farrer, A. M. Bowcock, J. M. Hebert, B. Bonn6-Tamir, I. Sternlieb, M. Giagheddu, P. St.George-Hyslop, M. Frydman, J. Lossner, L. Demelia, C. Carcassi, R. Lee, R. Beker, A. E. Bale, H. Donis-Keller, I. H. Scheinberg, and L. L. Cavalli-Sforza, Neurology 41: 992-999 (1991). 10. D. W. Cox, F. C. Fraser, and A. Sass-Kortsak, Am J Hum Genet 24: 646-666 (1972). 11. R. H. J.Houwen, G. R. Thomas, E. A. Roberts, and D. W. Cox, J H ~ u ~ u17: Z 269-276 (1993). 12. G. R. Thomas, E. A. Roberts, T. 0. Rosales, S. P. Moroz, M. A. Lambert, L. T. K. Wong, and D. W. Cox, Hum Mol Genet 2: 1401-1405 (1993). 13. P. C.Bull, J.A. Barwell, H. Hannah, S. E. Pautler, M. J. Higgins, M. Lalande, and D. W. Cox, Cyiogenet Cell Genet 64: 12-17 (1993).

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14. P. C. Bull and D. W. Cox, Genomics 16 593-598 (1993). 15. G. R. Thomas, P. C. Bull, E. A. Roberts, J. M. Walshe, and D.W. Cox, Am ]Hum Genet 54: 71-78 (1994). 16. K. E. Petrukhin, M.C. Speer, E. Cayanis, M. Bonaldo, U.

Tantravahi, M. Bento Soares, S. G. Fischer, D. Warburton, T.C. Gilliam, and J. Ott, Genomics 15: 76-85 (1993). 17. P. C. Bull,G. R. Thomas, J. M. Rommens,J. R. Forbes, and D. W. Cox, Nut ure Genet 5: 327-337 (1993). 18. C. Vulpe, B. Levinson, S. Whitney, S. Packman, and J. Gitschier, Nut ure Genet 3: 7-13 (1993). 19. J. Chelly, Z.Tumer, T. Tonnesen, A. Petterson, Y. Ishikawa-Brush, N. Tommerup, N. Horn, and A. P. Monaco, NutureGenet 3:14-19 (1993). 20. J. F. B. Mercer, J. Livingstone, B. Hall, J. A. Paynter, C. Begy, S.

Chandrasekharappa, P. Lockhart, A. Grimes, M. Bhave, D. Siemieniak, and T. W. Glover, Nut ure Genet 3: 20-25 (1993). 21. J. M. Rommens, B. Lin,G. B. Hutchinson, S. E. Andrew, Y. P. Goldberg, M. L. Glaves, R. Graham, V. Lai, J. McArthur, J. Nasir, J. Theilmann, H. McDonald, M. Kalchman, L. A. Clarke, K. Shappert, and M. R. Hayden, Hum. Mol. Genet. 2: 901-907 (1993). 22. P. C. Bull and D. W. Cox, Trends Genet 1 0 246-252 (1994). 23. R. E. Tanzi, K. E. Petrukhin, I.Chernov, J. L. Pellequer, W. Wasco, B. ROSS,D. M. Romano, E. Parano, L. Pavone, L. M. Brzustowicz, M. Devoto, J. Peppercorn, A. I.Bush, I.Sternlieb, M. Pirastu, J. F. Gusella, 0. Evgrafov, G. K. Penchaszadeh, B. Honig, I.S. Edelman, M. B. Soares, I.H. Scheinberg, and T. C. Gilliam, Nature Genet 5: 344-350 (1993). 24. Cox, D.W., Billingsley, G.D. The application of DNA markers to the diagnosis of presymptomatic Wilson disease, in Proceedings of:

Genetics of Psychiatric Diseuses Wenner-Gren International . Symposium, Stockholm(L. Wetterberg, Ed), Macmillan Press, London ,pp. 1988, pp. 167-177. 25. M. Sasaki, M. C. Yoshida, K. Kagami, N. Takeichi, H.Kobayashi, K. Dempo, and M. Mori, Rat News Lett 14: 4-6 (1985). 26. J. Wu, J. R. Forbes, H. S. Chen, and D. W. Cox, Nutrue Genet 7 541545 (1994).

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23 Zinc Therapy: An Advance in the Treatment of Wilson's Disease Tjaard U. Hoogenraad Department of Neurology, University Hospital, 4584 CX Utrecht, The Netherlands

I. INTRODUCTION In the past 30 years, it has become evident that zinc is an effective and safe remedy for the treatment of Wilson's disease.Zincsulfate was first used in the University Hospital of Utrecht for the treatment of Wilson's disease in 1976 and since then more than 50 patients have been treated with zinc sulfate. We learned to regard zinc sulfate as the treatment choice, of because this strategy combines effectiveness with safety, and comparison of our results with those obtained by others with penicillamine does not provide evidence that this chelation therapy is superior to the zinc strategy. In the following sections I will provide arguments supporting the concept that oral zinc is a better and more rational choice for the treatment of Wilson's disease than other agents. I will start with some historical facts about zinc therapy. Then I will summarize the major characteristics of Wilson's disease, so that the reader can appreciate the arguments when oral zinc therapy and treatment with penicillamine will be compared.

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362 11.

Hoogenraad HISTORY OF ZINC THERAPY

"Remedium, de cuius viribus experimentia aliquid boni the promittunt: the experiments promise something goodon zinc therapy, potentials of this remedy" Gaubius,about 1771. [ l ] The first scientific paperon zinc therapy dates back to 1771. It was written in Latin and is entitled "Luna fixata Ludemanni". The paper is a chapter of the book "Adversarium,

Figure 1

Hieronimua David Oaubiue (1704-1780)

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varii argumenti" written by Gaubius (Figure l). In this chapter Gaubius describes how he discovered that a mysterious drug, "luna fixata",sold by the renowned quack Ludeman, consisted of nothing else but zinc oxide [2]. Gaubius later describes how he studied the effect of zinc in the treatment of convulsions and spasms. He ends with the remark that it was to early to make statements about the reliability of the drug. "Probatae fuerint fidei...experientia constituator": "the reliability of the agents should be established by experience". In the 19th century oral zinc therapy became popular as an antiepileptic treatment. Zinc oxide was considered a very safe drug, even though the doses prescribed in those days were excessively high. 111. HEPATOLENTICULAR DEOEWBRATION (WILSON'S DISEASE)

Wilson's disease is a rare autosomal recessive disorder of copper metabolism resulting in a diminished biliary excretion of copper and retention of copper in the liver. Copper toxicosis develops when loosely bound (free) copper appears in the blood. Copper intoxication is dangerous and can lead to progressive hepatic, neurological, and psychiatric manifestations. Wilson (1912)[3] recognized the clinical syndrome caused by the disorder. He defined the new disease entity as being characterized by familial progressive lenticular degeneration and cirrhosis of the liver. Dysarthria, dystonia, and involuntary movements dominated in the clinical syndrome of the patients seen by Wilson. He had no experience of patients in whom cerebellar symptoms were predominant. Alzheimer (1912)[4] gave a detailed description of the neuropathological findings of a patient in whom cerebellar symptoms were found, and in whom the diagnosis pseudosclerosis had been made. Alzheimer recognized a new neuropathological entity characterized by abnormal glial cells in gray matter areas of basal ganglia, cerebellum and cortex. Several years later it would become evident that "familiar progressive lenticular degeneration with cirrhosis of the liver" and "pseudosclerosis" were one and the same disease [S], and that Kayser-Fleischer corneal rings were more or less specific for Wilson's disease. A. Clinical qndromerr

The first manifestations of Wilson's disease are hepatological in about 40 percent of patients. These manifestations most often appear between the dges of 6 and 12 years. Wilsonian hepatitis can be self-limiting, but it can also progress to chronic active hepatitis or fulminant hepatic failure. The latter form of hepatic Wilson's disease has a high mortality. In another 40 percent of patients the first symptoms are neurological. Movement disorders predominate. Three syndromes can be distinguished [6]. Firstly, a dystonic syndrome with involuntary movements and more sustained dystonic postures,

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and presentation before the age of 20 years. Secondly, a pseudocslerotic syndrome with postural and intention tremor, ataxia ofgait. often presenting after the age 20 years. Thirdly, a hypokinetic-rigid parkinsonian syndrome with rigidity and tremor at rest. In most patients the intelect remains intact even when there is severe neurological impairment. In a minority of patients psychiatric symptoms predominate. Other syndromes which may occur in Wilson's disease are hemolytic anemia, renal tubular dysfunction, osteochondritis dissecans, and cardiomyopathy. B Bi o. c h e m i s t r y

of

Wilson's

D i s e a( E s ee r e d i t a r y

Copper

Toxicosis) It was first recognized that copper toxicosis causes the symptoms of Wilson's disease when investigators found that the urinary excretion of copper was increased, and that levels of copper were raised in patients with the disease [ 7 1 . Several years later it was found that the concentration of ceruloplasmin, a major copper-binding protein, is decreased in Wilson's disease [ 8 ] and that the concentration of nonceruloplasmin bound copper is increased. Subsequently it was reported that the biliary excretion of copper is decreased 191, and a more recent finding makes it probable that there is a selective deficit in the excretion of a ceruloplasmin like copper-binding proteins in Wilson's disease [lo]. A decreased excretion of copper leads to an increased concentration of copper in hepatocytes, and this induces the synthesis of metallothionein in the nuclei of hepatocytes. Metallothionein is alow-molecular-weight copper binding protein that plays a key role in copper homeostasis. It is found in mucosal cells of the gut, hepatocytes, and glial cells of the brain. Copper bound to metallothionein is harmless, most of the copper in hepatocytes is bound to metallothionein. Recently we found that the concentration of metallothionein is greatly increased in gray and white matter of the cerebrum, cerebellum and brain stem of patients with Wilson'sdisease. In the normal brain, metallothionein is found the protoplasmic astrocytes in gray matter areas. The liver has an enormous capacity to accumulate copper without adverse effects. In healthy individuals the copper concentration is below 50 parts per million dry liver, in patients with Wilson's disease concentrations of up to 1200 parts per million dry liver are tolerated,and copper concentrations usually range from 250 to 3000 parts per million dry liver. Once the bindingsites are saturated, the patient will become symptomatic. When hepatocytes degenerate, the copper concentration in the liver may fall spontaneously. This is an important point because a decrease of copper Concentration is not nnecessarily a sign of amelioration of the copper status but may also occur in the natural course of the disease. Copper concentrations fall rapidly in the first year of treatment. Thereafter, there is no linear relationship between the duration of treatment and liver copper. poor compliers have a higher liver copper concentration than do good compliers [ll].

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The normal plasma copper concentration is 0.8-1.2 milligram per liter. In most patients with Wilson's disease the plasma concentration of copper is lower than this. This finding might seem paradoxical for patients with copper toxicosis, but is easily understood if one remembers that the concentration of the copper-binding protein ceruloplasmin is decreased in most patients with Wilson's disease, and below the normal level of 200 milligram per liter. Although the plasma copper concentration is decreased, the concentration of non-ceruloplasmin bound copper (free copper) is higher than normal (less than 0.1 milligram per liter) in most patients with Wilson's disease. The normal route of copper excretion is via the bile and the stools. Excretion of copper via urine is negligible and less than 0.1 milligram per 24 hours. In presymptomatic patients the level remains normal, but in symptomatic patients with Wilson's disease urinary copper excretion increases, and it is invariably above 0.1 milligram per 24 hours in these patients. The level is proportional to the level of free copper in the plasma. In severely ill patients with fulminant hepatitis, the urinary excretion of copper may increase to 6 milligrams per 24 hours. As a normal diet contains contains about 2 milligrams copper per day, the copper balance becomes spontaneously in these patients. C.

Diagnosis of Wilson's Disease

In many patients the diagnosis of Wilson's disease can be made on clinical grounds.. The coexistence of a movement disorder, liver disease, and Kayser-Fleischer rings is diagnostic. The Kayser-Fleischer ring is a golden-brown or greenish discoloration in the peripherial region of the cornea, it is caused by copper deposition in Descement's membrane. Virtually all patients with Wilson's disease with neurological findings have Kayser-Fleischer rings, but in many patients with hepatic manifestations the rings are absent. The diagnosis of Wilson's disease should be considered in every young adult patient with a movement disorder that cannot be explained, and in particular in patients below the age of 40 years who present with signs of parkinsonism, multiple sclerosis, or dystonia. The diagnosis should also be considered in young adults with behavioral and psychiatric problems resembling hysteria or schizophrenia; and in children or young adults with unexplained symptoms and signs of chronic active hepatitis, cirrhosis or acute hemolytic anemia. The concentration of ceruloplasmin in plasma is usually reduced. In 96 percent of patients with Wilson's disease the concentration of ceruloplasmin is less than 200 mg per liter. The total plasma copper concentration is usually reduced. The plasma concentration of non-ceruloplasmin bound or free copper can be determined from these two variables. Ceruloplasmin contains about 0.3 percent copper. The concentration of free copper in healthy individuals is less than 0.1 milligram per liter. In some patients a needle biopsy of the liver is needed

Hoogenraad t o establish the diagnosis. In untreated patients with Wilson's disease the hepatic copper concentration is generally markedly in excess of 250 microgram per gram dry liver (the normal limit is less than 50 microgram per gram). Urinary copper excretion is generally in excess of 0.1 milligram per 24 hours in almost all patients with neurological symptoms and signs. In difficult cases, measurement of the incorporation of radioactive copper into ceruloplasmin may resolve the diagnostic dilemma. D. Prevalence,

Oenetics and Zinc Fingers

Wilson's disease is rare and exhibits an autosomal recessive mode of inheritance. The prevalence at birth for the Netherlands has been estimated to be 1: 75.000 or about 13 per million. The often quoted figure of 1:35.000, or about 30 per million, is probably an overestimation [ 123. The gene for Wilson's disease has been localized on chromosome 13 and the locus for Wilson's disease has been found by making use of knowledge about the gene for Menkes e disease. The gene has all the aspects of a gene coding for a copper transporting P-type ATPase. Bow the gene interferes with metabolism so that the excretion of copper in the bile is disturbed and the incorporation of copper into ceruloplasmin becomes defective is unknown.

Induction of transcription by

Figure 2. Activation of metallothionein transcription by a transcription factor (TF) attached by its zinc fingers (ZF), to one of the metal responsive elements ( W )in the promoter zone of the gene, one of the nine upstream sites involved in expression of the metallothionein gene. Free copper (Cu), or another transition metal such as zinc, or cadmium, induces a morphological change in the inactive transcription factor.

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Genes also play a role in therapy of Wilson's disease, as some therapeutic strategies work by inducing metallothionein synthesis at the nuclear level. Activation of the transcription of the metallothionein gene results in the formation of metallothionein messenger RNA. Activation of receptor sites on the promoter zone of the metallothionein gene results in the binding of RNA polymerase to DNA. The receptor site on the promoter is called the metal-responsiveelement (MRE). It is a "transient response element" because activation stops when metallothionein has come to expression. The DNA fragments in the promoter zone can bind with an activating DNA-binding protein. This activator protein possesses very special structures called zinc fingers (Figure 2). The name refers to the fact that a zinc atom forms a complex with the polypetide chain in the DNA-binding region. The DNA-binding protein attaches with its zinc fingers to the metal-responsive element of the upstream site of the promoter zone of the metallothionein gene (Figure 3). IV. THERAPY OF WILSON'S DISEASE

Wilson's disease is one of the few chronic diseases of the liver and the brain for which an effective causal ther'apy

Figure 3. The transcription factor has a series of three zinc fingers, each with a characteristic pattern of cysteine (Cys) and histidine (His) residues that constitute the zinc-binding site.

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exists. The ultimate cause of the clinical signs and symptoms is the damage caused by the increased concentration of the free, diffusible, copper in the blood and tissues. Treatment aims at improvement or prevention of copper toxicosis. Therapeutic measures should normalize the plasma free copper concentration andprevent that they increase.Long-term treatment is aimed at decreasing the sequestration of copper, resulting inthe fading of Kayser-Fleischer rings anda decrease in the liver copper concentration. The main strategies for drug treatment of Wilson's disease which have been developed are: A) chelation therapy; B) oral zinc therapy. A. Chelation Therapy

-

Chelating agents (Greek: chela prehensile claw of a crab) form stable complexes with heavy metals and promote the excretion of copper in the urine. In general, the affinity of these agents for metal ions is stronger than that of endogenous proteins. Copper ions are toxic because they complex with endogenous proteins bearing thiol-groups (+H) or amino-groups (-NH2). Chelating agents detoxify by competitively complexing copper attached to these proteins. In the majority of patients chelation therapy 1ec.i~ to amelioration of clinical symptoms. In a minority of patients a paradoxical response occurs [13,14] 1 . Chelating Agents

Penicillamine is the most often used chelating agent. It was discovered by Walshe (1953)[15] discovered this chelating agent in the urine of patients with hepatic disease who were treated with penicilline. Penicillamine is R,R-dimethyl-cysteine is a monothiol and a derivative of penicilline [16]. It is an effective chelator of copper, mercury,. zinc, and lead, with which it forums stable, water soluble complexes, and promotes the excretion of these metals in theurine. Advocates of chelation therapy consider penicillamine the treatment of choice for Wilson's disease. When penicillamine does not work or side effects occur, then trientine can be used. British anti-Lewisite (BAL) is used as third-line chelating treatment for neurologlcal patients whodo not improve with peniicillamine and trientine. 2 . Mechanisms of Action of Penicillamine

The mechanism by which the drug works is not completely clear. The traditional explanation is that penicillamine induces decoppering by promoting the urinary excretion of copper to such an extent that the copper balance becomes negative [17201. The copper-penicillamine complexes are thought to have low toxicity. However, penicillamine might also induce the synthesis of metallothionein. It has been suggested that with the induction of metallothionein synthesis inthe liver,

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potentially noxious free copper ions in the hepatocytes might be converted in harmless copper-metallothionein complexes v91 Animal studies demonstrate that penicillamine increases the level of metallothionein messenger RNA in hepatocytes, by liberating copper from some intermediary ligand, thereby making copper available to induce metallothionein synthesis. This induction of metallothionein by penicillamine might be part of the therapeutic action of the chelator in the treatment of Wilson's disease. Thus patients with Wilson's disease may not be truly "decoppered" in thhe true sense but rather copper is accumulated in a nontoxic form by penicillamine-induced metallothionein [21-231. This putative mode of action of penicillamine is comparable with that of zinc. 3. Dosage of Penicillamine

Walshe [l91 advised starting treatment with relatively high dosages of 1.5 gram to 2.0 gram per day and to adjust the dose on the individual requirements. The drug should be taken in four dosages per day on an empty stomach, 30 minutes before meals. For maintenance therapy it was recommended that the dose be reduced the dose to 1 gram per day under cakefully monitoring of the free-copper serum fraction. Very high doses of 3 gram [ 19 3 or even 4 gram have been suggested for a few months only at the start of treatment in order to achieve rapidly a strongly negative copper balance. Other authors recommemd starting treatment with lower dosages. Scheinberg and Sternlieb [l51 advised a dose of 250 milligram four times per day. For younger children the daily dose of penicillamine should be 20 milligram per kilogram weight, rounded to the nearest multiple of 250 milligram. If neurological symptoms worsen after the start of penicillamine therapy or if serious side effects occur, the dose should be reduced and subsequently gradually increased over several weeks to months to the therapeutic level of 1 gram per day. Another approach is to start all patients, symptomatic and presymptomatic on low doses of penicillamine with gradual increments to the therapeutic range. Thus treatment is started with 125 mg per day during the first week. The daily dose is increased by 125 mg every week until a dosage of 1 .O-1.5 g per day is reached [24], but even this cautious regimen is no guarantee against "paradoxical" worsening [25]. A daily dose of 25 mg pyridoxine is usually added to the regimen because of a possible antipyridoxine effect of penicillamine. 4 . Effectivity of Penicillamine

Although treatment with penicillamine regularly leads to clinical improvement, it is slow, and during the first six to eight weeks the symptoms and signs may worsen. Most doctors who prescribe penicillamine advise their patients to continue treatment in spite of an apparent lack of clinical improvement. When treatment is started at an advanced stage of

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the disease, restoration of normal function should not be expected.If the patient survives the first months of treatment, significant neurological improvement with a marked decrease in rigidity and tremor occurs in the majority of patients. Similarly, clinical symptoms and signs of liver disease improve, and abnormal liver function tests gradually return to normal. The effectiveness of treatment can be monitored by measuring plasma copper and ceruloplasmin levels. As copper is excreted , these levels fall, because the plasma free copper fraction returns to normal. Although penicillamine is an effective cupriuretic agent it is rather slow in inducing clinical improvement in Wilson's disease. When treatment is started with dimercaprol (BAL), the clinical response is much more rapid. Nevertheless, after some delay, most patients will show a quite remarkable recovery of both hepatic and neurological function, and in the majority of patients the biochemical disturbances will improve after three months and the first signs of clinical improvement will appear a few weeks later [ 191. In general, it can be said that the degree of recovery will depend on the stage of the illness when treatment is started. If irreversible brain damage has already occurred or if contractures of the limbs have become established then, inevitably, recovery will be incomplete. Most patients can be expected to make a useful recovery and return to a normal way of life [26] 5. Paradoxical Worsening and other side effects

A major drawback of chelating agents is that they can induce paradoxical worsening of neurological manifestations. This phenomenon is called paradoxical because is occurs notwithstanding an increased urinary excretion of copper [13]. The phenomenon is not restricted to penicillamine or dimercaprol, or to patients with Wilson's disease. Kamerbeek and co-authors [l41 saw this paradoxical deterioration of neurological abnormalities intwo patients with thallium intoxication, who deteriorated while being treated with the chelating agent diethyldithiocarbamate. The authors argued that the paradoxical reaction is caused by a redistribution of the metal. Animal studies have shown that administration of the chelator results in the formation of metal complexes that can pass the blood-brain barrier and exert anoxious influence in the brain. Scheinberg and Sternlieb regard this paradoxical response of patients with neurological disease to penicillamine an important hazard of penicillamine therapy. They found that it occurred in about 10 per cent of patients with neurological manifestations 1131. These authors consider it essential to warn patients with neurological disease that deterioration may occur, in order to stop patients from abandoning the treatment in dismay. It is their experience that continued administration of the drug is almost always followed by steady improvement. It has also been described that asymptomatic patients with Wilson's disease become symptomatic during penicillamine treatment.

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Penicillamine is a typical orphan drug. Little was known about its adverse effects when the drug was introduced. Nowadays it is known that penicillamine has frequuent side effects although many of them are reversible when the drug is withdrawn. Gastrointestinal disturbances include anorexia, nausea, and vomiting may occur. Impaired taste is common. About 20 percent of patients develop early allergic side effects in the first month of treatment. The commonest are fever, rash, and lymphadenopathy. They are usually transient but temporary drug withdrawal and treatment with corticosteroids may be required. Penicillamine should be reintroduced at a low dose (0.25 gram daily) and then gradually increased to 1 gram daily over about a month. Steroids should be gradually withdrawn. A more serious early reaction is marked depression of bone marrow function with granulocytopenia and thrombocytopenia. Late reactions to penicillamine are seen after a year or so of treatment. The commonest late reaction is penicillamine dermatopathy, caused by damage of collagen andelastin, resulting in weakening of subcutaneous tissue so that slight trauma may cause bleeding and residual areas of brown papues as well as excessive wrinkling and thinning of the skin. Elastosis perforans serpiginosa most commonly appears on the neck and axilla. About 5 percent of patients develop proteinuria, which may progress to nephrotic syndrome. Other problems include the emergence of a form of systemic lupus erythematosus, thrombocytopenia, Goodpasture's syndrome and myasthenia gravis. Scheinberg and Sternlieb reported that these late side effects were recurrent in only 2 percent of their patients treated with penicillamine and were the reason to change patients to trientine. Discontinuation of penicillamine in patients with Wilson's disease has been reported to be dangerous and to result in rapid clinical deterioration, which is often fatal [27][Table l]. Table 1. Characteristics of Penicillamine

1. 2. 3. 4. 5.

Typical orphan drug Induces urinary excretion of copper Leads to slow clinical improvement Risk of worsening of neurological symptoms (10%) Early allergic side effects (25%) 6. Late side effects (10%) 7. Minor gastrointestinal disconfort

B. Zinc Therapy 1 . Schouwink's Report on E a r l y Copper Balance Studies in 1961

Zinc therapy was developed by Schouwink in The Netherlands shortly after penicillamine had been introduced by Walshe [28]. Two facts stimulated Schouwink (1961) to investigate the effect of zinc in Wilson's disease, namely that zinc

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supplements can induce copper deficiency in animals; secondly, he was familiar with the fact that zinc sulphate is a harmles drug, and that zinc is included in the Dutch pharmacopoeia as a safe drug without serious side effects. Schouwink was the first to describe that the administration of zinc sulphate can lead to a negative copper balance by increasing fecal copper excretion. 2. N-of-l Trial as

Prove

of Effectiveness of Zinc Sulphate

The University Department of Neurology in Utrecht took over the management of one of the two patients put on zinc sulphate by Schouwink. The patient has been using zinc sulphate in a dose of 100 mg three times daily for more then 1 0 years. He had no complaints. ais neurological condition had improved. The tremor had subsided. Dysarthria had improved. Most astonishing was that the Kayser-Fleischer rings had faded away. Before we concluded that zinc therapy was effective in this patient with Wilson's disease we conducted a "trial of therapy". Zinc therapy was discontinued and the patient was seen every six weeks. About a year after the cessation of zinc therapy, tremor returned and a hypokinetic rigid syndrome was found. Zinc sulphate was re-administered successfully [ 2 9 ] . Our experience with this patient, and especially the disappearance of the Kayser-Fleischer rings was reason for us to be convinced that oral zinc therapy is effective In Wilson's disease 1301. The disappearance of the corneal rings is a clear sign of effective decoppering as spontaneous disappearance of these rings has never been reported. 3. Induction of metallothionein synthesis by zinc

Zinc supplements antagonize the absorption of copper in the gut. The basis for this effect is the capacity of zinc to increase the concentration of metallothionein in the mucosa by up to 25-fold [ 3 1 ] . As the tissue content of metallothionein increases, the proportion of copper in the cytosol increases, either because copper is incorporated into metallothionein during the synthesis of the protein or because copper displaces zinc from binding sites on theprotein. As the liver is the main storage organ for copper, and metallothionein is one of the major copper-binding proteins in the liver, the induction of metallothionein synthesis, patients with Wilson's disease often have an increased concentration of in copper in the liver during zinc therapy [ 3 2 ] . It is probable that zinc therapy protects against copper toxicity in the liver by inducing the synthesis of hepatic metallothionein, which sequesters copper in a non-toxic form 1331

4. Decrease of copper absorption during zinc supplementation Since copper has a greater affinity than zinc for binding sites on metallothionein, it displaces zinc from the protein.

