LIBRARY llllllllllllllllllllllllllllllll l/ “i' ”an “ m w 3 1293 00667 6211 This is to certify that the thesis entitled CARBON TETRACHLORIDE METABOLISM: THE ROLE OF HEPATIC MICROSOMAL MIXED-FUNCTION OXIDASE COMPONENTS IN CARBON-HALOGEN BOND CLEAVAGE presented by Fredrick Oliver O'Neal has been accepted towards fulfillment of the requirements for Ph.D. degree in Biochemistry . l 7 '2 , ) g7 ,1 _ .V n f¢ Major professor 54/‘7‘3 Date 0-7639 University CARBON TETRACHLORIDE METABOLISM: THE ROLE OF HEPATIC MICROSOMAL MIXED-FUNCTION OXIDASE COMPONENTS IN CARBON-HALOGEN BOND CLEAVAGE By Fredrick Oliver O'Neal A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1978 ABSTRACT CARBON TETRACHLORIDE METABOLISM: THE ROLE OF HEPATIC MICROSOMAL MIXED-FUNCTION OXIDASE COMPONENTS IN CARBON-HALOGEN BOND CLEAVAGE By Fredrick Oliver O'Neal Carbon tetrachloride is activated to -CCl3 by the microsomal mixed-function oxidase system. The objectives of this research were (1) to define a role for each mixed-function oxidase component in CCl4 activation and (2) to determine the mechanisms involved in CCl4 acti- vation by this system. A model system was developed for determining the role of the flavoprotein, NADPH-cytochrome P450 reductase, in CCl4 activation. The system contained purified reductase, liposomes, and NADPH in the presence or absence of the electron acceptors, ADP-Fe+3 and EDTA-Fe+3. The enzyme cycles between its fully and half-reduced states during microsomal electron transfer reactions. Conditions favoring these states could be achieved in the presence and absence, respectively, of the electron acceptors. The CCl4-dependent stimula- tion of lipid peroxidation (malondialdehyde formation) or the anaerobic-covalent binding of 14 CCl4 to liposomes was used to assay CCl4 activation. The fully reduced reductase was shown to be incapable of C014 activation. Activation of CCl4 by the system favoring the half- reduced enzyme was observed. Further investigation of this Fredrick Oliver O'Neal activation showed a requirement for lipid hydroperoxides and a pro- oxidant. The reductase converts EDTA-Fe+3 2 to its pro-oxidant form, EDTA-Fe+ , which subsequently degrades lipid hydroperoxides to lipid radicals. These radicals activate CCl4 by an atom transfer reaction. Cytochrome P450(Fe+3) as well as hemin and other oxidized hemopro- teins will also serve as pro-oxidants in this system. It was con- cluded that the reductase does not activate CCl4. However, it func- tions indirectly via its ability to reduce cytochrome P450 and to form lipid hydroperoxides from polyunsaturated fatty acids when ADP-Fe+3 and 02 are present. The degradation of these hydroperoxides by pro-oxidants resultsinCCl4 activation. The role of cytochrome P450 in CCl4 activation was investi- gated in two systems. The first was liver microsomes from rats pre- treated with CoCl2 to alter cytochrome P450 levels. The second was a reconstituted system containing liposomes, cytochrome P450, and the reductase. In microsomes, both the rate and extent of CCl4-stimulated lipid peroxidation were related to the cytochrome P450 content. In the reconstituted system, an NADPH-dependent binding of ‘4 CCl4 was inhibited by an antibody to cytochrome P450 and by CO. Binding also resulted when dithionite was used as a source of reducing equivalents for cytochrome P450. Dithionite-reduced hemoglobin and hemin also activated CCl4. It was concluded that cytochrome P450 is the site of CCl4 activation within the mixed-function oxidase system. Also, the interaction of CCl4 with the reduced heme iron of cytochrome P450 is required for CCl4 activation to ~CC13. Fredrick Oliver O'Neal Liver microsomes from untreated rats or those pretreated with PB and 3—MC were utilized to determine the role of different cyto- chrome P450 hemoproteins in CCl4 activation. The initial rates of CCl4-stimulated lipid peroxidation and ‘4 CCl4 binding were expressed on the bases of microsomal cytochrome P450 content, turnover numbers. These values were consistently higher for untreated microsomes; how- ever, the relative magnitudes of these numbers for PB- and 3MC- microsomes depended on the method used to assay CCl4 activation. These differences are believed due to the relative amounts of «CCl3 and :CCl3 produced by each microsomal cytochrome P450 population. The former initiates lipid peroxidation as well as binds to lipid whereas the latter is only capable of binding. The turnover numbers for 14 CCl4 binding were more dependent on the reductase to cyto- chrome P450 ratios than on cytochrome P450's substrate specificity. This was confirmed by the fact that reconstituted systems containing the same amount of reductase and either cytochromes P450 or P448 gave similar rates of ‘4 CCl4 binding. In conclusion the following factors may account for observed differences in CCl4 activation and susceptibility of tissues to toxicity: the microsomal cytochrome P450 content and its rate of reduction; the relative amounts of -CCl3 or other toxic intermediates fermed ig_vivo; and cellular protection mechanisms. To My Parents ii ACKNOWLEDGMENTS I would like to express my appreciation to Dr. Steven D. Aust, my academic advisor, for the many contributions made during my graduate work. His welcome of a rather weathered sailor back I into his laboratory and his guidance and concern during this phase of my scientific career are especially appreciated. It is with grati- tude that I acknowledge the assistance of my Uncle Sam. His pro- viding me with the opportunity to acquire those sailing skills and financial assistance has made weathering the fiscal storm of gradu- ate school bearable. I also wish to express my thanks to other members of my guidance committee, Drs. Bruce A. Averill, David G. McConnell, William L. Smith, and John E. Wilson for their helpful discussions. I would also like to acknowledge the following members of the Aust Laboratory for their assistance and many helpful dis- cussions: Drs. John Buege, Robert Moore, and Ann Welton; Mr. Bruce Svingen; and Mrs. Poonsin Olson. Finally, I wish to acknowledge other members of the laboratory and of the Biochemistry Department for their moral support and friendship. TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . vii LIST OF FIGURES . . . . . . . . . . . . . . . 1X LIST OF ABBREVIATIONS . . . . . . . . . . . . . X INTRODUCTION . . . . . . . . . . . . . . . . I Background of the Study . . . . . . . . . 1 Organization of Dissertation . . . . . . . . 4 Chapter 1. LITERATURE REVIEW . . . . . . . . . . . . 5 Microsomal Mixed-Function Oxidase System . . . . 5 Purification of Mixed-Function Oxidase Components . . . . . . lO Reconstitution of Mixed- Function Oxidase Activity. . . . l4 Carbon Tetrachloride Metabolism and Toxicity . . l6 The Peroxidation of Membrane Lipids . . . . . 23 II. THE ROLE OF THE NADPH- CYTOCHROME P450 REDUCTASE COMPONENT OF RAT LIVER MICROSOMES IN CARBON TETRACHLORIDE ACTIVATION. . . . . . . . 27 Abstract . . . . . . . . . . . . . . 27 Introduction . . . . . . . . . . . 29 Materials and Methods . . . . . . . . . . 32 Material Sources . . . . . . . . . 32 Drug Pretreatment of Rats . . . . . . . . 33 Isolation of Rat Liver Microsomes . . 33 Purification of NADPH- Cytochrome P450 Reductase from Rat Liver Microsomes . . 34 Purification of Cytochromes P450. P443. .and b5 from Rat Liver Microsomes . . 35 Enzyme Assays and Analytical Procedures . . . 37 SDS-Polyacrylamide Gel Electrophoresis . . . . 4O Extraction of Lipids and the Preparation of Liposomes . . . . . . . . . . . . 40 iv Chapter Page Preparation of Lipid Hydroperoxides . . . . . 4l Deoxygenation of Argon . . . . . . . . . 41 Results . . . 42 Purification of NADPH- -Cytochrome P450 Reductase from Rat Liver Microsomes . . 42 Activation of CCl4 by NADPH- -Cytochrome P450 Reductase in Liposomes . . . 43 CCl4 Activation by NADPH- -Cytochrome P450 Reductase in the Presence of Added Electron Acceptors . . . 47 Determination of Optimal NADPH- -Cytochrome P450 Reductase and EDTA- Fe *3 Concentrations for CCl4 Activation in Liposomes . . . . 50 Activation of CCl in Liposomes Containing Ascorbate as a Source of Reducing Equivalents . . 55 The Role of Lipid Hydroperoxides in CCla Activa- tion by the Liposomal System . . . 56 Purification of Cytochromes P450, P443, and b5 from Rat Liver Microsomes . . 58 The Role of Cellular Pro- Oxidants in CCl4 Acti- vation: Implications of the Function of This Mechanism in Vivo . . 64 The Effect of the Electron Acceptors ADP- Fe+3 and EDTA- Fe+3 on CCl4 Activation by Rat Liver Microsomes . . . . . 69 Discussion . . . . . . . . . . . . . . 73 III. THE ROLE OF THE CYTOCHROME P45 COMPONENT OF RAT LIVER MICROSOMES IN CARBON TET CHLORIDE ACTIVATION . . . . . . . . . 79 Abstract . . . . . . . . . . . . . . 79 Introduction . . . . . . . . . . . . 80 Methods and Materials . . . . . . . . . . 83 Material Sources . . . . . . . . . . 83 Drug Pretreatments of Rats . . . . . . . . 84 Isolation of Rat Liver Microsomes . . . . . 84 Reconstitution of Mixed-Function Oxidase Activity from Purified Components . . . . . 85 Enzyme Assays and Analytical Procedures . . . 86 Dithionite Reduction of Microsomes, Hemo- proteins and Hemin . . . 87 Preparation of Antibody to Trypsin- -Solubilized Cytochrome P450. . . . . 87 Results . . , 37 The Relationship Between the Cytochrome P450. Content of Rat Liver Microsomes and CCl4- Dependent Lipid Peroxidation . . . . . . 87 V Chapter Activation of CCl4 by a Reconstituted Mixed- Function Oxidase System Containing Purified Components from PB- Microsomes . Activation of CCl4 by Dithionite- Reduced Cytochrome P450 and Other Hemoproteins The Role of Cytochrome b5 in the NADH- Dependent . Activation of CCl4 in Microsomes . The Effect of Hashing on Microsomal CCl4 Activation . . Discussion . IV. THE ACTIVATION OF CARBON TETRACHLORIDE BY CATA- LYTICALLY DIFFERENT CYTOCHROME P450 HEMOPROTEINS . Abstract Introduction Materials and Methods Material Sources . Drug Pretreatment of Rats Isolation of Rat Liver Microsomes . Enzyme Assays and Analytical Techniques Results . Activation of CCl by Microsomes from Untreated . and PB- and 3- M -Pretreated Rats CCl4 Activation by Reconstituted Mixed- Function . Oxidase Systems Containing Cytochrome P450 and P443 Hemoproteins from PB- and 3-MC-M1crosomes . Discussion . V. SUMMARY REFERENCES APPENDIX . vi Page 92 99 102 103 107 113 113 114 117 117 117 117 118 118 118 123 128 134 139 149 Table l. 10. 11. 12. 13. LIST OF TABLES The Purification of NADPH- -Cytochrome P450 Reductase from Rat Liver Microsomes NADPH- -Cytochrome P450 Reductase Catalyzed Oxidagion of NADPH in Liposomes Using CCl4 and EDTA-FE+ as Electron Acceptors . NADPH- Cytochrome P450 Reductase Catalyzed Activation of 14CCl4 in Liposomes in the Absence of Added Electron Acceptors . . . . . . . . CCl4-Stimulated Lipid Peroxidation in Liposomes . NADPH-Cytochrome P450 Reductase Catalyzed Activation of 14CCl4 in Liposomes in the Presence of Electron Acceptors . . . . . The Fate of NADPH- -Cytochrome P450 Reductase During CCl4 Activation in Liposomes . . The Dependence of 14CC14 Activation on NADPH-Cyto- chrome P450 Reductase Concentration in Liposomes . Ascorbate and Fe+2 Catalyzed Activation of 14CCl4 in Liposomes . . . . . . . . . Dependence of 14CCl4 Activation on the Lipid Hydroperoxide Contents of Liposomes Purification of Cytochrome P450 from Microsomes of PB-Pretreated Rats . . . . CCl4-Stimulated Lipid Peroxidation in Liposomes in the Presence of Hemoproteins and Hemin . Effect of Hemoproteins on 14CCl4 Activation in Partially Peroxidized Liposomes . . Effect of ADP- and EDTA-Chelated Iron on ‘4 CCl4 Activation by Rat Liver Microsomes . . vii Page 43 46 47 48 49 50 51 56 57 60 65 66 72 Table 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. The Effect of CoClz Pretreatment on Rat Liver Microsomal Enzymes . . . Reconstitution of Mixed- Function Oxidase Activity: Inhibition of Activity Nith Antibody Against Cytochrome P450. Effect of Inhibitors of Drug Metabolism on 14CCl4 Activation by a Reconstituted Mixed- Function Oxidase System . . . . . . . Activation of 14CCl4 by NADPH-Cytochrome P450 Reductase- and Dithionite-Reduced Cytochrome P450 in Microsomes . . . . . . . Activation of 14CCl4 by Dithionite-Reduced Hemoproteins and Hemin in Liposomes NADH- and NADPH- -Dependent Activation of 1“can; in Microsomes . . . . . The Effect of washing on the CO-Insensitive Mcc14 Activation by Liver Microsomes from PB- and CCl4-Pretreated Rats The Effect of Washing on Microsomal Enzymes from P8- and CCl4-Pretreated Rats The Effect of PB- and 3- MC Pretreatments of Rats on Microsomal Enzyme Content . . . . . The Effect of PB- and 3-MC Pretreatments on CCl4- Dependent Lipid Peroxidation in Rat Liver Microsomes . . . . . . . . . The Effect of PB- and 3-MC-Pretreatments on 14cc14 Binding to Lipid in Rat Liver Microsomes . . Benzphetamine and Beanyrene Metabolism by Reconsti- tuted Mixed- Function Oxidase Systems Containing Cytochromes P450 and .P448 from PB- and 3-MC- Microsomes . . . . viii Page 91 94 98 100 101 103 105 106 119 122 124 125 LIST OF FIGURES Figure 1. 10. 11. SDS- -Polyacrylamide Gel Electrophoresis of NADPH- Cytochrome P450 Reductase Purified from P8- Microsomes . . . . The Effect of EDTA-Fe 3 on 14cc14 Activation in Liposomes . . . . . . . . SDS-Polyacrylamide Gel Electrophoresis of Cytochromes P4 0, P , and b5 Fractions Purified from PB- ang Meffiicrosomes . . . . . . . Activation of 14CCl4 by Cytochrome P450 in Partially Peroxidized Liposomes 14CCl4 Activation by Rat Liver Microsomes . Stimulation of Lipid Peroxidation by CCl4 in Liver Microsomes from Unpretreated and CoCl2 Pretreated Rats . . . CHP-Dependent Lipid Peroxidation in Liver Microsomes from Untreated and CoCl2 Pretreated Rats 14CCl Activation by a Reconstituted Microsomal Ming Function Oxidase System . . Stimulation of Lipid Peroxidation by CCl4 in Liver Microsomes from Untreated, PB- and 3-MC- Pretreated Rats . . . 