a...» -1 ’ l 4 This is to certify that the dissertation entitled Role of Cytochrome P450 in Hepatic Microsomal Mixed Function Oxidase-Dependent Superoxide Production and Lipid Peroxidation presented by Lee Alan Morehouse has been accepted towards fulfillment of the requirements for Ph . D. degree in Biochemi stry Major professor :1 Date August 19, 1986 MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771 Ill Ill ll llllllillll llilllllll'lllifl'lllll :. 3 1293 00667 6203 )V1£SIEJ RETURNING MATERIALS: Place in book drop to LJBRARJES remove this checkout from -_ your record. FINES will be charged if book is returned after the date stamped below. .- .Ihrr— ROLE OF CYTOCHROME P450 IN HEPATIC MICROSOMAL MIXED FUNCTION OXIDASE-DEPENDENT SUPEROXIDE PRODUCTION AND LIPID PEROXIDATION By Lee Alan Morehouse ,A DISSERTATION Submitted to Michigan State University in partial fulfillment of the require-cute for the degree of DOCTOR OF PHILOSOPHY Depart-eat of Biochelintry 1986 C) '777 A V ABSTRACT ROLE OF CYTOCHROME P450 IN HEPATIC MICROSOMAL MIXED FUNCTION OXIDASE-DEPENDENT SUPEROXIDE PRODUCTION AND LIPID PEROXIDATION BY Lee Alan Morehouse NADPH-dependent lipid peroxidation (LP) requires the reduction of ferric ion prior to initiation. This work was undertaken to investigate the role of cytochrome P450 (P450) in this iron reduction step. Two mechanisms for iron reduction by the microsomal electron transport system and its components were examined: superoxide-(Oz:)-dependent and direct. NADPH—Cytochrome P450 reductase (reductase) generated very low rates of 027 production, indicating that oxygen is a poor substrate and 027 is not the likely reductant. Some ferric chelates (i.e., EDTA-Feat) were reduced by the reductase under anaerobic conditions and under aerobic conditions stimulated NADPH oxidation and 02? production. Other ferric chelates (i.e., ADP—Feat) were not reduced by the reductase, explaining previous results where ADP-Fe3+ did not promote LP in a model system dependent upon the reductase. Rat liver microsomes produced more 027 than the reductase, but also promoted anaerobic reduction of ADP- Fe3*. Lee Alan Morehouse NADPH-dependent peroxidation of microsomes required only ADP-Fe3+ and was not inhibited to any appreciable extent by SOD. Addition of P450 to reductase resulted in a competent electron transport chain that also reduced ADP- Fe3+ and stimulated 02: production. The degree of stimulation of 02: production depended on the particular P450 isozyme; maximum stimulation occurred at P450: reductase of 4 or 5:1. The role of P450 in LP was demonstrated by incorporating P450 isozymes with reductase in phospholipid vesicles. ADP-Fe3+ was the only chelate necessary to promote LP, mimicking the intact microsomal system. LP was inhibited by approximately 30-40 percent by SOD, indicating the existence of both Oat-dependent and independent iron reduction pathways but underscoring the direct reduction pathway as the primary reduction mechanism. The role of hydrogen peroxide (B202) in the initiation of microsomal LP was also examined. Ferrous is thought to reduce 8202 generating the hydroxyl radical, the proposed initiating species. However, contrary to the theory, 3202 inhibited LP whenever endogenous or exogenously-added catalase was inhibited by azide. To My Parents, Bobbie and Andrew ii ACKNOWLEDGMENTS Foremost, I must extend my gratitude to Steve Aust, my research advisor for his moral and financial support and his patience throughout my stay at MSU. It is not possible to mention all that he has done but providing funds for attending meetings, and giving me opportunities to write, review, and edit grants and manuscripts were Just two of the ways in which he aided my development as a scientist. Steve, thanks for everything. I would also like to thank Drs. Ian Gray, Mel Schindler, Bill Smith, and Jack Watson for serving on my Guidance Committee and providing many helpful suggestions. The generosity of Dr. Alfred Hang in making his EPR spectrometer available for my use is greatly appreciated. I am also