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In this way, copper from the gut and from the diffusible copper fraction in the blood is bound to the mucosal lining of the intestine. When the mucosal cells are sloughed off, (renewal occurs about every 3-6 days), copper is excreted via the stools. By inducing metallothionein synthesis, zinc not only impairs the absorption of exogenous copper but also the absorption of endogenous copper present in intestinal fluids, a form of copper which would otherwise be reabsorbed through the enterohepatic circulation [31]. Changes in the absorption of copper can be assessed with an oral radioactive copper loading test. In this test, the appearance of orally administered radioactive copper in plasma is monitored. Under normal conditions, a peak is seen approximately two hours after the dose is given. In healthy controls this peak is followed by a decline and a second rise in plasma radioactive copper, which is caused by the appearance in the circulation of radioactive copper incorporated in the liver in the plasma protein, ceruloplasmin. This second rise is absent in patients since they do not with Wilson's disease (Figure 4 ) , incorporate copper into ceruloplasmin. When a patient is treated with zinc sulphate, absorption from the ?U! is suppressed: the initial peak two hours after dosing dimlnlshes and may even disappear completely [30].

Oral radiocopper loading test

,

radiocop er in plasma ('6 dosis Cu / m1 x 10

ti

zinc sulfate:

"""""""""""""""""""""""""""""""""""-"""" " 0

Figure

***..*..*..*'.

Io time (hours)

4. Decrease in copper absorption during zinc supplementation in Wilson's disease. Oral radioactive copper loading test before and after oral administration of zinc sulfate, 3 x 200 mg per day.

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5. Inducement of copper

excretionvia the

stools

PatientswithuntreatedWilson'sdiseasehaveapositive copper balance because less copper is excreted in the stools than normal [28]. This is because the excretion of copper into bile is diminished. After the administration of zinc sulfate and after induction of mucosal metallothionein synthesis, the coppercontentoffecesincreasesbecause ofdecreased absorption of copper from the food and from intestinal fluids. Moreover, free diffusible copper from the blood is bound to the mucosal metallothionein and is excreted via the stools when the cells are sloughed off (Figure 5). 6. Zinc-induced

copper

deficiency

Chronic oral administration of zinc induces a negative copper balance, and can ultimately result in copper deficiency. The main features of this rare syndrome are anemia and neutropenia, both of which respond to copper supplementation

Copper balance in Wilson's disease m# Cu / 2 4 h

" "_

before zhc during zinc normal fiCu in feces W Cu jn urine Figuro 5. Influence of zinc sulfate on copper balance in a patientwithWilson'sdisease. A. Beforezinctherapy: excretion of copper via the stoolsisdecreased.Copper balance is positive although excretion of copper viathe urine is higher than normal. B. During zinc therapy with 3x200 mg zinc sulphate: copper balance is negative. Excretion of copper via stools is more than the daily intake of copper (1.5 mg per day). Excretion via urine has decreased. C. Normal control: balance is in equilibrium. Excretion of copper via urine is very low.

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[ 341. Anemia as part of the syndrome may easily be mistaken to be caused by iron deficiency.

7. Zinc sulfate as best choice for

Wilson*sdisease

The results of a study involving 27 patients and 142 patientyears were published in 1987. Nine patients were treated with zinc from the start of treatment, 8 were placed on zinc after they had developed intolerance to penicillamine and 10 were changed to zinc after they hadfirst been treated with penicillamine without developing signs of intolerance. The drug was administered in doses ranging from 300 to 1200 mg/day. In the first group 8 patients improved and 1 died from severe cirrhosis,in the second group, all patients improved, and in the third group 8 patients improved and 2 were changed back to penicillamine because of personal preference. Signs of intolerance to zinc sulfate were not observed [32],[Table 21. 8. Zinc sulfate: fonnule and mode of administration

Zinc sulfate, ZnS0.7H20, molecular weight: 287.5, is an odorless, white, crystalline powder. It is included in the pharmacopoeias of most countries. Zinc sulfate is usually administered in tablets or capsules. For the treatment of Wilson's disease, the usual dose is up to three times 300 milligram per day (200 milligrams zinc daily) for adults, and up to three times 150 milligram per day ( 1 0 0 miligrams zinc daily) for children. Table 2. Characteristics of Zinc T h e r a w

1. 2. 3. 4. 5.

Zinc sulfate is harmless Zinc sulfate induces fecal excretion of copper Clinical improvement is not slow No risk of paradoxical worsening No allergic side effects 6. No late side effects 7 . Minor gastrointestinal side effects

9 . Monitoring of zinc therapy

Careful assessment of the effect of therapy and monitoring of the patient is important in deciding whether the dosage is optimal. Patients should be seen regularly in the outpatient department, at least every 2 weeks in the first months and every six weeks or three months when long-term therapy is given. Patients should be monitored not only be on the basis of subjective .clinical evaluation and reliable laboratory tests. Experience has indicated that normalization of free plasma copper concentrations is a good intermediate goal of copper-removing treatment in Wilson's disease. Measurement of diffusible, non-ceruloplasmin-bound copper levels can give an indication of wether the treatment is working and whether the dose is sufficient. The concentration of diffusible copper is determined by subtraction of ceruloplasmin-bound copper (0.3%

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of ceruloplasmin) from the total plasma copper concentration. In untreated patients or in inadequately treated patients, the diffusible copper level is generally considerably higher than 0.10 mg/l and may exceed 0.50 mg/l. In adequately treated patients, it should be less than 0.10 mg/l. The level may normalize in the course of several weeks after the start of treatment. Treatment usually results inan improvement of the hepatic dysfunction. However, liver function tests are not a reliable means of assessing whether the copper-removing Table 3.

Comparison of Penicillamine and Zinc Sulphate Penicillamine

Dose, milligrams per day 3x300 Increased excretion + urinary copper fecal copper Decreased copper absorption + Metallothioneion induction + Proven clinical efficacy Type of evidence for proof in controlled trial N=1 trial Decreased copper accumulation: + -free copper in blood + -Kayser-Fleischer ring +/-liver copper +++ Effectiveness Adverse reactions +++ paradoxical worsening +++ sideeffects

--

-

Zinc sulfate 3x300

-

+ +++ +++ 4.

-

+ + + +/+++

-

+

treatment is adequate as many patients with severe Wilson's disease have a normal liver function and effective copper removal is not always accompanied by normalization of liver function. Liver biopsies provide valuable information about the effect of treatment, but can hardly be accepted as a routine procedure. We would not advocate the taking of liver biopsies more than once every two years. Biopsies should be investigated for histopathological abnormalities and analyzed quantitatively for copper and zinc. Interpretation of the results should take into account the fact that improvement of liver function may be accompanied by an increase in liver copper concentrations. We aim at adecrease in the copper/zinc ratio and increase thedosage when the ratio does not decrease. In general, the first effects of treatment can be expected in the first weeks after the start of treatment. We have found that the dosage has to be increased to three times 300 mg for the majority of patients.

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Two of our patients have now been on zinc sulphate therapy for more than thirty years. They are in good condition, their free-copper concentration is normal and Kayser-Fleischer rings have vanished.

10. Alternatives to Zinc Sulfate: Zinc Gluconate, Zinc

Lactate

We have used zinc gluconate in a patient who did not tolerate penicillamine or zinc sulfate. However, she did not tolerate zinc gluconate any better than zinc sulfate. Zinc lactate has also been studied for the treatment of Wilson's disease [35]. In the first reports on zinc lactate the drug was given every four hours,but later reports describe that the copper balance can be controlled effectively by a regimen of 50 mg zinc (as lactate) taken three times a day. Monitoring of patients on zinc lactate has been done in the same way as described for zinc sulfate. Several studies on the effect of zinc lactate on Wilson's disease have been published. The major importance of these reports on zinc lactate is that they confirm the effect of zinc on copper balance, copper absorption, and liver copper obtained with zinc sulfate. They provided no data suggesting that zinc lactate might be a better choice than zinc sulfate, butrather confirm that zinc is an advance in the treatment of Wilson's disease[36,31,38]. REFERENCES 1. E.D. Gaubius, Luna fixata Ludemanni,in: "Adversariorum varii argumenti", Luchtmans, Leiden, 1771. 2. T.U. Hoogenraad, Trace Elements Med. 1:47-49 (1984). 3. S.A.K. Wilson, Brain. 34:295-507 (1912). 4. C. Von E681in andA. Alzheimer, Zeitschr.Ges.Neuro1.Psychiatr. 8:183-209 (1912). 5. H.C. Hall, La degenerescence hepato-lenticulaire: maladie de Wilson-pseudosclerose, Masson, Paris, 1921. 6. C.D. Marsden, Quart.J.Med. 248:959-966 (1987). 7. J.N. Cumings, Brain 71:410-415 (1948). 8. I.H. Scheinberg and D. Gitlin, Science 116:484-485 (1952). 9. I. Sternlieb, C.J. Van-den-Eamer, A.G. Morell, S. Alpert, G.Gregordiadi.8, and I.E. Scheinberg, Gastroenterology 64:99-105 (1973). 10. V. Iyengar, G.J. Brewer, R.D. Dick, and O.Y. Chung, J.Lab.Clin.Med. 111:267-274 (1988). 11. K. Gibbs and J.M. Walshe, J. Gastroenterol. Hepatol. 5:420-424 (1990). 12. R.E. Houwen, J. van-Hattum, and T.U. Eoogenraad, Neth.J.Med. (1993). 13. I.E. Scheinberg and I. Sternlieb, Wilson's Disease. Volume 23 in the series Major Problems in Internal Medicine, Saunders W.B. Company, Philadelphia, 1984. 14. H.E. Kamerbeek, A.G. Rauws, M. Ten-Em, and A.N.P. Van-Eeijst, Acta Med. Scand. 189:149-154 (1971). 15. J.M. Walshe,Quart.J.Med. 22:483-507 (1953). 16. E.P. Abraham, E. Chain, W. Baker, and R. Robinson, Nature 151:107 (1943).

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17. J.N. Cumings, B r a i n 74:lO-22 (1951). 18. J.M. Walshe, Lancet 1:25-26 (1956). 19. J.M. Walshe, Wilson's disease (hepatolenticular deqeneration),.int "Handbook of Clinical Neurology, Metabolic and Deficiency Diseases of the Nervous System. Vo1.27, Part I", P.J. Vinken et al., eds., American Elsevier, New York, 1976, pp. 379-414. 20. A. Deiss, Ann.Int.Med. 99:398-400 (1983). 21. I.H. Scheinberg, I. Sternlieb, M. Schilsky, and R.J. Stockert, Lancet 2:95-95 (1987). 22. H.J. McArdle, P. Kyriakou, A. Grimes, J.F. Mercer, and D.M.Danks,Chem. Biol. Interact. 75:315-324 (1990). 23. A. McQuaid and J. Mason,J. Inorg. Biochem. 41:87-92 (1991). 24. S. Ivanova-Smolenskaya, S. Timerbayeva, and T. Mzhelskaya, Analysis of the effectiveness of long-term treatment of hepatolenticular degeneration with copper chelating agent and with zinc sulphate, in: "Proceedings of the 5th Symposium on Wilson's disease held at Madralin (near Warsaw, Poland) on june 20-23,1990", A. Czlonkowska et al., eds., Technical University, Delft (The Netherlands),l991, pp. 37-42. 25. C. Veen, C.J. Van-den-Hamer, and P.W. de-Leeuw, J.Intern.Med. 229:549-552 (1991). 26. J.M. Walshe, The treatment of Wilson's disease, with observations based on 134 patients presenting with neurological signs, in: "Proceedings of the 5th symposium on Wilson's disease held at Madralin (near Warsaw, Poland). on June 20-23,1990", A. Czlonkowska et al., eds., Technical University, Delft(The Netherlands),l991, pp. 5-10. 27. I.H. Scheinberg, M.E. Jaffe, and I. Sternlieb, N.Engl.J.Med. 317:209-213 (1987). 28. G. Schouwink,.De hepatocerebrale degeneratie, met een onderzoek naar de zinkstofwisseling. Ph.D. Thesis, (with a summary in English, French and German), Univ. Amsterdam, 1961. 29. T.U. Hoogenraad, R. Koevoet, and E.G.W.M. De-Ruyter-Korver, Eut. Neurol. 18:205-211 (1979) 30. T.U. Hoogenraad, C.J. Van-den-Hamer, R. Koevoet, and E.G. Korver, Lancet 2:1262-1262 (1978). 31. C.J.A. Van-den-Hamer, T.U. Hoogenraad, and E.R.K. Klompjan,Trace Elements in Med. 1:88-90 (1984). 32. T.U. Hoogenraad, J. van-Hattum, and C.J. Van-den-Hamer, J. Neurol. S c i . 77:137-146 (1987). 33. D.Y. Lee, G.J. Brewer, and Y.X. Wang, J.Lab.Clin. Med. 114:639-645 (1989). 34. A.S. Prasad, 'G. J.'Brewer, E.B. Schoomaker, and P. Rabbani, J A M A 240:2166-2168 (1978). 35. G. J. Brewer, G.M. Hill, k.D. Dick, T.T. Nostrant, J.S.Sams, J.J. Wells, and A.S. Prasad, J.Lab.Clin.Med. 109:526-531 (1987). 36. G.M. Hill, G.J. Brewer, A.S. Prasad, C.R. Hydrick, and D.E. Hartmann, Hepatology 7:522-528 (1987). 37. G.M. Hill, G.J. Brewer, J.E. Juni, A.S. Prasad, and R.D. Dick, Am.J.Med. Sci. 292:344-349 (1986). 38. G.J. Brewer, G.M. Hill, A.S. Praead, Z.T. Cossack, and P. Rabbani, Ann Intern. Med 99:314-319 (1983).

24 TranscriptionalRegulationand Function of Yeast Metallothionein Zhiwu Zhu, Mark S. Szaypka, and Dennis J. Thiele Department of BiologicalChemistry,TheUniversity of MichiganMedicalSchool,M5416 Medical Science 1, 1301 Catherine Road, Ann Arbor, MI 48109-0606

L INTRODUCTION The role of metal ions in biological systems, whether structural, catalytic or regulatory, is virtually governed by their chemical properties (1-4). Although the behavior of metal ions in biological systems has been studied in depth for decades, a comprehensive understanding and appreciation of their interactions with biological macromolecules has not yet been elucidated. Metal ions, as modulators for many biomolecules and cofactors in a variety of enzymatic reactions, can interact withbiologicalsystemsandthereforeinfluencethenormal growth,developmentandfunctions of livingorganisms by fulfdling versatile biological requirements (33. However, an elevated concentration of even essential metal ions such as Cu, as Cd and Fe or Zn, or non-biologically relevant metal ions such Pb are deleterious and toxic to living organisms. Toxic metal ions, due to their increasing abundance and bio-availability, can compete with essential metal ions for enzyme-protein active sites, ion channels, membranes and other important biological ligands. Consequently, the interactions of toxic metal ions with biological systems can alter cell growth, function and disrupt normal metabolism (2). Redox active metal ions such asCu and Fe can cause toxicity by reactingwith metabolites such as H202 or 02 to generate free radicals such as HO. and 0 2 . (6). Free 379

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radicals, due to their very reactive and readily diffusible nature, can oxidize biomolecules suchas proteins, DNA, lipids and other macromolecules which can eventually lead to disease states(7). Therefore, cells must possess effective mechanisms to manage metal ionsto avoid these undesirable effects. Copper is an essential element which can also be cytotoxic to living organisms (8). Due to its multiple oxidation states (Cu+ and C$+) and coordination by a varietyof biological ligands, Cu serves as a cofactor for as manyas thirty enzymes suchas Cu,Zn superoxide dismutase, which protects cells from oxidative stress by reducing free radicals, and cytochrome C oxidase, which plays a critical a role in electron transport during cell respiration(9). As opposed to its essential function, Cu is one of the most important reactants in the generation of free radicals within cells, via the following reactions(6):

or

The toxic yet essential nature of copper in biological systems demands that cells maintain a delicate balance between essential and toxic levels. Human genetic diseases havebeen described in which there are defects in copper homeostasis. Wilson's disease is characterized by excessive accumulation of copperin patient's liver andbrain.Menkes' diseasehasbeenascribedto the inadequate mobilization and transport of Cu which leads to improper cellular copper accumulation, resultingin a deficiency in theactivity of copperdependentenzymes.Recently,the familial form ofLou Gehrig's diseasehas been demonstrated to be associated with mutations in the human gene encoding Cu,Zn superoxide dismutase, which functions in free radical detoxification (10-15). In this review, we focus largely on the molecular response toCu in signal transduction to activate gene transcription. How do organisms strike a balance to accumulate appropriate levelsof essential metals, and yet prevent the elevated accumulation of toxic metals? Many organisms, from bacteriato humans, protect themselves from metal toxicity by synthesizing metallothioneins (MTs), low molecular weight, cysteine-rich metal-bindingproteins (16). It is believedthatMTsplay

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important roles in both metal homeostasis and heavy metal detoxification. In response to elevated concentrations of metal ions suchas copper, MT genes are biosynthetically inducedat the level of transcription (8,17). Although thefamily of MT genes in humans is transcriptionallyactivated by copper,nohuman copper-responsive transcription factors have been isolated. Two metal responsive transcription factors have been isolated from yeast S. cerevisiae and C. glabrata, ACE1andAMT1 respectively (18,19). Due to the relative experimental simplicity of yeast and its well characterized genetics, as a unicellular eukaryotic microorganism, this organism provides an excellent model for thestudyofmetal-regulatedtranscription.Two in the area of previous articles have given comprehensive reviews copper-mediated MTgene transcriptionalregulation(8,20). Therefore, in this chapter, we present a review of the current literature dealingwith copper-regulated MT gene transcription in yeast, and the impact of developments in the elucidation of homeostatic mechanisms.

IL THE BAKER'S YEAm Saccharomyces ceredsiae Metallothioneins (") are ubiquitous molecules which function to protect organisms from exposure to high environmental concentrations of metals (16). Many eukaryotic organisms possess metallothionein (MT)gene families whichare composed of several distinctMT genes. An excellent exampleof this phenomenon is found in micewhichhavea well characterized gene family containing MT-I, MT-11, MT-III and perhaps other MT genes (21-23). Expression of the MT-I and-11 genes is critical for protectionagainstexposure to high concentrations of cadmium (24). A number of studieshave established that MT biosynthesis in humans, mice and other organisms is induced at the level of transcriptionin response to a spectrum of metals including, cadmium (Cd), zinc (Zn) and copper (Cu) (8). In contrast to these organisms, in the baker's yeast, Saccharomyces cerevisiae, a single metallothionein gene (CUPI) has thus far been identified (16). Biosynthesis of the yeast CUP1 protein is induced in response to environmental copper and silver, however in contrast to mammalian cells, normal S. cerevisiae strains exhibit no significant biosynthetic as Cd induction of MT upon exposure to other heavy metals such (25).The CUP1 gene encodes a 61 amino acid polypeptide

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containing 12 cysteineresidues. NMR studies of silversubstituted yeast MT demonstrate that 10 of these 12 cysteine residues are involved in the tridental and bidental coordination of copper (26), bound as Cu(I) with an apparent stoichiometry of seven to one. CUP1 gene deletion experiments in S. cerevisiue have demonstrated that MT is not required for growth under standard laboratory conditions, however, CUP1 is critical when yeast cellare exposed to high concentrationsof exogenous copper (25). These observations confm that yeast M" is an important player in the copper detoxification pathway. Copper-activatedtranscription of the CUP1 gene is regulated in large part through the action of a copper binding transcriptionactivatorprotein ACEl (also known as CUP2) (28,29). ACEl resembles MT in that it contains 12 cysteine residues, and like MT undergoes a conformational change upon copperbinding (30). Thecopper-inducedstructuralchange facilitatesbindingof ACEl to cis-acting metal-regulatory elements located in theCUP1 promoter (31). This region of the CUP1 promoter has been designated as the CUP I upstream Activation Sequence (UAScUpI) (32). A series of biochemical experiments includingin vitro and in vivo DNase I footprinting, in vitro hydroxyl radical footprinting and in vitro DNA mobility shift assays, have clearly demonstrated that UAScupl the contains four protected regionsof DNA which support the binding of four monomeric ACEl molecules (Fig. 1) (31-34). Upon binding to the UAScup1, ACEl proteininduces CUP1 transcriptional activity, increasing steady state levels of CUP1 mRNA which results in elevated MT protein concentration (Fig.2) (18,35). It is generallyacceptedthat this increase in MT protein affords protection against the potential cytotoxic properties of this metal by chelating copper ions and thus neutralizing the reactive nature of this element (1634). However, recent studies have identified other roles forMT in S. cerevisiue (36-38). CUP1 transcription is induced in response to a variety of stresses in addition to the well documented ACEl mediated copper-activation. Genetic and biochemical experiments which addressed the biologicalsignificance of CUP1 transcription induction under conditions of oxidative stress have shown that copper M" [&(I)-MTJ or monkey MT-I or -11 can functionally substitute for the S. cerevisiue Cu,Znsuperoxidedismutase (SODI)and in addition has identified an anti-oxidant activity of purified yeast &(I)-MT (36). These data demonstrate that CumMT is important in the oxidative stress response and may suggest

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383

A.

B.

Collserwur

Blndlng Stte

ACE1 0 HSF m

TCY (M) GCTG GAA NW TTC

Figure 1 (A) Schematic diagram of the CUPl gene organization. ACEl and potential HSF binding sites in the UAS, are depicted. (B) ACEl and HSF binding site consensus sequences are shown. Y = pyrimidine; N = G, A, T or C.

Apo-ACE1

W

k

CU' Activation

Figure 2 Schematicrepresentation of Cu(I) activation of apo-ACE1and ACEl bindmg-mediated transcriptional activation of CUPl gene.

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an additional mechanismfor Cu(I)-MT in copper detoxification. Copper is a highly reactive metal which can participate in the Fenton-like reactions which generate superoxide and hydroxyl radicals (6). In addition to direct copper chelation, a potential mechanism employed by Cu(1)-MT in metal detoxification may be the protectionagainstsuperoxide or hydroxyl radicals generated by copper. This process may involve the direct binding or "scavenging" of these reactive species and/or possibly involve the utilization of an enzymatic dismutase reaction much like the mechanism which has been established for Cu,Zn superoxide dismutase (38,39). It is also entirely possible that Cu(1)-MT may as yet unidentified function in thiscapacitythroughsome mechanism. Recently, pulse radiolysis studies have determined second order rate constantsfor binding reactions between p d l e d S. cerevisiue Cum-MTandHO.and of 2.2~1011M-1 S-1and 7.5~106M-1 S-1 respectively (38). Theseobservations are consistent with the CuO-MT acting as a highly efficient free radical scavenger, as has been demonstrated in vitro for rabbit CdMT (37). Although the biological role for yeast Cu(I)-MT in protection against oxidative stress has been established in vitro and in vivo, the factor or factors responsible for the induction of CUP1 transcription underthese conditions remaintobe identified.

Otherconditionsandfactorswhichactivate transcription

CUP1

The potential link between MT and oxidative stress is postulated due to the radical generating capacity of copper and the genetic and biochemical data accumulated from a number of experiments addressing this phenomenon, however, the role of MT in protecting cells from heat shock is unclear. Although MT function in this process is not completely understood, the factor responsible for the induction of CUP1 transcription at elevated temperatures has been identified as the heat shock transcription factor (HSF) (40,41). DNA mobility shift assays using CUPI promoterfragmentsdemonstratethat HSF is able to bind efficiently to a DNA hgment which encompasses from bases -91 to -210 in the CUPI promoter (40). This fragment contains 2 HSF-binding site consensussequenceswhich are potential binding sitesfor HSF (Fig.l) (40). Experiments to further address the role of MT inthe heat shock response and the mechanism of HSF mediated activation ofCUPI are of extreme importancein

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understanding the complex nature this of promoter andto further our understanding of MT function in S. cerevisiae. Under exposure to copper, CUPI is transcriptionally activated by ACEl bindingto the UASCUpl.However, in copper-sensitive acel deletion strains which lack copper-mediated ACEl induced CUPI transcription,there is stilladetectable basal level of Cup1 mFWA (42). The ACE2 gene activator of CUP1 Expression whichencodesaputativezinc finger protein,wasisolated as a high copysuppresser of an ace1 deletion strain and was demonstratedto be required to maintain the observed basal level transcription of CUPI (42). Although ACE2 is important for C U P 1 basal level transcription, the mechanism through which ACE2 mediates this effect is not known.

a,

Potential regulatorsof copper bioavailability in S. cerevisiae

One of the major focuses ofall organisms is to maintaina homeostatic intracellularenvironment. When cells are faced with adverseenvironmentalconditions,such as exposure to high concentrations of environment metals, they must respond in a fashion which allows for the maintenance of the balanced state. Two of the major players in copper homeostasisin S. cerevisiae are the CUP1 and ACEl proteins, however, recent work has begun to identify additional cellular moleculesincluding membrane-associated copper transporters, additional putative metal regulatory transcription factors and organelles which are intimately involved in this process (Fig 3). These factors will undoubtedly play important roles in influencing and potentially regulating MT expression and functionin S. cerevisiae. Dancis and colleagues have identifieda gene encodingan integral transmembrane protein, designated Copper Transporter1 (CTRI), which is required for high affinity copper uptake (43). The presence and function ofboth low and high affiiity copper transporters, such as CTR1, at the plasma membrane or other intracellular organelles of yeast cells must by their intrinsic nature affect the availability of copper to ACEl andothermetalof activated transcription factors, thereby influencing transcription the CUP1 gene and other genes subject to copper regulation. ACEl also activates SOD1 gene transcription in response to high exogenouscopperconcentrations (44). Recentevidencehas implicated the vacuolein copper resistance inS. cerevisiae (45). In particular, strains which harbor a deletion of the VMA3 gene

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Figure 3 Cellular factors involved in copper homeostasis inS. ceratisiae and their location in the cell.

are hypersensitive to copper. VMA3 encodes a 16 kd subunit of thevacuolarH+-ATPasewhich is requiredforvacuolar acidification (45). The exact defect(s) which cause the copper sensitivephenotypeof VMA3 deletionstrains is notknown, however, it is possible that the loss of vacuolar function may directly or indirectly Muence the functiono f m , CTRI or other copper homeostatic elements at the level of transcription, protein synthesis or protein localization. A Factor bearing resemblance to the NHpend of both ACEl and AMTL MACl is a putative metal binding transcription factor which is distantly related to the metal-activated transcription factors ACEl and AMTl (46). TheactionofMAC1onthe CUP1 promoter has not been addressed, however, MAC1 has been shown tobe important for the regulation of several important stressresponsegenes.Although MAC1 is importantforthe response to a number of environmental stresses, the exact impact of MACl on MT function has yet to be determined.