146314 Activation by Reconstituted Microsomal Mixed-Function Oxidase Systems Containing Cytochromes P450 and P448 . . Schematic of Carbon Tetrachloride Activation by the Rat Liver Microsomal Mixed-Function Oxidase System . . . . ix Page 44 53 62 67 70 89 93 96 120 126 136 ADP BP BHT CHP Ci cpm DEAE di-l,2-GPC DDT EDTA G6Pase i.p. IgG LOOH MDA MeOH 3-MC NADH NADPH PB LIST OF ABBREVIATIONS Adenosine 5'-diphosphate 3,4-Benzpyrene Butylated hydroxytoluene Cumene hydroperoxide Curie Counts per minute Diethylaminoethyl Dilauroylglyceryl-3-phosphoryl choline l,l,l-Trichloro-2,2,-bis-(p-chlorophenyl)ethane Ethylenediaminetetraacetate Glucose-6-phosphatase Intraperitoneal Immuno gamma globulins Lipid hydroperoxides Malondialdehyde Methanol 3-Methycholanthrene B-Nicotinamide adenine dinucleotide, reduced form B-Nicotinamide adenine dinucleotide phosphate, reduced form Phenobarbital pCMB p-Chloro mercuribenzoate PCN Pregnenolone-lGa-carbonitrile PEG Polyethylene glycol s.c. Subcutaneous 5.0. Standard deviation SDS Sodium dodecyl sulfate SKF-525A 2-Diethylaminoethyl-2,2-diphenylvalerate Tris Tris(hydroxy methyl) amino methane xi INTRODUCTION Background of the Study Halogenated alkanes are quite prevalent and are being used for purposes ranging from medicinal to industrial. Millions of tons of these compounds are produced yearly and we are by various mechan- isms becoming increasingly exposed. It is of concern that many of these compounds, once believed to be innocuous, have been shown to be either toxic or can undergo metabolic activation into compounds that are. For example, several of the halogenated methanes which have been used as anesthetics, fire extinguishants, industrial sol- vents, and as aerosol pr0pellants were once believed to be quite stable and not readily metabolized. That they are metabolized has been amply demonstrated and a correlation made between their ability to form reactive intermediates and toxicity. Also, two of these halomethanes, CCl4 and CHCl3, have been shown to be carcinogenic in some laboratory animals. The hepatic microsomal mixed-function oxidase system is responsible for the metabolism of a variety of foreign compounds as well as endogenous substrates. It also has a major role in the dehalogenation and subsequent activation of haloalkanes. The mechanisms involved in the dehalogenation of these compounds to active intermediates and their subsequent interaction with cellular compounds leading to cell death are poorly understood. However, it has been demonstrated that the metabolism of some of these compounds initiates lipid peroxidation, a deleterious free radical mediated process, in microsomal membranes. Because of my general interest in toxicology and our laboratory's involvement with enzyme- catalyzed lipid peroxidation, I became interested in investigating the reactions involved in activating these compounds by the micro- somal mixed-function oxidase system. The general approach taken in these investigations was to characterize the metabolism of a model haloalkane in reconstituted systems containing purified mixed-function oxidase components. Carbon tetrachloride was suitable for these investigations because many of the conditions affecting its metabolism and toxicity had been determined and several metabolites identified. The metabolic activation of this compound initiates lipid peroxidation both in .yitrg and jn_yiyg5 there is also evidence suggesting the trichloro- methyl radical (-CCl3) is the metabolite responsible for this initiation. Unlike most of the reactions catalyzed by the micro- somal mixed-function oxidase system, the metabolism of CCl4 to ~CCl3 involves a reductive mechanism which can take place in the absence of oxygen. Only two reactions catalyzed by this system involving reductive mechanisms have been characterized, the reduc- tion of compounds containing azo and nitro groups to their cor- responding amines. Reducing equivalents can be transferred to the azo and nitro moieties of the compounds from both the flavoprotein, NADPH-cytochrome P450 reductase and the reduced hemoprotein, cytochrome P450, components of the hepatic microsomal mixed- function oxidase system. Similarly, evidence exists which suggests that either one or both of the mixed-function oxidase components are required for CCl4 activation, however; direct proof is lacking. Chapters 2 and 3 of this thesis include the investigations of the functional roles that NADPH-cytochrome P450 reductase and cytochrome P450, respectively, have in CCl4 activation. These investigations have been made possible by improvements in purifica- tion techniques for these components and the ability to reconstitute mixed-function oxidase activity; Model systems containing liposomes and one or both of the purified components have been developed to evaluate their participation in CCl4 activation free of interference from other microsomal constituents. The cytochrome P450 component of the microsomal mixed- function oxidase system of the liver represents a family of hemo- proteins with different but sometimes overlapping substrate specificities. The preferential induction of one or more of these hemoproteins by pretreatment with drugs has been shown to alter CCl4 toxicity. For example, PB pretreatment increases whereas 3-MC de- creases toxicity when compared to the controls. One possible explana- tion for these observed differences is that these hemoproteins differ in their ability to activate CCl4. Chapter 4 investigates this pos- sibility in microsomes as well as model systems containing catalytically different cytochrome P450 hemoproteins. Although there are several chemical reactions which form -CC13, it has not been determined how this may occur enzymatically. Chapters II and 111 include the investigations to determine the reac- tions in which the reductase and cytochrome P450 components parti- cipate to activate CCl4. Liposomal systems containing purified components were used here also. Organization of Dissertation This dissertation is divided into five chapters; the first and last contain the Literature Review and Sunmary, respectively. The inves- tigations of CCl4 activation by the hepatic microsomal mixed-function oxidase system have been divided into three areas. Each area of investigation is presented separately in Chapters II through IV with a format similar to that used in many scientific papers. These chapters are subdivided into the following sections: Abstract, Introduction, Materials and Methods, and Discussion. Chapter refer- ences as well as those for the Literature Review section have all been combined at the end of the dissertation. The Literature Review section presents a broad overview of the current knowledge concern- ing the function of the microsomal mixed-function system and its role in CCl4 metabolism and toxicity. A broad overview of lipid peroxidation is also presented since it is believed to have a key role in CCl4 toxicity. The introduction to each chapter is designed to provide a more specific background for that aspect of the research. Chapter V summarizes the data presented in Chapters 11 through IV. CHAPTER I LITERATURE REVIEW Microsomal Mixed-Function Oxidase System Mixed-function oxidases are generally defined as enzymes which catalyze the consumption of one molecule of oxygen per mole- cule of substrate with one oxygen atom being incorporated into the product and the other reduced to water (Mason, 1957). The endo— plasmic reticulum (microsomes) of rat hepatocytes has a membrane- associated mixed-function oxidase system which is involved in the metabolism of a broad spectrum of lipophilic substrates (Conney, 1967; Kuntzman, 1969; Mannering, l97l). Among these are the normal body constituents, steroids and fatty acids, and foreign compounds (xenobiotics) which include drugs, pesticides, carcinogens, indus- trial chemicals and food additives. In rat liver microsomes, mixed-function oxidase activity is associated with the NADPH- dependent electron transport chain. This chain contains two components: cytochrome P450, a hemoprotein so designated because of the characteristic absorption maximum at 450 mm for its reduced- CO difference spectrum (Omura and Sato, 1964), and NADPH-cytochrome P450 reductase, a flav0protein. The reductase transfers reducing equivalents from NADPH to cytochrome P450 which serves as the terminal oxidase for the system. In addition to oxygen, two general classifications of substrates will bind to cytochrome P450, Types I and II (Mannering, 1971). When bound to the oxidized (Fe+3) form of the hemoprotein each class of substrates will give its characteristic difference spectra. The binding sites for these substrate types are different with the Type II binding site being either at or closely associated with the heme moiety whereas the Type I binding site is not. Cytochrome P450 serves as the site for the formation of "active oxygen" which is subsequently incorporated into substrates of both classifications. A brief summary of the sequential events involved in cytochrome P450-mediated oxidations include: formation of a cytochrome P450(Fe+3)-substrate complex; a one electron reduction of the complex by NADPH-cytochrome P450 reduc- tase; oxygen binding to the reduced heme iron of cytochrome P450 forming a ternary complex; transfer of a second electron to form a short-lived "active-oxygen" complex; and the dissociation of the latter into the products, hydroxylated substrates and water, and cytochrome P450(Fe+3) (Estabrook, 1973). Cytochrome P450-containing mixed-function oxidases are of wide- spread occurrence in nature. They have been found in bacteria, yeast, plants, insects, fish, and mammals (Capdevila and Agosin, 1977; Katagiri et al., 1968; Mannering, 1971; Yoshida et al., 1977). In mammals mixed-function oxidase activity is greatest in liver microsomes but has been found also in microsomes from the kidney, lung, brain, placenta, intestine, and testis, and in adrenal cortex mitochron- dria (Conney, 1967; Sasame et al., 1977; Villarruel et al., l977). The mixed-function oxidase system of the liver functions primarily in the conversion of its lipophilic substrates, xenobiotics, to more water-soluble forms which facilitates their excretion into the urine, bile, and expired air (Cafruny, 1971; Munsen and Eger, 1971; Plaa, 1971). A wide variety of oxidative reactions are involved in the metabolism of these substrates. Included among these are the hydroxylation of aliphatic and aromatic compounds, epoxida- tions, 0- and N-dealkylations, N-oxidations, sulphoxidations and desulphurations (Mannering, 1971; Parke, 1975). Although the reac- tion products are varied, one unifying concept has been that these are all different types of hydroxylation reactions. Some of these reactions form unstable intermediates that quickly rearrange to the more stable products (Brodie et al., 1958). Reductive reactions, for example, the azo- and nitro-reductases, are also catalyzed by this system (Gillette, 1971a). The products of both the oxidative and reductive reactions may be either excreted directly or further metabolized. Microsomal and cytosolic enzymes can catalyze the con- jugation of many of these products with more water-soluble compounds such as glucuronic acid, glutathione, and sulfate (Mandel, 1971; Shuster, 1964). In general the reactions catalyzed by the microsomal mixed-function oxidase system function to detoxify xenobiotics but, unfortunately, they also have the capacity to increase the toxicity of relatively innocuous substances. Many cancerous agents and cellular oxidants are formed jn_yiyg from the rela- tively harmless pro-carcinogens and pro-oxidants, respectively (Jerina and Daly, 1974; Plaa and Witschi, 1976). Another charac- tersitic of the hepatic as well as other mixed-function oxidase systems is that they may be induced up to several fold by various compounds (Conney, 1967; Parke, 1975). The compounds which induce these enzymes are classified as either general or specific inducers depending on their abilities to induce a broad or limited range of activities (Conney, 1967; Gillette, 1971b). Phenobarbital (PB) is the classical prototype of the general inducers causing a proliferation of the cell's endoplasmic reticulum as well as an increase in its cytochrome P450 and cytochrome P450 reductase com- ponents. The polycyclic hydrocarbon, 3-MC, is the prototype for the specific inducers which in general only increase the hemoprotein component whose reduced-C0 difference spectrum has its absorbance maximum shifted to 448 nm. The induction of mixed-function oxidase activity has several important consequences which include an increased tolerance to therapeutic drugs, altered metabolism of steroid hormones, and increased activation of the potentially toxic substances, pro-carcinogens and pro—oxidants (Hall, 1976; Jerina and Daly, 1974; Plaa and Witschi, 1976). There are two electron transport chains associated with rat liver microsomes, the NADPH-dependent chain previously described and an NADH-dependent chain which functions in the desaturation of fatty acids (Oshino, 1966, 1970; Strittmatter, 1974). The compo- nents of this chain include the flavoprotein,NADH-cytochrome b5 reductase, cytochrome b5.and the desaturase enzyme. Although these two electron transport chains have dissimilar functions, they are believed to interact as shown schematically below: exogenous cyt c 1. :e RH + 02 + 2H+ g‘ NADPH-cyt P , cyt P NADPH reductase 450 fam11§50 ROH + H20 _ /f e /e / fatty acyl CoA A / - +02+2H+ fiN DH- -cyt b e fatty acid NADHfi reductase 5 cyt b5 desaturase mono-unsaturated fatty acyl CoA + 2 H20 The NADPH-dependent chain contains NADPH-cytochrome P450 reductase and a family of cytochrome P450 hemoproteins which confer broad substrate specificity on the mixed-function oxidase system. The reductase can transfer electrons to exogenous acceptors such as cytochrome c and various forms of chelated iron and to the endogenous acceptors, cytochromes P450 and b5 (Archakov et al., 1975; Noguchi and Nakano, 1974; Pederson et al., 1973; Yasukochi and Masters, 1976). Although cytochrome b5 is reduced by NADPH-cytochrome P450 reductase, the rate is much slower than that involving NADH-cytochrome b5 reduc- tase (Archakov et al., 1975). Microsomal mixed-function oxidase reactions involve a two electron reduction of molecular oxygen and it has been hypothesized by Hildebrandt and Estabrook (1971) that cytochrome b5 serves as the source of the second electron. 10 Several lines of evidence suggest that reducing equivalents are transferred from the reduced cytochrome b5 to cytochrome P450. These include the following: the NADH/NADPH synergism in the micro- somal metabolism of several substrates (Cohen and Estabrook, 1971; Mannering, 1974); the substrate-dependent increase in cytochrome b5 oxidation (Fujita and Peisac, 1977; Mannering, 1974); and the metabolism of 3,4-benzpyrene in a reconstituted system containing cytochrome b5, NADH-cytochrome b5 reductase, lipid and cytochrome P448 (Lu et al., 1974a). Although some interaction between the two microsomal electron transport chains has been demonstrated in yjtgg, some uncertainty still exists concerning the function of this interaction jg_yjyg_($chenkman and Jansson, 1974). The metabolism of several drugs by a reconstituted system containing only cytochrome P450 and cytochrome P450 reductase suggested that cytochrome b5 was not an obligatory component of the microsomal mixed-function oxidase system (Guengerich, 1977). Purification of Mixed-Function Oxidase Components Mixed-function oxidase components are tightly associated with microsomal membranes. Until recently, this property has made them difficult to purify and characterize. Earlier attempts at purification led to either a deactivation or incomplete resolution of these components from other membrane proteins and lipids. How- ever, newly developed techniques have led to the purification of the cytochrome P450 and NADPH-cytochrome P450 reductase components in 11 forms that retain their catalytic activity. The use of hydrophobic (Shaltiel, 1975) and affinity (Cuatrecasas, 1970) chromatography in the presence of ionic and non-ionic detergent mixtures has made possible the purification of both components in good yields (Guengerich, 1977; Imai, 1976; Yasukochi and Masters, 1976). SDS-polyacrylamide gel electrophoresis utilizing both heme and protein staining techniques has been a very useful analytical tool in determining the molecular weights and homogeneity of purified mixed-function oxidase components and has aided in the identifica— tion of the cytochrome P450 hemoproteins (Fairbanks, 1971; Moore et al., 1978). NADPH-cytochrome P450 reductase had been solubilized and purified earlier by proteolysis (Williams and Kamin, 1962; Pederson et al., 1973) and more recently by affinity chromatography in the presence of non-ionic detergents (Yasukochi and Masters, 1976). Both procedures resulted in enzymes with similar flavin contents and each was able to reduce the exogenous substrates, cytochrome c and K3Fe(CN)6. The major differences between the two preparations were that the protease-solubilized reductase was approximately 8,000 daltons smaller and had lost its ability to reduce cytochrome P450 (Lu et al., 1969; Welton et al., 1973). The proteolysis of microsomal membranes separates the catalytically active hydrophilic region of cytochrome P450 reductase, an amphipathic molecule, from the non-catalytic hydrophobic region. The latter functions as an anchor attaching cytochrome P450 reductase to the hydrophobic 12 microsomal membrane matrix (Peterson et a1 . , 1976) and probably func- tions similarly in reconstituted systems, facilitating its reduction of cytochrome P450 in the liposomal membranes. The detergent- solubilized reductase is capable of reducing not only different cytochrome P450 hemoproteins from the same animal but those of other species as well (Coon et al., 1977; Lu and Levin, 1974). It was generally believed that NADPH-cytochrome P450 reduc- tase wasa single enzyme (Welton, 1974); however, Coon