indebted to Dr. Shelagh Ferguson-Miller’s laboratory for helpful suggestions regarding phospholipid vesicle preparation and for providing the asolectin. Thanks also to Rich Voorman, Libby Pulsford, and John Bumpus for supplying purified isozymes of cytochrome P450. In addition, the secretarial assistance of Ann Alchin in the preparation on this dissertation is gratefully acknowledged. I would also' like to thank the members of the laboratory both past and present with whom I had the pleasure of working. Their advice and especially their camaraderie made the lab an enjoyable place to be, even on iii nights and weekends. Sheer numbers prevent me from mentioning them all, but special thanks must go to the ”LP boys": John Bucher, Ming Tien, Morio Saito, Giorgio Minotti, and especially Craig Thomas with whom I shared a lab aisle, authorship, and innumerable good time both in and out of the lab. Lastly, I am eternally grateful for the support of my wife, Bob. By continuing to work at a job she grew to dislike, she kept the household afloat financially. It was her willingness to shoulder the yeoman’s share of domestic responsibilities especially in the last six months that made the completion of this dissertation possible. iv TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . . . LIST OF FIGURES . LIST OF ABBREVIATIONS . . . . . . . . . . . . . INTRODUCTION REVIEW OF LITERATURE . . . . . . . . . . . . . . . . Chemistry of Dioxygen and its Reduction Products. Dioxygen . . . . . . . . . . . . . . . Superoxide Hydrogen Peroxide Hydroxyl Radical . . Measurement of Oxygen Radical Production Superoxide . . . . . Hydrogen Peroxide . . . . . . . . . . Bydroxyl Radical . . . . . . . . . . Cellular Sites of Oxygen Radical Generation . . . Microsomal Electron Transport System Uncoupling of Microsomal Electron Transport Induction of Cytochrome P450 . . . . . . Lipid Peroxidation . . . . . Cellular Antioxidant Defenses Initiator Formation . . . . . . Haber- -Weiss Reaction . . . . Objections to the Haber- Weiss Reaction . . Alternate Initiators . . . . NADPR- -Dependent Lipid Peroxidation . Roles of Cytochrome P450 in Lipid Peroxidation. LIST OF REFERENCES . . . . . . . . . . . . . . . CHAPTER I: Ferric Chelates Stimulate NADPH Oxidation by NADPH-Cytochrome P450 Reductase: A Superoxide-Independent Iron Reduction Activity . . . . . . . . . . . . . . . Abstract . . . Introduction .g. . . . ... . . . . . . . . . Materials and Methods . . . . . . . . . Chemicals . . . . . . . . . . . . . . . . . Preparation of Microsomes . . . . . . . . . Purification of Enzymes . . . . . . . . . . Assays . . . . . . . . . . . . . . . . . . . EPR Measurements . . . Iron Reduction Assays . . . . . . . . . . Page viii xiii O) 54 55 57 60 60 60 61 62 63 Results . . . Stimulation of NADPH- -Cytochrome P450 Reductase- -Dependent NADPH Oxidation by Ferric Chelates . . . . . . . . EPR Spin Trapping . . Anaerobic Iron Reduction by NADPH- Cytochrome P450 Reductase . . . Stimulation of Microsomal NADPH Oxidation by Ferric Chelates . Anaerobic Iron Reduction by Microsomes and a Reconstituted Mixed Function Oxidase System . . . . . Discussion . List of References CHAPTER II: Superoxide Production by Microsomal Mixed Function Oxidase Components Abstract . . Introduction . . . . . . . . . . . . . Materials and Methods Purification of Cytochrome P450c and P450d Acetylation of Cytochrome c . . . Acetylated Cytochrome c Reduction . Hydrogen Peroxide Production . . . Results Reduction of Acetylated Cytochrome c by Xanthine Oxidase and NADPH Cytochrome P450 Reductase . . . . . . Superoxide Generation by Xanthine Oxidase: Effect of Iron Chelates . . . Superoxide Production by Purified NADPH Cytochrome P450 Reductase: Effect of Iron Chelates . . . . Superoxide Generation by Rat Liver Microsomes: Effect of Iron Chelates. Superoxide Production by the Reconstituted Mixed Function Oxidase System Pitfalls in Measuring Oxygen Radical Production by Microsomes Stoichiometry of Microsomal Oxygen Radical Production .'