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HI. YEAST Candida glabreta Studies of copper-mediated transcription in S. cerevisiue has provided rich insights into physiological responses to altered environmental metal concentrations. However, interpretation of data obtained from experiments inS. cerevisiue has been limited by the fact that higher eukaryotes contain MT multi-gene families, whereas only a singleMT gene has been reported in S. cerevisiae (8, 20). To use yeast as a model to investigate metal-mediated multi-MT.gene transcriptional regulation, and to understandthe mechanism through which eukaryotes coordinate multi-MT gene transcription in the defense against metal toxicity, additional modelsystemscan be utilized. C. glubrutu, an opportunistic pathogenicyeast,presents an opportunity to studymetaldependent gene transcription and function of metallothioneins in copper detoxification, due to recent development of genetic techniques with this organism and the presence of a MT multigene family which is similar to higher eukaryotic MTgene families (47,17). Theyeast C. glubrutu hasa MT geneorganization, containing two known classes: MT-I and MT-II. M" includes two sub-isofonns: MT-IIu and M7"I.b (48). Interestingly, the MT" and h4 T - I I genes havebeenmapped to distinct chromosomes in C. glubrutu, whereas MT gene isoforms in highereukaryotes are localized on thesamechromosome (16,48,49). Compact gene organization often reflects an efficient mechanism for coordinatedregulation (50). Thedifferences observed in MT gene organization from S. cerevisiue to C . glubrutu to humans may reflect evolutionary development from yeast to higher eukaryotic organisms. MT-I and M T - I I encode 62 and 57 amino acid polypeptides, containing 18 and 16 cysteine residues, respectively. The cysteine residues are arranged in Cys-X-Cys and Cys-X-X-Cys motifs, which are characteristic of all known metallothioneins. As stated previously, 10 of the 12 cysteines residues in S. cerevisiue MT protein participate in chelating Cum andformasingleCu-thiolatecluster.However, in higher eukaryotic MTs, 7 m 12 metal ionsaxe segregated intotwo metalthiolate clusters intwo independent domains ( a and B ) (51-53). Like MT-I in C.glubrata, crustacean MTs have 18 cysteines and bind 6 divalentmetalionssuch as Zn2+ and Cd2+ or 12 monovalent metal ions such as Cu+ (54-57). N M R studies have established that crustaceanMTs have two metal-thiolate clusters

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occurring in two isolated domains (Do andBn) (58,59). Interestingly, examination ofC.glabratu MT-I protein sequence reveals an organization of cysteine residues similarto that found in crustaceans in that the first nine cysteines (within residues 428) are located in the N-terminal half of the MT-I, while the second nine cysteines (33-61)are found in the C-terminal half of the protein. Similarly, the 16 cysteinesin C.glabrata MT-IIa and MT-IIb are evenly distributed throughout the proteins, with 8 cysteines located in both the N- and C-terminal halves. Titration ions and MT-IIs studies have established that MT-I bind 12 bind 10 (60). Considering the fact that MT-I and MT-IIs have a distribution of cysteine residues similar to crustacean MTs and similar Cu-MT stoichiometry,it is very possible thattwo copperthiolate clusters and two independent domain structures occurin the yeast C.glabrata MTs. Although, there is limited sequence conservation between C. glabruta MTs and crustaceans MTs,the abovehypothesis is supported by therecentfindingthat metallothioneins have a variety of abilities to coordinate metal ions and still fulfill overall tight folding of the 30 amino acid residuesaboutthecorecluster(59).Preferentialbindingof different metal ions to the different domains has suggested that two independent domains in metallothioneins may play different physiological roles, withthe 4-metal cluster a-domain sequestering toxic metal ions and the 3-metal cluster B-domain participating in essential metal metabolism (61-63). Therefore, it is very likely thatC.glabrata is not only a model system to study metal mediated multi-MT genes transcriptional regulation but also a potential model to testin vivo the hypotheses that MT isoforms or the two distinct domains of a single MT isoform have different functions. Therefore, the yeast C. glabratu is an excellent model to study structure-function relationships of metallothioneins. Even though located on distinct chromosomes, C.glabrutu M7"I and MT-IIs are coordinately regulated by a single Metal Responsive Transcription Factor(MRTF), AMTl (64). Similar to ACE1, the amino-terminal amino acid residues (1- 115) contain 11 cysteines with Cys-X-Cys and Cys-X-X-Cys motifs binds Cu(I) viacysteinylthiolates to form aCu-cluster.ThisCu-cluster formation confers a DNA-binding function to AMTl while the carboxyl-terminal domain (1 16-265) presumably provides a transactivation function(64). The cis-acting control elements that are likely to confer copper dependent transcription on C. glabrutu MT-I and MT-I. promoters have been mapped by in vitro DNaseI footprinting and Cu+

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Figure 4 Model of thecellularhomeostaticmechanismsthatoccur in Candida glabrata leading to transcriptional autoactivationof the AMTl gene.

methylationinterferenceanalysis (Fig.4) (64). The AMTl binding consensus GCTG among the promoters is consistent with sequence a specific DNA-protein interaction model. Also, the sameconsensus GCTGbetween AMTl and ACEl binding sequences reflects the structural similarities between AMTl and ACEl proteins, mainly both have two domains consisting of a DNA-binding domain comprised of a Cu-thiolate cluster and an activation domain (20). The localization of the Cu- and DNAbinding functions of AMTlwithin the same N-terminal region (1proper conformation and 115) establishes that Cu-binding confers therefore directs the sequence specific DNA-AMT1 interactions, and also establishes the role of AMTl as a copper sensory molecule (64). The induction of MT-I and MT-II mRNA in C. glubrutu by Cu(1) and iso-electronic Ag(I) and not by Cd(II) further establish the structural requirement in the DNA binding and transcriptional regulation(65). Despite protein structural and DNA-binding similarities, AMTI differs from ACE1 in that AMTI gene transcription is regulated by its own product (65). In contrast, A C E l in S. cerevisiae is constitutively expressedat a constantlow level; this

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continued presence of ACE1 is thought to facilitate a rapid transcriptional response to toxic copper ion concentrations (28). A single copper responsive regulatory element located in the AMTI promoterwhichsequence-specificallyinteractswitha monomeric AMTl molecule has been established by DNase I footprinting and site-directed mutagenesis(65). This homologous cis-acting regulatory element shares the same consensus of GCTG within the AMTl binding sequence on the heterologous AMTl targetgenes, MT-I and MT-II. Kineticstudiesrevealedan extremely rapid induction of AMTI steady state mRNA levels. The accumulation of AMTl mRNA reached maximal steady-state levels after approximately 5 minutes exposure to copper (65). One would ask why should AMTI regulate its own expression ? As a copper sensory molecule, AMTl activates at least two defense lines, synthesis of MT-I and "l-II. The apo-MT-I and apo-MT-11 proteins bind intracellular copper ions and modulate thecopperconcentration. AMTl proteinitself is acopperbinding molecule. Copper is both essential and toxicfor cells and this dual nature mainlyis differentiated by copper concentration. Therefore, AMTl not only functionsas a transcription factor but alsofunctions as amodulatortoadjustintracellularcopper concentrations and maintain a delicate balance between copper essentiality and toxicity. However, the precise mechanism(s) of the auto-activation of AMTI and AMT1-mediated co-regulation of the MT-I and W - I I genes has not yet been established. One potential mechanism for the rapid auto-activation of A M T I , as compared to the MT genes,is a potentialsignificant difference in the binding affinity for the respective AMTl binding sites. The AMTl binding site on its own promoter is approximately 10 bp larger thanthe AMTl binding sites withinthe M T gene promoters, as determined by DNase I footprinting (65). Whether this site representsan extremely high afffity M 1 binding site, relative to AMTl sites in the MT gene promoters is currently unknown. Interestingly,immediatelyadjacent to the AMTl binding-site in the AMTl promoter lies an unusual stretch of 16 consecutive adenine residues. Previously,it has been shown that repeated oligo-d(A)*d(T) tracts tend to bend DNA (66). DNA bending is thought to be instrumental in the assembly of large transcription initiation complexes by facilitating the interaction betweentranscriptionfactorsand the generaltranscriptional machinery. Alternatively, the energy generated by DNA bending could be used to promote formation of an open transcription complex or cause the dissociation of theRNA polymemse in the

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Function of Yeast Metallothionein Genes

39 1

transition from initiation to elongation (67). Another possibility is that this poly (A) stretch may modify the chromatin structure and facilitate a favorable interaction between AMTl and its binding site on the AMTI promoter. Previous work has demonstrated that poly (A)stretchescan cause the absence of nucleosomes potentially rendering DNA moreaccessible to transcription factors and therefore enhancing the kinetics of gene transcription in respondtobiologicalsignals. An additional putative mechanism may involveother transcription factor(s) which bind a single-stranded DNA formed by the A16 stretch, since A-T-rich DNA stretches tendto form single-stranded DNA molecules. This has beenobserved with the yeast PUB1 proteinwhich specifically binds T-rich single stranded DNA (68). However, whether this poly (A)stretch in the A M T I promoter plays any role in the rapidAMTI auto-activation is currently unknown. The functional importance of A M T I auto-activation in copper detoxification hasbeen demonstrated by the recent finding that a copper-sensitive amtl-I mutant strain transformed with a plasmid containing an wild type AMTI gene which contains a mutation in the AMTI binding site on its own promoter, showed dramatic decreases in copper tolerance (65). Interestingly, the umtl mutant gene also displayed a marked loss of MT-II gene expressionwhileMTI mRNA levels were not significantly affected (65). A model for copper homeostatic control through gene regulation inC. glabrutu is shown in Figure 4.

W. SUMMARY Studies of metal-regulated metallothionein gene transcription in yeast have provided us with a great deal of information on metal homeostasis and the cellular response to toxic metal ions. Tissue specific expression of MT genes in higher eukaryotic organisms has suggested additional roles for S. MT other than metaldetoxification.Recentstudiesusing cerevisiue have identified an additional rolefar MT, in protection against oxidative stress. Oxidative stress has been implicatedin the process of aging andis associated with defects observedin in vitro embryogenesis (69). Therefore, the observation thatMT can function to protect cells against oxidative damage, pointsto an important biological role for MT in aging and other oxidative stress related disease states. The recent discoveries of copper transport proteins in yeast and a putative copper transporter in humans, which is

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associated with Menke's disease, has opened new avenues to investigatecopperinbiologicalsystems.However,copper metabolism is a complex process, involvingits uptake, transport, efflux and compartmentalization. The emergence of C. glabrata asa newmodelsystem toinvestigatemetal-regulatedgene transcription has contributed to a greater understanding of metal induced gene transcription. C. glabrata MT gene organization is in higher eukaryotic organisms and therefore, closer to that found future studies of this system will begin to give new insights into the complex mechanisms which take place in organisms with MT gene families and contribute to to a better understanding of metal homeostasis in higher eukaryotes.

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Transcriptional Regulation of the MetallothioneinGene:MetalResponsive Element and Zinc RegulatoryFactor Shinji Koizumi Department of Experimental Toxicology, National Institute of Industrial Health, 6-21-1 Nagao, Tama-Ju, Kawasaki 214, Japan Fuminori Otsuka

Teikyo University, Kanagawa,Japan

I. INTRODUCTION Metallothioneins W s ) are low molecular weight heavy metal-binding proteins whose biologicalroles have been postulated to be the protection against toxic substances including heavy metals and the homeostatic regulation of essential metals [1,21. Mammalian MTs are induced by several heavy metals such as Zn and Cd, and the mechanisms involved in this regulation have been investigated for these 15 years. It is now known that theinduction occivs mainly at thetranscriptional level. In this chapter, we will describe the findings with regard to the heavy 397

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metal-dependent cis-acting control elements of mammalian MT genes and ourrecent experimental results about a nuclear protein specifically interacting with these elements. 11. MRE, THE HEAVY METAL-DEPENDENT REGULATORY

ELEMENT OF MAMMALIAN METAUOTHIONEIN GENES Heavy metals suchas Zn and Cd are known h, control the expression of several mammalian genes, including those coding for MT [1,2], heat shock protein 131, heme oxygenase[41, Jun [5] and Myc [5]. Most of the works for this kind of regulation have been done on MT genes, originating from the studies of heavy metal-induced accumulation and synthesis of MT proteins in a variety of organs of experimental animals [6-81and cultured cell lines [g-111. The induced synthesis of M" mainly

consensw

TGCRCNCGGCCC

hw-IIA

a b C

d

e f g

-54 -82 -139 -153 -162 -246 -306

gctttTGCACTCGTCCCggctc ctgccTGCACACGCCCCgcgct cccagTGCGCGCGGCCGggtgt ccgggTGCGCCCGGCCCagtgc ggctcTGCACLCGGGCCgcggg cccgcTGCACCCAGCCCcttcc agctgTGCACACGGCGGaggcg

-43 -93 -1 28 -142 -1 73 -235 -295

N R

-51 -59 -128 -146 -171

cccttTGCGCCCGGACTcgtcc gggttTGCACCCAGCAGgcggt aaaagTGCGCTCGGCTCtgcca agctcTGCACTCCGCCCgaaaa cgctgTGCACACTGGCGctcca

-40 -70 -1 17 -135 -1 60

N R N N N

N N

R N N

mMT-I 8

b C

d e

Figure 1 MREs of mammalian MT genes. M R E s in the upatream region o f the human (h) MT-IIA and mouse (m) MT-I genes are shown with their start and end positiona and normal 0 and reverse (R) orientations. Flanking sequences are ale0 ahown by lower case letters. The MRE core ie indicated by boldface typea.

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results from the activation of transcription [10,12,131. After the genes coding for MTs had been cloned [14,151,their upstream sequences were found to activate transcription from heterologous promoters linked to them in thepresence of heavy metals [16,171,suggesting the presence of a control DNA element required for this regulation. Deletion mapping experiments [M-201identified such an element, which was named metal responsive (or regulatory) element (MRE). MRE is usually found in multiple imperfect copies in theupstream region of any MT-I and MT-11 genes, as described later. Palmiter’s group showed that a chemically synthesized MRE sequence by itself can mediate the heavy metalinducedtranscriptionalactivationwhen placed upstream of a heterologous promoter, demonstrating the regulatory function of MRE [21,223. The consensus sequence for MRE (Figure 1) consists of the highly conserved core andflanking GC-rich sequences. A detailed analysis of point mutations introduced in MFtEd of the mouse MT-I (mMT-I) gene revealed that thecore sequence, TGCRCNC (R,purine; N, any nucleotide), is particularly important for heavy metaldependent transcriptional activation [231. On the other hand, we found that MREa of the humanM T - I I A (hMT-IIA)gene requires a few additional bases 5’ of the MRE core for its activity. Each of these two MREs has been shown to be able to transmit several heavy metal signals by itself, although multiple copies are required for ita functioning. Multiple MRE sequences are found in theupstream region of any mammalian MT-I and MT-I1 genes thus far reported (Figure 2). In many of these genes, two MREs are positioned in opposite orientations between -40 and -100 bases from the transcription start site, and several MREs are clustered between -100and -200bases. Among these sequences, MREa, MREb, MREc, ,and MREd ofthe mMT-I gene [211and MREa of the hMT-IIA gene 1241 were confirmed to be functional as heavy metal-responsive regulatory elements, when placed upstream of heterologous promoters. A single copy of these MREs has undetectable or very low transcriptional activity, whereas their activity is enhanced when repeated. We observed that Zn-dependent transcriptional activity of hMT-IIA MREa is raised with increasing copy numbers, but the activity is not simply proportional to the number of repeats. Two copies of MREa have very low activity, whereas four copy repeats show a significant activity. Two additional copies give a further several fold increase of the activity as compared to the four copy repeats. These results suggest that there are interactions between MREs, and their positions relative to each other may be important. The activities of

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-400

-300

-200

-1 00

mMT-II "1

~

rbMT-l2 4 "

sMT-la 4 4

Figure 2

Amqpment of MREs in the upstream region of mammalian MT genes.

MREs in the 5' flanking region of mammalian MT-I and MT-II genes thus far reported are shown bythe arrows. Thesegenes include human(h) MT-IA[B] MT-IB , [39], MTIE [40], MT-IF [40], MT-IG [41], MT-IIA [161, mouse (m)MT-I 1141, "T-II [42], rat (r) MT-l 1431, MT-2 [441, Chinese hamster (ch) MT-11 [461, rabbit (rb) M"-I 1461, sheep (S) MT-Ia [471, MT-Ib 1481, "-IC [48] and "-IT 1481 genes. Some of the MREs shown do not correspond to ones described in the original reports, since the consensus sequence of the MRE core (TGCRCNC) is used ae the key sequence forsearch. MREs are usually named MREa, MREb, etc. in the order of proximity to the transcription etart

site.

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individual MREs are not the same.For example, MREd has the strongest activity among MREs located upstream of the mMT-I gene

m .

III. ZRF, AMRE-BINDING PROTEIN "RE was expected to be the target site of a sequence specific DNAbinding protein acting as a transcriptional regulator, like othercontrol DNA elements. By mobility shift assay using a chemically synthesized, 32P-labeled oligonucleotidecontaining the hMT-11~MREa sequenceas a probe, we searched for "binding activity in nuclear extracts of HeLa cellsderived from human cervical carcinoma. A sequence-specific protein-DNA complexwas detected, which appears only when Zn ions are added to the binding reaction [241. As other heavy metals fail to supportthis complex formation, the DNA-binding proteinwas designated Zinc Regulatory Factor (ZRF). A significant level of this activity is detected in the nuclear extract of cells cultured without exogenously added Zn, although a small increase of the activity is observed in cells exposed to heavy metals for several hours 1243. By mobility shift assay using the same probe and a mouse L cell nuclear extract, we also observed the formation of a sequence-specific and Zndependent protein-DNA complexthat shows an electrophoretic mobility similar to that of the human ZRF-DNAcomplex. Another binding reaction between a 32P-probe containing the mMT-I MREd sequence and HeLa nuclear proteins also yields a proteinDNA complex with similar properties [251.

W.PURIFICATION OF ZRF We then purified ZRF from the nuclei of HeLa cells, using "binding activity in mobility shift assay as an index. We first attempted to isolate the protein by conventional chromatography techniques with DEAE-Sepharose, CM-cellulose, heparin-agarose and Sephacryl S-300 columns as well as an affinitycolumnwithmultimeric MRE oligonucleotides as ligands. However, the results were unsatisfactory in both purity and yield, possibly becauseof nonspecific binding of proteins to, and loss of ZRF by irreversible absorption to the column matrices. In the affinity column, hindrance of the complex formation between ZRF

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and fixed DNA ligands may also be a problem. We then tried another strategy, the biotin-streptavidin method 1261, which was expected to overcome these difficulties. After the binding reaction between HeLa nuclear proteins and a MREa oligonucleotide probebiotinylated a t one end, the ZRF-DNA complexes formed were trapped by streptavidinagarose beads and recovered as precipitates. ZRF was then extracted with a high-salt buffer. This method was found to be efficient in both removal of nonspecific contaminants and recovery of ZRF (Figure 3). By repeating this procedure three times, a nearly homogeneous protein with molecular weight of 116,000 was obtained. We confirmed that this 116 kDa protein has ZRF activity by the following experiments. i) The recovery of ZRF activity in the biotin.?

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Figure 3 Purification of ZRF by the biotin-streptavidin method, Crude HeLa nuclear extract (lanes 1-31and proteins obtainedby one cycleof the biotin-streptavidin 16-fold mlative to the crude extract;lanes 4-6) were affinity purification (concentrated assayed for ZRF activity bymobility shift assay using a 32P-labeled 28-bp oligonucleotide pmbe containinghn4T-Q m a . Lanes: 1and 4.0.26 cI1; 2 and6,O.S pl;3 and 6 , l plof the protein solutions. The arrowhead and F indicate the ZRF-DNA complex and fiw probe, respectively.

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streptavidin purification depends on Zn ions added to the binding reaction and the DNA sequence of the probe. In these criteria, the 116 kDa protein showed behaviors identical with those of ZRF activity. ii) The complex formed between ZRF and a MREa probe labeled with 32P at the ZRF-binding site was cross-linked by ultraviolet irradiation. After removing unbound portions of the probe by nuclease digestion, 32P-labeled proteinswereanalyzed by SDS-polyacrylamide gel electrophoresis. Only a 116 kDa protein band was detected, dependent on Zn and thesequence of probe. iii) MRE-binding proteins in theHeLa nuclear extract were detected by South-western blotting procedure using a 32P-labeled MREa probe. Only a 116 kDa protein band was detected when Zn was included in the binding reaction. From these results, we concluded that ZRF activityresidesinthe 116 kDa polypeptide.

V. CHARACTERISTICSOF ZRF The characteristics of ZRF were studied using the preparations obtained by the biotin-streptavidin purification repeated twice. In mobility shift gene assay, a 28-bp oligonucleotide containing MREa of the ~MT-IIA was end-labeled with 32P and used as theprobe. A. Dependency on Heavy Metale In mobility shift assay, ZRF binds to MREa in thepresence of Zn, while not in the absence of Zn (Figure 4, lanes 1 and 2). The extent of DNA binding depends on the concentration ofZn. The maximal binding activity is observed at 100 pM,and higher concentrations of Zn are. slightly inhibitory. By contrast, other heavy metals that can induce MTs including Cd2+, C@+, Hg2+, Ni2+, C$+and A p do not support the complex formation. In thecase of Cd, no ZRF-MRE binding is observed at the concentrations of 0.01 through 200 pM. B. Specificity for DNA Sequence Recognition

Oligonucleotidescontaining various mutations within or near the MREa sequence were used as competitors in mobility shift assay to examine the bases essential for ZRF-MREa binding. As described in section 11, MRE consists of the core andflanking GC-rich sequences. An

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oligonucleotide with a single base exchange in the GC-rich region inhibits the complex formation as a wild-type MREa competitor, while one with a mutation within the core shows no effect(Figure4, lanes 36). Another experiment using a series of mutant oligonucleotides with consecutive four base exchanges over the MREa and flanking sequences codlrmed the importance of the MRE core for ZRF-MRE binding. In addition, the bases just 5’ of MREa were found to be also important for the DNA-binding of ZRF, while those 3’ of MREa were not. The requirement of bases for ZRF-MRE interaction is consistent with that for Zn-inducedtranscriptional activation described in section II.

Figure 4 Sequence-apeciTk and Zndependent DNA-binding of ZRF. ZRF purified by two cyclea ofthe biotin-streptavidin procedure was assayed an in Figure 3. &SO4 (100 and cold oligonucleotide competitors (130-fold excess over probe) were added as indicated below. Competitora used m:wt,wild type MREa; m l , mutant MREa with a single base change at the firet T;m2, mutant MREa with a single base change at the ninth T. Additions to the binding r e d i n are as follows. Lanes: 1, none; 2, Zn ; 3, Zn and wt;4, Zn and m l ; 5, Zn and m2. The armwhead and F indicate the ZRFDNA complex andfree probe, respectively.

a)

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C. Requirement for M@+

ZRF requires M g ions for its Zndependent binding to MREa. The effect of Mg is dose-dependent, reaching a maximum at 2-5 mM. The doseresponse curve between Mgconcentration and the ZRF activity in crude nuclear extracts shows a sharp peak a t 2 mM [241, probably affected by coexisting nuclease activity enhanced a t high concentrations of Mg. D. Requirement for Dithiothreitol Dithiothreitol @'IT), a SH-reducing agent, is also essential for the DNA-binding of ZRF in thepresence of Zn. The optimal concentration of D" is 10 mM. Such a requirement of relatively high concentration of D" may suggest that free SH residues in ZRF are important for its function.

E. Effect of Chelaters

The ZRF-MRE complex already formed is easily dissociated by the addition of heavy metal chelaters such as ethylenediaminetetraacetic acid (EDTA) and o-phenanthroline.Theminimalconcentration of EDTA required for the dissociation is 20 mM,whereas that of 0 phenanthroline which has a higher specificity for Zn is 2 mM. These results suggest that Zn ions are essential for the maintenance of ZRFMRE complex. F. Specificity for Individual MREs To examine the binding affinity of ZRF to individual MREs present in the upstream region of the hMT-IIA gene, oligonucleotides each containing one of five MRE sequences proximal to the transcription start site (MREa-MREe; Figure 1) were used as competitors in the mobility shift assay with the MREa probe. This experiment showed that ZRF binds most strongly to MREa and MREb. MREd and MREe have an intermediate affinity to ZRF,whereas MREc shows no affinity.

G. Footprints DNase I footprints on MREs have not been shown for Zn-dependent MRE-binding factors including ZRF 80 far. In our previous studies

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using ZRF-containing nuclear extracts, no footprint was observed on hMT-IIA MREs. However, purified ZRF gives Zn-dependent and sequence-specificfootprintsmostclearlyonMREa and MREb, consistent with the resultsobtained by the mobility shift assay. It is not clear whether the concentration of ZRF in the crude extracts is not enough to give footprints in uitro, or the extractscontain an inhibitory factor. VI.

RELATIONSHIPS BETWEENZRF BINDING FACTORS

AND OTHERMRE-

Although a number of MRE-binding proteins have been reported (for review, see Ref. 271, direct relationships between them have been ambiguous. In our laboratory, another MRE-binding factor named MREBP has been identified [28,291. MREBP is different from ZRF in several characteristics: it binds to hMT-IIA MREs in the absence of heavy metals with a low affinity, and the binding is abolished by high concentrations of heavy metals. From its DNA-binding properties, MREBP maynot have a principal role in theactivation of MT genes by heavy metals [271. ZRF can be physically separated from MREBP by glycerol gradient centrifugation, and the purified ZRF does not show even a trace of MREBP activity. These observations indicate that these two factors are distinctproteins. As the MRE-binding factors sharing similar characteristics with ZRF,human MTF-1 1301 and rat ZAP 1311 have been reported. These three factors require the MRE core for their DNA-binding, although thereare minordifferences intheirrequirements for flanking sequences. All of these factors are activated by exogenously added Zn ions in binding to MRE, while not by other heavy metals. It is thus possible that these three factors are homologous proteins. Recently, cDNA coding for mouse MTF-1 (mMTF-1) has been cloned, and its amino acid sequence was deduced from the primary structure of DNA [321. On theotherhand, we determined thepartial amino acid sequences of purified ZRF, and found that these show a high degree of homology with the partial sequences of mMTF-1. This finding suggests that ZRF is a human counterpart of mMTF-1. In fact, human MTF-1 detected by South-westem blotting shows an electrophoretic mobility similar to that ofZRF 1321. Since the peptide fragments of ZRF sequenced so far correspond to several parts only in theamino-terminal

Regulation of Mammalian Metallothionein Genes

407

half of mMTF-1, it remains unclear whether these two proteins are homologous overthe entirepolypeptide sequences or have only a limited homology.