et a1. (1977) have recently obtained evidence for two forms in rabbit and rat liver microsomes. Two detergent-solubilized NADPH-cytochrome P450 reduc- tases have been isolated from the liver microsomes of both PB pre- treated species. Although the purification methods for each form were different, they resulted in homogeneous preparations with simi- lar flavin contents but different specific activities when cyto- chrome c was used as the electron acceptor and different electropho— retic mobilities during SDS-polyacrylamide gel electrophoresis. Microsomal proteins with electrophoretic mobilities corresponding to molecular weights of 78,000 and 76,000 daltons in the rat and 74,000 and 68,000 daltons in the rabbit were found to be identical to those of the purified reductases. Both NADPH-cytochrome P450 reductases were present in untreated and PB microsomes in equal amounts suggesting that neither form was preferentially induced. Both forms could also reduce several cytochrome P450 hemoproteins. Neither gel pattern was affected when the microsomes or enzyme prepara- tion contained the general protease inhibitor, phenylmethane 13 sulfonyl fluoride. Trypsin treatments of the purified proteins convert both rat enzymes to a 69,000 dalton species, lower than either detergent-isolated form. Both findings suggest proteolytic artifacts during isolation procedures are not responsible for the different reductases. The existence of more than one cytochrome P450 reductase has interesting implications. I_.!jgg, where the reductase component may be limiting, preferential reduction of cyto- chrome P450 hemoproteins by specific reductases would greatly affect the overall rate of metabolism for specific substrates. This matter is the subject of further investigation by that laboratory. Cytochrome P450 has been the mixed-function oxidase compo- nent most difficult to purify. Earlier attempts at purification either resulted in incomplete resolution from membrane proteins or its conversion to the inactive, cytochrome P420, form (Lu and Levin, 1974). Techniques for purifying this hemoprotein in a catalytically active form have now been developed in several laboratories (Coon et al., 1977; Guengerich, 1977; Lu and Levin, 1974). Generally, these procedures involve solubilization with ionic detergents, initial fractionation by either ammonium sulfate, polyethylene glycol, or hydrophobic chromatography, and further purifications involving ion exchange chromatography in the presence of non-ionic detergents. It is generally accepted that multiple cytochrome P450 hemoproteins exist and that this accounts for the rather broad 14 substrate specificity of the microsomal mixed-function oxidase system. Using various drug pretreatments and the above purification procedures, Guengerich (1977) has been able to demonstrate at least eight cytochrome P450 hemoproteins in the rat liver microsomes and at least six in rabbit liver microsomes. Neither should be taken as a final number in that more are likely to be found as techniques improve. Of the cytochrome P450 hemoproteins purified to date, differences have been found in some but not all of the following: molecular weight; amino acid content; substrate specificity (most were found to have overlapping but different substrate specifici- ties); pH optima for reconstitution of mixed-function oxidase activity; antigenic determinants; and reduced-CO difference spectra (Coon et al., 1977; Guengerich, 1977; Lu et al., 1976). Reconstitution of Mixed-Function Oxidase Activity Our understanding of the metabolism of drugs by the NADPH- dependent mixed-function oxidase system has been greatly increased by the successful reconstitution of its activity from purified components. Lu et a1. (1969) found a requirement for a heat-stable lipid fraction in addition to partially purified cytochrome P450 and cytochrome P450 reductase for mixed-function oxidase activity. It was later determined that this fraction could be replaced by synthetically prepared phosphatidyl cholines or by detergents (Lu et al., 1974b; Strobel et al., 1970). The lipid fraction facili- tates the reduction of cytochrome P450, probably by mimicking the 15 hydrophobic membrane matrix of microsomes allowing for proper posi- tioning and/or protein conformations favorable for electron transfer (Lu et al., 1974b; Lu and Levin, 1974). The order of mixing the mixed-function oxidase components and lipid is important. Maximum rates of drug metabolism are achieved only'when the lipid, cyto- chrome P450 reductase, and cytochrome P450 fractions are thoroughly mixed prior to the addition of the buffer and other cofactors (Lu and Levin, 1974). Several investigators have now been able to reconstitute mixed-function oxidase activity from components purified from a variety of sources (Coon et al., 1977; Kamataki et al., 1976; Lu and Levin, 1974). The substrate specificities and reaction products are similar to those of the microsomes from which the cytochrome P450 components were isolated. It was observed that the ratio of the protein components, reductase to cytochrome P450, required for maximum activity in these reconstituted systems was generally higher than that found in microsomes (Kamataki et al., 1976; Lu et al., 1972). Cytochrome P450 reductase is the limiting component in microsomes generally present at a ratio of one molecule of reductase to twenty molecules of cytochrome P450 (Peterson et al., 1976). Most of the reconstituted systems contain cytochrome P450 as the limiting component. This is reflected in the fact that turnover numbers, mole of product formed per minute per mole of cytochrome P450, are higher for a number of substrates in the 16 reconstituted system than in microsomes (Lu and Levin, 1974; van der Hoeven and Coon, 1974). The reconstitution of microsomal mixed-function oxidase activity has provided a very useful tool for investigating the metabolism of a variety of compounds. Information regarding the specificity, product formation, and mechanisms involved in the metabolism of substrates in mixed-function oxidase systems con- taining various cytochrome P450 hemoproteins can be obtained. A means is also provided for the evaluation of requirements for other microsomal constituents in mixed-function oxidase reactions. Mixed-function oxidase as well as other reconstituted membrane systems will be used in several investigations reported in this thesis. Carbon Tetrachloride Metabolism and Toxicity Carbon tetrachloride is one of several halogenated alkanes which has had many useful applications within the last century. Included among these applications have been its use as an indus- trial and dry-cleaning solvent, a fire-extinguishing agent, an anesthetic, and as an antihelminthic agent for the treatment of hookworms (von Oettingen, 1955). The toxicity of this compound has been well established and the symptoms of its poisoning include the following: severe liver and kidney dysfunction; central nervous system depression; increased clotting time; decreases in lymphocytes, leucocytes, and blood platelets (Bini, 1976; Back, 1977; von Oettingen, 1955). Although the 17 effects of CCl4 poisoning are apparent to some degree in other tissues, the most severe damage occurs in the liver. Because the CCl4-induced liver damage is similar to that caused by other hepato- toxins and by certain forms of liver diseases, CCl4 is often used as a model compound in investigations of mechanisms for general liver dysfunction (Goldblatt, 1972; Rees, 1976). Several theories had previously been developed to account for CCl4 toxicity; however, it is now generally accepted that its metabolism to a reactive intermediate by the microsomal mixed- function oxidase system is required (Recknagel and Glende, 1973; Recknagel, 1967). Some of the observations leading to this hypothe- sis are briefly mentioned below. Exposure of laboratory animals to 14CCl4 leads to the expiration of small amounts of 14C02 and to the covalent binding of the label to cellular lipids and proteins (Benedetti et al., 1977; Gillette et al., 1974; Gordis, 1969; Villarruel and Castro, 1975). Cytotoxicity occurred only in those tissues with high mixed-function oxidase activity, e.g., the liver and kidney, even though significant amounts of CCl4 were present in other tissues as well (Villarruel, 1971). Animals such as chickens which are resistant to CCl4 toxicity generally have low hepatic microsomal mixed-function oxidase activity (Diaz Gomez et al., 1975; Recknagel and Glende, 1973). The evidence which more directly implicates the microsomal mixed-function oxidase system includes the following: initial damage is localized within the microsomes resulting in a loss of cytochrome P450, GGPase, 18 membrane lipids, and alteration of other microsomal functions; gen- eral inducers of mixed-function oxidase activity increase toxicity whereas its inhibitors decrease toxicity; and that CCl4 forms a Type I substrate binding spectrum when added to microsomes, sug- gesting the involvement of cytochrome P450 in its metabolism (Reck- nagel and Glende, 1973). Characterization of the products of CCl4 metabolism has been undertaken by several investigators to determine the identity of its toxic intermediate. The discovery that free radical trappers and antioxidants protect animals from the toxic effects of CCl4 led to the suggestion that this intermediate was a free radical (Recknagel, 1967; Reynolds and Moslen, 1974). Recknagel and Ghoshal (1966) demonstrated that CCl4 increased NADPH-dependent microsomal lipid peroxidation, a free radical mediated process jg_ 313:9, They also demonstrated a CCl4-dependent increase in the conjugated diene byproduct of lipid peroxidation in microsomes from CCl4-treated rats. This provided the first indication that CCl4- dependent lipid peroxidation occurred in gig. Confirmation of this finding came later when the ethane and pentane byproducts of lipid peroxidation were found in the expired breath of CCl4-treated rats (Riely et al., 1974). Pretreatment of these animals with anti- oxidants decreased alkane evolution and a correlation was found between the CCl4-dependent expiration of alkanes and hepatic mixed-function oxidase activity (Dillard et al., 1976; Hafeman and Hoekstra, 1977; Lindstrom and Anders, 1978; Riely et al., 1974). 19 The detection of significant quantities of CHC13 and CCl3-CC13in animals exposed to CCl4 led to the hypothesis that the trichloromethyl radical ('CC13) was the toxic intermediate (Bini et al., 1975; Fowler, 1969). Chloroform would be the expected product of the abstraction of a hydrogen atom from cellular compo- nents by °CC13. Hexachloroethane would be the product resulting from the dimerization of two -CC13. Both reactions are common to many free radical mediated processes (Pryor, 1976). More recently, Villarruel and Castro (1975) chemically generated ~CC13 in the presence of polyunsaturated fatty acid methyl esters and demon- strated a similarity between the labeled products and those isolated from rats previously injected with ‘4 CCl4. This binding could occur by two reactions, one involves a termination reaction with a lipid radical and the other, an addition to the olefin bonds resulting in a trichloromethylated lipid radical (Benedetti et al., 1977). It is now generally accepted that 'CC13 formation does occur during CCl4 metabolism; however, the reactions involved 13_ yjy9_have not been characterized. Chemical reactions which cata- lyze -CC13 formation are known; those which may be biologically important are as follows (Gregory, 1966; Pryor, 1966): (1) R° + CC14---+ °CC13 + RC1 (2) *e‘ + cc14——-» occ13 + c1' 20 Since free radicals are normal intermediates of many biological reactions, activation may occur by the "atom-transfer" reaction in equation (1). It is of interest that several flavoproteins form the flavin semiquinone radical intermediate in many of their reac- tions (Dixon, 1971b;Iyanagi et al., 1974; Slater, 1972). The possible activation of CCl4 by the flavoprotein NADPH-cytochrome P450 reductase by this reaction will be one of the questions addressed in this thesis. Reaction (2) is that proposed by Reynolds (1966) who coined the term "electron capture" to describe its mechanism. This reaction has been demonstrated to take place in the gas phase with a sourcetrf "high-energy" electrons available. Reynolds has suggested that this reaction could occur in biological systems and that the source of electrons would be those "loosely bound" to cellular electron transfer components. Support for this hypothesis stems from the observations that the reduced forms of some transition metals, heme compounds, and hemoproteins interact with various halogenated compounds yielding free radical inter- mediates (Asscher and Vofsi, 1963; Stotter et al., 1977; Wade and Castro, 1973; Wade et al., 1969). The implications of these find- ings with regard to CCl4 activation by the microsomal mixed- function oxidase system will be discussed in more detail later. Reactions (1) and (2) are similar to the chemical reac- tions which involve classical 5N2 mechanisms (Morrison and Boyd, 1971). Pryor (1976) has used the designation S 2, for H substitution, homolytic-bimolecular, to distinguish it from that 21 involving the nucleophilic reactant. A bimolecular intermediate complex may be formed between CC14 and either a free radical or cellular electron transfer component. Dissociation of this inter- mediate would involve cleavage of a carbon-halogen bond and sub- sequent °CC13 formation. The extent to which this reaction occurs would to a large part be dependent on the dissociation energy of the bond being broken. That reactions (1) and (2) may be biologi- cally significant is further supported by the relationships between the hepatotoxicity of compounds in the chloromethane series and their bond dissociation energies (Recknagel and Glende, 1973; Reynolds and Yee, 1967). In order of decreasing C-Cl bond dissociation energy. the toxicity of halomethanes increases as follows: CH C1 > CH C1 3 2 2 > CHCl > CC1 > CBrC1 3 4 3 Recknagel et al. (1977) and Koch et a1. (1974) demonstrated increased toxicity and a much greater increase in diene conjugation in microsomal lipids from rats treated with CBrCl3 than with CCl4. This correlates well with the bond dissociation energies of 49 and 68 kcal/mole for CBrCl3 and CCl4, respectively. The events which mediate CC14 activation and cytotoxicity are complex, making it difficult to pinpoint the causes of cell necrosis. Some of the earlier effects of CC14 poisoning include the following: binding of CC14 to cellular lipids and proteins; increased peroxidation of microsomal lipids; destruction of cyto- chrome P450, GSPase, and subsequent loss of mixed-function 22 oxidase activity; polyribosome disaggregation; decreased synthesis of protein and phospholipid; triglyceride accumulation; and loss of NADPH, NADP, ascorbate, vitamin E, and glutathione (Halbreich and Mager, 1969; Recknagel, 1967; Recknagel and Glende, 1973; Slater, 1972). Damage to other organelles, such as mitochondria and lysosomes, occurs later in the course of CC14 poisoning, after evidence of cell necrosis has appeared (Recknagel, 1967). There are two general hypotheses to account for CC14 toxicity: that proposed by Castro et a1. (1972) which emphasizes the importance of binding to critical cell components, and that of Recknagel (1967) which emphasizes lipid peroxidation. Some support has been found for both hypotheses. Villarruel et a1. (1977) and Diaz Gomez et a1. (1975) investigated hepatotoxicity of CC14 in several animal species as well as general tissue damage within the same animal. Both studies demonstrated that tissue damage corre- lated better with binding to cellular components than with diene conjugation. Since arachidonic acid is destroyed during lipid peroxidation, Villarruel et a1. (1976) also examined the effect of CCl4 on the arachidonic acid contentlof microsomes from animals that showed moderate and high susceptibility to CCl4-hepatotoxicity. Again, they were not able to correlate hepatotoxicity with lipid peroxidation. The species most susceptible to CCl4-hepatotoxicity showed no change, whereas the species which was least susceptible showed significant changes in arachidonic acid content. 23 Recknagel (1967) and Slater (1976) developed the lipid peroxidation hypothesis to explain the widespread damage caused by CC14 within the cell. They suggested that binding hypothesis was unlikely due to the reactivity of -CC13. It would be expected to have a very short biological half-life and would not migrate any significant distance from its site of formation in the micro- somal membrane to damage other subcellular organelles. By-products of lipid peroxidation would be more stable and their toxic effects have been demonstrated (Dianzani et al., 1976; Gammage and Matsu- shita, 1973; Matsushita et al., 1970; Recknagel and Turocy, 1977; Ugazio et al., 1976). The question as to which hypothesis is correct has not been resolved and remains an active area of investigation by several laboratories. What is certain is the fact that the metabolism of CC14 by the microsomal mixed-function oxidase system does initiate lipid peroxidation jg_vitro as well as jn_vivo. Due to my general inter- est in enzyme-catalyzed lipid peroxidation, a further investigation of some aspects of its metabolism and the relationship between the activation reactions and lipid peroxidation was undertaken. The Peroxidation of Membrane Lipids Biological membranes contain significant quantities of polyunsaturated fatty acids which are important for both their structural integrity and function (Slater, 1972). A common property of polyunsaturated fatty acids is their ability to undergo peroxi- dative destruction in the presence of oxygen and free radical 24 initiators. The reactions involved are complex but can be simpli- fied into three general categories common to all free radical chain reactions (Pryor, 1976): l. Initiation LH--:fl;-+'L- 2. Propagation L- + 02:---+ L00- L00- + LH-—-—-—+ LOOH + L: 3. Termination 2 L- ---+ L - L L° 4' L00° —-* LOOL 2 L00- -——-» LOOL + ‘02 In biological systems the initial hydrogen abstraction to form the lipid radical, reaction (1), can be catalyzed by free radi- cals. These may be produced during either normal cell reactions or from the enzymatic activation of pro-oxidants, compounds whose metabolism or interaction with other compounds results in free radi- cal formation (Plaa and Witschi, 1976). Another possibility involves the enzymatic reduction of ADP- or pyrophosphate-chelated iron and its subsequent reaction with oxygen to form the perferryl ion, a proposed initiator of lipid peroxidation (Pederson and Aust, 1975; Svingen et al., 1978). Polyunsaturated fatty acids would be especially susceptible to attack because they all contain reactive hydrogens located on the methylene carbon shown below. Hydrogen abstraction from this structure would result in the formation of resonance stabilized radicals. Reaction of these with hydrogen donors forms lipids containing conjugated dienes detectable by their absorption of light at 233 nm (Recknagel and Goshal , 1976). In the pres- ence of oxygen, lipid hydroperoxides and more lipid radicals are formed as shown by reaction (2). The lipid hydroperoxides can be degraded by several cellular constituents with pro-oxidant activity including heme, hemoproteins, and transition metals which readily undergo one electron oxidation-reductions (O'Brien, 1961 ; O'Brien and Little, 1969; Wills, 1965). Products of this degradation are numerous and have yet to be thoroughly characterized for the more complex poly- unsaturated fatty acids. Those which have been identified include lipid and alkoxy radicals, aldehydes (MDAL.ketones,lipid alcohols, epoxides, and alkanes (Gardner, 1975; Gardner et al., 1974; Hamburg, 1974). Techniques for measuring lipid hydroperoxides directly or the degradation products, malondialdehyde, ethane, and pentane, are currently used to detect lipid peroxidation jn_yjyo. and jg_yit£g (Dillard et al., 1976; Slater, 1972). The formation of lipid hydroperoxides and their subsequent degradation by cellular pro-oxidants have important biological consequences. Since lipids are required for membrane integrity, 26 peroxidative destruction would disrupt membrane function and destroy many membrane-associated enzyme activities. Several degra- dation products of lipid hydroperoxides have been shown to be capable of crosslinking proteins, inhibiting several enzymes, causing red blood cell lysis, and killing microorganisms (Dianzani et al., 1976; Pfeifer and McCay, 1971; Recknagel and Turocy, 1977; Ugazio et al., 1976). Lipid hydroperoxides can also react directly with enzymes resulting in a loss of activity (Gamage and Matsu- shita, 1973; Matsushita et al., 1970). Fortunately, under normal conditions, mechanisms exist which control the extent of lipid peroxidation. In addition to the termination process, reaction (3), the cell is protected to some extent by its relatively low oxygen content and the presence of both hydrophilic and lipophilic antioxidants (Recknagel, 1977). The reactions involved in propagation are believed to be blocked by many antioxidants which function as either radical trappers or ‘alternate hydrogen donors. Radicals which result from the inter- action with antioxidants are much less reactive and more likely to participate in termination reactions (Demopoulos, 1973; Mead, 1976). In addition, the cell contains enzymes with peroxidase activity which in the presence of suitable electron donors will reduce the lipid hydroperoxides to lipid alcohols and water. Two such enzymes involved in these reactions are glutathione peroxidase and cyto- chrome P450 (Hrycay and O'Brien, 1973; McCay, 1976; Sies and Sumner, 1975). CHAPTER II THE ROLE OF THE NADPH-CYTOCHROME P REDUCTASE 450 COMPONENT OF RAT LIVER MICROSOMES IN CARBON TETRACHLORIDE ACTIVATION Abstract A model membrane system was developed for determining the role of the flavoprotein, NADPH-cytochrome P450 reductase, in CC14 activation. The system contained purified cytochrome P450 reductase, liposomes, and NADPH in the presence or absence of the electron 3 and EDTA-Fe+3. acceptors, ADP-Fe+ Conditions which favor the fully reduced and half-reduced flavoprotein could be achieved in the absence and presence, respectively, of the electron acceptors. The CCl4-dependent stimulation of lipid peroxidation or the anaerobic- covalent binding of 14 CC14 to liposomes was then used to assay CC14 activation by both forms of the enzyme. The ability of the fully reduced NADPH-cytochrome P450 reductase to activate CC14 was investigated. CC14 addition to the liposomal system in the absence of electron acceptors did not cause 3 the oxidation of NADPH. However, addition of EDTA-Fe+ to the same system resulted in a substantial rate of NADPH oxidation. The fully reduced enzyme also did not cause the covalent binding of 14CC14 to liposomes. 27 28 To determine CC14 activation by the half-reduced NADPH- 3 3 cytochrome P450 reductase both ADP-Fe+ and EDTA-Fe+ were included in the system. CCl4-stimulated NADPH-dependent lipid peroxidation (MDA formation) and an NADPH-dependent binding of 14CC14 to lipo- somes were observed. Binding was also observed when either ascor- 2 bate or Fe+ were added to the system in the absence of NADPH and NADPH-cytochrome P450 reductase. Activation by NADPH-cytochrome 3 3 P450 reductase in the presence of NADPH, ADP-Fe+ . and EDTA-Fe+ was not observed unless the liposomes contained lipid hydroperox- ides. The reductase was required to reduce EDTA-Fe+3 oxidant form, EDTA-Fe+2. to its pro- The latter catalyzes the degradation of lipid hydroperoxides into lipid radicals which activate CC14 by atom transfer reactions. Cytochrome P450(Fe+3) as well as other oxidized hemoproteins will also serve as pro-oxidants in this system. The results of these studies suggest that NADPH-cytochrome P450 reductase does not directly serve as a site of CC14 activation in the microsomal mixed-function oxidase system. Cytochrome P450 reduc- tase participates primarily in CC14 activation through its reduction of cytochrome P450 and, alternatively, through its ability to gen- 3 and oxygen. erate lipid hydroperoxides in the presence of ADP-Fe+ The hydroperoxides subsequently activate CC14 in the presence of cytochrome P450(Fe+3) or other cellular pro-oxidants. 29 Introduction The metabolism of CC14 by the microsomal mixed-function oxidase system of the liver has been previously established (Reck- nagel et al., 1977). Unlike the majority of substrates for this system, the metabolism (activation) of CC14 can proceed by reductive reactions requiring NADPH but not oxygen (Glende et al., 1976; Uehleke et al., 1973). The reductive reactions involved and the role of each of the microsomal mixed—function oxidase components in CC14 activation have not been characterized. In other reductive reactions catalyzed by the mixed-function oxidase system, the azo- and nitro-reductases, both the cytochrome P450 and NADPH- cytochrome P450 reductase components will transfer reducing equiva- lents to the substrates (Gillette, 1971a). Evidence which suggests that these components function similarly in CC14 activation has been previously presented (Recknagel et al., 1977; Shah and Carlson, 1975; Slater, 1972). The evidence which supports the view of cyto- chrome P450 reductase being the locus of CC14 activation within the mixed-function oxidase system will be presented below. Although a role for NADPH-cytochrome P450 reductase in CC14 activation has not been clearly established, it is believed to be the site of oCCl3 formation within the mixed-function oxidase system (Sla- ter, 1971b). The general reactions by which this may occur, atom transfer and electron capture, were previously discussed. That NADPH-cytochrome P450 reductase also activates CC1 4 by one of these reactions is a possibility. Slater (1972) has pr0posed that an 30 electron capture reaction involving the fully reduced flavoprotein, NADPH-cytochrome P450 reductase, may be responsible for CC14 acti- vation. An atom transfer reaction involving this enzyme is also possible. Masters et a1. (1965) have shown that cytochrome P450 reductase shuttles between its fully and half-reduced states during the transfer of electrons to cytochrome P450 and other electron acceptors. In its half-reduced state, cytochrome P450 reductase contains the flavin semiquinone radical (Iyanagi et al., 1974) which may participate in CC14 activation directly or through formation of secondary radicals (Slater, 1974; Slater and Sawyer, 1971b). Evidence which supports the hypothesis that NADPH-cytochrome P450 reductase is the locus of CC14 activation within the microsomal mixed-function oxidase system is as follows. Slater and Sawyer (l97la,b) used the CCl4-dependent stimulation of microsomal lipid peroxidation, MDA production, as an indicator of -CC13 formation. They found that compounds which interact with cytochrome P450 to inhibit mixed-function oxidase activity, e.g., SKF-525A, CO, and nicotinamide, did not decrease the CCl4-dependent stimulation of lipid peroxidation. This was true even though these compounds caused a significant, 50 to 60%, inhibition of mixed-function oxi- dase activity, aminopyrine demethylation. Masuda and Murano (1977) developed a similar system but used EDTA-washed microsomes to greatly reduce endogenous rates of lipid peroxidation and to thereby increase the magnitude of CCl4-stimulated lipid peroxidation. In their system CO inhibited aminopyrine demethylation by 91% but did 31 not decrease CCl4-stimulated lipid peroxidation. Mixed-function oxidase substrates or inhibitors, aminopyrine and SKF-525A, were inhibitory only at relatively high concentrations where they are believed to function as antioxidants rather than competitive inhibitors. CCl4-stimulated lipid peroxidation was completely inhibited, however, by low concentrations of substances which inter- act with cytochrome P450 reductase, pCMB, K3Fe(CN)6, cytochrome c, and vitamin K3. This agreed with the earlier hypothesis which sug- gested that inhibition was due to the competition of these agents with CC14 for reducing equivalents from the fully reduced cytochrome P450 reductase (Slater and Sawyer, 1971b). It was also pointed out that the destruction of NADPH observed during CC14 intoxication was due to its interaction with -CC13 at the activation site, NADPH- cytochrome P450 reductase (Slater and Sawyer, 1977). Because of these findings an investigation was undertaken to assess the role of NADPH-cytochrome P450 reductase in CC14 activation. At the beginning of this investigation the objectives sought were to determine if the enzyme was capable of CC14 acti- vation and, if so, to determine the reactions involved. A model membrane system consisting of liposomes prepared from extracted liver microsomal lipids, purified NADPH-cytochrome P450 reductase, and NADPH in the presence of absence of electron acceptors (ADP-Fe+3 and EDTA-Fe+3) was used in these investigations. The 14 stimulation of lipid peroxidation by CC14 and the binding of CC1 4 to liposomes was used to estimate -CC13 formation. 32 Materials and Methods Material Sources Male Sprague-Dawley rats obtained from Spartan Research Ani- mals, Haslett, Michigan, were used. Rats pretreated with P8 and 3-MC ranged in weight from 225 to 250 g and from 90 t0125 g, respectively. Soybean lipoxygenase (Type I), hemoglobin (Type IV), hemin (Type I), cytochrome c (Type VI), and bovine serum albumin (BSA) (Type V) were obtained from the Sigma Chemical Company, St. Louis, Missouri. Bromelain was a gift from the Dole Company, Honolulu, Hawaii. Butylated hydroxytoluene (BHT), dithiothreitol (reagent grade), sodium cholate, Sepharose 4-B, sodium dodecyl sulfate (SDS), thiobarbituric acid, Tris base, ADP (Grade III), NADPH, Dowex chelating resin, 1,4-bis [2-(5 phenyloxazolyl)] benzene (POPOP), 3-methylcholanthrene(3-MC), polyethyleneglycol 400, and Brilliant Blue R (Coomassie blue) were obtained from the Sigma Chemical Company, St. Louis, Missouri. Glass-distilled toluene was obtained from Burdick and Jackson Lab., Inc., Muskegon, Michigan. 2,5-Diphenyl oxazole (PPO) was obtained from Research Products International, Elk Grove, Illinois. Deoxycholic acid, 1,8-diamino octane, cyanogen bromide, and anthraquinone 2-sulfonic acid were obtained from the Aldrich Chemical Company, Milwaukee, Wisconsin. EDTA and sodium hydrosulfite (dithionite) were obtained from the Fisher Scientific Company, Fairlawn, New Jersey. All electrophoresis reagents were from Canalco, Inc., Rockville, Maryland. Sucrose was from the Schwartz-Mann Division of Becton-Dickinson and Co. , Orangeburg, 33 New York. Emulgen 913 was a gift from Kao-Atlas, Ltd. , Tokyo, Japan. Phe- 14 nobarbital (P8) was from Merck & Co. , Inc. , Rahway, New Jersey. CCl4 and [MC] toluene were from Amersham Searle, Arlington Heights, Illinois . 14 CCl4was also obtained from New England Nuclear, Boston, Massachusetts. Dilauroylglyceryl-3-phosphoryl choline (di-l ,2-GPC) was obtained from Cal-Biochem, San Diego, California. 