VII. ZRF AS A TRANSCRIPTIONALREGULATOR The role of ZRF in thetranscriptional regulation of the ~ M T - I I Agene is suggested by the following findings. i) In the presence of Zn, ZRF specifically protects MREs in the hMT-IIAgene upstream region in DNase I footprinting in vitro. It has been shown byin vivo footprinting experiments that MREs are protected in heavy metal-exposed cells 133,343. Theseobservationssupport that ZRF acts as a positive regulator of the hMT-IIA gene. ii) The MREa core and flanking 5' sequences are particularly important for recognition byZRF. The same sequences are also essential for the ability of MREa to mediate Zndependenttranscriptionalactivation.Such a coincidence inthe sequence requirement also supports the role of ZRF as a transcriptional regulator. iii) ZRF showshigh homology with rnMTF-I, which has been shown to be a transcription factor 1323. The observation that Zn ions are required for the maintenance of ZRF-MRE complex suggests that ZRF has a metal-chelating structure, like those of Zn-finger transcription factors 1351 and the regulator of the yeast MT gene,, ACE1 1361. The requirement ofDTT for ZRF-MRE complex formation may reflectthat SH residues possibly involvedin the formation of this structure should take a reduced form. Furthermore, ZRF may have Zn-finger structures, assumed by the homology with the Zn-finger region of mMTF-1[321. Based on these findings, Zn appears to be an integral part of the functional ZRF molecule. We first expected that the activation of ZRF by Zn observed in DNA-binding reaction in vitro reflects the principal mechanism for the heavy metal-induced transcription of the hMT-II~gene in uiuo. A similar mechanism has been shown to be responsible forthe Cu-induced activation of the yeast MT gene 1363. However, the fact that ZRF binds to MRE in response only to Zn, but not to other heavy metals which can also activate MT genes in vivo, argues against this hypothesis. Since MREa by itself responds to multiple heavy metal signals in vivo 1241, the limited response onlyto Zn in vitro appears to be a characteristic of ZRF, rather than that of the target MREa sequence. Recently, it has been proposed by Palmiter 1371 that various heavy metals may replace

408

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Zn loosely bouilu to cellular components, resulting in theactivation of MT genes by the released Zn. This mechanism allows heavy metals other than Zn to activate However, it is also possible that the Zn dependency of ZRF observed in vitro represents only the structural requirement for a functional ZRF molecule, and the transcriptional regulation by heavy metals is achieved by another mechanism. It has been shown that mMTF-1, which is related to ZRF, constitutivelyactivates MREmediated transcription in the absence of Zn, when overexpressed in cultured cells 132,373. This suggests that m"F-l itself is not regulated byZn, and its overproduction overwhelms a negativeregulatory mechanism, for example, inactivation of mMTF-1by binding of a heavy metal-sensitive inhibitor. There are some supporting evidences for the presence of such an inhibitor of mMT,F-1[371. Cloning and analysis of cDNA encoding ZRF are now in progress. These works will finally answer to the questions about its structural similarity to mMTF-1 as well as its putative metal-binding structure, and also provide us means to understand its role in thetranscriptional regulation of the M" gene.

ZRF.

ACKNOWLEDGMENT We thank our collaborators, Dr. H. Yamada, Mrs. K. Suzuki (National Institute of Industrial Health), Mr. A. Iwamatsu (CentralLaboratories for KeyTechnology, Kirin Brewery Co., Ltd.) and Dr. D. Hamer (National Institute of Health, U. S. A.). This work is supported in part by a Grant-in-Aid fromthe Science and Technology Agency,Japan.

REFERENCES 1. J. H. R. Kligi, and Y. Kojima (Eds.), Metallothionein 11,Birkhiiuser

Verlag, Basel, 1987. 2. D. H. Hamer, Ann. Rev. Biochem. 55: 913-951 (1986). 3. B. J. Wu, R. E.Kingston, and R. I. Morimoto, P m .NatZ. Acd.Sci. USA83: 629-633 (1986). 4. J. Alam, S. Shibahara, and A.Smith, J. BWZ. Chem. 2 M 63716375 (1989). 6. P. Jin, andN.R.Ringertz, J. BWZ. Chem. 265:14061-14064 (1990).

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D. R.Winge, R.Premakumar, andK.V. wagopalan, Arch. Biochem. Biophys. 170 242-252 (1975). 7. G. S. Pmbst, W. F. Bousquet, and T. S. Miya, Toxicol.Appl. P h u r w o l . 3 9 51-60 (1977). 8. M. G. Cherian, J. Nutr. 107: 965-972 (1977). 9. H. A,' Hidalgo, V.Koppa, and S. E. Bryan, Biochem. J. 170 219-

6.

226 (1978). 10. S. Koizumi, T.Sone, N. Otaki, and M. Kimura, Biochem. J. 227 879-886 (1985). 11. S. Koizumi, T. Sone, and M. Kimura, J. Cell. Physiol. 125 223-228 (1985). 12. D. M. Durnam, and R D. Palmiter, J. Biol. Chem. 256 5712-5716 (1981). M. Karin, R.D. Andersen, and H. R. Herschman, Eur. J. Biochem. 13. 118: 527-531 (1981). 14. N. Glanville, D. M. Durnam, and R.D. Palmiter, Nature 292: 267269 (1981). 15. M. Karin, and R.I. Richards, Nature 299 797-802 (1982). 16. K E. Mayo, R.Warren, and R D. Palmiter, Cell 2 9 99-108 (1982). 17. M. Karin, A.Haslinger, H. Holtgreve, G. Cathala, E. Slater, andJ. D. Baxter, Cell 3 6 371-379 (1984). 18. G. W. Stuart, P. F. Searle, H. Y.Chen, R.L. Brinster, andR.D. Palmiter, P m . Natl. &d.Sei. USA 81: 7318-7322 (1984). 19. A. D. Carter, B. K Felber, M. Walling, M. F. Jubier, C. J. Schmidt, and D. H. Hamer, Proc. Natl. had.Sci. USA81: 7392-7396 (1984). 20. M. Karin, A. Haslinger, H. Holtgreve, R.I. Richards, P. &auter, H. M. Westphal, and M.Beato, Nature 308 513-519 (1984). 21. G. W.Stuart, P. F. Searle, and R.D. Palmiter, Nature 317: 828-831 (1985). 22. P. F.Searle, G. W. Stuart, and R D. Palmiter, Mol. Cell. Bwl. 5: 1480-1489 (1985). 23. V. C. Culotta, and D. H Hamer, Mol. Cell. Biol. 9 1376-1380 (1989). 24. S. Koizumi, H Yamada, K Suzuki, and F. Otsuka, Eur. J. Biochem. 210 555-560 (1992). 25. F.Otsuka, M. Ohsawa, and S. Koizumi, Ind. Health 31: 133-142 (1993). 26. L.k Chodosh, Purification of DNA-binding proteins using

biotidstreptavidin affinity systems, in Current Protocols in Molecular Biobgy (F.M. Ausubel, R Brent, R E. Kingston, D. D.

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Moore, J. G. Seidman, J. A. Smith, and K.Struhl, Eds.), Greene Publishing and Wiley-Interscience, New York, 1991, pp.12.6.1-12. 6.9. 27. S. Koizumi, and F. Otsuka, Factors involved in thetranscriptional regulation of metallothionein genes, in Metallothionein ZZZ (K. T. Suzuki, N. Imura, andM. Kimura, Eds.), Birkhauser Verlag, Basel, 1993, pp.457-474. 28. S. Koizumi, F. Otsuka, and H. Yamada, Chem. -BWl.Interact. 80 145-157 (1991). 29. S. Koizumi, K Suzuki, and F. Otsuka, J. Biol. Chem. 267 1865918664 (1992). 30. G. Westin, and W. Schaffner,EMBO J. 7 3763-3770 (1988). 31. P. F. Searle, Nucleic Acids Res. 18 4683-4690 (1990). 32. F.Radtke, R Heuchel, 0. Georgiev, M. Hergersberg, M. Gariglio, 2. Dembic, and W. Schafher, EMBO J. 12:1355-1362 (1993). 33. P. R Mueller, S. J. Salser, and B. Wold, Genes Deu.2:412-427 (1988). 34. R.D. Andersen, S. J. Taplitz, S. Wong, G. Bristol, B. Larkin, and H. R.Herschman, Mol. Cell. Biol. 7 3574-3581 (1987). 35. R.M.Evans, and S. M. Hollenberg, Cell 52: 1-3(1988). 36. D. J. Thiele, Nucleic Acids Res. 20: 1183-1191 (1992). 37. R D. Palmiter, Proc. Natl. Acad. Sci. USA 91:1219-1223 (1994). 38. R. I. Richards, A. Heguy, and M. Karin, Cell 3 7 263-272 (1984). 39. A. Heguy, A.West, R. I. Richards, and M. Karin, Mol. Cell. Biol. 6: 2149-2157 (1986). 40. C.J. Schmidt, M. F. Jubier, and D. H. Hamer, J. Biol. Chem. 260 7731-7737 (1985). 41. R.Foster, N. Jahroudi, U. Varshney, and L.Gedamu, J. Biol. Chem. 263: 11528-11535 (1988). 42. P. F. Searle, B. L. Davison, G.W. Stuart, T. M. Wilkie, G. Norstedt, and R. D.Palmiter, Mol. Cell. Bwl. 4 1221-1230 (1984). 43. R.D.Andersen, B. W.Birren, S. J. Taplitz, and H. R. Herschman, Mol. Cell. Biol. 6 302-314 (1986). 44. R.D.Andersen, EMBL M11794. 45. D. L.Grady, EMBL X55064. 46. Y. C.Tam, A. Chopra, M. Hassan, and J. P. Thirion, Biochem. Bwphys. Res. Commun. 153:209-216 (1988). 47. M.G. Peterson, and J. F. B. Mercer, Eur. J. Biochem. 160 579-585 (1986). 48. M.G. Peterson, F. Hannan, and J. F. B. Mercer, Eur. J. Biochem. 174:417-424 (1988).

26 Gene Disruption of the Transcription Factor MTF-1 Leads to Loss of Metal Regulation of Mouse Metallothionein Genes Freddy Radtke, Rainer Heuchel, Oleg Georgiev, and Walter Schaffner 190, CHInstitut fir Molekularbiologie 11, Universitiit Zurich, Winterthurestrasse 8057 Zurich, Switzerland Gerlinde Stark and Michel Aguet Institut f3r Molekularbiologie I, Universittit Ziirich, CH-8093 Ziirich, Switzerland

Abstract Metallothioneins (MTs) are small cysteine-rich proteins whose structure is conserved from fungi to man. MTs strongly bind heavy metals, notably zinc, copper and cadmium. Upon exposure of cells to heavymetaland other treatments, MT gene transcriptionis strongly enhanced. We havepreviouslydescribedandclonedafactor(MTF-1)thatbinds specifically to heavy metal-responsive DNA sequenceelementsinthe enhancer/pmmoter region of metallothionein genes. MTF-1 is a proteinof 72.5 kDa that contains six zinc fingers and multiple domains for transcriptional activation. Here we report the disruption of both alleles of the MTF-1 gene in mouse embryonicstem cells by homologous recombination. The resulting null mutant cell line fails to produce detectable amounts of MTF-1. Moreover, due to the loss of MTF-1 the endogenous metallothionein-i and 11genes are silent, indicating that "'F-1 is required for both their basal and zinc-induced transcription. In addition to zinc, other heavy metals including cadmium, copper, nickel, and lead also fail to activate metal-responsive promoters in null mutant cells. However, cotransfection of an MTF-1 expression vector and metal-responsive reporter genes yields strong basal transcription that canbe further boosted by zinc treatment of cells. These results demonstrate MTFthat 1 is essential for metallothionein gene regulation. Finally, we present evidence that MTF-1 itself is a zinc sensor. The DNA binding activity of MTF-1 are modulated either by zinctreatment of the cellsor by changingzinc concentration inDNA protein binding reactions. 41 1

412

Radtke et al.

Introduction Heavy metal detoxification in eukaryotes is mediated by small cysteine-rich proteins, known as metallothioneins. They have a high affmity for metals, in particular for zinc, cadmium and copper. Metallothionein gene expression is induced bya great numberof stimuli, most notably by adverse conditions such as heavymetalload,viral infection, UV- andX-irradiation [l, 21. Consequently,theseproteinshavebeenimplicatedinheavymetal radical scavenging. Metal detoxification detoxification, metal homeostasis, and I has been unambiguously demonstrated, since elimination of metallothioneins and I1 by targeted gene disruption results in mice are thatparticularly sensitive to cadmium [3,4]. In addition to the ubiquitously expressed metallothionein genes I and 11, there are two additional metallothioneins,111 and IV, whose expression is restricted to brain and squamous epithelial tissues, respectively [5; R.D. Palmiter, personal communication]. Palmiter and colleagues originally noted the presence of conserved DNA sequence motifs in the promoters of a number of metallothionein genes, so-called metal responsive elements (MREs) which can confer heavy metal inducibility to heterologous reporter genes [6,7]. We have inserted metallothionein upstream sequences into the SV40 genome, thus creating metal-dependent viruses, and also found that these upstream sequences could act over large distances as metal-responsive transcription ours, have described proteins that bind enhancers [8]. Several groups, including to metal-responsiveDNA elements [g-131. The candidate transcription factor (MTF-1)thatspecificallybindstothemetalresponsiveelementsof metallothionein-Ipromoters [ g ] , wassubsequentlycloned[14].Deletion analysis of the MTF-l cDNA identifieda zinc finger-type(C2H2) DNA binding domain and transcriptional activation domains. When expressed in transfected mammalian cells, "'F-1 could activate transcription from metal-responsive promoters. However, activation was mostly constitutive, i.e. basal level rather than zinc-induced transriptionwas increased. These results could be explained by the assumption that the endogenous metal-response system was already saturated and unable to respond to additional "F-1. However, despite the fact that no other MRE-binding factor was isolated, we could not conclude with certainty that MTF-l was involved in heavy metal-induced transcription.

To investigate the role of" I F 1 in metallothionein gene regulation we have constructed an embryonic stem (ES) cell strain that has lost MTF-1 expression bymeansoftargetedgenedisruption.Usingthiscelllineweobtained conclusive evidence that MTF-1 is required not only for heavy metal induction but also for basala transcriptionof metallothionein I and I1 genes. We find no evidence for additional members an of "F-l-related factor family, nor fora ES cells. MTF-1 is backup system of metal regulation by other factors in these not only essential for induction by zinc but also mediates the response to other heavy metals suchas cadmium, copper, nickel and lead.

Gene Disruption of the Transcription Factor MTF-1

413

Finally, we also present data showing that the DNA binding properties of MTF-1 are modulated by zinc treatment, indicating that this factor can respond to changesin intracellular zinc concentration.

Results In order to mthe role of=-l in metallothionein gene regulation we decided to ablate the MTF-1 gene expression by knocking out the MTF-1 alleles. For targeted disruption of both alleles of the MTF-1 gene, we constructed two targeting vectors where most of the first zinc finger exon was replaced by a selection marker gene in reverse orientation. This exon was chosen because it would eliminate DNA binding, in case residual protein was produced. In addition, as was found later, the strongPGK promoter of the selection marker MTF-1 gene gene, transcribed in reverse orientation, apparently interferes with transcription with the effect that no transcripts were observed anymore in a RNA mapping analysis (data not shown). The neomycin phosphotransferase of the first. and second allele, and hygromycin genes used for disruption respectively, yielded homologous insert colonies at frequencies of 1/8 and 1/16, ES cellswere respectively(Fig. 1; see alsoMaterialsandMethods). electroporated consecutively with these two vectors and homozygous mutant (-/-) ES cell colonies were identified byPCR and confirmed by Southern blot analysis (datanot shown). To examine whether the-/-ES cells obtained by this procedurestill contained any residual MTF-l-like binding activity, perhaps due to a related and/or redundant protein,we performed gel retardationanalyses. Nuclear extractsfrom control +/+ ES cells (wildtype for the MTF-1 locus but G418-resistant due to nonhomologous vector integration) and -/- ES cells had indistinguishable amounts of the ubiquitous transcription factorSpl. However, only the+/+ ES cell contained any MRE-binding activity. " F - l binding activity was increased several fold by zinc treatment of the cells, while the signal intensity of Spl -/remained unchanged;this effectwill be addressed in more detail below). The ES cells showed no detectableMTF-l, either before after or zinc treatment (data not shown). This result confirms the successful disruption ofm the-1locus are no additional proteins binding to the MRE-site and also indicates that there oligonucleotidestested.

mRNA levels of endogenous metallothioneinI and II genes were measured in order to investigate the role of MTF-1 in metallothionein gene regulation. shown in Fig. 2, the mRNA level of MTF-1 itself is only marginally byheavymetal treatment,essentiallyruling out anytranscriptional autoreflation. As expected. levels of metallothionein-1 and I1 gene transcripts were greatly increased upon treatment with zinc, cadmium or other heavy

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416

Radtke et al.

metals like copper, nickel, and lead (Fig. 2, lanes 1 vs. 2-6). Inmarked contrast, both before and after treatment of the cells with zinc or other heavy metals, the -/- ES cells contained undetectable amounts ofMTF-1 transcripts and little, if any metallothionein mRNA (less than 1%of basal level in wild type cells)(Fig. 2, lanes 7-12). By introducing the MTF-1 cDNAintonullmutantES cells in transient transfection experimentswe sought to restore transcription of metal-inducible promoters. Wild type and null mutant cells were transfected with reporter genes under the control of either 4 copies of the strong metal-responsive element " l(4xMREd OVEC), or the complete mouse metallothionein-I promoter (MT-I OVEC). Treatment of +/+ ES cells with 400 p m zinc resulted in a 10

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Gene Disruption

of the Transcription Factor

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fold increase of transcription from a transfected 4xMREd reporter gene (Fig. 3A,lanes 1 & 2). Cotransfection of 2.5 pg MTF-1expressionvector dramatically increased both basal and zinc-induced transcription (Fig. 3A, lanes -/- ES cell line, reporter gene expression was barely 3 & 4). By contrast, in the detectable either with or without zinc treatment (Fig. 3A, lanes 5 & 6). Only to the after cotransfection of the cloned MTF-l gene was transcription restored levels observedwith MTF-1 cotransfected+/+ ES cells (Fig. 3A, lanes3,4 and 7,8). Similar results were obtained using a reporter gene driven by the complete metallothionein-I promoter instead of the 4xMREd promoter; increasing concentrations of "'F-l resulted in increased levels of both uninduced and induced transcription (data not shown). Finally, we have addressed the question whether zinc-induced DNA binding activity ofMTF-1isduetothesame, or to different mechanisms when observed in vitro versus in vivo.Figure 4 shows that in nuclear extracts which are not supplemented with zinc," F - 1 is incompletely zinc-saturated, and that binding activitycan be greatly increased by zinc addition. This effect is much morepronouncedthanwith Spl, anothermammalianzincfingerfactor, indicating thatMTF-1 requires a higher zinc concentration for optimal DNA binding than doesSpl. cells prior to extract preparation, We had also noted thatafter metal treatment of there is always a several fold increasein MTF-1 bandshift activity in nuclear extracts (Fig. 4 A vs. B). It was unclear whether this was due to a higher

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PM ZnS04 - 100 200 400 800 1200 Fig. 4 Zinc-induced DNA binding of MTF-1 fromuntreatedand zinc-treated cells. Nuclear extracts were prepared from untreated mouse 3T6 cells (A) and cells treated with 100 pM zinc sulfate for hr 4 prior to harvest and nuclear extract preparation Lanes 1-6,increasing amountsof zinc ions were added to the the binding buffer before the DNA-protein binding reaction and gel electrophoresis. The left panel shows bandshifts with the MREd oligonucleotide, which binds both to Spl and "F-1. The right panel shows control bandshifts with an "octamer" site oligonucleotide and Oct-l factor, to whose DNA binding domaindoes not contain zinc fingers and is known be unaffected by zinc concentration [13.

(B).

418

Radtke et al.

starting concentration of zinc ions in the extracts, to ora separate mechanism of some kind. Here we show that the latter is the case. Increasing amounts of zinc added to either the extracts of untreated or zinc-pretreated cells does not level out the several fold difference between them,ateven zinc concentrations that largely exceed the conditions of cell treatment (Fig. 4). Thus we have of active MTF-l: identified two separate ways to yield higher concentrations Firstly, the proteinis sub-optimally saturated with zincin vitro, and upon zinc addition an increasing fraction canbe converted toa DNA binding form. From this it appears likely that the zinc fingers of MTF-l are, directly or indirectly, involved in metal induction (see Discussion): Secondly, there is another mechanism in zinc-treated cells that mobilizes a severalfold elevated DNA binding activity, perhaps as a result of de novo protein synthesis, or by the modification/release of a preexisting " F - 1

P "

DISCUSSION MTF-l gene disruption

We have previously cloned and characterized a transcription factor (MTF-l) that specifically binds to metal-responsive elements (MRE) of metallothionein promoters [g, 141. When transfected into mammalian cells, MTF-1 strongly stimulated expression of MRE-containing promoters. However, activation was largely constitutive, and some queries remained about the role of" W 1 in metal induction.To settle this question, we have eliminatedMTF-1 expression @S cells) of the by means of targeted gene disruption in embryonic stem cells mouse. Our results showthat both basal and metal-inducible transcriptionfrom natural is lost upon disruption of the MTF-1 and synthetic metal-responsive promoters gene and can be restored to a large extent by cotransfection of the MTF-l expression vector. The fact that no bandshift activity was detectable using cells indicates that no other factors with related DNA extracts from null mutant binding properties existin these cells. From this we conclude that MTF-l is the crucial transcription factor for metallothionein gene induction. In addition, MTF-1 is also absolutelyrequiredforbasalleveltranscription of metallothionein genesI and II. This is surprising, becausein vivo footprintingdata of Barbara Wold and her colleagues have shown that binding sites for constitutive factors Spl and USF in the absence in the metallothionein I gene promoter, are fully occupied even [lo]. Thesefactorsapparentlycannotsupportbasal ofheavymetal transcription; they may nevertheless have an auxiliary role in concert with MTF-1. The finding thatMTF-1 is also necessary for the basal transcription of metallothionein genes implies a role forMTF-l (and metallothioneins) under nonstressed, physiological conditions.

Gene Disruption

of the Transcription Factor

MTF-1

419

Zinc-induced DNA binding of MTF-1 Zinc-treatment of nuclear extracts or zinc treatmentof living cells prior to extract preparation was found to increase DNA binding of MTF-1 to metalresponsive elements. Upon investigation we found that MTF-l factor in vitro is more sensitive to the environmental zinc concentration than Spl factor (Figure 4). Spl does not mediate aheavy metal response, althoughit harbors the same type of His2Cys2 zinc fingers as MTF-l, and can be reversibly inactivated by zinc withdrawal [15-171. Taken together, our studies reveal a differential sensitivityof factors to zinc in vitro, which may also reflect the situation in vivo: Octamer factor 1 that binds DNA via a POU homeodomain, is completely insensitiveto zinc. RNA polymerase I1 contains essential zinc but is less sensitive to zinc levels than Spl, which in turn is less sensitive than MTF-1. Thus onecan envisage a scenario whereby classes of transcription factors are regulated by zinc availability[g, 17. MTF-1 would only bind zinc, and thus become DNA binding-competent,at high zinc load (Figure 5A). This model, proposed originally by Westin and Schaffner [9], does not take into account the largely constitutive activation of transcription by transfected mouse MTF-l,unless one considers a saturation effect due to MTF-1 overexpression [14; in this contextit is worth mentioning that human MTF-l was also cloned by us and for unknown reasons confers a more pronounced zinc response in transfectedcellsthanthemousefactor; 181. This modelproposed for mammalian MTF-l, has been shown independently to be correct for the yeast copper metallothionein systems[g, 19,201. as Another possible model involves a specific zinc dependent coactivator illustrated in Figure 5B. It mightalsobepossiblethatmetalregulated transcription of metallothionein genes isin fact a combinationof both models as shown in Figure 5C. We have also discovered an independent mechanism that leads to elevated MTF-l binding activity. Extracts from cells pretreated with zinc always contain several fold more DNA-binding MTF-1 than control extracts. irrespective of the amount of zinc added to the binding reaction (Figure 4 A vs. B). This MTF-1 it accumulation is relatively slow, over several hours (data not shown). Thus cannot explain the rapid induction of metallothionein gene transcription, it but may nevertheless contribute a delayed boost. This slow increase in MTF-1 binding activityis presumably due to zinc-induced de novo protein synthesis, however a conversion of some inactive, or less active precursor form of MTF-l to a more active or higher affinity one can not be excluded. In any case, transcriptional activation of the "F-1 gene itself is ruled out, since MTF-1 mRNA is barely affected by metal treatment of cells (Figure 2, lanes 1 vs. 2-6). Recently R. Palmiter [21] has also reported that MTF-l induced constitutive transcription upon stable cell transformation, and he obtained evidence for a negative regulatory mechanism. He proposed that MTF-1 is a constitutive factor controlled by a zinc-sensitive inhibitor, tentatively designated MTI, which would be titrated out in the transformation experiments. It was thus suggestedthatthekeytounderstandingmetalregulationwouldbethe identification of such an inhibitor and determination of howit interacts with

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Fig. 5 Models for metal-induced transcription of metallothioneingenes by MTF-1. (A) Allosteric zinc finger model. Transcriptional inductionof the metallothionein gene is solely dependent on promoter occupancyby MTF-1. Zinc acts as a coinducer in that it transforms the "loose" zinc finger structures of MTF-1 into a DNA binding-competent form.

(B) Coxtivator model. In this model, a specific coacthator binds zinc upon increase of the intracellular

MTF-l proteinstofullyinduce zincconcentrationandinteractswith transcription of the metallothionein genes. (C) Combination of model A and B. Further possibilities such as metal-induced removalof an inhibitor, rather than recruitment of a coactivator are not shown. 420

l

Gene Disruption

of the Transcription Factor

MTF-1

421

"'F-l and respondsto meW. Our data presented before [l41 and here also are compatible with negative regulation by an inhibitor that controls MTF-l availability. However, we believe that the direct influence of zinc on the DNA binding properties of MTF-1 in vitro strongly suggest that MTF-1is itself a zinc sensor, orat least part of the in vivo sensing mechanism, rather than being acted upon by a putative metal sensing mechanism. Within the framework of such a mechanism, an inhibitor of MTF-l would not necessarily need to be regulated itself by hevy metal. The problem of metallothionein induction notwithstanding, it will be of great interestto see whether MTF-l, which is highly conserved betweenhuman and mouse, regulates additional cellular genes other than those for metallothioneins.