2' ,5'-ADP-Sepharose 4B was obtained fromll L. Biochemicals, Milwaukee, Wisconsin. Hydroxyapatite (Biogel- HTP) , calcium phosphate gel, and Biobeads SM2 were obtained from Biorad, Richmond, California. Argon and nitrogen gases (99.99% purity) and oxygen (99.5% purity) were obtained through General Stores, Michigan State University, East Lansing, Michigan. CO (PCP grade, 99.5% purity) was obtained from Matheson, E. Rutherford, New Jersey. All other reagents used were of analytical grade. All aqueous solutions were prepared from water which had been distilled and passed through a mixed-bed ion exchange column. Cholic and deoxycholic acids were treated with activated charcoal and recrystallized from 95% ethanol prior to use. Drungretreatment of Rats Rats were treated with P8 by including it in their drinking water at a concentration of 0.1% for 10 days. Rats were treated with 3-MC by i.p. injection of a dose (20 mg/kg, dissolved 10 mg/ml in polyethyleneglycol-400) at 36 and 24 hours prior to sacrifice. Isolation of Rat Liver Microsomes Rats were fasted 18 hours prior to killing by decapita- tion. Microsomes were prepared by the differential centrifugation 34 procedure of Pederson (1973) except as modified below. Microsomes to be used for the activation of 14CC14 were washed by resuspending in 1 mM Tris-HCl (pH 7.5 at 25°C) to a final protein concentration of from 3 to 5 mg/ml and recentrifuging as described. Microsomes were stored in argon-saturated 0.05 M Tris-HCl (pH 7.5 at 25°C) containing 50% glycerol as described but BHT was omitted. Stock buffers of Tris-HCl were treated with Dowex chelating resin to remove trace amounts of iron. These "chelexed" buffers were subse- quently used to prepare the Tris buffers mentioned above. Micro- somes isolated in this manner were used within two days. Microsomes used for cytochrome P450 and P448 isolations were prepared as above but were isolated and washed in 1.15% KCl containing 10 mM EDTA and stored in the presence of 0.01% BHT. Microsomes used for the preparation of NADPH-cytochrome P450 reduc- tase were isolated and stored in the presence of BHT according to Pederson (1973) but were washed prior to use according to Yasukochi and Masters (1976). All microsomal preparations were stored under argon at -20°C. Purification of NADPH-Cytochrome P450 Reductase from Rat Liver Fficrosémes Two procedures were used to isolate cytochrome P450 reduc- tase from the microsomes from P8 pretreated rats. The procedure of Dignam and Strobel (1975) was used without modification; that of Yasukochi and Masters (1976), with the modifications included below. 35 Gel filtration on LKB ultrogel AcA 34 was omitted and treatment with Biobeads SM2 for 90 minutes at O-4°C (Holloway, 1973) was used for detergent removal instead of DEAE-cellulose chromatography. Purification of Cytochromes P450, P443, and b5 from Rat Liver Microsomes A column (2.6 x 40 cm) of 8-aminoocty1 Sepharose 4B was prepared according to the procedures of Imai and Sato (1974) and Cuatrecasas (1970). This column was subsequently used to purify cytochromes P450, P448’ and b5 according to the combined procedures of Imai (1976) and Guengerich (1977). The latter method was used to purify cytochromes P450 or P448 from the microsomes of P8- or 3-MC- pretreated rats, respectively. Modifications of the procedure are included below. All buffers were adjusted to the proper pH at room temperature prior to the addition of dithiothreitol. The deionized-distilled water used to prepare buffers for hydroxyapatite chromatography was degassed to remove CO2 traces and help maintain good flow rates. Biobeads SM2 treatment (90 minutes, O-4°C) fol- lowed by adsorption, washing, and elution from calcium phosphate gel, was used to remove excess detergent, Emulgen 913. The DEAE- cellulose chromatography was omitted because it did not improve the specific content and resulted in even lower yields of cyto- chrome P450 (P448)' Peak fractions which eluted from the hydroxy- apatite column with 90 and 150 mM "phosphate buffers" were pooled and concentrated four to six-fold by ultrafiltration on an Amicon 36 PM30 membrane. Detergent was removed as described and the fractions were dialyzed against 30 volumes of 10 mM Tris-acetate (pH 7.4 at 25°C) containing 20% glycerol and 0.1 mM EDTA. The dialyzed frac- tions were centrifuged at 20,000 x g or higher for 20 minutes to remove any precipitate and stored under argon at -20°C. If a sub- stantial precipitate forms, as has happened in the purification of cytochrome P448’ a 0.1 M potassium phosphate buffer, pH 7.25, con- taining 20% glycerol, 0.1 mM EDTA, and from 0.01 to 0.05% Emulgen 913 can be used for redissolving and storage. The P448 remained stable and could be used in experiments in which detergent contami- nation would not interfere. Cytochrome b5 was prepared by subsequent elution from the octylamine column by the method of Imai (1976). The column was first washed with 0.05 M potassium phosphate buffer (pH 7.25) con- taining 20% glycerol, 0.15% deoxycholate, and 0.35% cholate (approxi- mately 1 to 1.5 liters) to remove an NADPH-cytochrome P450 reductase-containing fraction with specific activity ranging from 10 to 15 units/mg protein. Cytochrome b5 was eluted with a 0.05 M potassium phosphate buffer (pH 7.25) containing 20% glycerol, 0.35% deoxycholate and 0.15% cholate. Peak fractions were pooled, con- centrated by ultrafiltration, treated with Biobeads SM2, dialyzed as previously mentioned, and stored. The detergents deoxycholate and cholate were still contaminating this preparation and were not significantly decreased by further Biobead treatment or dialysis for 2 days against the "Tris-acetate" buffer. Unless otherwise 37 stated, all hemOprotein preparations were run over a Sephadex 625 column, equilibrated in 0.1 M Tris-HCl (pH 7.25 at 25°C and chelexed) buffer containing 20% glycerol, before being used in experiments described in this thesis. When required the octylamine column was regenerated by passing approximately five column volumes of 0.1 M potassium phos- phate buffer (pH 7.25) containing 20% glycerol, 0.2% Emulgen 913, 0.6% cholate, and 1 mM EDTA. The column was then washed with three column volumes of the equilibration buffer as described. Enzyme Assays and Analytical Procedures Cytochrome P450, P448’ and b5 (Omura and Sato, 1964) and NADPH-cytochrome c reductase (Pederson, 1973) were assayed as pre- viously described. Hemoglobin was treated with potassium ferri- cyanide as described (Antonini and Brunori, 1971), and subjected to column chromatography on Sephadex 625. The concentration of the methemoglobin formed was determined spectrally in 0.1 M potassium phosphate buffer (pH 6.5) using an extinction coefficient of 179 1 '1 for the peak at 405 nm (Winterhalter, 1974). NADH- mM' cm- cytochrome b5 reductase activity was measured by determining the reduction of K3Fe(CN)6. The 1 ml assay contained 0.1 M potassium phosphate buffer (pH 7.4), 1 mM K3Fe(CN)6, 0.25 mM NADH, and from 0.02 to 0.4 mg protein. The change in absorbance at 420 nm was 1 1 followed with time and an extinction coefficient of 1.02 mM' cm" used for reduced ferricyanide. All assays were performed at 25°C unless stated otherwise. 38 The activation of 14 CC14 was determined by measuring its covalent binding to lipid. The 2 ml liposomal and microsomal assay systems differed in their initial preparation but not extraction and subsequent analytical procedures. All reactions were run under anaerobic conditions, which were established as follows: all buf- fers and reagents were thoroughly saturated with argon by bubbling for approximately 30 min (0-4°C) prior to use; microsomes or other reaction components were added to 10 m1 erlenmeyers under a stream of argon and capped with gas-tight rubber caps (either caps from Vac-u-tainer blood-collecting tubes or serum caps were used); and each flask was evacuated and flushed five times with de-oxygenated argon and left under slightly positive argon pressure. 14 CC14 and reagents required for the initiation of the reactions (NADPH, Fe(NH4)2(SO)2, ascorbate, or partially peroxidized lipid) were added via glass syringe. All of the above procedures were performed at 0-4°C. To conduct the reactions, flasks were preincubated for 3 min (at 37°C) and the reaction initiated and run for a designated period of time with constant shaking (90 to 100 rpm). Reactions were stopped with the addition, via glass syringe, of 2 ml of cold nitrogen-saturated chloroform-methanol (CHC13:Me0H) (1:1 v/v) con- taining 0.01% BHT and placed on ice with caps in place. When all reactions were completed the flasks were allowed to reach room temperature before extraction proceeded. The contents of each flask were quantitatively transferred to 20 m1 screw-cap culture tubes, 2.4 ml CHC13:Me0H (0.01% BHT) was then used to rinse each 39 flask and added to the previous fraction to achieve a final CHC13: MeOH:H20 ratio of 2:2:1.8 used in the lipid extraction procedure of Bligh and Dyer (1959). The tubes were capped, vortexed for 50 sec, and centrifuged. After discarding the aqueous phase, 0.2 volumes of Folch-salts (Folch et al., 1956) were added to the 2.4 ml organic phase and the procedure repeated. The washed organic phase was quanti- tatively transferred to tared scintillation vials and evaporated to dryness at 45°C undera stream of nitrogen. To the residue were added successively 0.2 and 0.1 ml aliquots of CCl4and absolute ethanol, respectively, and the mixture taken to dryness after each addition. Reactions were conducted and extractions performed under subdued lighting conditions, whenever possible. The weights of the lipid residues were determined and prepared for scintillation counting. To prepare the extracted lipid for scintillation counting, 15 ml of toluene containing 0.5% PPO and 0.03% POPOP were added to the scintillation vials. The optimum instrumental conditions were 14C and where required were quench- determined for counting corrected by the Channels Ratio method (Bush, 1963). Reactions were generally performed in triplicate and the Student t-test used to determine significance of differences between the means (Klugh, 1974). CC14 activation was also measured by its ability to enhance lipid peroxidation. For these reactions lipid and hemOproteins were mixed first, 10 min at 0-4°C, prior to addition of other reagents. Other components were then added as described in each figure legend, 40 at 0-4°C. The mixture was subsequently preincubated for 2 min at 37°C prior to initiation of the reaction. MDA was determined from l-ml aliquots as described elsewhere. Only the protease-solubilized NADPH-cytochrome P450 reductase was used for these experiments. 3 and EDTA-Fe+3 Unless otherwise stated, ADP-Fe+ were previously mixed in 0.05 M Tris HCl and pH adjusted to 7.5 at 25°C; their respective molar ratios to iron were 16.6:1 and 1:1. Buffers treated with Dowex chelating resin were used for all reactions in which CC14 activation was determined. Malondialdehyde (MDA) and lipid hydroperoxides (Buege and Aust, 1978) and total lipid phosphate (Bartlett, 1959) were deter- mined as previously reported. Protein was determined by the method of Lowry et a1. (1951) and standardized with bovine serum albumin 1% cm at 280 nm equal to 6.6 (Rutter, 1967). using E SDS-Polyacrylamide Gel Electrophoresis Electrophoresis was carried out according to the procedure of Welton and Aust (1973) with the following modifications: samples were dialyzed overnight against 50 volumes of distilled-deionized water and 1yopholyzed; and 1.5% SDS was included in the prepared sample to ensure migration of all protein into the gel matrix. Extraction of Lipids and the Preparation of Liposomes The procedures of Pederson (1973) were used for the extraction of lipid from the microsomes of untreated rats and for the preparation of liposomes. Liposomes were prepared in 41 argon-saturated deionized-distilled water. When synthetic lipid was used a small quantity was weighed, distilled water added, and sonicated as before. Preparation of Lipid Hydroperoxides Soybean lipoxygenase was used to generate lipid hydroperox- ides in a reaction containing 0.05 M borate buffer (pH 9.0), 0. 1 mM EDTA, 0.3% sodium deoxycholate, 0.10 mg lipoxygenase/ml, and from 2.5 to 5.0 umole lipid-P04/ml of reaction. Reactions, 20 ml, were carried out in 250 ml erlenmyer flasks under a stream of oxygen with constant agitation. The reaction was stopped after 30 min by the addition of CHCl3zMe0H (1:2) to form a single phase consisting of CHC13zMeOH:H20 in a ratio of 1:2:0.8. A sufficient volume of CHC13:H20 (1:1) was subsequently added to yield CHCl3zMeOH:H20 ratios of 2:2:1.8 as required by Bligh and Dyer(1959) for total lipid extraction. The resulting mixture is centrifuged and the organic phase washed with 0.2 volume Folch-salts solution (Folch et al., 1956). The organic phase was concentrated by evaporation under a stream of nitrogen at 45°C and stored under argon at -20°C. Storage under these conditions resulted in little or no loss of hydroperoxides for periods up to 5 days. Deoxygenation of Argon Argon was used as the gas phase in all of the reactions conducted anaerobically. Deoxygenation was according to the method of Vogel (1958) with a saturated solution of lead acetate included 42 in the line to trap any H2S which may be evolved. Thick-walled rubber tubing was used thought to minimize oxygen leakage or diffu- sion into the system (Dixon, 1971a)- Results Purification of NADPH-Cytochrome P45“ Reductase from Rat Liver Microsomes In order to determine the role of cytochrome P450 reductase in CC14 activation, the pure enzyme is required. During the course of these investigations two purification procedures were used, that of Dignam and Strobel (1975) and of Yasukochi and Masters (1976). An excellent preparation resulted from both procedures; however, since the latter preparation was used in the majority of these studies, only data concerning its purification will be presented. Table 1 summarizes the purification of NADPH-cytochrome P450 reduc- tase from liver microsomes of PB-pretreated rats. The specific activity obtained, 47.7 umoles cytochrome c reduced/min/mg protein, was comparable to that reported when measured under their assay conditions (same as Methods section of this chapter but at 30°C). The peak fractions eluting from the 2',5'-ADP-sepharose affinity column were sufficiently pure and therefore the final gel filtration step on Ultrogel AcA-34 was omitted. The purified reductase con- tained a minor contaminant as shown in Figure l. 43 TABLE 1.--The Purification of NADPH-Cytochrome P450 Reductase from Rat Liver Microsomes. Protein Total Spec. Act. Yield Fraction (mg) Units* (units/mg) (%) Washed PB-microsomes 1,600 705.6 0.44 100 DEAE column (peak fractions) 80 314.2 3.93 45 2',5'-ADP affinity column (peak fractions after -- -- 24.36 -- Sephadex G25 treatment) Affinity column fractions after Biobeads SM2 treat- 4.5 164.2 36.50** 23 ment and dialysis *1 unit = l umole cytochrome c reduced/min when assayed as described in Methods. **47.7 units/mg when assayed at 30°C. Activation of CCl4 by NADPH- Cytochrome P450 Reductase in Liposomes Slater (1972) has proposed a reaction for the activation of CC14 by NADPH-cytochrome P450 reductase involving electron capture from the fully reduced flavoprotein. Two approaches were used to investigate this possibility. The first involved a determination of NADPH oxidation in a model membrane system containing only NADPH- cytochrome P450 reductase and liposomes prepared from the saturated lipid, di-l,2-GPC. Table 2 contains the results of this investigation. NADPH oxidation, as determined by the decrease in absorbance at 340 nm, was not observed in the presence of CCl4. However, the addition of EDTA-Fe+3 (1:1 molar ratio), a known 44 Figure 1.--SDS-Polyacrylamide Gel Electrophoresis of NADPH-Cytochrome P450 Reductase Purified from PB-Microsomes. NADPH-cytochrome P 50 reductase was purified as described in Methods. A sample contain1ng 10 ug of protein was applied to the gel and electrophoresis conducted in the presence of 1% SDS as described in Methods. The gel was stained for protein with Coomassie blue and photographed. Fig. 1 45 46 TABLE 2. --NADPH- Cytochrome P450 Reductase Catalyzed xidation of NADPH in Liposomes Using CCl4 and EDTA- Fe+ as Electron Acceptors. Reactions contained 0.1 mg di-1,2-GPC/ml, 0.025 units/ml NADPH- -cytochrome P450 reductase, + 0. 2 mM EDTA with 0.2 mM FeCl , +1 ul CC1 4/ml, and 0.1 mM NADPH in 0.1 M Tris- HCl, pH 7. 4 at 37°C The reactions were conducted at 37°C and 00340 measured. Description nmole NADPH Oxidized/min/ml Complete system 0 Complete system + CCl4 0 +3 Complete system + CCl4 + EDTA-Fe 1.70 electron acceptor (Noguchi and Nakano, 1974), resulted in a signifi- cant rate of NADPH oxidation. These results suggest that CC14 is not a good electron acceptor and hence not activated significantly under these experimental conditions. A more sensitive and direct assay was used to exclude the possibility that CC14 was being activated at rates too low to be measured by following NADPH oxi- dation. The covalent binding of ‘4 CC14 to liposomes under anaerobic conditions was used to indicate CC14 activation. Anaerobic conditions were used to exclude the interference of oxygen with binding (Uehleke et al., 1973; Wood et al., 1976) and to exclude the possibility of its competing with CC14 for electrons from the fully reduced enzyme. Oxygen is capable of accepting electrons from the fully reduced enzyme and would be expected to decrease its steady-state levels (Dixon,197lb). ‘The results of Table 3 confirm the findings of the previous experiment suggesting that the 47 TABLE 3.--NADPH-Cytochrome P45 Reductase Catalyzed Activation of 4CCl4 in Liposomes 9n the Absence of Added Electron Acceptors. Reactions cpntained 1.0 umole lipid-P0 /ml, 0.01% sodium deoxycholate, 1 in 4CCl4/ml (0.25 m Ci/ml 01:14): 0.3 mM NADPH in 0.1 M Tris-HCl, pH 7.4 at 37°C. The reactions were conducted under anaerobic conditions as described in Methods for 30 min at 37°C. Description cpm/mg Lipid i 5.0. Liposomes (-) NADPH (-) NADPH cytochrome 515 + 10 P reductase ‘ 450 Liposomes + 0.3 mM NADPH + 0.025 units reductase/ml 496 i 28 Liposomes + 0.3 mM NADPH + 0.100 units reductase/ml 529 i 73 hypothesis of CC14 activation involving electron capture from the fully reduced enzyme is incorrect. The experimental conditions favored the full reduction of NADPH-cytochrome P450 reductase; how- ever, CC14 was not activated at either enzyme concentration used. CCl4 Activation by NADPH-Cytochrome P450 Reductase in the Presence of Added Electron Acceptors A property of NADPH-cytochrome P450 reductase which has led to its proposed function in CC14 activation is its ability to form semiquinone radicals during its electron transfer reactions (Iyanagi et al., 1974). A plausible mechanism for CCl4 activation would involve atom transfer from either the flavin semiquinone radical or perhaps secondary radicals generated as a result of its 48 formation. One suitable system in which to investigate CC14 activa- tion via the half-reduced enzyme would be that developed by Noguchi and Nakano (1974) and Pederson and Aust (1973) for the characteriza- tion of NADPH-cytochrome P450 reductase-dependent lipid peroxida- tion. This well-characterized system contains NADPH-cytochrome 3 and EDTA-Fe+3 P450 reductase, liposomes, and ADP-Fe+ , as electron acceptors. The formation of -CC13 can be estimated from the stimu- lation of lipid peroxidation, MDA formation. Table 4 presents the initial experiments to demonstrate CC14 activation by this system. A small but reproducible stimulation of MDA production resulted from the addition of CCl4. To increase the sensitivity of this assay, 14 the anaerobic binding of CC14 to liposomes, prepared from extracted microsomal lipid, was again utilized as an indicator of CC14 activation. These experiments resulted in a small but TABLE 4.--CCl4-Stimulated Lipid Peroxidation in Liposomes. Reactions contained 0.5 umole lipid-POq/ml, 0.02 units/ml NADPH-cytochrome P450 reductase, 1.66 mM ADP with 0.1 mM FeCl3, 0.05 mM EDTA, 0.1 M NaCl, 1 ul CCl4/m1 i 0.25 mM NADPH in 0.1 M Tris-HCl, pH 7.4 at 37°C. Reactions were carried out for 15 min at 37°C and MDA determined as described in Methods. Description nmoles MDA/15 min/ml Liposomes (-) NADPH 0.12 Liposomes (+) NADPH 3.80 Liposomes (+) NADPH (+) CC14 4.58 49 statistically significant increase in labeling in the presence of NADPH (Table 5). TABLE 5.--NADPH-Cytochrome P450 Reductase Catalyzed Activation of 4CCl4 in Liposomes in the Presence of Electron Acceptors. Reactions contained 1.0 nmole lipid-PO /ml, 0.02 unit/m1 NADPH-cytochrome P452 reductase, 1.66 mM ADP w1th 0.1 mM FeCl , . 0.05 mM EDTA, 1 in CCl4/ml (0.25 m Ci/mlCC14).:t 0.3 mM NAD H 111 0.1 M Tris-HCl, pH 7.4 at 37°C. Reactions were conducted under anaerobic conditions as described in Methods for 30 min at 37°C. Description cpm/mg Lipid i S.D. Expt'l.-Cont. Liposomes (-) NADPH 484 i 36 Liposomes (+) NADPH 621 1 54 137* *Significantly different from control, p < 0.05. It was of interest to determine the fate of cytochrome P450 reductase during activation by this system. If the mechanism of activation involves an atom transfer reaction between CC14 and the flavin semiquinone radical, one should expect chlorination of the flavin moiety and perhaps inactivation of the enzyme. That inacti- vation does not occur is indicated by the results presented in Table 6. Using similar reaction components and assay conditions as in the previous experiment, cytochrome c was added to these incuba- tions at the end of 30 minutes and tested for enzymatic activity, the reduction of cytochrome c. In neither of these reactions was the reduction of cytochrome c by the enzyme decreased. The presence of oxygen (and hence lipid peroxidation) also did not alter enzy- matic activity. These results are in agreement with the 50 TABLE 6.--The Fate of NADPH-Cytochrome P450 Reductase During CCl4 Activation in Liposomes. Reactions contained 0.1 umole lipid-PO4/ml, 0.018 units/ml NADPH-cytochrome P45 reductase, 1.66 mM ADP with 0.1 mM FeCl3, 0.05 mM EDTA, 1 ul C814/ml, t 0.1 mM NADPH in 0.1 M Tris-HCl, pH 7.4 at 37°C. Reactions were conducted as follows: the 0.8 m1 reaction mixture was incubated under anaerobic or aerobic (99% 02) conditions for 30 min at 37°C after which it was placed on ice. Reactions were allowed to reach room temperature and NADPH and cyto- chrome c added to final concentrations of 0.2 mM and 75 uM, respec- tively. Cytochrome P450 reductase activity was determined according to Methods section. nmoles Cytochrome c Description Reduced/min/ml Experiment 1 (anaerobic conditions) Complete system (-) NADPH 3.6 (+) NADPH 4.1 (+) NADPH + CC14 4.4 Experiment 2 (aerobic conditions) Complete system (-) NADPH 4.6 (+) NADPH 6.1 (+) NADPH + CC14 7.0 observations that reductase is not destroyed in CCl4-poisoned ani- mals or during the in vitro metabolism of CC14 by microsomes (Glende et al., 1976; Recknagel and Glende, 1973). Determination of Optimal NADPH—Cytochrome P450 Reductase and EDTA-Fe+3iConcentra- tions fer CCl4 Activation in Liposomes Having established that CC14 activation does occur in the liposomal system, a determination of the optimal experimental 51 conditions was pursued. If the flavin semiquinone of NADPH-cytochrome P450 reductase participates in CC14 activation, binding should in- crease with the enzyme content of the assay. The results of an experi- ment in which the dependence of activation on NADPH-cytochrome P450 reductase concentrations were determined are shown in Table 7. A correlation between activation and reductase content of the assay was 3 alone was included 3 not observed in this experiment in which EDTA-Fe+ as the electron acceptor. The same was also true when both ADP-Fe+ 3 and EDTA-Fe+ were added as electron acceptors (data not included). It therefore became apparent that a factor other than NADPH- cytochrome P450 reductase was limiting. 3 The concentration of EDTA-Fe+ used for the above experi- ment is lower than that found optimal for the NADPH-cytochrome P450 TABLE 7.--The Dependence of 14CCl4 Activation on NADPH-Cytochrome P450 Reductase Concentration in Liposomes. Reactions contained 1.0 umole lipid—PO4/ml, 0.1 mM EDTA with 0.1 mM FeCl3, 0.25 M NaCl, 0.05% sodium deoxycholate, 1 ul CCl4/ml (0.25 m Ci/ml CCl4), 0.3 mM NADPH in 0.1 M Tris-Hcl. pH 7.4 at 37°C. The reactions were conducted under anaerobic conditions as described in Methods for 30 min at 37°C. Description ' cpm/mg Lipid i S.D. Liposomalg§ystem 0 unit/ml NADPH-cytochrome P450 reductase 1,638 i 110 0.005 unit/ml NADPH-cytochrome P450 reductase 1,528 i 129 0.020 unit/m1 NADPH-cytochrome P450 reductase 1,557 1 28 0.100 unit/ml NADPH-cytochrome P450 reductase 1,521 i 143 52 reductase-dependent lipid peroxidation in liposomes. Of the elec- 3 is reduced most readily by the tron acceptors present, EDTA-Fe+ enzyme (Noguchi and Nakano, 1974); its increase would also increase the number of enzyme molecules in its half-reduced, flavin semi- quinone form. The results of the experiment to determine the 3 effect of EDTA-Fe+ on CC14 activation is presented in Figure 2. A substantial increase in activation was obtained at EDTA-Fe+3 levels ranging from 0.05 to 0.5 mM. The relationship between CC14 activation and EDTA-Fe+3 concentration suggests a role for the flavin semiquinone radical of NADPH-cytochrome P450 reductase in CC14 activation; however, alternative interpretations are possible. As mentioned in the Literature Review of this thesis, some transi- tion metals, including iron, are oxidized in the presence of halo- alkanes resulting in free radical formation (Asscher and Vofsi, 1963; Castro and Kray, 1966). Though these reactions required non-physiological conditions, chelation by EDTA could sufficiently alter the properties of iron to make this a plausible mechanism. Another possibility stems from the fact that the reduced chelate, EDTA-Fe+2, functions as a pro-oxidant catalyzing the degradation of lipid hydroperoxides (Svingen et al., 1978). Since free radicals are included among these products, CC14 may be subsequently acti- vated through atom transfer reactions (O'Brien, 1969; O'Brien and Little, 1969). Although care was taken to exclude 02 from both the liposomal preparation as well as the assays themselves, one cannot exclude completely the presence of some endogenous lipid 53 .uoum um ewe om ace mcoguwz cw umawLUMmu we mcowuwucoo ownocmocm gone: umpuzucou mam; mcowpommm .UONM ea 4.“ 1a ._oz-m_ee z F.o em :aoaz :2 m.c A .Aepuu _e\eo e mN.oV _e\epoow P: _ .mpumd :5 _.o saw: m+md1ao< :5 om.F .Aowumc m—os PaPV m+mu1woo< 4.88 +oa-one .N can P mcszyoo soc» um_ooav p Fm.o om m.¢ mama—m :5 omp .csapou upmumamaxocux: m mo.m me_ m.e_ opa=_o as om .ee=Foo oomoaaezxoceaz new o_ mw.a mam _.oM ooazpo :5 om .ee=.oo ooeoaaeAXOLeAI on— em om.m moo._ Fa_ m 5555 .meowooace ee=_oo oeAEa_»ooc mm ma.e omm AN. a 3555 .meowooacc ee=_oo messepxooo “a mo.~ mam.~ 5.5.2 oeaoaeeaaaa eo~w_ee=_aa ooa_o;o a x ooo.- so, Nm._ maa.~ mmm._ aoeomoco2e-ma 55:55: ARV Ame\mwposcv AmmpoE:v Away epoa> oeooeou oeceooam oeaoeoo Faooe meeooeca eoeeoeca .mumm umummcumca1ma mo mwsomocomz scam omen msoggoouzu we covumuweweza11.op m4mwuo<11.¢ mczmwu 68 + Cyt P450 Cyt P450 pgdu 6w / LUdD Time min Fig. 4. 69 tested also increased binding; however, hemin was the more effec- tive pro-oxidant. The Effect of the Electron Acceptors ADP-Fe+3 and EDTA-Fe+3 on 0014 Acti- vation by Rat Liver Microsomes Activation of CC14 by the pro-oxidant reactions requires sources of lipid hydroperoxides and pro-oxidants. In microsomes lipid hydroperoxides can be generated by the NADPH-dependent reduc- tion of ADP- or pyrophosphate-chelated iron (Pryor, 1976; Svingen et al., 1978). The oxidized microsomal hemoproteins, cytochromes P450(Fe+3) and b5 (Fe+3), have pro-oxidant activity and conditions which increase steady-state levels of this form therefore favor CC14 activation by pro-oxidant mechanisms. Other electron accep- tors, K3Fe(CN)6 and cytochrome c, are believed to inhibit CC14 activation by channeling electrons away from cytochrome P450 (Glende and Recknagel, 1969). If the same were true for ADP-Fe+3 1 vivo, the pro-oxidant mechanism would be favored. Since cyto- chrome P450 is concomitantly destroyed in these reactions, the net effect of this iron form would be inhibitory. It was therefore desirable to test the effect of the chelated iron on the rate of microsomal CC14 activation under anaerobic conditions. Figure 5 shows that the reaction was fairly linear in control rat liver microsomes for up to approximately 20 minutes. Fifteen-minute 3 3 assays were used to determine the effects of ADP-Fe+ and EDTA-Fe+ 3 on CC14 activation (Table 13). ADP-Fe+ did not decrease CC14 7O .uomm we :AE om cu m soc; mvocumz cw umawcommv mm mcowuwucoo o_pocwmco Lona: vmuoavcou mam: mcowuommm .mmouuavmg Omen mEoL;60pxo1:aow4 gem An covuw>mpo< epuu 11.m mczmwm up 71 EE 2:; cu cc AK ow AM om oH — . n b p P p 5.5: $.22- «.1 Imomo pom An comum>wuo< e—uu up :0 :OLH umumpw501w4 cw «Poo xn cowumuwxocma uwgwg yo :oAAm—zamum11.m venom; 90 .o .3”— EE 2:: 8 AN 8 2 2 m N o . p p . p . . zaeaz- ET no 58 a o .. 1592+ . 58 f In. 92.. 50+ 1 A68 .. 1522+ .. o - SA fiw Juan 1 + #060 - fin NBoo 1 and 98 «So A u e .. v.2 u I It Dwuu< epuuepul.m mesmwu 97 In 32 + 55 2:: T ‘TUTIIIIII1TTII m .m: omm UJdD omm 98 TABLE 16.--Effect of Inhibitors of Drug Metabolism on 14cc14 Activa- tion by a Reconstituted Mixed-Function Oxidase System. Experiment 1. Reactions contained 50 ug/ml di-l,2-GPC, 0.150 units/ml NADPH-cytochrome P450 redufitase, 0.4 nmole/ml cyto- chrome P450 (4 nmole cyt P450/m9), 1 ul CCl4/ml (0.25 m Ci/ml CCl4), and other components described in Methods. Experiment 2. Reactions contained di-l,2-GPC, NADPH- cytoc rome P4 0 reductase, cytochrome P450 (9.88 nmole cyt P450/mg), 1 pl 4CCl4/m1 (0.25 m Ci/ml CCl4) and other components as described in Methods. Experiments 1 and 2 were run for 60 and 30 minutes, respectively, under anaerobic conditions according to Methods. 190 content expressed as ratio of mg IgG:nmole cyt P450. Description cpm i 5.0. Expt'l.-Cont. Experiment 1 Complete - NADPH 876 i 112 -- Complete + NADPH 1,269 i 131 393* Comp1ete + NADPH + C0 937 t 71 61 Complete + NADPH+ Preimmune IgG (2:1) 1,012 i 61 136 Complete + NADPH + Immune 196 (2:1) 846 i 140 < 0 Experiment 2 Complete - NADPH 2,509 i 55 -- Comp1ete + NADPH 2,756 i 134 247* Complete + NADPH + C0 2,641 i 154 132 Complete + NADPH+ Preimmune IgG (10:1) 2,663 i 61 154 Complete + NADPH + Immune IgG (10:1) 2,491 i 164 < 0 *Significantly different from control, p < 0.05. 99 inhibited 93 and 56% of benzphetamine demethylation by the recon- stituted systems used in Experiments 1 and 2, respectively. Both 14 preimmune and immune IgG inhibited CCl4 binding, however, the latter was consistently the more effective inhibitor. That the preimmune IgG was also inhibitory is not unexpected since it can 14 compete with lipid for 0014 binding. Inhibition by CO was vari- 14 able but in both experiments decreased CCl4 activation to levels not significantly different from the non-enzymatic labeling. Activation of CCl4 by Dithionite- Reduced Cytochrome P450 and Other7Hemoproteins Under normal conditions cytochrome P450 is reduced by the NADPH-cytochrome P450 reductase-mediated transfer of reducing equivalents from NADPH. Guengerich et a1. (1975) have demonstrated that dithionite will serve as a source of reducing equivalents in the metabolism of some drugs, though less efficiently than when cytochrome P450 was enzymatically reduced. It is also possible that dithionite may serve as a source of reducing equivalents for 0014 activation, thereby further demonstrating that cytochrome P450 is responsible for this activity. Although some investigators have reported little or no CC14 activation by dithionite-reduced micro- somes, the activation of alkyl halides by other chemically reduced hemoproteins encouraged further pursuit of these experiments (Wade and Castro, 1973, 1974). Table 17 shows an experiment where excess dithionite was added to an anaerobic suspension of washed rat liver microsomes and 100 TABLE l7.--Activation of 14CCl4 by NADPH-Cytochrome P450 Reductase- and Dithionite-Reduced Cytochrome P450 in Microsomes. Reactions contained 2 2 mg/ml washed microsomal protein from unpretreated rats, 1 pl 1 C01 4/ml (0. 25mCi/ml C014), and either + 0. 25 mM NADPH or + 50 uM dithion1te in 0.1 M Tris-H01, pH 7.4 at 35° C. Reactions were conducted under anaerobic conditions as described in Methods for 20 min at 37°C. 1+ Description cpm/mg Lipid S.D. Expt'l.