Materials and Methods Targeteddisruption of theMTF-1gene: Using the 520 bp KpnI-BspHI probe harboring the complete zinc finger region of the mMTF-l cDNA, a 17 kb DNA fragment was isolated from a genomic (AB-l; [22]) h GEM-l1 library(kindgiftfromDr. U. Mueller).Inthe targeting vector, based on pBluescript KS (Stratagene), the 146 bp KpnI-BamHI zinc finger exon fragment was replaced by either a blunt ended neomycin phosphotransferase or hygromycin expression cassette [23,24] in the antisense orientation. Into the polylinker EcoRI site 5' of the neo expression cassette, the blunt ended NcoI-Sal1 fragment of the upstream mouse sequence (UMS; [24, 251) was insertedto reduce unwanted readthrough of the targeted IvfI'F-l gene. 5' are 1.2kband5.5kbofintronsequences, and3'ofthemutatedexon respectively. The 3' intron region is followed by the herpes virus thymidine [X],which resulted ina 4.5-fold enrichment kinase gene for double selection for G418-resistant andtk- GS-ES cell colonies (GS-ES cellsare derived from mouse strain Agouti 129/SV) after selection with G418 (0.4 mg/ml) and FIAU (0.2 ClM) for 9 days. Identification of GS-ES cell clones with homologous recombination of the replacement vector was done as described [27] with the first specificprimer (GEN131) 5TGTGTATGTCTICITGGGGATGGAACC3' corresponding to genomic sequence upstream of the targeting vector sequence and the secondprimer (P6;[U] SAlTCGCAGCGCATCGCCTTCTATCGCCTcorrespondingto the 3' end ofthe neecassette.Cycling was done with an initial denaturation at 95°C for 3', then 38 cycles at 94°C for 0.5 min, 63°C for 0.5min. 65°C for 2 min and final extension at 65°C for 10 min. By Southern blot analyses, all PCRpositive clones were foundto be heterozygous for the MTF-l locus, without additional integrations of the targeting vector. The heterozygous clone 34F6 was elecmporated with the hygromycin targeting vector. After 8 days of selection (100 pg hygromycin B/ml and 0.2 pM FIAU/ml) resistant clones were picked and analysed by PCR as before, with the first primer (GEN136)

422

Radtke et al.

SATGTCTTCTTGGGGATGGAACC3'andthesecondprimer (p7;[24] S'CCGGGACTGTCGGGCGTACACA3'. Doubleselectionyielded5-fold ES cell clone outof 16 was correctly targeted.As before, enrichment, and one PCR-positive clones were verified by genomic Southern blot analysis. Culturing conditions for +I+ and 4- ES cells: ES cells weregrownonirradiated SNL76/7 feeder cells [23]without antibiotics. 24 hours before harvesting, cells were trypsinized and depleted of hour each on non-gelatinized cell culture feeder cells by two passages for one dishes. This method ensuresa greater than90%depletion of feeder cells. The remaining ES cells were plated on gelatine(1%) pretreated cell culture dishes for approximately 12 hours. For heavy metal-induction ES cells were treated with 400 pM ZnSOq or 80 pM CdC12 in growth medium (DMEM, 20% FCS, 0.1 mM 2-mercaptoethanol) as indicated. Please note that 400 p.M ZnSO4 or 80 pMCdC12used forinductionarehigherthantheusual concentrations for metal-induction of cultured cell lines. However, substantial amounts of heavy metal is probably bound to the highly concentrated serum proteins of ES cell medium. Reporter DNAs, transfections, transcript mappings and gel retardation assays: Metal-responsiveOVEC reporter genes, transfections, and S1 nuclease mapping of transcripts were as described previously [14]. Mapping of transcripts from M"F-l. Spl, metallothionein-1 andI1 genes: Preparation of cytoplasmicRNA was according to Radtke et al., [14]. RNase protection was performed as described in Mitchell et al., [27]. MTF-l antisense probe: full length,355 nts; 287 nts protected, corresponding to positions +373 (SnaBI) to+ M O (BamHI). Metallothionein-I antisense probe: full length, 259 nts; 105 nts protected, corresponding to positions +l to +l05 Metallothionein-II antisense probe: full length, 274 nts; 67 nts protected, corresponding to positions +340 to 4 0 7 relative to the transcriptionstart site [28]. Spl antisense probeis described in Mitchell et al. [27]. The signal intensities of protected RNA fragments were quantified using a PhosphorImager (Molecular Dynamics) and data were normalized against Spl transcript signals including the specific activity of the individual probes. This sensitive detection method allowedtousdetermine that in MTF-1 -/- ES cells, transcript levels of metallothioneinIand I1 genes were less than 1% of the basal level inMTF-1 +/+ cells. The autoradiograph from easy reference. one experiment was cut into strips and the bands arranged for Preparation of nuclear extracts and gel retardation assays, including the [13]. oligonucleotidesused, are described in Radtke et al.,

Acknowledgements We are indebtedto Fritz Ochsenbein for preparation of the Figures and Dto r. Ulrike Miiller for providing the genomic ES cell library. Wealso thank Drs. Pamela Mitchell, John Silke and Christopher Hovens for critical of reading the manuscript and valuable comments.

Gene Disruption of the Transcription Factor MTF-1

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REFERENCES 1. Kaegi, J. H. R. (1991) Methods in Enzymol. 205, 613-626. 2. Andrews, G. K (1990) Progress In Food And Nutrition Science 14, 193-258. 3. Michalska. A.E. and Choo, K.H.A. (1993) Proc. Natl. Acad. Sci. USA 90, 8088-8092. 4. Masters, B A . Kelly, E.J.. Quaife, C.J., Brinster. R.L. and Palmiter, R.D. (1994) Proc. Natl. Acad. Sci. USA 91. 584-588. 5. Uchida, Y.. Takio, K., Titani, K., Ihara, Y.& Tomonaga, M. (1991) Neuron 7, 337-347. 6. S t u q G. W.,Searle, P. F., Chen, H.Y.. Brinster, R. L. & Palmiter, R. D. (1984) Proc. Natl. Acad. Sci. USA. 81. 7318-7322. 7. Stuart, G. W., Searle. P. F. & Palmiter, R. P.(1985) Nature 317. 828-831. 8. Serfling. E., Luebbe, E., Dorsch-Haesler, K and Schaffner. W. (1985) EMBO J . 4. 3851-3859. 9. Westin,G. & Schaffner.W. (1988a) EMBO J 7. 3763-3770. 10. Mueller, P. R.. Salsa, S. J. & Wok, B. (1988) Genes Dm. 2,412427. 11. SSguin, C. and PrSvost, J. (1988) Nucleic Acids Research 16, 10547-10560. 12. Searle, P.F. (1990) Nucleic Acidr Research 18. 4683-4690. 13. Andersen, R.D.. Taplitz, S.J., Obezbauer, A.M..Calame, K.L. and Herschman, H.R. (1990) Nucleic Acids Research 18, 6049-6055 14. Radtke. F., Heuchel, R.. Georgiev, 0.. Hergersberg, M.. Gariglio, M.. Dembic. S. & Schaffner,W. (1993) EMBO 12, 1355-1362. 15. Westin, G. and Schaffner, W.(1988b) Nucl. Acids Res. 16, 5771-5781. 16. Kadonaga, J.T., Camer. K.R.. Masiarz, F.R. and Tjian, R. (1987) Cell 51. 1079-1090. 17. k g . J., Heuchel, R., Schaf€ner, W. and Kaegi, J.H.R. (1991) FEBS Lett. 279.310-312. 18. Brugnera, E.. Georgiev, 0..Radtke, F.. Heuchel, R., Baker, E., Sutherland, G. R. and Schafher, W. (1994) Nucl. Acids Res. in press. 19. Fuerst, P.. Hu, S.. Hackett, R., and Hamer, D. (1988)CeN 55, 705-717. 20. Hu, S., Fuersf P. and Hamer. D.H. (1990) New. Biol. 2. 1-13. 21. Palmiter, R.D. (1994) Proc. Natl. Acad. Sci. USA 91. 1219-1223. 22. McMahon,A. P. & Bradley, A. (1990) Cell 62, 1073-1085. R. & Bradley, A. (1991) Cell 64, 23. Soriano, P.. Montgomery. C., Geske. 693-702. 24. Ruffner. H., Reis, L.F.L.. Niif, D., & Weissmann C. (1993) Proc. Natl. Acad.Sci. USA 90. 11503-11507. 25. Heard, J.-M, Herbomel, P., Ott, M.-0.. Mottura-Rollier, A., Weiss, M., & Yaniv, M. (1987) Mol. Cell. Biol. 7, 2425-2434. 26. Mansour, S. L.,Thomas, K. R. & Capecchi. M. R. (1988) Nature 336, 348-352. 27. Mitchell, P. J., Timmons, P. M.. H6bert. J. M., Rigby, P. W. J. &Tjian, R. (1991) Genes Dm. 5. 105-119. 28. Sear1e.P.F.. Davison, B.L., Stuart, G.W.. Wikie, T.M., Norstedt, G. and Palmiter, R.D.. (1984) Mol. Cell. Biol. 4, 1221-1230.

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27 CharacterizationandPurification of MEP-l, a Nuclear Protein Which Binds to the Metal Regulatory Elements of Genes Encoding Metallothioneins Simon Labbk, Lucie Larouche, Jacinthe Prkvost, Paolo Remondelli, and Carl SCguinCentredeRechercheenCancCrologiede1'UniversittLaval,1'HBtelDieu de Quebec, 11 CBtedu Palais, Quebec G1R 2J6, Canada

I. INTRODUCTION

Heavy metals can have a profound deleterious effect on intracellularhomeostasis.Cellsrespond to fluctuationsinthe concentrations of various metals in the extracellular environment bymodulatingtheexpressionofspecific sets ofgenes.The metallothionein 0 genes havebeen the most intensively studied and best understood examples of metal-regulated genes and thus, provide useful a model system for understanding how an eukaryotic gene modulates its expression in response tometal ions, and for characterizing the signal transduction pathway involved in thisactivation. All vertebratesexamined contain two ormore distinct M" isoforms which are grouped into four classes, MT-I through MT-N (reviewed in [1,2]). MTs have been identified in a wide range of species and are present in various tissuesand cell types. MTs are inducible at the transcriptional level by a wide variety of agents that include metal ions such as Cd2+,Zn2+ and Cu2+,hormones, cytokines, alcohols, herbicides, and by a number ofstresses,including UV irradiation,heatandcoldexposure, to oxidative stress, or tissue injury resulting from the exposure turpentine, carbon tetrachloride, or bacterial endotoxin. Metals are the most general and potent of these inducers. The ability of vertebrateMT genes to be induced by metals is controlled in cis by a short DNA sequence (metal regulatory 425

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Labbd et al.

element or "MRE") present in multiple imperfect copies in the 5' flanking region of the MT genes (reviewed in [4]). Figure 1 shows thearrangementofthesix M W elementsonthemouseMT-I gene,theG-richsequencethatinteractswiththemajor late transcriptionfactorMLTFIUSF [5] and thetwo Spl [6] sites. Different MREs have different transcriptional efficiencies. MREd is the strongest, MREa and MREc are 50 to 80% weaker, MREb is very weak and MREe and MREf are apparently nonfunctional [7,8]. Detailed point mutation analysis of mouse MT-IMREa and MREd shows that the highly conserved core sequence, 5'TGCRCNC-3'@,purine; N, any nucleotide),iscrucialfor induction by metals(reviewedin [9]). At least two MREs are required for efficient metal induction and these elements can be present in different orientation. In metazoan, very little is known about the mechanismsby which the cell senses the metal concentration and transduces the information to the gene. It has been suggested that the ability of MREs to modulate transcription in response to metals depends on

MREe

-132

-150

-175

MREd

-

MREb MREI

MREt

MREe

"

-94

+l

"

-70

-54

I

1

-+ MT- 1

TATAAA

foolprint

...

-127

0.0.

...I

0.0.

GOCGCTCCAQ~ACICTCT~~~~~?~~QTCICGCTC~CTCTQ

I

I

MREd

L

MREc

Figure 1 Arrangement of the sixmetal regulatory elements (arrows)of the mouse MT-I gene, thebinding sites for thetranscription factor Spl, theG-richsequenceinteracting transcription factor MLTF andtheTATA

with the

box. The sequence around MREd is shown with

brackets above the sequence indicating thearea protected from ExoIII and brackets below the sequence indicating the positions of MREC and

MREd. Dots above the sequence indicate the

wnserved MRE core sequence TGCRCNC. Marks below the sequence indicate homology to the consensus Spl binding site; black dots indicate agreement with unique nucleotides, white dots indicate agreement with ambiguous nucleotides and the cross ambiguous site.

(X) indicates disagreement at an

MEP-l Interactions with Metallothionein Gene Promoters

427

the ability ofa getallozegulatory panscription factor(s) (MRTF) to bindto the sequencein the presence of metalsandinduce A critical step the in transcription (reviewed [3]). in characterization of the signal transduction pathway by which metal ions regulate gene expressionis to isolate a true MRTF regulating metalresponsivegeneexpression. The molecularcloningand characterization of a nuclear regulatory factor which can act as a to sense and translateinorganic metal-responsiveswitchable signals into changes in metabolism is central to define the signal transduction pathway mediating metal-regulated transcription.

II. IN VITRO FOOTPRINTING STUDIES We have used the mouse MT-I geneas model system to study MT gene transcription. Using an in vivo competition assay, we have shown that a limiting cellular factor for MT gene induction is a positive activator that, in the presence of metals, binds to the same sequencesrequiredformaximaltranscription [lo]. Using an Exonuclease111 (ExoIII) footprinting assay, we have shown one that or more nuclear factors, present in extracts from L cells bind to the mouse MREd (Figure 2, lane U, Figure 3, lanes 0, and Figure 4) [l 1,121. While the addition of exogenous CdCI, to the buffers of noninduced-cell extracts slightly increased the signal (Figure4, compare lanes 7-9with 10-12), the chelating agents EDTA (Figure 2, lane 0, and Figure4,lane 2) and 1,lO-phenanthroline[l21 selectively inhibit the binding of this proteinto the MRE.Binding activity could be restored by Zn2+,but none of the other cations tested (Cd2+,Cu2+,Mn2+,Mg2+,Ca2+)restored binding activity to the treated extracts (Figure 2). This shows that Zn2+ions are required for specific in vitro DNA binding of the MREd-binding protein and suggeststhattheMREdbindingcomponentis a metalloprotein which requires bound Zn2+ for the integrity of its DNA-binding domain. The nucleotide sequence recognized by this factor is the same as the one required for in vivo transcriptional activity of MREd, since individual nucleotide substitutions introduced in the core region (5'TGCACTC3', nucleotide -147- to 141) at either residues G (-146) or C (-143) completely abolished binding activity as assayed by competition experiments (Figure 3) 1121. Interestingly, extracts prepared with heavy metal-resistant L cells (L50) containapproximatelyfourtimesmore MREd-

428

Labbe et al. EDTA 0.1 mM

" "

(-1 53)

e

FEgure 2 Ability of different cations to restore extracts binding activity at the MREd region

followingEDTAchelation

as assayedby ExoIII footprinting analysis.Samples of extracts

prepared from L50 cells were dialysed overnight againsta buffer containing 0.1 mM EDTA as indicatedandthenincubatedin

the presence of increasing concentrations of ZnClz, CdCl,,

CuSO,, CaCl,, MnCl, or CdCI, plus ZnCl,.

After treatment, the binding activity was assayed

in footprinting reactions. Lanes:N. No extract. U. Untreated extract. The arrow indicates the protected band at nucleotide position -153 in the MREd region. (From Ref. 12).

binding activity than the extracts prepared from L cells (Figure4, compare lanes 1and 6) thus showing that an activation or increase of the synthesis of the MRE-binding factor had O C C U K ~ ~in the L50 cells. Southem-blothybridization,using L50 and L cell genomicDNAs and a mouse MT-1 cDNA, did notreveal any amplification of the MT genesin L50 cells (unpublished data). Genetically selected and naturally occurring lines of both cultured mammaliancellsandyeastexhibitsubstantialvariationsin MT synthesis and heavymetalresistance [13]. Experimentsutilizing cloned MT gene hybridization probes have demonstrated that such differences can becausedbychanges in the copynumber or

MEP-l Interactions with Metallothionein Gene Promoters MREd MUT-2 0

20

40

429

MUT-5 100

0

20 100

0

20

40

100

ng of

(-1 53)

t

Figure 3 Competition experiments in Exon1 footprinting assays. Competition was performed with double-stranded unlabeled oligos correspondingto a wild-type MREd (5'-CGATGCACTCCGCCCGA-3'.the

MRE core consensus sequence is

*

underlined) and to hvo mutants, "5 (S'-CGATCI'CI'GCAT"GCCCGA-3') and MUl"

*

2 (S'CGATCTQACACrCCGCCCGA-3'). Asterisks (*) indicate the positions of nucleotide

substitutions inthe mutated sequences. The amount of competitorused in each reactionis shown over the lanes and the probe concentration was approximately 1 nglreaction. Arrowas in Figure 2. (From Ref. 12).

methylation status of MT genes. Whiles enhanced MT synthesis was attributed to increased copy number of MT genes In some resistantcells [14], increased Cd2+ resistancedoesnotrequire increased copy number of MT genes [13,15] and may involve an

increase in the synthesis of other factors such as transcriptional regulatory proteins. The four-fold increase in the MW-binding activity detected by the footprinting assay in L50 cells may be the consequenceof an increase of the intracellular concentrationof the protein(s)bindingtothiselement or, alternatively, it maybe caused by an increase of the affinity toward the binding site as a

430

Labbd et al.

0 1 2 3 4 5 6 7 8 9101112

4

-153

Extracts prepared from the cadmium-resistantL cell line L50 show increased MREd-binding activity. ExoIII footprinting experiments were performed with10 pg (lane 3), 25 pg (lanes 4,

7, lo), 50 pg (lanes 5, 8, 11) or 100 pg (lanes 1, 2, 6, 9, 12) of extracts from mouse L cells

(lanes 1,2, 7-12) or L50 cells (lanes 3-6). Extracts were prepared from noninducedells with a final dialysis buffer containing no added EDTA (lanes 7-12) or 0.1 m M EDTA (lane 2) or

fromCd*'-induced

cells (lanes 1, 3-6). Lanes 10-12, the binding reaction was performed in

presence of 40 FM CdCI,. 0, no extract. The arrowhead to the right indicates the protected band at position -153 in the MREd region.

consequence ofa mutation. The cloning ofthe cDNA encoding this MRE-binding protein in L and L50 cells will be required to answer this question. The MREd element has the cyacity to respond to the same spectrum ofmetalions (Cd2+, Zn andCu +) as does the complete MT genepromoter [161, suggestingthatall MRE elements are responsive to the different metals and act together to facilitate a strong induction response. However, itis not yet known whether metal induction of MT genetranscriptioninvolves different factors binding to different MRE sequences or a single factorbindingtodifferent MREs withdifferentaffinities.To determine if different MRE elements can bind common nuclear factors, we performedcompetitionexperimentsin an Ex0111 footprinting assay, using synthetic oligodeoxynucleotides (oligos) correspondingtothemetalregulatoryelementsMREa, MREb, the MREc, MREd and MREe ofthemouseMT-Igene.Allof MREs testedcouldcompete with the protein(s)bindingto the +

43 1

MEP-1 Interactions with Metallothionein Gene Promoters

1

+ aMTgal + mMT-I mMREdl

"t mMREds

f-f

WE4

-8-

t M R E a

0 0

10

20

30

0

40

20

40

60

80

100

COMPETITOR (ng)

Fiere5 Graphic qmentations of ExoIII footprinting

competition experiments performed with

induced LSoCell nuclear extracts and double-stranded unlabelled

oligos corresponding to the

various mouse ( m m ) , human (hm or) trout (tMRE) MRE elements, as indicated, or with the same DNA fragment

(mm-I) used as a probe in the ExoIII assay. The probe was used

at a concentrationof approximately 1 ng per assay. The DNA competitoramgal is aplasmid

in whichmost of the promoter sequences have

been replacedby a fragment of the human

U-globin gene[IO]; no MRE element is present in this DNA. The intensity of the band at -153 (arrow in Fig. 2 and 3) relative to that with no competitor DNA (relative activity) is shown as a function of the concentration of competitor DNA. (From Ref. 17).

mouse MREd region, indicating that the same cellular factor binds to a ll ofthese MRE elements(Figure 5, leftpanel); M m , MREa, MREb and MREc showedsimilarcompetitionstrength, while MREe, a weakelement in vivo, was approximately 50% weaker [17. Competition experiments were also performed with synthetic oligos corresponding to the MFEa of the trout MT-B gene and to a regionofthe human MT-IIAgenecoveringthe M m element and the 5' endportionof MRE3. Allofthese oligos competed equally well and as strongly as MREd (Figure 5, right panel).Thus,theprotein@)binding to themouse MREd region can bind the otherMREs present in the mouse geneas well as MREs from MT genes of other swies.

432

Labbd et al.

III. SOUTHWESTERN ANALYSIS Using a protein blotting procedure (Southwestern) and synthetic oligos, we have shown that a nuclear protein of 108 m a , termed MEP-l, binds specifically toMREd (Figure 6) [181. In addition to the conserved MRE core sequence TGCRCNC, the MREd region contains a putative binding site (5'-YCCGCCC-3') (Y = T or C) for the transcription factor Spl. In order to assess the specificity MEP-l, we usedmutatedoligoscontaining ofthebindingto mutated version of the MRE oligo probe. In one oligo, five of the moststronglyconservednucleotidesofthe MRE consensus

.

MEP-l Interactions with Metallothionein Gene Promoters

433

sequence werealteredwhileinanother, five of the conserved nucleotides of theSpl site were changed.We showed that boththe MRE and Spl consensussequencesTGCAC and CCGCC are MREd to MEP-l (Figure 6). requiredforefficientbindingof Mueller et al. [6] have shown that, in vitro, purified Sp-l weakly binds to this region. The Spl factor purified as two polypeptides of 105 and 95 kDa [19]. The possibility that MEP-l was in fact a Spl was tested by using, in theproteinblottingprocedure, specific DNA probe known to bind Spl; a DNA fragment of SV40 whichcontainssix Spl bindingsitesdidnotbind MEP-l as assayed by Southwestern analysis (Figure 7) nor did it compete for the protein(s) that protected theMREd region against digestion by 1

2

3

4

Fire 7 southwestern analysis; biding of various '*P-labelled DNAs to nuclearproteins.

Lanes: l, MREd oligo; 2, SV40 DNA fragment; 3, MT promoter DNA fragment; 4, both MREd oligo and SV40 fragment. Heavy arrows correspond to MEP-l and thii arrows indicate

nuclear protein species that migrate with apparent Mrs 95,OOO and 105,OOO.(From Ref. 18).

434

Labbe et al.

theExoIIIenzyme(Figure 8). Similarly,a Spl oligodidnot compete for the proteins binding to the MREd region as assayed byExoIII(Figure 8 ) or DNase1 (LabbC, S and SCguin, C., unpublished data) footprinting analyses. Thus, despite the presence of a seven out of a eight base pair match withthe Spl binding site consensussequence (C/T)CCGCCC(C/A),the Spl transcription factordoesnotinteractwith the MREdprobe and therefore is different from MEP-1. We also performed Southwestern experiments to assess the bindingpropertiesofMEP-1todifferentMREelements.Our results show thatMEP-1 which specifically binds with high affinity to MREd also binds to theothermouseMREelements,with different affinities, mimicking their relative transcriptional strength invivo:MREd 2 MREa = MREc > MREb > MREe > MREf (Figure 9) Similarly, MREa of the trout MT-B gene and MRE4 of the human MT-IIA gene bind to MEP-1 [171. We have also detected a proteinsimilarin size toMEP-1inHela-cell nuclear extracts using mouse MREd or human M W oligo as the probe [17]. The nonfunctionalcore mutant MUT2,which is inactive invivo, does not compete forthe MREd-binding protein@) in an ExoIII footprintingassay(Figure 3), and did not bind to the relative MEP-1 [171. These results correlate very well with strength as metal-dependent cis-acting transcriptional elements of these MREs in vivo and suggest a central role of MEP-1 in the regulation of MT gene transcription.

[la.

1 2 3 4 5 6 7 8 9 10 11 1213 . . ..

1415 16 17 18 19 20 21 22

. . .

4

-153

(MREd)

Figure 8 Competitionexperiments in an ExoIII footprinting assay. The arrowhead indicates the ExoIJI stop at the -153 boundary. Competitors used in competition experiments. Lanes 1 4 , the oligo Spl(0,20.40, 100 ng);5-8, MREd (0,20,40,100 ng);9-13, the MT promoter DNA fragment(-200 to +a)(0, 1, 10, 50, 100 ng); 14-18, the SV40 DNA (0, 3.5, 35, 175, 350 ng); 19-22, a 309 bppBr322- Mspl fragment (0, 17, 34, 51 ng). (From Ref. 18).

MEP-l Interactions with Metallothionein Gene Promoters

435

MRE

dl

a

b

I

C

e

+ 92.5 69

Figure 9

-

Binding of oligo MRE variants to L S k l l nuclear proteins as assayed by

Southwestern. Mouse MRE oligo probes MREd to MREe as designated above the lanes. A

shorter exposure of the band corresponding to MEP-1 (arrows) is shown at the bottom of the figure. (From Ref. 17).

IV. W CROSS-LINKINGANALYSIS UV cross-linking experiments were performed with WO-cell crude nuclear extracts. The major protein species complexed with the MREd oligo (and cross-linked by UV irradiation) migrated on a denaturating gel with an apparent Mr of 115 000 [171. Moreover, using this UV irradiation assay, all of the mouse MRE elements tested were able to form the same complex of 115 kDa. Since the covalent attachment of short oligos to proteins has only a minor effect on the mobilityof these proteinsin SDS-polyacrylamide gel a protein of electrophoresis,theseexperimentsindicatethat

Labbe et al.

436

approximately 115 kDa bindsto the "binding sites. The molecular weight of this protein is consistent with that of MEP-l. Furthermore, while the chelating agent EDTA led to a 45 prcent inhibition of complex formation with the MREd oligo, Zn2 could restore the binding activity of chelated extracts to 135 percent of the control.

V. PURIFICATION OF MEP-l Wehavepurified MEP-1 to homogeneityandcharacterized its binding properties [20]. MEP-l has been purified from L50 cells using footprinting, Southwestern andUV cross-linking techniques to assayits binding activity. The purification scheme, starting from crudenuclearextracts,involvedacombination of heparinSepharose and MRE-DNA affinity chromatography (Table1). The enrichment of MEP-1 activity was estimated about 700-fold. The of purified protein preparation showed a single polypeptide band 108kDaonpolyacrylamidegelelectrophoresis,and2D-gel analyses revealed the presence of a protein species migrating as a singlepopulationof 108 m a . MEP-l doesnotappear to be glycosylatedsinceitelutedwithtfieflow-throughon a Wheat TABLE 1 Purification of mouse MEP-I'

Fractions

Total protein (W)

Nuclear extract Heparin-Sepharoee DNA affinity 1 DNA affinity 2

Total Specific activity activity (Unit)b (Unit/pg)

4 430,000 30 400,000 0.2 500 80,000 0.001 25,000 2 5 , 0 0 0

120 15

Purification (fold)

1 7 130 6,800

a) Results obtained in a typical experiment, starting witha crude nuclear extract prepared from 6 1 of L50 cells (5 x 10s cells/ml). b) One thousand units of activity is defined as the amount of protein required to obtain the signal produced by the quantity of MEP-1 present in 270 pg (30 pl) of a standard crude nuclearextract,under the conditions of our Southwestern assay, after a 1 hr exposure at - 8 K , using 1 x 10s c p d m l of a mouse MREa oligo probe freshly labelled at 1500 cpdfinol. Source: Ref. [20].