-Cont. Control Microsomes Control - NADPH 40 i 8 -- Control + NADPH 2,343 i 82 2,303* Control + 50 uM dithionite 295 i 12 255* *Significantly different from control, p < 0.01. the extent of activation determined. Some labeling was achieved although it was only 10% of that for NADPH-reduced microsomes over the same 20-minute time period. Table 18 contains the results of experiments in which 0014 activation by several hemoproteins and hemin was investigated. Dithionite gave some background labeling; however, each of the hemoproteins tested resulted in labeling above this background. The labeling obtained in the presence of methemo- globin and hemin suggests that a specific substrate binding site is not required but interaction with the heme iron is required for 0014 activation. 101 .mo.o v a .Aauccowgucu + masomoaw_v Foepcou sate “cacmcccu »_~=muwcwcmwme w-.~ o_F.. a mmo.¢ cwea; _E\a_oe= o + acccow;u_u :1 om + masomoawb ~_N Pm“ H Nem.~ cwao_moea;pas Fs\mpoa= m + mpwcowgp_u :1 om + meOmoa_4 we awe a cam._ meea “so Ps\mpos= o + mpmcowgpwe :1 om + mmEOmoawb smma.. mam a mmm.m omea “xv _s\apos= o + muccowguwo :1 om + masomoacs somo.. New 5 cmw.N omen “so _e\a_05= m + upweowgumu :1 om + masomoa_4 -- mm_ a omm._ mu_=owguwu :1 om + mmsomoawb .. mu A mwp.p mwsomoawb .ucou-._.paxm .o.m n uwawb mE\Eau cowaawgommo .mmsomoawA cm swam: can mcwmuogaoem: cmuzummlmawcowgumo x3 vpuu .Uomm um cps om L0$ muocumz cm umnwLUmmu mm mcowuwccou ownogmmcm Luvs: umuuaucou mew: meowuummm .uoum an 13:. 2 To 5 3235.6 2: om A .559. .8 233395; .8528 act 2.3.5; 29:: ago m .238 :55 =_mm.ov Ps\a_uu¢_ _: _.auapoguxxomu saweom x_o.o ._e\¢oa-uwaw. 8.05: o.. umcwm“=ou mcopauamm vp 4.“ :8 .puz co =o_pa>puu<--.m_ m4mFe+3x + R- 3 Fe+ X' + solvent-———>Fe+3(solvent) + X' 2R°-—-*R 110 By monitoring either the rates of hemoprotein oxidation or of dimer formation, they were able to demonstrate the dehalogenation of several compounds. Dehalogenation was catalyzed by all hemoproteins tested in which the axial positions of the heme iron were accessible to the substrates. That similar reactions are involved in the cytochrome P450 catalyzed metabolism of CC14 was suggested by Wolf et a1. (1977). They detected CO formation from various halomethanes upon anaerobic incubation with NADPH- or dithionite-reduced micro- somes. The reactions proposed to account for C0 formation were as follows: 2 (1) cx4 + Fe+ ———+ [Fe+3-CX3] + x" (2) [Fe+3-CX3] + e-—+ [Fe+2-CX3] ‘__.__"" [1:846:09] (3) [Fe+3:cx§] + e' -——> [Fe+2:cx§] ——-> [Fe+2:CX2] + x' 2 <4) [Fe*2:cx21 + H20——> Fe+ + to + 2 HX The dissociation of ~CX3 from cytochrome P450, reaction 1, would account for the increases in lipid peroxidation and for part of the binding to microsomal lipid and protein observed during CCl4 intoxi- cation. The formation of the carbene intermediate has been sup- ported by Mansuy et al. (1977). They were able to demonstrate the formation of a stable carbene complex with the metalloporphyrin 5,10,15,20-tetraphenylporphinato iron (II). 111 The formation of a -CC13 intermediate is also of interest in that its reaction with oxygen would provide a means by which both the toxic alkylating agent phosgene, C0C12, and its hydrolysis prod- uct C02 may be formed (Rebbert and Ausloss, 1976; Slater, 1972). Although the detection of phosgene has not been reported, the expira- tion of 14 14 CO2 by animals administered CC14 has been demonstrated (Seawright and McLean, 1967). Dichlorocarbenes are also capable of binding to olefins forming cyclopropane derivatives (Kleveland et al., 1977). This in part may account for some of the observed bind- ing to microsomal unsaturated lipids. This is not likely, however, to account for the observed toxicity in that not all halomethanes capable of carbene formation, for example, CFC13, are toxic (Cox et al., 1972; Wolf et al., 1977). From this series of reactions, three possible reactive inter- mediates result from the cytochrome P450-mediated reduction of CCl4. -CC13, C0C12, and :CClz. The relative proportions of each produced would be related to €014 toxicity. For example, toxicity can result from the binding of -CCl3 or C0Cl2 to cellular components or from the ability of -CC13 to initiate lipid peroxidation. Wolf et a1. (1977) demonstrated that CO formation was at a rate approximately one-third that observed for NADPH oxidation. Whether the remaining intermediate products primarily consist of ~CCl3 or :CCl2 (and C0Cl2 under aerobic conditions) has not been determined. Another observation of interest was the fact that microsomes from BP- pretreated rats produced CO at a faster rate than did those from 112 PB-pretreated rats. Whether these differences in cytochrome P450 hemoproteins can also account for differences in hepatoxicity will be discussed in Chapter IV. Although cytochrome P450 has a major role in 0014 activa- tion, other microsomal components may also participate. The lack of inhibition of CCl4-stimulated lipid peroxidation by mixed- function oxidase inhibitors (Masuda and Murano, 1977; Slater and Sawyer, 1971b) suggest this possibility. Cytochrome b5, for example, may be capable of CCl4 activation. The increase in NADH- dependent CC14 activation in microsomes in the presence of C0 (Table 19) supports this possibility. Activation by the reduced cytochrome b5 would, however, be expected to be of little conse- quence in mediating CC14 toxicity when compared to the cytochrome P450-dependent activation rates. In addition to activation by cytochrome b5 another, C0-insensitive, component was found to be involved in CC14 activation. This component was removed by wash- ing and may be or functions similarly to ADP-Fe+3 (Table 13). The identity of this component and its mechanism of action are not known. CHAPTER IV THE ACTIVATION OF CARBON TETRACHLORIDE BY CATALYTICALLY DIFFERENT CYTOCHROME P450 HEMOPROTEINS Abstract Liver microsomes from untreated rats or those pretreated with PB and 3-MC were utilized to determine if differences in CC14 activation and toxicity were related to the substrate specificites of their cytochrome P450 hemOproteins. Activation was determined by measuring both the CCl4-dependent lipid peroxidation and the covalent binding of 14 0014 to microsomal lipid. The turnover num- bers for CC14 activation by microsomes from untreated and pretreated rats were different. The relative magnitudes of these numbers for P8- and 3-MC-microsomes depended on the method used to detect CC14 activation. These differences are believed due to the relative amounts of -CC13 and :CCl2 produced by each microsomal cytochrome P450 hemoprotein population. For example, the more ~CC13 produced as an intermediate, the greater the turnover number from the assays which detect lipid peroxidation. Binding of ‘4 CC14 to microsomal lipid is a better indicator of the rate of CC14 conversion to both ~CC13 and :CC12. The former initiates lipid peroxidation as well as binds to lipid whereas the latter is only capable of binding. 113 114 The turnover numbers for covalent binding of 14 CC14 to microsomal lipid were more dependent on the NADPH-cytochrome P450 reductase to cytochrome P450 ratios than on qualitative differences in the cytochrome P450 hemoproteins. This was confirmed by the fact that reconstituted mixed-function oxidase systems containing catalyti— cally different cytochrome P450 hemoproteins and the same amount of NADPH-cytochrome P450 reductase gave similar rates of ‘4 CCl4 binding. It is concluded that several factors other than the dif- ferences in substrate specificity of the cytochrome P450 hemopro- teins account for CC14 activation and toxicity. Included among these are the following: the microsomal content of cytochrome P450; the rate of cytochrome P450 reduction; the relative amount of -C013 formed as an intermediate during CC14 metabolism in_yjyg: and cellular protective mechanisms. It is believed that either all or a combination of these factors are crucial in determining the susceptibility of various tissues and animal species to CC14 toxicity. Introduction The hypothesis which assigns cytochrome P450 as the site of CC14 activation within the liver microsomal mixed-function oxi- dase system (Recknagel and Glende, 1973; Recknagel et al., 1977) has been confirmed by the experimental results of Chapter III. A relationship between CC14 activation and relative amounts of micro- somal cytochrome P450 has been observed by others (Recknagel and 115 Glende, 1973; Carlson, 1975),and the data presented in Chapter III, Figures 6 and 7, agrees with this observation. Mixed-function oxidase systems that activate CC14 have been demonstrated in a variety of tissues within the rat. However, the extent of activa- tion as measured by ‘4 CCl4 binding to microsomal lipids does not correlate with cytochrome P450 levels in these tissues (Villarruel et al., 1977). This was true even though CC14 levels in these tissues were of similar magnitude. Diaz Gomez et al. (1975) examined the livers of several species for their susceptibility to cc14 toxicity. They were not able to correlate either ‘4 CC14 binding, CCl4-dependent increases in diene conjugation, or 0014- hepatotoxicity with cytochrome P450 levels. Species differences in the spectral binding constants for CC14 with cytochrome P450 were also unrelated to CC14 activation and hepatotoxicity. There are several plausible interpretations of these findings; one is that the multiple forms of cytochrome P450 differ in their ability to activate CCl4. Another possibility would be due to differences in defense mechanisms which protect against CC14 hepatoxicity, for example, the cellular antioxidant content. Several chemical compounds will induce cytochrome P450 hemoproteins with different substrate specificies. Pretreatment of animals with these compounds has been demonstrated to alter CC14 hepatotoxicity. For example, PB, PCN, and 3-MC induce cyto- chrome P450 hemoproteins in rats which are catalytically different from each other as well as from those in untreated rats. 116 PB enhances hepatoxicity whereas PCN and 3-MC, an inducer of cyto- chrome P448’ afford some protection (Castro et al., 1973; Recknagel and Glende, 1973; Suarez et al., 1975; Tuchweber et al., 1974). It is of interest that BP, which also induces cytochrome P448, enhances rather than protects against CCl4 hepatoxicity (Pitchumoni et al., 1972). The interpretation of these observations is complicated by the fact that they also alter several microsomal components, some of which may affect CC14 metabolism. For example, both PB and PCN cause a marked proliferation in the smooth endoplasmic reticulum and induce both of the mixed-function oxidase components and a broad spectrum of mixed-function oxidase activities (Parke, 1975; Tuchweber et al., 1974). Treatment with 3-MC or BP does not result in this proliferation and the spectrum of enzymatic activities induced is more limited. Both also induce the cytochrome P448 hemoprotein but only the latter enhances the cytochrome P450 reduc- tase component (Parke, 1975). Metabolites of BP and 3-MC are antioxidants and if present in significant amounts would reduce peroxidative damage and perhaps toxicity (Pederson, 1973; Suarez et al., 1975). The investigations presented in this chapter address two questions concerning CC14 activation: (1) Are the differences in CCl4 toxicity observed in various tissues or animal species due to the substrate specificities of their cytochrome P450 hemoproteins? and (2) Are there other factors which influence CCl4 toxicity in various tissues? The ability of microsomes from rats pretreated 117 with various compounds and of reconstituted systems containing cytochrome P450 purified from these microsomes to activate CC14 has been used in these investigations to answer these questions. 14 The CCl4-dependent lipid peroxidation and the binding of CC14 to liposomes have been used as an estimate of CCl4 activation. Materials and Methods Material Sources 3,4-Benzpyrene was obtained from the Aldrich Chemical Co., Milwaukee, Wisconsin. The sources of other reagents have been listed in Chapters II and III. Drug Pretreatment of Rats 3-MC pretreatments have been described in Chapter II. PB (80 mg/kg in saline) was given to rats by daily injections, i.p., for 5 days prior to sacrifice. The microsomes isolated from rats injected with the saline or PEG vehicle or with P8 or 3-MC are referred to as control (saline)-, control (PEG-), PB- and 3-MC- microsomes, respectively. Isolation of Rat Liver Microsomes Rats were starved for 16 hours prior to sacrifice and their liver microsomes isolated and washed as described in Chapter II but with the exceptions listed below. Livers were perfused with and homogenized in argon-saturated solution containing 1.15% KCl and 10 mM EDTA, pH 7.5. Microsomal pellets were washed once with 118 argon-saturated 1.15% KCl and stored without BHT as previously described, Chapter II. Microsomes were used within two days of preparation. Enzyme Assays and Analytical Techniques NADPH-cytochrome P450 reductase (Chapter II), benzphetamine demethylase (Chapter III), benzpyrene hydroxylase (Welton et al., 1975), and cytochromes P450 and b5 (Omura and Sato, 1964) were assayed by previously described techniques. Benzphetamine and BP metabolism were determined at 37°C. All other assays were carried out at room temperature. MDA was determined as described in 14 Chapter II. CC14 activation was determined by covalent binding to microsomal lipids as described in Chapter II with the modifica- tion that 2 mg/ml microsomal protein was used and the assays run 14 anaerobically for 5 minutes. Activation of CC14 in liposomes was as described in Chapter III. Results Activation of CC14 by Microsomes from Untreated and PB- and 3-MC-Pretreated Rats The failure of CCl4 activation and toxicity to correlate with the cytochrome P450 levels found in various tissues and animal species may be related to qualitative differences in these hemo- proteins. To test this hypothesis, rats were pretreated with compounds which alter both liver microsomal cytochrome P450 levels and substrate specificity and the activation of C014 by these 119 microsomes determined. Two methods were used to estimate CC14 activation; one involved the stimulation of NADPH-dependent lipid peroxidation by C014 and the other measured binding of 14 C014 to microsomal lipids. The effect of these pretreatments on microsomal enzymes and CCl4-stimulated lipid peroxidation are shown in Table 22 and Figure 9. Endogenous rates of NADPH-dependent lipid peroxi- dation have been greatly reduced by the microsomal isolation and washing procedures described in the Methods section. This allows for better quantitation of the lipid peroxidation stemming from CC14 activation. PB pretreatment increased whereas 3-MC pretreat- ment of rats decreased the CCl4-dependent lipid peroxidation when TABLE 22.--The Effect of PB- and 3-MC Pretreatments of Rats on Microsomal Enzyme Content. The pretreatment of rats, microsomal isolation and washing procedures, and enzymatic analysis were as described in Methods. Pretreatment P4 0 Reductase (nmole/mg) (nmole/mg) 1units/mg)* Microsomes Control (saline) 0.756 0.385 0.295 PB 2.973 0.477 0.439 Control (PEG) 0.667 0.415 0.272 3-MC 1.157 0.476 0.264 *1 unit = umole cytochrome c reduced/minute. 120 .muosumz cm cmnpsummc mm emcee -Lmumu w4 cw vpuu x2 cowumcmxoema uwawg mo cowumpaepumii.m assume 121 r =aowpo< ¢F \epuu _: _ .Pe .Ame\wwosc mm.uv c ¢.o .mmmuuzvme cowuummm ucou uue_--.op mesmee 127 SE 2:: me am mg - P p - b b Iaow4 pom mg» »n cowpm>wuo< mvpeoFcumepm» :oaemo mo uwuwemgumui.pp mgamwm N8. :8 .2863? N8. + -8 A255; 5 137 :N .88- Natema NE .N\ :8 Are v2: 5 SN 78 N+e via .3 N8. .: .3“. No + E + :08 $8 - .22 Tel 3.838 2:. 