MEP-l Interactions with Metallothionein Gene Promoters

437

GermLectinSepharosecolumn.Some transacting regulatory proteins such as Spl [21] and HNFl [22] are glycos lated posttranslationally, and Wheat Germ Sepharose, a lectin afmity matrix which recognizes N-acetyl-D-glucosamine, is used for the purification of RNA polymerase I1 transcription factors. Chelating Sepharosechargedwith asuitablemetalwillselectivelyretain proteins when amino acid residues (i.e., cysteine, histidine) which have an affinity for the chelated metal ions are exposed on the ChelatingSepharose proteinsurface. MEP-l is retained ba matrix charged with zinc while most o! f the protein was found in the flow-through [20]. Purified MEP-l binds specifically to MRE sequences and produces a footprint identical to that produced by the factor@) presentin crude nuclear extracts (Figure 10). As for

r

12 34 5 6 7 . I

I

Figure 10 DNase1 footprintinganalyses.Analysis

of chromatographicfractions.

Fifteen

microliter aliquots of heparin-Sepharose fractions (lanes 3-5) or of the second affinity column fractions (lanes 6 and 7) were added to the different reactions. Lanes: 1, no extract; 2, crude nuclear extract (10 pl); 3 and 6, flow through; 4 , 5 and 7, 650 mM salt fractions. The positions of the hfRE elements and of the USFMLTF binding siteare indicated on the leftas determined

by Mm-Gilbert sequencing. (From Ref. 20).

1

ExoIII f

2

3

~ analyses ~ of affinity purified ~ MEP-1 ~ following g ~m~titio with n

(A) or chelation with 1,lO-phenan~oline(B). A) Competition was various ~ m p e ~ t DNAs or ~

~

o with ~ unlabelled e d double-~~ oligos d ~ c o ~ e s ~ n to ~ na gwild-

(lanes 3-6) and to the mutant, MUT-5 (lanes 7-10). In each case 0, 20, competitors were used. Lanes: 1, probe alone without extract or digestion with EkoIII; 2, probe digested with Ex0111 in absence of extract. B) Ability of different cations.to rest0 binding activity at the MHZd following 1,10-phenanthrolinechelation. Samplesof puri 1 were treated without (lane 2) or with 500 pM (lanes 3 to 6) 1,lO-phenanthroline and then incubated in the presence of 100 p M ZnCl, (lane 4), 50 pM CdCl, (lane 5) or 50 p (lane 6), and assayed in f ~ ~ r i n t i experiments. ng Lane 1, no protein. Arrows as in Figure 2 and 3. (From Ref. 20).

Labbe et al.

440

Table 2 MRE-binding proteins Description Proteins

MTF-1

ZAP P39 MBF-1

ZRF

MREBP MRE-BF1R MafY

MREBP-34 P33

1)

2)

HeLacells;detectedbyelectrophoresismobility shift assay (EMSA) with mouse MREd; zinc-inducible; cDNA cloned; zinc finger protein; MW = 72,5 kDa; would be for basal expression, a constitutive activator responsible or wouldinteractwithanonDNA-bindinginhibitor (MTI) acting as a metal sensor [30-321. Nuclei from rat liver cells; EMSA with mouse MREa and M W ; zinc-inducible [8]. RatFAOcells;EMSA;needs two MRE elements for binding; cadmium-inducible; M W = 39 kDa [28]. MouseL-cells; MW = 74 kDa;cDNAclonedand corresponds to RP-A [33, 341. HeLacells;EMSAwithhuman MTIIA MREa; zinc inducible; purified; corresponds to MTF-1 [25l. HeLa cells; MW = 112 kDa; binds to several MREs of the human MTIIAgene promoter; partially purified [24]. Present in different human cell lines and interact with MREa of the human MT-I genes; MW = 86 and 28 kDa 1291. Mouse cells; cDNA cloned in yeast by genetic complementation;approximately 10 m a ; includes a single zinc finger sequence; constitutively expressed in L cells;inducestranscriptioninametal-independent fashion [27]. Cultured human fibroblastes and HeLa cells; EMSA with human MTIIA MREa; DNA binding enhanced by Cu; MW = 34 kDa [26]. Liver nuclear extracts; EMSA with =c’; MW = 33 m a ; involved in basal transcription (231.

There is a good correlation between the binding affinity of MEP-l towards the different mouse MREs, as assayed in vitro by southwestern analyses, and their relative strength for transcriptional metal induction in vivo. The nucleotidesequence recognized bypurified MEP-l is

MEP-l Interactions with Metallothionein Gene Promoters

441

the same as that required forin vivo transcriptional activity of mouse M W . 3) ThebindingofpurifiedMEP-l to MRE elements is metaldependent.MEP-lbindingactivity is inhibitedby the chelating agent 1,lO-phenanthroline andcan be restored by zinc ions. MEP-l is sufficient to produce a specific footprint 4)Purified on MRE elements. 5) Nuclearextractspreparedfrommetal-resistant U 0 cells containedapproximatelyfourtimesmoreMREd-binding activity than extracts prepared from induced L cells. The assessmentof MEP-l as a metal regulatory protein and of the possible interactionswith other MRE-binding proteins will require the cloning of the corresponding cDNA, and cotransfection experiments usinga MEP-l expressing vector together with a reporter plasmid. The purification of MEP-l provides an essential tool to answer these questions. ACKNOWLEDGEMENTS This project is supported by grants number 1 R01 CA6126141 from the National Cancer Institute, NIH, and MT-12468 from the Medical Research Council. CS is a Scholar from the Fonds de la recherche en sand du Quebec.

REFERENCES l. G.K. Andrews, Prog. Food Nutrition Science 1 4 1 9 3 (1990). 2. M.P. Waalkes and P.L. Goering, &m. Res.Toxicol. 3:281 (1990). 3. D.J. Thiele, Nucleic Acids Res. 2&1183 (1992). 4. D.H. Hamer, Annu. Rev. Biochem. 55:913 (1986). 5. R.W. Carthew, L.A. Chodosh, and P.A. Sharp, Genes & Dm. 1:973 (1987). 6. P.R. Mueller, S.J. Salser, and B. Wold, GenesDev. 2:412 (1988). 7. G.W. Stuart, P.F. Searle, and R.D. Palmiter, Nature 31Z828 (1985). 8. P.F. Searle, Nucleic Acids Res. 184683 (1990). 9. J. Imbert, V.C. Culotta, P. Fiirst, L. Gedamu, and D.H. Hamer, Adv. Inorg. Biochem. 8 1 3 9 (1990).

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10. C. SCguin, B.K. Felber, A.D. Carter, andD.H. Hamer, Nmre 312:781 (1984). 11. C. SCguin and D.H. Hamer, Science 2321383 (1987). 12. C.SCguin, Gene 97:295(1991). 13. M.D. Enger, C.E. Hildebrand, J.K. Griffith and R.A. Walters, in Metabolism of trace metaZs in man, vol. 2 0.M. Rennart and M. Y. Chan, Eds. CRC Press, Boca Raton, 1984, pp.7-24. Palmiter, Proc.Nutl.Acad. Sci. USA 14. L.R.BeachandR.D. 78:2110 (1981). 15. A. Chopra, J. Thibodeau, Y.C. Tam, C.Marengo, M. Mbikay, and J.P. Thirion, J. cell Physiol. 142:316 (1990). 16. V.C. Culotta and D.H. Hamer, Mol. cell. Biol. 91376 (1989). 17. S. LabbC, J. PrCvost, P. Remondelli, A. Leone,andC.SCguin, Nucleic Acids Res. 19:4225 (1991). 18. C. SCguin and J. Prevost, Nucleic Acids Res. 1610547 (1988). Tjian, Science 19. M.R.Briggs, J.T. Kadonaga, S.P. Bell,andR. 23447 (1986). 20. S. LabbC, L. Larouche, D. Mailhot, and C. Seguin, Nucleic Acids Res. 21: 1549 (1993). 21. S.P. Jackson and R. Tjian, Cell 52125 (1988). 22. S. Lichtsteiner and U. Schibler, Cell 521179 (1989). 23. P.K.Dattaand S.T. Jacob, Cell. Mol. Biol. Res. 39439 (1993). 24. S. Koizumi, K. Suzuki, and F. Otsuka, J. Biol. &m. 267: 18659 (1992). 25. S. Koizumi, H. Yamada, K. Suzuki,and F. Otsuka, Eur. J. Biochem. 210555 (1992). 26. S.H. Hahn andW.A.Gahl, Biochem. Med. Metab. Biol. 50346 (1993). 27. C. Xu, DNACell Biol. 12517 (1993). 28. R.D. Andersen, S.J. Taplitz, A.M. Oberbauer, K.L. Calame, and H.R. Herschman, Nucleic Acids Res. I86049 (1990). 29. M.Czupryn, W.E. Brown, and B.L.Vallee, Proc. Natl. Acud. Sci. USA 8910395 (1992). 30. G. Westin and W. Schaffner, EMBO J. Z3763 (1988). 31. F. Radtke, R. Heuchel, 0. Georgiev, M. Hergersberg, M. Gariglio, 2. Dembic, and W.Schaffner, EMBO J. 12: 1355 (1993). 32. R.D. Palmiter, Proc. Natl. Acud.Sci. USA 91:1219(1994). 33. J. Imbert, M. Zafarullah, V.C. Culotta, L.Gedamu,and D.H. Hamer, Mol. Cell. Biol. 95315 (1989). 34. K. T. Suzuki, N. Imura, and M. Kimura, Metallothionein III: Biological roles and medical implications,Birkauser, Basel, 1993.

CopperAccumulation,and MetallothioneinStabilityand DevelopmentalRegulation,inthe Toxic Milk Mouse Jim Koropatnick, Greg Stephenson, and M.George Cherian Department of Oncology,LondonRegionalCancerCentre, 790 CommissionersRoad East, London, Ontario N6A 4L6, Canada

I. INTRODUCTION A. Copper homeostasis Copper is a trace metal, widely distributed in nature,is both that essential for [l]. It isarequiredcofactor for life and toxic under some circumstances oxidativeenzymes(catalase,peroxidase,cytochromeoxidase,andothers), tyrosinase (involved in the formation of melanin), superoxide dismutase, amine of iron.Copper oxidases,anduricase,and is essentialfortheutilization deficiency leads to hypochromic, microcytic anaemia resulting from defective hemoglobinsynthesis.Dietarycopper is absorbedinamannernormally

443

444

Koropatnick et al.

regulated by body stores [2] and subsequently transported in serum bound initially to albumin and later araeruloplasmin. to Most copperis stored in liver andbonemarrow:thebile is thenormalexcretorypathway,andplaysa primary role in copper homeostasis.The toxic effects of copper may be due to its participation in the formation of oxygen radicals through Fenton and HaberWeiss reactions (discussedby Cai et al., this volume): these reactive species have the capacity todamage cell membranes, mitochondria, proteins and DNA

131. The dual capacity of copper to both nurture and damage living cells suggests thatgenetically-perturbedhomeostaticcontrolwouldlead to pathological changes. In fact,there are at least two inheritedinborn errors ofcopper metabolism in humans that lead to disease. Menke's disease and Wilson disease (hepatolenticulardegeneration) are bothcausedbyadisruption in copper transport [4]. Menke's disease ( "kinky hair" syndrome) is characterizedby peculiar hair,failure to thrive, low liver and brain coppex levels, and excessive copperaccumulation in othertissues: the P-type ATPase putativecopper has recently been described [5transporter gene defective in Menke's patients 71. Defects in a similar but distinct gene have alsobeen reported for Wilson disease [8], which is characterized by failure to incorporatecopperinto The caeruloplasmin in the liver, and to excrete copper from the liver into bile. consequence is excessivehepaticcopperaccumulation.Aspectrumof histopathologic changes have been reported in the livers of Wilson disease fatty metamorphosis,followedby patients. The initialchangesconsistof necrosis and fibrosis ofthe parenchyma with gradual progressionto cirrhosis [9,10]. The hepatic damagemay be related to the accumulation of copper [ll]: the localization of copper in hepatocytes changes with the stage of hepatic damage. Copper is present mainly in a diffuse form in hepatocytes during initial fatty changes, whileit is present in lysosomes at later stages of fibrosis and cirrhosis [121. The hepatic cirrhosismay be caused bythe toxic ionic form ofcopper in the liverbecauseofthesaturationofbindingsites of metallothionein with that metal [13]. In a previous report, we showed that in Wilson metallothionein is one of the major hepatic copper-binding proteins in copper levels above normal disease [131. An approximately 50-fold increase controls was observed in two patients who underwent liver transplantsat the

Effects of Copper Accumulation in the Toxic Milk Mouse

445

University Hospital in London,Ontario. Control human liver had high levels [14]. In of zinc-containingmetallothionein, as comparedtootherspecies Wilsonsdisease,therewasathree-foldincreaseinMTlevelsabove this already high MT level. Unlike MT from normal livers, it was. saturated with copper.Theseresultssuggestthatmetallothioneinaccumulationandmetal association are altered in Wilson disease: there are few suitable experimental animal models to study these changes in copper metabolism and liver damage. The copper transporter genes implicated in Menke's and Wilson disease are similar to previously identified prokaryotic heavy metal transpoiters [8] and are described in detail elsewhere in this volume. Because of the importance of copper for normal metabolismandcell function, and the alteredcopper homeostasis and transport associated with human disease, increased insight into the handling of copper in singlecells and whole organismsis essential.

B. Animal Models to Study Copper Metabolism 0

Long-Evans cinnamon rat: accumulate hepatic copper and metallothionein 0 spontaneous jaundice, hereditary hepatitis, and hepatocellular carcinoma potential model for Wilson disease

0

Brindled mouse: X-linked elevated kidney copper 0 decreased liver copper potential model for Menke's disease

0

Blotchy mouse Macular mouse Inherited copper toxicosis in Bedlington terriers Toxic milk mouse

0 0 0

Figure l Animal models of abnonnal copper metabolism

Several mutations exist in animals that involve copper toxicity (Figure 1). The Long Evans cinnamon (LEC) rat exhibits abnormally high hepatic copper

446

Koropatnick et al.

accumulationandincreasedhepatitis [15]), harboursamutationintherat homologue ofthe putative Wilson disease gene, and may be a good modelfor m al., and Suzuki et al., discussed elsewherein this volume). that disease (Cox The X chromosome-linked bbtchy and brindled alleles [l61 have been described in rhice: brindled mice may provide a model for Menke’s disease. Macular mice with altered copper accumulation [l71 and Bedlington terriers with inherited copper toxicosis[l81 have also been described. An autosomalrecessivemutationwhichalterscopperhomeostasis in C57BL/6 mice was first described by Rauch in1983, who dubbedthe mutation toxic milk because oflow copper contentin milk of relatively young (1-2 month old) affected dams (19,201. Toxic milk miceexhibittwo kinds of copperrelateddefects.Juvenilemice are copperdeficient anddisplaymarkedly reducedhepaticcopperconcentration,hypopigmentation,reduced growth, abnormal locomotor behaviour, tremors, and ultimately death at 2 weeks of manifest themselvesbecausethe age:thepathologicaleffectsapparently diminished hepatic copper reserve in embryonic mice remains uncorrected by copperdepleted milkfromaffectedmothers.If,however,theprogenyof affected dams are fostered to lactating normal females, or are administered copper (by injection or gastric intubation) during the neonatal period toraise hepatic copper to normal levels, they survive into adulthoodwith few symptoms of the uncorrected disease during the first 2-4 months of life. Maternal mice greater than2-3 months of age also appear to supply sufficient copper in their milk to allow survival of offspring (J. Koropatnick, unpublished observation). Although copper-rescued toxic milk mice are relatively normal early in life, they do not entirely escape the effects of early copper deprivation. They exhibit markedly decreased fertility beginning between 3-5 months of age, enlarged spleenswithsignificantlyhighermetallothioneinandcopperaccumulation (Table 1) and develop alopecia, mild tremors, andi n c m e d growth of incisor teeth (indicative of poor feeding), all of which lead to overall degeneration 8-12 monthsofage(unpublishedobservations). requiringeuthanasiaby (SOD) activity has been reported, while Increased hepatic superoxide dismutase [20] andhepatic caerulop~asminremains serum CaerUlOphsmin islow unchanged [21]. They also exhibit an enormous increasein hepatic copper by one month of age ([22] and Table 2) which is accompanied by degenerative

447

Effects of Copper Accumulation in the Toxic Milk Mouse

changes. The earliestandmostpronouncedeffect is in theendoplasmic reticulum (which forms concentric whorls) and in the integrity of nuclear membranes resulting in invaginations by cytoplasmic elements. Heavily copperloaded n o d mice of thesamestrainthatgaveriseto toxicmilk mice exhibited none of the same pathological changes, suggesting thattoxic the milk defect (and not copper toxicity alone) was involved[22]. At 6 months of age or more, they reported that nodules appeared in liver surrounded by fibrotic bands with proliferating bile ducts and round cell infiltrates (predominantly lymphocytes) observable by light microscopy: only a minority of homozygotic toxicmilk mice developedgrosslyvisible .modules ([22] andKoropatnick, unpublished observations). The visible nodules havebeen reported to contain apparently regenerating hepatocytes with fewer pathological changes than

Table 1 Biochemical Changesin Control and MutantAdult Mouse Spleen Mice

WEIGHT

SOD

MT

(g)

w/mg

W&

cu (Irgh3)

Zn (Ccgk)

protein) 27.2 f

0.38 f

23.8 f 6.0 (3) 61.5 f

3.5 (2) 16.1 f

22.0 f

Spleensweredissectedandhomogenized in 0.25 M sucrose.Superoxide dismutase (SOD)activity was assayed by the method of Sun et al, I988 [26], metallothionein (MlJ by the Ag-heme method [ 2 7 and Cu and Zn by atomic absorption spectrophotometryin an air-acetylenecflameP8J. l’he average age for control andmutantgroups was 6.6 f 1.0 and 7.0 f 0.7 months, respectively. llu?results are expressed as means f SE. l’he asterisk denotes dtrerence signifcandy dl#erentfrom wild type. l’he number in parentheses is the number of animals assessed.

448

Koropatnick et al.

surroundingnecrotichepatictissue,andcharacteristicallyhomogeneous electrondense lysosomesthatresemblethelysosomalgranulescapable of sequestering excess copper in the hepatocytes of patients with advanced Wilson disease [12,231 and in Bedlington terriers affected by copper toxicosis [24,25].

II. METALLOTHIONEIN IN ToxlC MILK MICE Metallothioneins(MTs) are lowmolecular-massmetal-bindingproteins involved in resistance to heavy metals [29,30]. They are bound primarily to zinc and copperin humans and rodents untreated with heavy metals[14], and are encoded by a familyof genes in primates (a single functional MT-2 gene, a brain-specificMT-3gene,and at leastfivefunctionalMT-1genes)and rodents(by single copiesofMT-1,MT-2andMT-3genes).MTgene expression is induced by heavy metals and a variety of other agents, including hormones and y and U.V. irradiation [31-341. MT genes are developmentally regulated in rodents [35-373 and humans [38], and are overexpressed in some solid human tumours [12]. Because of the wide range of conditions affecting been MT expression, a homoeostatic role in zinc and copper regulation has postulated for the protein [30]. In order to understand this homoeostatic role and relate it to human diseases known to involve abnormal metal metabolism, it is desirableto study organisms with altered metallothionein expression. Some cultured cells with naturallyoccurring high (B16F1 mousemelanoma cells [40]; IGR 39 human melanoma cells [34]) or low (S49.1mouse lymphoma and W7 mouse thymoma cells [41]) MT expression, or high MT expressiondue to transfection of transcriptionally active foreign MT genes[41-44]exist.However,apartfromthe LEC rat (discussed by Suzuki et al., this volume), animals with mutations resulting in altered MT tissue levels have not been available. Because of the altered copper has shown to be a major retention in toxic milk mice, and the fact that MT been hepatic copper-binding protein in Wilson disease [13,45], we examined tissue MT levels as well as the rate of decay of newly-synthesised MT and non-MT proteins, in toxic milk mice and the unaffected wild-type animals from which they were derived. Adult (2-3 month old)toxic milk mice had more than 100-fold more

Effects of Copper Accumulation in the Toxic Milk Mouse

449

Table 2 Metallothionein, copper, zinc and cadnrium in toxic milk and wUdtype mice content (jlg/g of tissoe)

MT

cu

zn

Cd

10.3 f 3.3 (8)

4.8 f 1.5 (8)

33.6 f 2.7 (8)

N.D. (4)

174 f 6

7.0 f

(8)

0.3 (8)

44.4 f 8.4 f 1.5 (8) (8) 1.1

20.8 f 0.4 (7)

4.6 f 0.4 (7)

19.1 f 0.9 (7)

0.2 f 0.1 (7)

25.9 f 5.6 (7)

5.7 f 0.4 (7)

18.8 f 0.9 (7)

13.2 f 8.6 (7)

2215 f 127 (12)

325 f 116 (12)

71 f 6 (12)

0.22 f 0.13(4)

1969 f 141(11)

246 f 16 (1 1)

60 f 43 (1 1)

10.0 f 0.2 (11)

35.5 f 9.3(12)

9.0 f 0.7 (11)

27.5 f 0.4 (11)

0.4 f 0.2 (4)

48.0 f 10.1(11)

11.6 f 0.8 (11)

20.2 f 0.8 (1 1)

2.7 f 0.2 (11)

Results are means f S.D. for the numbers of animals given in parenthesa. 2hsue samples were collected 24 hours afier i.p. treatment with 5 mgkg cd (“CdCI,”) or iso-osmotic saline (“Controln). MT, copper, zinc and cadmium were measured as describedfor Table 1. N.D = not detected. metallothionein protein(asmeasured by the Ag-haem method)in the 10,OOO g supematantfraction of liverprotein than wild-typemice in theabsence of exogenousinducingagents (P<0.0002)(Table 2). Because metallothioneins remain soluble even after heat denaturation[27], assay of soluble proteins was

le t o ~ i ~c

icm

i copper 2 ~ levels § i ~ n i ~ c

6.2 f 1.2 (8) 2.3 f 0.5 (4) 2.9 f 0.3 (8)

.~2)(resultsnot shown). Liver zinc levels were gh in toxic milk than in ~ild-typemice (Table issue copper levels were also elevated in toxic miZk

o signi~cantdifferences in kidney and liver glutathione (GSH) levels were

roteins were separat

452

Koropatnick et

al.

Metallothionein protein from toxicmilk mouse livers was associated primarily with copper and, to a lesser extent, zinc. It was separableon a Sephadex G-75 column in a manner indistinguishable from purified MT isolated from normal mouse livers (Figure 3a). Greater than 8096 of hepatic copper in toxic milk mouse, and approximately50% of hepatic cytosolic zinc, was associated with a protein peak co-migrating with purifiedMT. Therefore MT was the major copper storage proteinin mutant mice. Treatment with cadmium depletedthe cytosolic metallothioneinantainingprotein fraction of both zinc (decreased 49 9 6)and copper (decreased 1196). Copper and zinc in the high molecular mass non-MT fraction were changed only slightly: zinc content wasdecreased 7% andcoppercontentwasincreased 5% (Figure3b).Cadmiumtreatment increased hepatic cytosolic cadmium levels from undetectable to 4.0 ppm, almostexclusivelywithinthe MTantaining columnfraction (Figure 3b). Therefore, hepatic metallothionein intoxic milk mice was capable of binding administered cadmium, presumablyby displacing MT-bound zinc (to a major degree) and copper(to a minor degree) in a manner similar to that observed in neonatal rats [SO].

m.

DEVELOPMENTALREGULATIONOFMETALLOTHIONEININ TOXIC MILK MICE

The copperdeficiency in juvenile mice, coupled with pathological copper accumulation in adult mice, suggested that metallothionein levels might be correlatedwithabnormal metalaccumulation.Wemeasured copper and metallothionein protein and mRNA accumulation during early and late postnatal developmentin mutant and wild-type miceto determine ifthis was hue. All animals assessed were suckled from dams providing sufficient copper to sustain development. Hepatic copper was, indeed, significantly depleted in toxic milk mice prior to weaning and enhancedone at and two months of age (Figure 4A). Liver metallothionein protein levels were similarlywellamelated with copper content. Intriguingly, thetoxic miZk mutation was sufficientto ovemde the normal signalling events determining the well-characterized early developmental regulationof metallothionein protein accumulation [35-371:MT protein was significantly decreasedin mutant mice three days after birth (Figure

Effects of CopperAccumulation

in the Toxic Milk Mouse

453

FRACTION

Figure 3 Copper, zinc and cadmiumcontent of metallothionein in toxic milk mouse liver. In vivo association of toxic milk mouse liver metallothionein with copper, zinc and cadmium 24 hours after treatment with iso-osmotic saline (A) or 5 mgkg CdCI, (B).Liver cytosols were prepared and separated using as described in Table a Sephadex G-75 column, and metal content determined 1. The arrow indicates the positionas which purified rat liver MT-2 [49] and peak rabbit liver MT-l and MT-2 (Sigma) was eluted fromthe column and the of cadmium content in liver cytosol isolated from wild-type mice 24 h after treatment with 5 mgkg cadmium.

454

Koropatnick et al.

4B). MT-l plus MT-2 mRNA accumulation prior to weaning correlated well with protein and copper accumulation, with MT low levels on theday of birth (3.6-foldlessthan in wild-type animals) risingto n o m 1 by 8 dayspostin partum. Because assessed animalsbegan to receivesufficientcopper maternal milk immediately after birth, it is conceivable that the rapid rise in MT mRNA during the first weekof life wasduetoinduction andlor sequestration of copperMT. However, in one andtwo month old mice, there was no significant difference inMT-l + MT-2 mRNA accumulation in wildtypeandmutantmice(Figure 5). Thus, toxic milk mice had altered MT, and MT mRNA. The profile developmental regulation of hepatic copper, of wild-type mouse MT mFWA accumulation (elevated until weaning, with decreased levels thereafter) was consistent with that observed in several other mouse strains [35,37].

IV.POST-TRANSCRIPTIONALCONTROL OF METALLOTHIONEIN ACCUMULATION IN TOXIC MILK MICE The lack of correlation between MT mRNA leveland MT protein accumulation in adult mutantmice is outlined in Table 4. There was no significant difference between MT mRNA levels in mutant and wild-typemice uninducedbymetals, or aftertreatmentwithcadmium or zincmetal salts (P=0.4), nor were there sex-related differences in the MT mRNA level in control toxic milk (P=0.4) or wild-type (P=0.2) mice (data not shown). The size of the MT mRNA from induced mutant micewas identical with that seen in wild-type liver RNA after hybridization to the radiolabelled MT-l/MT-2 probe (data not shown). Thus MT gene control elements(for MT-1, MT-2, or both) were responsive to metal inductionin both toxic milkand wild-type mice, resulting in increased mRNA accumulation, presumably due to increased gene transcription. Cadmium treatment also resulted in increased MT protein accumulation in kidneys of both wild-type andtoxic milkmice, and in livers of wild-type mice. However, no significantincrease in mutantmouseliver MT proteinwas e c a u s e : a) MT mRNA synthesized in detectable (Table 2). This could be b response to cadmium induction might not be translated into MT protein in

Toxic Milk Mouse

Effects of CopperAccumulationinthe

455

mutantmice, or b)increasesinhepatic MT resultingfrom MT gene transcription might be too small to be apparentwithinmeasurement error associated with the extremely high constitutive hepatic MT protein levels in toxic milk mice (Table 2). We hypothesized that MT protein in mutant mice was degradedat a lower rate than in wild-type mice, resulting in the observed enhanced accumulation. To test this possibility, the decay rate (Kd) of MT before and after induction with cadmium was measured in both mutant and wild-type mice. The protocol to radiolabel cellular proteinsin vivo with %-Lcysteine and follow the decay of radiolabelled metallothionein specificallyis outlined in Figure 6. A

LIVER COPPER

'"[

0

8

9

1

-0

0 +I+ 0 txltx

10

20

30 40 50 AGE (days)

60

LIVER METALLOTHIONEIN

10

20

30

40

50

70

B

60

70

AGE (days)

Figure 4 Developmentalchanges inhepaticcopper (A) and metallothionein o(B in) toxic milk (&l&)and wild-type (+/+) mice. Values are f SE of estimations from 3 mice sampled at each developmental stage.

456

Koropatnick et al.

SDSIPAGE gel electrophoresis successfullyseparated radiolabelled MT from other proteins: the putative low molecular weight MT band competed for

I

I

I

I

0

I

I

I

Toxic milk (n= 4) Wild type (n =4)

I

I

I

I

I

I

0

10

20

30

40

50

f 60

Figure 5 Liver metallothionein mRNA in toxic milk and wild-type mice during development. Total cellular RNA was isolated by a standard guanidinium isothiocyanatehot phenol method and separated by agarose gel electrophoresis. The northern blot was hybridized, in sequence, to a radiolabelled 11 kb mouse probe containing complete genomic MT- 1 and MT-2 DNA, and an 18s rRNA probe. The ratio of MT mRNA/18S rRNA was calculated from data derived from densitometer tracing of resulting autoradiographs. The mean ratio f SE (n=4 mice) is plotted.

Effects of Copper Accumulation in the Toxic Milk Mouse

Table 4

457

Metallothionein mRNA accumulation in wild-type and toxic milk mice M T mRNA/@-actinmRNA* Treatment

I Control Liver

Kidney

zincs

*

I

I

* Wild-type

Toxic milk

0.3 f 0.1 (8)

0.25 f 0.004 (6)

1.9 f 0.2 (6)

2.0 f 0.2 (5)

0.20 f 0.02 (5)

0.20 f 0.04 (5)

1.9 f 0.2 (6)

2.2 & 0.3 (5)

0.9 f 0.3 ( 5 )

1.2 f 0.1 (6)

From densitometry of autoradiographs of total tissue RNA dot blots hybridized to MT and 0-actin cDNA probes; results are means & SE for the numbers of animals given in parentheses; MTIP-actin mRNA ratios were measured in six individual RNA dots for each tissue sample. t Animals treated with 1 mg of CdC12/kg i.g. 6 hours prior to sampling. $ Animals treated with 20 mg of ZnC12/kg i.p. 6 hours prior to sampling.

binding to rabbit anti-(rat MT-2) antibody and contained a high proportion of 35S-cysteinelabel (Figure 7), as expected considering the high cysteine content of metallothionein. The specific activity of biochemically separated MT was used to measure the decay rate (the slope of the regression of the natural logarithm of [35S]cysteinedpm versus time. MT half-life (t%)was calculated from decay rate by the relationship [50,51]: t N =(M)/fractional

decay rate

Figure 8 illustrates the loss of radioactivity in MT (indicating MT decay) over

458

Koropatnick et al.

time in both cadmium-induced and wild-typemice. Assuming singleexponential decay rates [28], hepatic metallothionein in toxic milk mice decayed significantly more slowlythan in wild-type animals (Table 5). Cadmium treatment decreasedthe half-life ofMT to a minor degree in both toxic milk and wild-type mice, but the d i f f m c e in degradation rate between the two m b e d significant (Figure 8 and Table5):the differencein MT halflife betwean wild-type and mutant mice after cadmium induction (77%) was equal to that in animals untreated with cadmium(79%). In addition, decreased loss of MT in mutant mouse liver appears to be specific to M"', or a small

Tirne(h)

0

I

6

1

24

48

72

96

I

I

I

I

I

l

l

l

I

or isotonic saline nontadioactlve

L-cysteine

120

I

Figure 6 Experimental protocol tomeasuremetallothioneinstability in toxic mUk and wild-type mice. Both mutant and wild-typemice were injected i.p. with isotonic salineor cadmium chloride, and with radiolabelled Lcysteine 6 hours later (during the peak of induced MT production). Cold L-cysteine(in approximately lob-fold excess) was injected at 24 hours post-induction and the experiment to minimk every 24 hoursthereaileruntiltheendof reincorporation of radiolabel into MT.

Effects

Of

Copper Accumulation in the Toxic Milk Mouse

RELATIVE MT LEVEL (RIA)

[35S]dpm (X l o 2 )

LANE

2

3

459

4

0

5

101520

Figure 7 Sepamtionand iden@%ation of mdiohbelhd hepatic MT. Soluble hepatic protein fractions from one representative toxic milk mouse 24 h after p S ] cysteinetreatment(lane 3) or awild-typemouse 24 hour after PS] cysteine treatment and 30 h after cadmium induction (1 mg/kg)(lane 4) were separated on a polyacrylamide gel, stained, and radiolabel was measured in of increasingmolecular mass is proteinfractions.Radiolabelinproteins depicted on theright. M" protein was measured byradioimmunoassayin pooled fractions 1-8, 9-16, and 17-24 and the results are shown on the left. Commercial rabbitMT-1 (lane 1) and MT-2 (lane 2) were m as standards; the indistinctbands between 18.5 and 27.5 kDa are due to contaminatingnon-MT protein in the commercial preparations.

460

Koropatnick et al.

fraction of hepatic proteins of which MT is a part: non-MT proteins from wildtype and toxic milkmice decayed with half-lives of 76.2 f 11.1 hours and 70.0 f 9.3 hours, respectively. Cadmium treatment did not introduce differences ([28] and data not shown). Thus, non-MT proteins were equally stable in toxic milk and wild-type mice.

Table 5

Fmctional decay mtes (K,,)and hav-lve 1% of metallothionein in control and cadmium-inducedwild-type and toxic milkmouse livers

Animals

Wild type

Treatment

K*

Control

-0.034 f 0.007 (? = 0.87)

20.4 f 3.1

-0.019 f 0.002

16.4 f 4.1

Cadmium

(? = 0.89) Control

-0.042 f 0.004

36.5 f 1.5

(? = 0.91) Toxic milk

Cadmium

' ~

-0.024 f 0.003 (? = 0.94)

29.2 f 3.8

Correlation coeflcients (?) were calculated as the SE of the estimate of the slope. Cadmium = 1 mg CdClJkg i.p. 24 hours prior to thefirst sample being taken.

It is unclear whether the increased stability of MTin mutant mouse liver is due to structural differences in MT resulting from increased association with copper (similar to the approximately 4-fold i n c m in Cd-MT half-life in comparison with Cu,Zn-MT[48,52-561). Since those studies reported &creased

Effects of Copper Accumulation in the Toxic Milk Mouse

46 1

toxic milk wild type

.-> .-> W W

Cd-induced

l*-

1

0

g .-U

II-

E

10-

v)

In 0 -

-c

7i 9-

tw[tx; = 29;.

6

1

f 1.8 h

t,Iwtl = 16.4 f 4.1 h

0

20

40

60

80

100

120

Time after [35S]cysteine treatment(hours) Figure 8 Degmaktion of hepatic merallothwnein in toxic milk and wild-type mice. Toxic milk (0) and wild-type (0)mice were treated with cadmium (1 mgkg 6 hoursprior to radiolabeling)("Cd-induced"), or iso-osmoticsaline ("Controln).Specific activity of MT separated by SDS-PAGE was determined between 24 and 120 hours as described in the text.

462

Koropatnick et al.

rather than increased stability for Cu-MT (the predominant species in toxic milk mice) this seems unlikely. Altered MT gene structure, or decreased activity of proteinase@),remaincandidatemechanismstoaccount for altered MT degradation, and are actively being explored. remains The underlying metabolic defect@) leading to reduced proteolysis undefined. IncreasedMT half-life intoxic milk mice does not imply that altered metallothionein regulationis the specific defect leading to pathological changes in mutant mice. Changes in degradation of other proteins,or in regulation of could also contribute to the toxic milk phenotype: other proteins or &As, (as altered regulationof copper transport through a specific transporter protein described for Wilson disease) mustbe considered in this regard. The similarities between human Wilson disease and the consequences of the toxic milk mutation in mice (excessive hepatic copper, low caeruloplasmin, [la], high hepatic reduced rate of incowration of copper into caeruloplasmin MT [13,45],and toxic changes in livers in both toxic milk mice and patients withWilsondisease [57,58] make t h w mice apossiblemodeltostudy perturbedcoppermetabolisminthisdisease. It is not yetclearwhether human disease is caused by MT increased MT and copper accumulation in the induction by excess copper, MT expression induced by toxic stress effects [29,59] co-incidentwith or causedbyexcesscopper, or altered MT metabolism. It is important to note that Cu-MT enhances lipid peroxidation in the presence of pro-oxidantsmore effectively than equimolar inorganic copper [60]: therefore, the accumulation of copper-associated MT in toxic milk mice and Wilson disease may enhance rather inhibit the toxic effects of the metal. We suggest that the increased MT protein stability and altered developmental MT protein and mRNA accumulation in toxic milk mice may be a model to help in understanding human disorders (Wilson disease, for example) involving altered copper metabolism.

V. ACKNOWLEDGMENTS This researchwassupportedby grants to MGC and JK by the Medical Research Council of Canada, and grants from the London Regional Cancer Centre Research Fund and the Victoria Hospital Research Development Fund

Effects of Copper Accumulation in the Toxic Milk Mouse

463

to J.K. We are indebted to Dr. Harold Rauch for providing breeding pairsof toxic milkmice. We thankSusan Vesely andJane Pearson-Sharpefor excellent technical assistance.

REFERENCES 1. R.A. Goyer, ToxicEffects of Metals, in Casarett andDoull's Toxicology (M.O. Amdur,J.Doull,andC.D. Klaassen, Eds.), PergamonPress, 1991, pp. 623-680. 2. B. Sarkar, J.-P. Laussac, and S. Lau, Transport forms of copper in human

serum, in Biological Aspects of Metals and Metal-Related Diseases (B. Sarkar, Ed.), Raven Press, New York, 1983, pp. 23-40. 3. G.J. Brewer and V. Yuzbasiyan-Gurkan, Medicine 71, 139-164 (1992). of CopperTransport,in MetabolicBasis of 4. D.M.Danks,Disorders Znherited Disease (A.L. Beaudet,W.S.Sly,andD.Valle, Eds.), McGraw-Hill, New York, 1989, pp. 1411-1431. 5. C. Vulpe, B. Levinson, S. Whitney, S. Packman and J. Gitschier, Nature Genet. 3, 7-13 (1993). 6. J.F.B. Mercer et al., Nature Genet. 3, 20-25 (1993). 7. J. Chelly et al., Nature Genet. 3, 14-19 (1993). 8. P.C. Bull et al., Nature Genet. 5, 327-337 (1993). 9. P.J. Anderson and H. Popper, Am. J. Pathol. 36, 483 (1960) 10. F. Schaffner et al., Am. J. Pathol. 41, 315 (1962) 11. S. Goldfischer, et al., Am. J. Pathol. 99, 715 (1980). 12. S. Goldfischer and I. Sternlieb, Am. J. Pathol. 53, 883 (1968). 13. N.O. Nartey, J.V. Frei, andM.G.Cherian,Lab.Invest. 57,397-401 (1987). 14. J. Chung, N.O. Nartey, and M.G. Cherian, Arch. Environ. Health 41, 319-23 (1986). 15. N. Sugawara et al., Experientia 47, 1060-63 (1991). 16. D.M. Danks, J. Med. Genet. 23:99 (1986). 17. N. Shiraishi et al., Toxicol. Appl. Pharmacol. 110, 89-86 (1991). 18. G.F. Johnson et al., Hepatology 1, 243 (1981). 19. H. Rauch, J. Heredity 74, 141 (1983).

464

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20. H. Rauch et al., Hepatic copper and superoxide dismutase activity in toxic

milk mutant mice,in Superoxide and Superoxide Dismutase in Chemistry, 1986, p. Biology and Medicine (G. Rotilio, Ed.), Elsevier, New York, 304. 21. J.F.B. Mercer et al., J. Nutr. 121, 894-899 (1991). 22. L. Biempica et al., Lab. Invest. 59, 500-508 (1988). 23. I. Sternlieb, Gastroenterology 78,1615(1980). 2 4 . L.-C. Su et al., Am. J. Physiol243, G231 (1982). 25. D.C. Twedt, I. Sternlieb, and S.R. Gilbertson, J. Am. Vet. Med. Assoc. 175, 269 (1979). 26. W. Sun, L.W. Oberley and Y. Li, Clin. Chem. 34,497-500 (1988). 27. A.M.ScheuhammerandM.G.Cherian,Toxicol.Appl.Pharmacol. 82, 417422 (1986). 28. J. Koropatnick and M.G. Cherian, Biochem. J. 296-449 (1993). 29. D.H. Hamer, Annu. Rev. Biochem. 55, 913-951 (1985). 30. J.H.R. Klgi and B.L. Vallee, J. Biol. Chem. 235, 3460-64 (1960). 31. J.H.R. KSgi and A. Schlffer, Biochemistry 27, 8509-15 (1988). 32. J. Matsubara, Y. Tajima, and M. Darasawa, Radiat. Res. 111, 267-275 (1987). 33. A.J. Fornace,H.Schalch,andI.Alama,Mol.Cell.Biol. 8, 4716-20 (1988). 34. J. Koropatnick, M.E.I. Leibbrandt, and M.G. Cherian, Radiat. Res. 119, 356-365 (1989). 35. A.J. Ouellette, Dev. Biol. 92, 240-246 (1982). 36. G.K. Andrews, E.D. Adamson, and L. Gedamu, Dev. Biol. 257,294-303 (1984). 37. J. Koropatnick and J.D. Duerksen, Dev. Biol. 122, 1-10 (1987). 38. N.O. Nartey,D.Banerjee,andM.G.Cherian,Pathology 19,233-238 (1987). 39. N.O.Nartey,M.G.Cherian,andD.Banerjee, Am. J.Pathology 129, 177-182 (1987). 40. J. Koropatnick and J. Pearson, Somat. Cell Molec. Genet. 16,529-537 (1990). 41. K.E. Mayo and R.D. Palmiter, J. Biol. Chem. 256, 2621-25 (1981).

Effects of Copper Accumulation in the Toxic Milk Mouse

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42. S.L. Kelley et al., Science 241, 1813-15 (1988). 43. B. Kaina et al., P m . Natl. Acad. Sei. USA 87, 2710-14 (1990). 44. H. Lohrer et al., Carcinogenesis 11, 1937-41 (1990). 45. C.D. Bingle, S.K. Srai, and 0. Epstein, J. Hepatol. 15, 94-101 (1992). 46. M.E.I. Leibbrandt et al., Biol. Trace Element Res. 30, 245-256 (1991). 47. F. Tietze, Anal. Biochem. 27, 502-522 (1969). 48. K. Cain and D.E. Holt, Chem.-Biol. Int. 28, 91-106, 1979. 49. S.K. Kremski et al. Biochem. J. 255,483-491 (1988). 50. J.C.Waterlow,P.J.Garlick,andA.J.Millward, Protein turnover in mammalian tissuesand the whole animal, Amsterdam:NorthHolland (1978). 51. D.E. Laurin, D.M. Barnes, and K.C. Klasing, P m . Soc. Exp. Biol. Med. 194, 157-164 (1990). 52. S.L. Feldman, M.L. Failla and R.J. Cousins, Biochim. Biophys. Acta 4, 638-646 (1978). 53. M. Karin and H.R. Herschmann, J. Cell. Physiol. 103, 35-40 (1980). 54. S.L. Feldman and R.J. Cousins, Biochem. J. 160, 583-588 (1976). 55. I. Bremuer et al., Biochem J. 174, 883-895 (1978). 56. K.-S. Min, Toxicol. Appl. Pharmacol. 113, 299-305 (1992). 57. I.H. Scheinberg and I. Sternlieb, Majorproblems in internal medicine, vol. 23; Wilson’sdisease, W.B. Saunders, Philadelphia, pp. 1-27 (1984). 58. I. Sternlieb, in Progress in liver diseases,vol.

(H.

4 Popperand F. Schaffner, Eds.), Grune and Stratton, New York, p. 511 (1972). 59. M. Karin, Cell 41, 9-10 (1985). 60. G.F.Stephenson,H.M.Chan,andM.G.Cherian,Toxicol.Appl. Pharmacol. 125, 90-96 (1994).

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29 MetallothioneinSynthesis Is Selectively Enhanced by Copper in the Liver of LEC Rats Kazuo T. Suzuki Faculty of Pharmaceutical Sciences, ChibaUniversity, Yayoi, Inage, Chiba 263, Japan

I.

INTRODUCTION

Long Evans rats with cinnamon-like coat color (LEC) were established from Long Evans agouti (LEA)rats as an inbred strain showing hereditary hepatitis and hepatic carcinoma (1). This strain develops acutehepatitis at the age of a b u t 4 months. Those rats that survive the acute hepatitis will suffer from chronic hepatitis and develophepatoma after about 1 year (1). Thecausesand mechanisms leading to chronic hepatitis and/or hepatic carcinoma have been of great concern and LEC rats are recognized to be a valuable experimental model animal to study the whole process of spontaneous hepatic carcinoma.

A.

Accumulation of copperin the liver of LEC rats

The liver of LEC rats was found to accumulate much greater amounts of copper (Cu) than the original type strain (2). The Cu accumulating in the LECrat liver is mostly bound to metallothionein (MT) (2-7). Although the mechanisms how the Cu accumulating in the liver in a form bound to MT causes acute hepatitis and hepatic carcinoma are notknownyet,themetalisthought tocause spontaneous hepatitis and hepatoma. In fact, it was demonstrated 467

468

Suzuki

that LEC rats can be effectively cured from otherwiselethal signs by removing Cu selectively from theliver with tetmthiomolybdate (TTM) treatment (8). As a result, the LEC rat appears to be an interesting experimental model to study not onlyspontaneous hepatic carcinoma but also the toxicity and regulation mechanism of Cu (1- 10). B

LEC rats as an experimentalmodelanimal Wilsondisease

for

Wilson disease is recognized as an inherent disease of abnormal Cu accumulation in the liver (11-14). Patients of Wilson disease are cured of the hepatitis by treatment with chelating agents ( 12- 14). Likewise, Cu in the LEC rat liver was decreased by the administration of chelating agents, andthis treatment also inhibited the development of hepatitis (8),suggesting that the Cu accumulating in the liveris the cause for the toxicity both in Wilson disease and in LECrats. These to observationstogetherwiththeirpathogenicsimilaritylead recognize LEC rats as an experimental model animal for Wilson disease (2) 11. DISORDERED BIOLOGICAL REGULATION FOR COPPER METABOLISM Cu is an essential trace metal and is regulated homeostatically by fundamental metabolicprocesses to avoid any toxic effect by its excessive presence. The liver isa key organ in the regulationof Cu metabolism (11-14). Cu ion is preferentially transported in the blood as a complex of albumin (16), and the metal incorporated in excess into the liverinduces MT and binds to this protein. Cu in the liver is secreted to blood in ceruloplasmin binding form (17-19). However, the metabolic process for Cu homeostasis in the liveris still unclear. Disordered Cu metabolism in hereditay diseases certainly gives a clue to understand the normal functioning mechanism of Cu. LEC rats may demonstrate toxicity due to some disorder@) in the metabolic processes of Cu (2-10). A . Defective expression of the transporter gene efflux of copper in Menkes and Wilson diseases

for

Recently the cause of abnormal Cu regulation for Menkes disease has been suggested to be a lack in normal functioning CudependentATPase (20-22), whichmayindicatethatCuis

Metallothionein Synthesis Selectively Enhanced

by Copper

469

accumulated in the cell owingto the lack in the efflux mechanism of Cu and thatMT is induced to sequester the excessiveCu in thecell. Although thetwo genetic diseases appear to be quite differentin their symptoms, the cause of the other genetically disodered disease, Wilson disease is alsosuggested to be a lack in expressionof normal functioning Cu-dependent ATPase (23-25). Although this transporter for efflux of Cu has only been suggested from the gene sequences similar to that encoding bacterial Cu-dependent ATPase (26), the lack in normal functioning efflux system for Cu certainly results in abnormal accumulation of Cu inthe cell. Accumulation of Cu is toxic to the cell and thecell induces the synsthesis of MT to sequester free Cu ion in a form bound to MT. Therefore, theaccumulation of Cu in a form bound to MT in the liver of Wilson diseasepatients is thought to be the result of abnormal Cu accumulation. Cu does not accumulate in the liver of Menkes disease patients andthesymptomisthat of Cu deficiency.However, cultured Menkes cells accumulate Cu by exposure to Cu (27, suggesting the defective expression of the exporter gene for efflux of Cu at the absorption site which results in prevention of the deliveryof Cu to the liver (28). B Imbalancebetweenthesynthesis metallothionein

and degradation of

An abnormal accumulation of Cu in a form of M" can be caused by a different mechanism from that suggested by a lack in normal functioning transporter for efflux of Cu, Cu-dependent ATPase.Namely, anabnormalaccumulation of Cu-binding MT owing to accelerated induction or restricted degradation of MT may explain an abnormal accumulation of Cu in the liver becauseof the lack in anactive secretion of MT from the cell. The abnormal Cu accumulation ina form bound to MT in the liver of toxicmilk mice is suggested to be caused by an imbalance of MT metabolism by restricted degradation of MT (29). In the liverof this strain, MT is synthesized normally, but degraded at a slower rate than the control, which results in the accumulationof Cu bound t o

MT.

C. Accumulation of copper metallothionein

in a form other than

470

Suzuki

Indianchildhoodcirrhosis is known to be causedby an abnormal accumulationof Cu in the liver. However, the inducibility of MT synthesis by Cu and other heavy metals is lower than the normal level and the Cuaccumulating in the cultured fibroblast is not (30). presentin a formbound to MT inthisgeneticdisease Therefore, Cu seems to accumulate bya mechanism different from those mentioned above, suggesting the third mechanism that may not involve MT in itsaccumulation. 111.ACCUMULATION LEC RATS

OF COPPERIN THE LIVER O F

2.0

300

C' 250

Y

1.5

.I

.-.

-g 200 3 3 .CI

W

1.o

150

gS

9

Y

0

CO

E

c,

E

Y

5

Q E

U

100 0.5

8

50 0.0

0 2 4

6 8 l0 12 14 16 18

20

Age (weeks)

Figure 1. Changes in concentrations and content of Cu in the liver of LEC rats with age. Livers were homogenized in 4 volumes of extraction buffer and thehomogenates were centrifuged at 105,000g for 60 min. Cu levels in the liversof female LEC rats at 2,4,6,9, 12, 15, 16, and 16-18weeks of age were expressed by concentration (pg/g liver, open square) and content (mgliver, closed square) (From Ref. 31).

Metallothionein Synthesis Selectively Enhanced by Copper

471

Since it was suggested that hereditary hepatitis and hepatic carcinoma are caused by abnormal accumulation of Cu in the liverof LEC rats, attention has been focused on LEC rats from the two standpoints; One isthe mechanism leading to acutehepatitis and the whole process causing spontaneous hepatic carcinoma, and the other is the mechanism of disordered metabolism of Cu in relation to that in Wilson disease. Our approach from the latter standpoint is to be described in thepresent communication (8-10,28,31,33).

A. Accumulation of copperintheliver age

of LEC rats with

Although the Cu accumulating in the liver of LEC rats was reported to be present in a form bound to MT (2-3, those were as to of Cu accumulation and certainaspectsofthewholeprocess information about theinteraction with other metals suchas zinc (Zn) and iron (Fe) wasalso not sufficient. Therefore, we tried to find, at first, the whole processof Cu accumulation in the liver togetherwith thoseinthe bloodplasma,kidneys,andurine.Changes in concentrations of Zn and Fe in the liver, kidneys, and blood plasma were also traced to find the interaction of Cu with those metals (3 1). Figure 1 shows the whole process of Cu accumulation in the live of LECrats (31). Concentration of Cu in the liver is alreadyas high as approximately 200 pg/g liver at 2 weeks of age before weaning. Although Cu appears to decrease during and after weaning when the Cu level is expressed by concentration (pglg liver), the metal is shown to increase steadily in the liver when it isexpressed by content (mg/whole liver) (31). The Cu accumulating in the liver was distributed mostlyin thecytosol fraction and the cytosolicmetal was present mostly in the form bound to MT and Cu,Zn-superoxide dismutase (Cu,Zn-SOD) (9,31). The Cu level decreased suddenly after 16 weeks of age in accordance with the onset of jaundice. The Zn level started to decrease one week earlier than the Cu level did (data notshown). An abrupt increase of the Fe level in the liver was observed with the onset of jaundice (datanot shown) (31). B.

Changes in copper distribution in the plasma and kidneys of LEC rats following acute hepatitis

472

Suzuki

Following the accumulationof Cu up to the limit in theliver, concentration of the metal in theblood plasma increased dramatically and the metal boundto ceruloplasmin (Cp) increased, suggestng that holoceruloplasmin (holo-Cp) was excreted from the liver instead of apoceruloplasmin (apo-Cp) (10,3 1,32). This result implies further that Cu is supplied differentially to the two Cu-enzymes (Cp and SOD) synthesized in the liver. In addition to the increased distibution of Cu to Cp, the Cu increased in the blood plasma was shown to be present in the forms bound to MT and albumin toward the onset of jaundice (31). MT in the blood plasma is known to be filtered from the glomerulus and reabsorbed by the proximal tubules. In accordance with the increase of MT-bound Cu in plasma, concentrations of Cu in the kidneysand urine increased rapidly (10,31), indicating that Cu overflew from the liver to plasma in the form of MT when the metal accumulated to the limit. The details of the age-related changes in concentrations of Cu, Zn, and Fe are reported elsewhere (31) with intensive observationat the onset of jaundice(9, 10). IV. ENHANCED SYNTHESIS OF METALLOTHIONEIN IN THE LIVER OF LEC RTAS (28,33)

W e aimed to clarify how the LECrat liver accumulates Cuin a form bound to M" at a high concentration. For this purpose, the induced synthesis of MT by Cu was compared in vivo and in vitro between LEC andLEA rats (the original strain of LEC rats). A . Enhanced synthesis of metallothioneinin LEC rats (33)

the liver of

Among possible mechanisms for the accumulation ofCu in a form bound to MT in the liverof LEC rats, we tried to compare the As inducibility of MT by Cu from the following consideration; discussed above in 11. Disordered biological regulation for copper metabolism, accumulationof Cu in a form bound to MT is explained either by the result or by the cause. MT synthesis is tobe induced to sequester free Cu accumulating in the liver owing to imbalance between influx and efflux in the former case, while the protein accumulates in the liver owingto imbalance between synthesis and degradation of MT, resulting in an accumulation of Cu in the latter case.