88:85-2 22 4522 138 damage to the microsomal membranes and perhaps other cellular components. Other minor routes of CC14 activation that do not involve the microsomal mixed-function oxidase components also occur in the cell. The reduced form of cytochrome b5 can activate CC14 by an electron capture reaction. In_yjyg_this contribution is expected to be of little consequence. Also, a CO-insensitive CC14 activa- tion, which is removable by washing, exists in rat liver microsomes. This activity is minor and has not been characterized. It is also 3 possible that ADP-Fe+ , or other weakly chelated iron forms, enhances microsomal CC14 activation jg_vivo as it does i vitro. The causes of this enhancement have not been determined. REFERENCES REFERENCES Antonini, E. and Brunori, M. (1971). Hemoglobin and Myoglobin in Their Reactions with Ligands. North-Holland Research Monographs, Frontiers of Biology 23, 41. Archakov, A. I., Devichensky, V. M. and Karjakin, A. V. (1975). Arch. Biochem. Biophys. log, 295. Archakov, A. I. and Karuzina, I. I. (1973). Biochem. Pharm. 22, 2095. Asscher, ii. and Vofsi, D. (1963). J. Chem. Soc. 1887, #350. Back, K. C. (1977). Ann. Rev. Pharmacol. Toxicol. 12, 83. Bartlett, G. R. (1959). J. Biol. Chem. 224, 466. Benedetti, A., Casini, A. F., Ferrali, M. and Comporti, M. (1977). Chem. Biol. Interactions 12, 151. Bini, A. M., Burdino, E., Cessi, C., Pagnoni, V. M. Ugazio, G. and Vannini, V. (1976). Panminerva Medica 12, 403. Bini, A., Vecchi, G., Vivoli, G., Vannini, V. and Cessi, C. (1975). Pharm. Res. Comm. 2, 143. Bligh, E. G. and Dyer, W. J. (1959). Can. J. Biochem. Physiol. 21, 911. Brodie, B. B., Gillette, J. R. and LaDu, B. N. (1958). Ann. Rev. Biochem. 22, 427. Buege, J. A. and Aust, S. D. (1978). Methods in Enzymol. 51, 302. Bush, E. T. (1963). Analytical Chem. 25, 1024. Cafruny, E. J. (1971). Fundamentals of Drug Metabolism and Drug Disposition. B. N. LaDu, H. G. Mandel and E. L. Way, ed., pp. 119, Williams and Wilkins Co., Baltimore, Md. Capdevila, J. and Agosin, M. (1977). Microsomes and Drug Oxidations. V. Ullrich, A. Hildebrandt, R. W. Estabrook, 1. Roots and A. H. Conney, ed., pp. 144, Pergamon Press, N.Y. 139 140 Carlson, G. P. (1975). Toxicology 5, 69. Castro, J. A., Cignoli, E. V., de Castro, C. R. and de Fenos, 0. M. (1972). Biochem. Pharm. 21, 49. Castro, J. A., de Castro, C. R. D'Acosta, N., Diaz Goemez, M. I. and de Ferreyra, E. C. (1973). Biochem. Biophys. Res. Comm. 5_o, 273. Castro, C. E. and Kray, W. C. (1966). J. Am. Chem. Soc. 55, 4447. Cohen, 8. S. and Estabrook, R. W. (1971). Arch. Biochem. Bi0phys. 143, 46. Conney, A. H. (1967). Pharmacological Reviews 15, 317. Coon, M. J., Vermilion, J. L., Vatsis, K. P., French, J. 5., Dean, W. L. and Haugen, D. A. (1977). Drug Metabolism Concepts. ACS Symposium Series, No. 44. D. M. Jerina, ed., pp. 46. Cox, P. J., King, L. J. and Parke, D. V. (1972). Biochem. J. 159, 87 p. Cuatrecasas, P. (1970). J. Biol. Chem. 215, 3059. Demopoulos, H. B. (1973). Fed. Proc. 52, 1903. Dianzani, M. V., Gabriel, L., Gravela, E., and Paradisi, L. (1976). Panminerva Medica 15, 310. Diaz Gomez, M. I., de Castro, C. R., D'Acosta, M., de Fenos, 0. M., de Ferreyra, E. C. and Castro, J. A. (1975). Toxicol. Applied Pharmacol. 55, 102. Dignam, J. 0., and Strobel, H. W. (1975). Biochem. Biophys. Res. Comm. _6_3_, 845. Dillard, C. J., Dumelin, E. E. and ‘Pappel, A. L. (1976). Lipids 12, 109. Dixon, M. (1971a). Biochim. Biophys. Acta 225, 241. Dixon, M. (1971b). Biochim. Biophys. Acta 225, 269. Fairbanks, G., Steck, T. L. and Wallach, D. F. H. (1971). Biochemistry 19, 2606. Folch, J., Lees, M. and Stanley, G. H. S. (1956). J. Biol. Chem. gag, 497. 141 Fowler, J. S. L. (1969). Br. J. Pharmac. 51, 733. Fujita, S. and Peisac, J. (1977). Biochem. Biophys. Res. Comm. 15, 328. Gamage, P. T. and Matsushita, S. (1973). Agr. Biol. Chem. 51, l. Gardner, H. W. (1975). J. Agr. Food Chem. 25, 129. Gardner, H. W., Kleiman, R. and Weisleder, D. (1974). Lipids 5, 696. Gillette, J. R. (1971a). Handbuch der Exp. Pharmakol., Concepts in Biochem. Pharmacol. 25 (part 2), 347. Gillette, J. R. (1971b). Annals N.Y. Acad. Sci. 115, 43. Gillette, J. R., Mitchel, J. R. and Brodie, B. B. (1974). Ann. Rev. Pharmacol. 11, Glende, E. A. (1972). Biochem. Pharm. 21, 1697. Glende, E. A., Hruszkewycz, A. M. and Recknagel, R. 0. (1976). Biochem. Pharm. 25, 2163. Glende, E. A. and Recknagel, R. O. (1969). Exp. Mol. Path. 11, 172. Go1db1att, P. J. (1972). Sub-Cell. Biochem. _1_, 147. Gordis, E. (1969). J. Clinical Inves. 15, 203. Gregory, N. L. (1966). Nature 212, 1460. Guengerich, R. P. (1977). J. Biol. Chem. 252, 3970. Guzelian, P. S. and Bissell, D. M. (1976). J. Biol. Chem. 251, 4421. Hafeman, D. G., and Hoekstra, W. G. (1977). J. Nutrition 151, 656. Halbreich, A. and Mager, J. (1969). Biochem. Biophys. Acta 151, 584 . Hall, P. F. (1976). Adv. Exptl. Med. Biol. 11, 303. Hamburg, M. (1974). Lipids 15, 87. Heicklen, J. (1969). Adv. Photochem. 1, 57. Hildebrandt, A. G. and Estabrook, R. W. (1971). Arch. Biochem. Biophys . 1433.: 56 . 142 Holloway, P. W. (1973). Anal. Biochem. 55, 304. Hrycay, E. G. and O'Brien, P. J. (1973). Arch. Biochem. Biophys. E1, 7. Horecker,B. L. and Kornberg,A. (1948). J. Biol. Chem. 1E, 385. Imai, Y. (1976). J. Biochem. 55, 267. Imai, Y. and Sato, R. (1974). J. Biochem. 15, 689. Iyanagi, T., Makino, N. and Mason, H. S. .(1974). Biochemistry 15, 1701. Jerina, D. M. and Daly, J. W. (1974). Science 155, 573. Kamataki, T., Belcher, D. H. and Neal, R. A. (1976). Mol. Pharmacol. 12, 921. Katagiri, M., Ganguli, B. N. and Gunsalus, I. C. (1968). J. Biol. Chem. _24_3_, 3543. Kleveland, K., Skattebol, L. and Sydnes, L. K. (1977). Acta Chem. Scand. 51, 463. Klugh, H. E. (1974). Statistics: The Essentials for Research, pp. 203, John Wiley & Sons, Inc., N.Y. Koch, R. R., Glende, E. A. and Recknagel, R. 0. (1974). Biochem. Pharm. _2_3_, 2907. Koster, U., Albrecht, D. and Kappus, H. (1977). Tox. Applied Pharm. 11, 639. Kuntzman, R. (1969). Ann. Rev. Pharmacol. 5, 21. Lever, M. (1977). Anal. Biochem. 55, 274. Lindstrom, T. D. and Anders, M. W. (1978). Biochem. Pharm., in press. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). J. Biol. Chem. 155, 265. Lu, A. Y. H., Junk, K, W. and Coon, M. J. (1969). J. Biol. Chem. 293. 3714. Lu, A. Y. H., Kuntzman, R., West, 5., Jacobson, M. and Conney, A. H. (1972). J. Biol. Chem. 211, 1727. Lu, A. Y. H. and Levin, W. (1972). Biochem. Biophys. Res. Comm. 59, 1334. 143 Lu, A. Y. H. and Levin, W. (1974). Biochem. Biophys. Acta 511, 206. Lu, A. Y. H., Levin, W., West, 5. B., Vore, M., Ryan, 0., Kuntzman, R. and Conney,A. H. (1974a). Adv. Exptl. Med. Biol. _55, 447. Lu, A. Y. H., Levin, W. and Kuntzman, R. (1974b). Biochem. Biophys. Res. Comm. 55, 266. Lu, A. Y. H., Ryan, 0., Kawalek, J., Thomas,P.,West,S. 8., Huang, M. T. and Levin, W. (1976). Biochem. Soc. Trans. 4, 169. Mandel, H. G. (1971). Fundamentals of Drug Metabolism and Drug Disposition. B. N. LaDu, H. G. Mandel, and E. L. Way, ed., pp. 149, Williams and Wilkins Co., Baltimore, Md. Mannering, G. J. (1971). Fundamentals of Drug Metabolism and Drug Disposition. B. N. LaDu, H. G. Mandel, E. L. Way, ed., pp. 206, Williams and Wilkins Co., Baltimore, Md. Mannering, G. J. (1974). Adv. Exptl. Med. Biol. 55, 405. Mason, H. S. (1957). Advan. Enzymol. 15, 79. Mansuy, D., Lange, M., Chottard, J., and Guerin, P. (1977). J.C.S. Chem. Comm., p. 648. Masters, B. S. S., Kamin, H., Gibson, 0. H., and Williams, C. H. (1965). J. Biol. Chem. 215, 921. Masuda, Y. and Murano, T. (1977). Biochem. Pharm. 25, 2275. Matsushita, S., Kobayashi, M. and Nitta, Y. (1970). Agr. Biol. Chem. 54, 817. McCay, P. 8., Gibson, D. D., Fong, K. and Hornbrook, K. R. (1976). Biochim. Biophys. Acta 151, 459. Mead, J. F. (1976). Free Radicals in Biology. Vol. I. W. A. Pryor, ed., pp. 51, Academic Press, N.Y. Moore, R. W., Welton, A. F. and Aust, S. D. (1978). Mthds. Enzymol. 51, 324. Morrison, R. T. and Boyd, R. N. (1971). Organic Chemistry, 2nd ed., pp. 470, Allyn and Bacon, Inc., Boston, Mass. Munson, E. and Eger, E. (1971). Fundamentals of Drug Metabolism and Drug Disposition. B. N. LaDu, H. G. Mandel, and E. L. Way, ed., pp. 106, Williams and Wilkins Co., Baltimore, Md. 144 Nash, T. (1953). Biochem. J. 55, 416. Noguchi, T. and Nakano, M. (1974). Biochim. BiOphys. Acta 555, 446. O'Brien,1h J. (1969). Can. J. Biochem. 11, 485. O'Brien, P.J. and Little, C. (1969). Can. J. Biochem. 11, 493. O'Brien. P.J. and Rahimtula, A. (1975). J. Agr. Fd. Chem. 25, 154. Omura, T. and Sato, R. (1964). J. Biol. Chem. 255, 2370. Orrenius, S. and Ernster, L. (1974). Molecular Mechanisms of Oxygen Activation. O Hayaishi, ed., pp. 215, Academic Press, N.Y. Oshino, N., Imai, Y. and Sato, R. (1966). Biochim. Biophys. Acta 128, 13. Oshino, N., Imai, Y. and Sato, R. (1970). J. Biochem. 55, 155. Parke, D. V. (1975). Enzyme Induction, pp. 207, Plenum Press, London and New York. Pederson, T. C. (1973). Ph.D. thesis, Michigan State University, East Lansing, Michigan. Pederson, T. C. and Aust, S. D. (1973). Biochem. Biophys. Res. Comm. 52, 1071. Pederson, T. C. and Aust, S. D. (1975). Biochem. Biophys. Acta §§§, 232. Pederson, T. C., Buege, J. A. and Aust, S. D. (1973). J. Biol. Chem. 215, 7134. Peterson, J. A., Ebel, R. E., O'Keefe, D. H., Matubara, T. and Estabrook, R. W. (1976). J. Biol. Chem. 251, 4010. Pfeifer, P. M. and McCay, P. B. (1971). J. Biol. Chem. 215, 6401. Pitchumoni, C. S., Stenger, R. J., Rosenthal, W. S. and Johnson, E. A. (1972). J. Pharm. Expt'l. Therap. 1§l, 227. Plaa, G. L. (1971). Fundamentals of Drug Metabolism and Drug Disposition. B. N. LaDu, H. G. Mandel and E. L. Way, ed., pp. 131, Williams and Wilkins Co., Baltimore, Md. Plaa, G. L. and Witschi, H. (1976). Ann. Rev. Pharm. Tox. 15, 125. 145 Poyer, J. L. and McCay, P. B. (1971). J. Biol. Chem. 215, 263. Pryor, W. A. (1966). Free Radicals, pp. 10, McGraw-Hill, N.Y. Pryor, W. A. (1976). Free Radicals in Biology, Vol. I, pp. 1, Academic Press, N.Y. Rebbert, R. E. and Ausloos, P. J. (1976). J. Photochemistry 5, 265. Recknagel, R. O. (1967). Pharmacol. Rev. 15, 145. Recknagel, R. O. and Ghoshal, A. K. (1966). Lab. Invest. 15, 132. Recknagel, R. O. and Glende, E. A. (1973). CRC Critical Rev. Toxicology 2, 263. Recknagel, R. 0., Glende, E. A. and Hruszkewycz, A. M. (1977). Free Radicals in Biology. Vol. III. W. A. Pryor, ed., pp. 97, Academic Press, N.Y. Recknagel, R. O. and Turocy, Y. (1977). Exp. Mol. Path. 21, 93. Rees, K. R. (1976). Panminerva Medica 15, 289. Reynolds, E. S. and Moslen, M. T. (1974). Toxicol. and Applied Pharm. _25, 377. Reynolds, E. S. and Yee, A. G. (1967). Lab. Invest. 15, 591. Riely, C. A., Cohen, G., and Lieberman, M. (1974). Science 155, 208. Rutter, W. J. (1967). Methods in Developmental Biology. F. H. Wilt and N. K. Wessels, eds. pp. 675, Thomas Crowell Co., N.Y. Sasame, H. 0., Ames, M. M. and Nelson, 5. D. (1977). Biochem. Biophys. Res. Comm. 15, 919. Schenkman, J. B. and Jansson, I. (1974). Adv. Exptl. Med. Biol. 58, 387. Schenkman, J. B., Remmer, H. and Estabrook, R. W. (1967). Mol. Pharmacol. 3, 113. Seawright, A. A. and McLean, A. E. M. (1967). Biochem. J. 155, 1055. Shah, H.C. and Carlson, G. P. (1975). J. Pharm. Expt'l. Therap. m, 231. 146 Shaltiel, S. (1975). Federation of European Biochem. Soc. 15, 117. Shuster, L. (1964). Ann. Rev. Biochem. 55, 571. Sies, H. and Summer, K. (1975). Eur. J. Biochem. 51, 503. Sipes, I. G., Krishna, G. and Gillette, J. R. (1977). Life Sci. 2_0, 1541. Slater, T. F. (1972). Free Radical Mechanisms in Tissue Injury, pp. 48, 118, 198, Pion Ltd., London. Slater, T. F. (1974). Pathogenesis and Mechanisms of Liver Cell Necrosis. D. Keppler, ed., pp. 209, MTP. Slater, T. F. (1976). Panminerva Medica 15, 381. Slater, F. and Sawyer, B. C. (1971a). Biochem. J. 125, 805. Slater, F. and Sawyer, B. C. (1971b). Biochem. J. 125, 815. T. T. Slater, T. F. and Sawyer, B. C. (1977). Chem.-Biol. Interactions 16, 359. Smuckler, E. A. (1976). Panminerva Medica 15, 292. Stotter, D. A., Thomas, R. D. and Wilson, M. T. (1977). Bioinor- ganic Chem. 1, 87. Strittmatter, P., Spatz, L., Corcoran, 0., Rogers, M. J., Setlow, B. and Redline, R. (1974). Proc. Nat. Acad. Sci. 11, 4565. Strobel, H. W., Lu, A. Y. H., Heidema, J. and Coon, M. J. (1970). .1. Biol. Chem. 255, 4851. Suarez, K. and Bhonsle, P. (1976). Tox. Applied Pharm. 51, 23. Suarez, K. A., Carlson, G. P. and Fuller, G. C. (1975). Tox. Applied Pharm. 51, 314. Suarez, K. A., Carlson, G. P., Fuller, G. C. and Fausto, N. (1972). Tox. Applied Pharm. 25, 171. Suriyachan, D. and Thithapandha, A. (1977). Toxicol. Applied Pharm. 11, 369. Svingen, B. A., O'Neal, F. O. and Aust, S. D. (1978). J. Photochem. Photobiol., in press. 147 Thiers, R. E., Reynolds, E. S. and Vallee, B. L. (1960). J. Biol. Chem. 255, 2130. Tuchweber, B., Werringloer, J. and Kourounakis, P. (1974). Biochem. Pharm. 25, 513. Uehleke, H., Hellmer, K. H. and Tabarelli, S. (1973). Xenobiotica 3, l. Ugazio, G., Torrielli, M. V., Burdino, E., Sawyer, B. C. and Slater, T. F. (1976). Biochem. Soc. Trans. 1, 353. van der Hoeven, T. A. and Coon, M. J. (1974). J. Biol. Chem. 215, 6302. Villarruel, M. C. and Castro, J. A. (1975). Res. Comm. Chem. Path. Pharm. 15, 105. Villarruel, M. del C., de Toranzo, E. G. D. and Castro, J. A. (1976). Res. Comm. Chem. Path. Pharm. 11, 193. Villarruel, M. del C., de Toranzo, E. G. D. and Castro, J. A. (1977). Toxicol. and Applied Pharmacol. 11, 337. Villarruel, M. C., Diaz Gomez, M. I. and Castro, J. A. (1975). Tox. Applied Pharm. 55, 106. Vogel, A. I. (1958). Elementary Practical Organic Chem., pp. 785, John Wiley & Sons, N.Y. von Oettingen, W. F. (1955). The Halogenated Hydrocarbons Toxicity and Potential Dangers. U.S. Dept. of Health, Ed. and Welfare, Public Health Serv. Pub. #414, pp. 77-101, U.S. Govt. Printing Off., Wash., D.C. Wade, R. S. and Castro, C. E. (1973). J. Am. Chem. Soc. 55, 231. Wade, R. S. and Castro, C. E. (1974). J. Am. Chem. Soc. 55, 231. Wade, R. S., Havlin, R. and Castro, C. E. (1969). J. Am. Chem. Soc. 21, 7530. Walling, C. (1957). Free Radicals in Solution, pp. 460, John Wiley & Sons, N.Y. Welton, A. F. (1974). Ph.D. thesis, Michigan State University, East Lansing, Michigan. Welton, A. F. and Aust, S. D. (1974). Biochem. Biophys. Acta 515, 197. 148 Welton, A. F., O'Neal, F. 0., Chaney, L. C. and Aust, S. D. (1975). J. Biol. Chem. 255, 5631. Welton, A. F., Pederson, T. C., Buege, J. A. and Aust, S. D. (1973). Biochem. Biophys. Res. Comm. 51, 161. Williams, C. H., Jr., and Kamin, H. (1962). J. Biol. Chem. 251, 587. Wills, E. D. (1965). Biochem. Biophys. Acta 55, 238. Winterhalter, K. H. (1971). Clinical Biochemistry-~Principles and Methods, V01. 2. H. C. Curtis and M. Roth, eds., pp. 1308, W. de Gruyter, Pub. Wolf, C. R., Mansuy, D., Nastainczyk, W., Deutschmann, G. and Ullrich, V. (1977). M01. Pharm. 15, 698. Wood, C. L., Gandolfi, A. J. and van Dyke, R. A. (1976). Drug Metab. Disp. 1, 305. Yasukochi, Y. and Masters, 8. S. S. (1976). J. Biol. Chem. 251, 5337. Yoshida, V., Aoyama, Y., Kumaoka, H. and Kubota, S. (1977). Biochem. Biophys. Res. Comm. 15, 1005. APPENDIX APPENDIX Publications Welton, A. F., O'Neal, F. 0., Chaney, L. C., and Aust, S. D. (1975). Journal of Biological Chemistry 255, 5631-5639. "Multi- plicity of Cytochrome P450 Hemoproteins in Rat Liver Micro- somes: Preparation and Specificity of An Antibody to the Hemoprotein Induced by Phenobarbital." Svingen, B. A., O'Neal, F. 0., and Aust, S. D. (1978). Journal of Photochemistry and Photobiology, in press. "The Role of Superoxide and Singlet Oxygen in Lipid Peroxidation." Abstracts Moore, R. W., O'Neal, F. 0., Chaney, L. C., and Aust, S. D. (1975). Federation Proceedings 51, 623, Abstr. 2289. "Specificity of Antibody to the Cytochrome P-450 Hem0pr0tein Induced by Phenobarbital." Peters, J. W., Cook, R. M., O'Neal, F. 0., and Aust, S. D. (1975). Federation Proceedings 34, 784, Abstr. 3198. "Microsomal Mixed-Function Oxidase AEtivation of Aflatoxin B1." Buege, J. A. Svingen, B. A., O'Neal, F. 0., and Aust, S. D. (1977). Federation Proceedings 55, 843, Abstr. 3013. "The Mechan- ism of Microsomal NADPH-Dependent Lipid Peroxidation." Svingen, B. A., Buege, J. A., O'Neal, F. 0., and Aust, S. D. (1977). Federation Proceedings 36, 998, Abstr. 3835. "Relationship Between Liver Microsomal Cyt. P-450-Dependent Drug Metabolism and Lipid Peroxidation." O'Neal, F. 0., and Aust, S. D. (1978). Federation Proceedings 51, 561, Abstr. 1829. "Carbon Tetrachloride Activation: The Role of Microsomal Mixed-Function Oxidase Components." 149