Metallothionein Synthesis Selectively Enhanced by Copper

A

a .-

473

250

3 200 3

c 150 0

1-

c1

m

L: 100

.5

8

8

B

50

h

L

al

.-> S I

3

lo

t

1 0 1 2 3 4 5 6 7 8 9 Cu admistration (days)

Figure 2. Changes in concentrations of Cu and Zn in the whole livers and supernatants of Cu-loaded LEA and LEC rats. LEA rats were injected subcutaneously withCuC12 daily at a doseof 3 mglkg body weight for 2 , 4 , 6 and 9 days. LEC rats were killed without any treatment. Livers were homogenized in 3 volumes of extraction buffer and the homogenates were centrifuged at 170,000 g for 1 hr. Concentrations of Cu (A) and Zn (B) were determined by AAS. The closed circle and triangle indicate the concentrations in the liver and supernatant of LEA rats, respectively, while the open circle and triangle indicate thosein the liver and the supernatant in 5-week-old LEC rats, respectively. The concentration in the supernatant (pglg liver) was calculated by multiplying the value inthe supernatant by 4 (pglml). Data are expressed as means S.D. of 3 samples (From Ref. 33).

*

474

Suzuki

We observed that Cu accumulating in the liverin a form bound to MT can be removed selectively by treatment with a chelating agent, TTM (8). This result suggests that Cu bound to MT is not secreted from the liver, while Cu not bound to MT can be secreted from the liver, indicating that accumulation of Cu ina form bound to MT may be the cause in the liver of LEC rats. Then, we tried to find whether the accumulation of MT is due to enhanced synthesis or restricted degradation (33). For this purpose, the induced synthesis of MT by Cu and the distribution profiles of Cu and zincZn in the liverof LEA rats after injections of Cu wereexamined, and they were compared with those of LEC rats. 1. Accumulation of copper in the liver of LEA rats with injections of copper and comparison with those in LEC rats (33)

Concentrations of Cu in the LEA rat liverand the supernatant gradually increased with consecutive injections of CuC12 as shown in Fig. 2A. The formerconcentration was similarto that in fiveweekold control LEC rats after 4 injections (about 120 pg/g). Although the Cu present in the LEC rat was mainly recovered in the liver supernatant (Fig. 2A), only aboutone half of Cu in the LEArat liver was recovered in the supernatant (about 60 pg/g, Fig. 2A). The Cu concentration continued to increase in thewhole liver with injections reaching more than 200 pglg after 9 injections. However, only 40 % of Cu in the liver wasrecovered in the liver supernatant (about 90 pglg), which was still lower than that in the LEC rat liver supernatant (about 120&g). These results indicate that Cu was mainly retained in soluble forms in the LEC rat liver, but more than a half of Cu accumulating in theLEA rat liver was not recovered in the supernatant and remained in the non-cytosolic fraction. The concentrations of Zn in the whole liver and the supernatant of LEA rats increased slightly after Cu injections for 2 days, but it was less than one half of Zn concentration in LEC rats and was not elevated any moreby additional injections (Fig. 2B).

2. Disribution profiles of copper accumulatting in the liver of LEA rats by injections of copper (33) Chemical forms of the Cu accumulating in the LEA and LECrat livers were determined together with those of Zn by gel filtration chromatography (by HPLC-ICP onan SW column) as shown in Fig.

Metallothionein Synthesis Selectively Enhanced by Copper

475

LEA rat

1

"

10

15

20

l . . . . I .

10

15

...

.... 25

I . . . . l

20

LEC rat

LEA rat

MT-2,

I

.... I ....I

10

15

.

20

.

I . . . . I . . . . l . . . . ,

10

15

20

Retention time (min)

Figure 3. Changes in distribution profiles of Cu and Zn in the liver supernatants of Cu-loaded LEA and LEC rats. The distribution profiles of Cu (A) andZn (B) in the liver supernatants that correspond to those in Fig. 2 were determined by the HPLC-ICP method on an SW column. The vertical bar indicates the detection level for each element at0.05 pglml (From Ref.33).

476

Suruki

3. In the profile of control LEA rats, superoxide dismutase (Cu,ZnSOD) was eluted as distinct Cu and Zn-bindingpeaks at a retention time of 15.1 min (Figs. 3A and 3B). Injections of Cu into LEA rats induced the synthesisof MT in the liver, and Cu was sequestered by the protein. Two isoforms of MT (MT1 and MT2) were eluted at retention times of 19.4 and 18.1 min, respectively, MT2 being the major isoform in the Cu-loaded LEA rat liver. The amount of MT was gradually increased by 4 consecutive injections of Cu, but was not elevated by anyadditional injections. Excessive Cu not bound to MT was distributed to unidentified proteins which were eluted as high molecular weight proteins between the void volume of the column and MT peaks. The amountof SOD stayed constant during consecutive loadings of Cu to LEA rats. Cu in the liversupernatant of LEC rats was mainly bound to MT. Unlike in LEA rats, MT1 was the major isoform in the LECrat liver. The elution profiles of Zn changed slightly with loadingof Cu to LEArats (Fig. 3B). The small Zn peakin the MT fraction observed in the control rat liver was gradually decreased by the loading with Cu. In LEC rats, Zn was mainly recovered in the MT fraction, and MT1 was also the major component containing Zn. 3. Expression of MT and MT mRNA inthe LEA rat liver by injections of copper (33)

The amount of MT induced in the liver was estimated by measuring the concentration of Cu bound to MT. The amountof Cu bound to MT was gradually increased with injections of Cu up to 4 times, but did not increase afteradditional injections (at maximum, about 22 pglg of liver), approximately 40 % of Cu in the liver supernatant being bound to MT (data not shown). Hepatic MT mRNA was analyzed by Northern blotting. The MT mRNA content in the LEA rat liver was elevated by 2 injections of Cu, but did not increase beyond this level if additional Cu was injected. The MT mRNA content in the LEA rat liver was much lower than in the LEC rat liver (Fig. 4) (33). Lactate dehydrogenase (LDH) activity was used as an indicator for the cytotoxicity caused by Cu administration. LDH activity was not changed by Cu administration, indicating that Cu did not damage the rat liverunder the conditions used in the present experiment. The results raised a question why the Cu accumulating in the liver of LEA

Metallothionein Synthesis Selectively Enhanced

by Copper

477

l 2 3 4 5 6 7

Figure 4. Amounts of MTmRNA in the livers of Cu-loaded LEA and LEC rats. LEA rats were injected subcutaneously with CuC12 daily at a dose of 3 mglkg body weight. Total RNA was extracted from the liversof LEA rats 1 day after thelast injection at 2,4 and 6 days,andalsofromtheliver of LECratswithouttreatment. Amounts of MT mRNA were determined by Northern blot analysis using a mouse MT1 cDNA probe. Lanes 1 and 2; LEA rats without treatment: Lanes 3,4, and 5; LEA rats with 2, 4, and 6 injections, respectively: Lanes 6 and 7 ; LEC rats without treatment (From Ref. 33).

rat liver inthe forms not bound to MT is not toxic as judged bythe LDH activity (33).

The in vivo studydescribed above in this section suggests that MT synthesis is induced only to a limited extent in the liverof LEA rats even if Cu is taken up andaccumulates to the same level or more than that in the LEC rat liver without any signs of liver damage. In other words, MT synthesis in the liver of LEC rats is enhanced and Cu accumulating in the liver ispreferentially sequestered in a form bound to MT.

B.

Selective enhancement of metallothionein mRNA expression by copper in primary cultured liver parenchymal cells of LEC rats (28) The in vivo study described in the preceding section indicates that Cu was taken up dose-dependently by the liver of LEA rats, however, only a limited amount of Cu was present bound to MT (33). These results suggest that the high MT level is the cause for the

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0

5

10

15

20

25

30

Cu treatment @M)

Figure 5. Uptake of CUby primary cultured liver parenchymal cells prepared from LEAandTTM-treatedLECrats. Liver Parenchymal cells prepared from LEA rats (open circles) and TT” treated LEC rats (closed circles) were exposed to 0.125, 0.25, S, 10 and 25 PM CUfor 6 hr, and concentrationsof Cu in the cells(pg/mg Protein) were plotted againstCu concentration in the medium (From Ref. 28).

abnormal Cu accumulation in the liver of LEC rats. The low MT level in LEArats was reflected in the low MT mRNA level, while the high MT level in LEC rats correlated with the high MT mRNA level. These results suggest thatCu is accumulated in the liver of LEC rats because of enhanced MT synthesis. To clarify the mechanism functioningin LEC rats, we further compared the inducibility of MT synthesis between the mutant LEC of Cu and MT rats and the control LEA rats by determining the levels

mRNA in liver parenchymal cells prepared from LEC and LEA rats. Levels of MT and MT mRNA in the liver parenchymal cellsof LEC rats were reduced to the same levels as those of LEA rats before preparing the cells by removing Cu selectively with repeated injections of tetrathiomolybdate (TTM) into LEC rats(8). Then, the MT mRNA level was compared in the two cells by exposure to inducers of MT: cadmium (Cd), Cu, dexamethazone (Dex), and Zn (2%. 1. Uptake of copperandexpression of metallothionein mRNAin primary cultured liver parenchymal cells prepared from LEA and ITM-treated LEC rats

The amounts of Cu and MT mRNA were comparable in both up liver parenchymal cells before exposure to Cu. Cu was taken dose-dependently by both cells and the amount was comparable between LEA and LEC rat cells, although the mutant cells seemed to take up moreCu by exposure to a dose higher than 10 p M (Fig. 5). MT mRNA was expressed dose-dependently by exposure to Cu in both parenchymal cells (Fig. 6). However, spots on the gel by Northern blotting shown in Fig. 6A were more dense in the cells prepared from TTM-treated LEC rats than those prepared from LEA rats, and this relation was clearer in Fig. 6B after analysis with an image analyzer. Fig. 6B indicates that MT mRNAwas expressed than in LEA cells by exposure to Cu, both in more efficiently in LEC a dose-dependent manner up to 10 pM, beyond which there was a leveling off. Expression of &actin mRNA was constant by exposure to cu ( 2 8 ) .

2.

Selectiveexpression of metallothwnein mRNA by copper

Expression of MT mRNA by exposure to typical MT inducers including Cu was examined in both parenchymal cells to determine whether the response to Cu is specific to the genetic disorder. MT mRNA expression by the three inducers was comparable in both parenchymal cells as shown in Figs. 7A and 7B, indicating that response to MT inducers other thanCu is not affected by mutationin LEC rats, but is specifically enhanced by Cu(28).

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A

I

LEA

1 2 3 4 5 6 '

B

I

LEC

7 8 91Ollld

8000 n .c) .L

C

1

L

ie

4

4000

Figure 6. Amounts of M" mRNA expressed by exposure to Cu in primary cultured liver parenchymal cells prepared from LEA and of MTmRNA inboth liver TTM-treated LECrats.Amounts parenchymal cells exposed to Cu for 6 hr were analyzed by Northern blotting for the cellsused in Fig. 5. LEA (lanes 1 - 6) and LEC cells (lanes 7 - 12) were exposedto CuC12 at concentrations of 0 (lanes 1, 7), 1.25 (lanes 2, €9,2.5 (lanes 3, g), 5 (lanes 4, lo), 10 (lanes 5, 1l), and 25 pM (lanes 6, 12). The amounts were expressed by spot density on a gel (A)and by arbitrary units after processing with an image analyzer(B)(From Ref. 28).

Metallothionein Synthesis Selectively Enhanced

A

LEA ' 1 2 3

4 " 5

48 1

by Copper

LEC 6 7

8 '

B

Control

Zn Cd Treatment

Dex

Figure 7. Amounts of MT mRNA expressed by exposure to Zn, Cd and dexamethazonein primary cultured hepatocytespreped from LEA and TTM-treated LECrats. Amounts of MT mRNA in the liver parenchymal cells exposed to Zn (25 PM), Cd (2 PM) and dexamethazone (Dex, 10 PM) for 6 hr were analyzed by Northern blotting. The amounts were expressed by spot density on a gel (A) and by arbitrary units after processing with an image analyzer (B). Control (lanes 1 and 9, Zn (lanes 2 and 6), Cd (lanes 3 and 7 ) , and Dex (lanes4 and 8) (From Ref. 28).

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CONCLUSION

V.

Cu in the liver of LEC rats is already much higher than in the liver of control LEA rats before weaning. The liver continues to take up Cu from foods andwater and accumulates constantly with age ina form bound to MT up tothe limit of approximately250 pglg liver. Thus, the Culevel was correlated with MT level and, then, the MT level was correlated with MT mRNA level in the liverof LEC rats. Further, MT synthesis in the liver of LEC rtas was shown to be enhanced selectively by exposure to Cu. These results suggest that the accumulation of Cu in the liver of LEC rats is caused by the enhanced synthesis of M T and that theenhanced synthesis of IvlT is the cause of the accumulation of Cu in the liver of LEC rats. The conclusion mentioned above indicates that the mechanism for abnormal accumulation of Cu in the liverof LEC rats is different from thatof the defectiveexpression of the exporterfor effluxof Cu. If the mechanism in LEC rats is the same as that explained by the defective expression of the gene of Cu-dependent ATPase, then a reasonable explanation for the enhanced expression of MT mRNA by Cu in LEC rats must exist. One possible such explanation may be that the transcription factor for the MT gene was present in the nucleus even after the corresponding MT mRNA disappeared by TI" treatment. However, this transcription factor yet to be characterized (28).

REFERENCES 1.

2. 3.

4.

5.

N. Takeichi, H.Kobayashi, M. C. Yoshida, M. Sasaki, K. Dempo, and M. Mori, Acta. Pathol. Jpn. 38: 1369-1375 (1988) Y. Li, Y. Togashi, S. Sato, T. Emoto, J-H. Kang, N. Takeichi, H. Kobayashi, Y. Kojima, Y. Une, and J. Uchino, J. C h . Invest. 87: 1858-1861 (1991). Y. Li, Y. Togashi, S. Sato, T. Emoto, J-H.Kang, N. Takeichi, H. Kobayashi, Y. Kojima, Y. Une, and J.Uchino, Jpn. J. Cancer Res. 8 2 490-492 (1991). N. Sugawara, C. Sugawara, M. Sato, M. Katakura, H. Takahashi,and M. Mori, Res. Commun. Chem.Pathol. Pharmacd. 72: 353-362 (1991). N. Sugawara, C. Sugawara, M. Sato, M. Katakura, H. Takahashi, and M. Mori, Experientia 47: 1060-1063 (1991).

Metallothionein Synthesis Selectively Enhanced by Copper

6.

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H.Sakurai, A. Fukudome, R. Tawa, M. Kito, S. Takeshima, M. Kimura, N. Otaki, K. Nakajima, T. Hagino, K. Kawano, S. Hirai, and S. Suzuki, Biochem. Biophys. Res. Commun. 184: 1393-1397 (1992).

7.

H. Sakurai, H. Kamada, A. Fukudome, M. Kito, S. Takeshima, M. Kimura, N. Otaki, K. Nakajima, K. Kawano, and T. Hagino, Biochem. Biophys. Res. Commun. 185: 548552 (1992).

K. T. Suzuki, K. Yamamoto, S. Kanno, Y. Aoki, and N. Takeichi, Toxicology 83: 149-158 (1993). 9. K. T. Suzuki, S. Kanno, S. Misawa, and Y. Sumi, Res. Commun. Chem. Pathol. Pharmucol. 82: 217-224 (1993). 10. K. T. Suzuki, S. Kanno, S. Misawa, and Y. Sumi, Res. Commun. Chern. Pathol. Pharmacol. 82: 225-232 (1993). 11. I. H.Scheinberg, and I. Sternlieb, in Truce Elemenb in Human Health and Disease. (A. S. Prasad, Ed.), Academic Press, New York, 1976, pp. 415-438. 12. I. Bremner, Nutritional and physiological significance of metallothionein in Metallothionein JZ. (J. H. R. Kagi, and Y. Kojima, Eds.), BirkhauserVerlag,Basel, 1982, pp. 81-107. 13. D.M. Dank, in Metabolic Basis of Inherited Disease. I . (C. R. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle, Eds.), McGraw-Hill Information Service Company, New York, 1989, pp. 1411-1431. 14. I. Sternlieb, Hepatology 1 2 1234-1239 (1990). 15. I. H.Scheinberg, M. E. Jaffe, and I. Sternlieb, N. Engl. J. Med. 317: 209-213(1987). 16. K. T. Suzuki, A. Karasawa, and K. Yamanaka, Arch.Biochem. Biophys. 273: 572-577(1989). 17. R. J. Cousins, Physiol. Rev. 6 5 238-309 (1985). 18. I. Bremner, J. Nutr. 117: 19-29 (1987). 19. T.Yamada, T. Agui, Y. Suzuki, M. Sato, And K.Matsumoto, J. Biol. Chern. 268: 8965-8971 ( 1993). 20. C. Vulpe, B. Levinson, S. Whitney, S. Packman, and J. Gitschier, Nature Genet. 3: 7-13 (1993). 21. J. Chelly, Z.Tiimer, T. Tonnesen, A. Petterson, Y. IshikawaBrush, N. Tommerup, N. Horn, and A. P. Monaco, Nature Genet. 3: 14-19 (1993). 22. J. F. B. Mercer, J. Livingston, B. Hall, J. A. Paynter, C.Begy, S. Chandrasekharappa, P. Lockhart, A. Grimes, M. Bhave, 8.

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D. Siemieniak, and T. W. Glover, Nature Genet. 3:20-25 (1993). 23. P. C. Bull, G. R. Thomas, J.M. Rommens, J. R. Forbes, and D. W. Cox, Nature Genet. 5: 327-337 (1993). 24. K. Petrukhin, S. G. Fischer, M. Pirastu, R. E. Tanzi, I.

Chernov, M. Devoto, L. M. Brzustowicz, E. Cayanis, E. V i d e , J.J.Russo, D. Matseoane, B. Boukhgalter, W. Wasco, A. L. Figus, J. Loudianos, A. Cao, I. Sternlieb, 0. Evgrafov, E. Parano, L. Pavone, D. Warburton, J. Ott, G. K. Penchaszadeh, I. H. Scheinberg, and T. C. Gilliam, Nature Genet. 5: 338-343 (1993).

25. R. E. Tanzi, K. Petrukhin, I. Chernov, J. L. Pellequer, W.

Wasco, B. Ross, D.M. Romano, E. Parano, L. Pavone, L. M. Brzustowicz, M. Devoto, J. Peppercorn, A. I. Bush, 1. Sternlieb, M. Pirastu, J.F. Gusella, 0. Evgrafov, G. K. Penchaszadeh, B. Honing, I. S. Edelman, M. B. Soares, 1. H. Scheinberg, and T.C. Gilliam, Nature Genet. 5: 344-350 (1993). 26. F. Portillo, and R. Serrano, EMBO J., 7, 1793-1798 (1988). 27. D. M. Danks, J. Camakaris, S. Herd, J.R. Mann, and M.

Phillips, Copper Transport inMenkes' Disease, in Biological Aspects of Metals and Metal-related Diseases. (B. Sarkar, Ed.), Raven Press, New York, 1983, pp. 133-146. 28. S. Kanno, J. S. Suzuki, Y. Aoki, and K.T. Suzuki, Res. Commun. Chem. Patlwl. Pharmacol. in press (1994). 29. J. Koropatnick, and M. G. Cherian, Biochem. J. 296 443-

449 (1994). 30. S. H.Hahn, M. L. Brantly, C. Oliver, M. Adamson, S. G. Kaler, and W. A. Gahl, Pediatr. Res., 35: 197-204 (1994). 3 1. K. T.Suzuki, S. Kanno, S. Misawa, and Y.Aoki, Toxicology, in press (1994). 32. M. Sato, N. Hachiya, Y. Yamaguchi, J.Kubota, Y. Saito, Y.

Fujioka, H. Shimatake, Y. Takizawa, T. Aoki, Life Sei., 53: 141 1-1416 (1993). 33. S. Kanno, Y. Aoki, J. S. Suzuki, N. Takeichi, S. Misawa, and K. T. Suzuki, J. Inorg. Biochem., in press (1994).

Index Antimutagenic effects, 87 Antioxidant, 21,22 Arsenite, cytotoxicity of, 93 Ascorbate, 37 ATPases, 5,305,356,357,366, 444,468 Bleomycin: cobalt, 190 copper, 190 iron, 185 Blotchy mouse, 445 Brindled mouse, 445 British Anti-Lewisite(BAL),368 Cadmium, 3 , 9 C-fos, 3, 13 c-jun, 3, 13 c-myc, 3, 13 Calf thymus DNA, chromium binding to, 43 Calmodulin, 2,13 Catalase, 22,102,134 Catecholamines. 329,332,339 Ceruloplasmin, 276,309,319,332, 364,446 Chelation therapy, 368 Chernobyl children, 21 Chromatin, 102,132 oxidative DNA modifications, 132 Chromium(VI), 37,39 485

Chromosome aberrations,25,26 Cobalt, 69 copper: copper deficiency, 305 copper exchange, 307 copper transport, 324 excretion, 333,365 toxicosis, 277 Copper histidine: bioavailability,312 biochemical response to, 330 formulation for treatment, 308 in human serum, 306 Menkm disease treatment, 305, 317,323 stability constant, 306 structure, 306 transport, 324 uptake, 323 Cross-linking, 69,70 CytochromeC oxidase, 275,312, 318,324,380 Cytochrome P-450,156 Cytotoxicity, 102, 121 D-penicillamine,329,340 DNA cleavage: by cationic metalloporphyrins, 153 by i r o n 0 EDTA, 217 by iron finger,243

486

DNA damage: by benxne metabolites, 133 by caffeic acid, 139 by copper, 103 by H202,135 by iron.103 metal-induced, 101 nickel-induced,64 by non-mutagenic carcinogen, 145 by o-phenylphenol, 136 oxidative, 131 by pentachlorophenol, 141 by tryptophan metabolites, 143 DNA depurination,80 DNA hypermethylation,53 64 DNA methylation, nickel-induced, DNA-protein complex, 217 DNA-protein crosslinks, 53 DNA strand breaks,90 Dopamine-p-hydroxylase,275,324, 339,340,312 Ehlers-Danlos type V, 297 Endoplasmic reticulum,57,277 Esterase D, 345 Estrogen receptor, 239 Fenton reaction, 101,112,132,154, 219,229 Footprinting, 427 Free radical,21,22, 101,275 Free radical scavenging, 87 Gene amplification, 88 Gene expression, 2, 13,278

Index

Gene regulation, 102 Genetic toxicology: Hg2+, 3 lead, 3,9 Genotoxic carcinogens,53 Genotoxic effects,73 Genotoxicity: chromium0, 37 cobalt@), 69 nickel(@, 69 oxygen radical,102 Glutathione peroxidase,22,102 Glutathione reductase, 22 Hepatitis, 363 Heterochromatic DNA,53.57 HIV-1, 167,255 Holliday junction, 227 Hydroxyl radical, 22,71, 101, 113, 154, 186,219,224,229,243 Indian childhood cirrhosis, 275 Iron@) EDTA, 217 Irradiation, 22 Kaiser-Fleischer ring,345,368 Lanthanideo, as synthetic nuclease, 173 LEC rats, 355,445,467 Leukocyte, 24 Linkage analysis, 277 Lipid peroxidation, 121 Lipid peroxides,73 Lipoic acid, 23

487

Index

Lou Gehrig’s disease, 380 Lysyl oxidase, 276,312,318 Macrocyclic complexes, 174,201 Mannitol, 308,318 Maxam-Gilbert sequencing, 202 Menadione toxicity, 92 Menkes disease, 275,285,305,307, 323,325 380,444,468 Menkes disease gene, 275 Menkes disease therapy, 305 Metal regulatory elements, 367,398, 425 Metallothionein, 3, 13,87, 101,276, 364,379,425,443,454,467, 469,472 Metallothionein gene,397,411 Metallothionein mRNA, 479 Missing nucleoside experiment, 220 Mottled mouse, 293 MRE, 426 =binding factors: MEP-1,425 MTF, 406,411 W ,406 ZRF,401 MREbindmg proteins,4.40 Mutagenesis, 89,102,101,279 Ni(cyclam), 211 Nickel, 53,69,201 Nickel-promoted oxidation, 202 Nuclear: control of calcium, 4 Wet, 3 uptake of metals, 9

Occipital horn syndrome,288,292 Oxidative damage,l02 Oxidative stress, 96 Oxygen radicals, 53 Phagocytosis, 53 Positional cloning, 277 Protein-protein dimerization, 237 Protein+rotein interaction, 256 P-type ATPase, 280,285,305,356, 366,444 Retinoblastoma, 345 Retinoic acid receptor, 239 Retinoid X receptor, 239 Reverse transcriptase,3 RNA cleavage, 176,201 RNA polymerase,3 Sarcoplasmic reticulum, 13 Selenite, 124, 125 Southwestern analysis, 432 Spl, 417,433 Splicing mutations, 319 Steroid hormone receptor, 237 Strand break, 38,44,53,69,80, 121, 157, 185, 196 Superoxide, 22,71, 122 Superoxide dismutase, 22, 102, 134, 276,380,446 Superoxide radicals, 112 Tat protein, 255 =-DNA complex, 224 Toxic milk mouse, 443

488

Index

Transactivation response element

PAW,256 Transcription factorIIIA, 217 Transcriptional regulation: ACEl,385,407 ACE2.385 AMTl, 386 CUPl, 384 MEP-l, 425 MTF, 406.41 1 ZAP,406 ZRF,401,407 Translocation, 276,277,278 Triplex DNA, 227 Tyrosinase, 276, 312,318

UV cross-linking analysis,435

Vitamin C, 102 Vitamin E, 23,102 Wilson disease, 275,281,343,345, 354,361,365,380,444,468 Wilson disease gene,345 Wilson disease therapy,361

X chromosome, 277 X-linked copper disturbances, 286 Yeast artificial chromosomes,278, 347

Zform of DNA, 227 Zif268,224 Zinc finger, 224,237,243,255,366, 407,412 metal replacement,237,267 Zinc therapy, 361,368