. . . Discussion . . . List of References . CHAPTER III: The Roles of Superoxide and Hydrogen Peroxide in NADPH-Dependent Microsomal Lipid Peroxidation . . . . . . . Abstract . . Introduction . . . . . . . vi Page 64 64 64 73 73 76 87 91 95 96 98 103 103 103 104 105 107 107 107 112 114 118 g 129 134 140 150 156 157 158 Page Materials and Methods . . . . . . . . . . . . . . 160 Chemicals . . . . . . 160 Preparation of Phospholipid Liposomes . . . 160 Lipid Peroxidation Assays . . . . . . . . . 160 Results . . . . . . . . . . 162 Role of Hydrogen Peroxide in Microsomal Lipid Peroxidation . . . . . . . 162 Effect of Catalase on Lipid Peroxidation . . 167 Role of Superoxide in NADPH- -Dependent Lipid Peroxidation: Comparison with Xanthine Oxidase-Dependent Peroxidation . . . . . . . . . . . . . 178 Discussion . . . . . . . . . . . . . . . . . . 181 List of References . . . . . . . . . . . . . . . 186 CHAPTER IV: Cytochrome P450-Dependent Lipid Peroxidation of Phospholipid Vesicles . 188 Abstract . . . . . . . . . . . . . . . . . . . . 189 Introduction . . . . . . . . . . . . . . . . 190 Materials and Methods . . . . . . . . . . . . . . 193 Chemicals . . . . . . . . . . . . . 193 Preparation of Enzymes . . . . 193 Preparation of Phospholipid Vesicles. . 194 Results . . . . . 195 Requirements for NADPH—Dependent Peroxidation . . . . 195 Effect of Cytochrome P450 Induction on Microsomal Lipid Peroxidation . 195 Effect of Cytochrome P450 on NADPH- Cytochrome P450 Reductase- Dependent Peroxidation of Liposomes . . . . . . . . . 198 Incorporation of Cytochrome P450 and NADPH- Cytochrome P450 Reductase into Phospholipid Vesicles . . . . 198 NADPH-Dependent Peroxidation of Phospholipid Vesicles . ; . . . . 200 Discussion . . . . . . . . . . . . . . . . . . 209 List of References . . . . . . . . . . . . . . . 213 SUMMARY 0 O O O O O O O O O O O O O O O O O 0 O O O O 215 APPENDIx . . . . . . . . . . . . . . . . . . . . . . . 219 vii LIST OF TABLES Page Effect of Ferric Chelates on the Rate Of NADPH Oxidation by NADPH-Cytochrome P-450 Reductase . 65 Rates of Reduction of Ferric Chelates by NADPH- Cytochrome P450 Reductase under Anaerobic Conditions . . . . . . . . . . . . . . . . . . . 74 Effect of Ferric Chelates on Rates of NADPH Oxidation by Rat Liver Microsomes. . . . . . . . 75 Rates of Reduction of Ferric Chelates by Rat Liver Microsomes under Anaerobic Conditions . . 81 Rates of Reduction of Ferric Chelates by the Reconstituted Mixed Function Oxidase System under Anaerobic Conditions . . . . . . . . . . . 86 Superoxide Production by Xanthine Oxidase . . . 111 Superoxide Production by NADPH-Cytochrome P450 Reductase . . . . . . . . . . . . . . . . . 113 Superoxide Production by Rat Liver Microsomes. . 117 Requirements for Superoxide Production by the Reconstituted Mixed Function Oxidase System. 122 Stoichiometry of Oxygen Reduction by Rat Liver Microsomes . . . . . . . . . . . . . . 136 Effect of Butanol Pretreatment on NADPH Oxidation and Superoxide Production by Liver Microsomes Isolated from Isosafrole Induced Rats . . . . . . . . . . . . . . . . . 138 Effect of Washing and the Addition of Exogenous Catalase on Microsomal Lipid Peroxidation . . . . . . . . . . . . . . . . . . 173 Requirements for NADPH-Dependent Peroxidation of Rat Liver Microsomes . . . . . . . . . . . . 196 Effect of Cytochrome P450 Induction on the Rates of NADPH-Dependent Microsomal Lipid Peroxidation . . . . . . . . . . . . . . . . . . 197 viii 15 16 17 Requirements for the NADPH—Dependent Peroxidation of Phospholipid Liposomes Promoted by the Reconstituted Mixed Function Oxidase System . . . Requirements for the NADPH-Dependent Peroxidation of Cytochrome P450-Containing Phospholipid Vesicles . . . . . . . . Summary of Results ix Page 199 201 216 Figure 10 11 12 13 LIST OF FIGURES Schematic of Cytochrome P450- Dependent Oxidations . . . . . . . . . . Stimulation of NADPH-Cytochrome P450 Reductase-Dependent NADPH Oxidation by EDTA-Fe3+ and DTPA-Fe3+ . . . . . . . . Effect of Superoxide Dismutase on Xanthine Oxidase-Dependent Hydroxyl Radical Formation . . . . . . . . . . . . Effect of Superoxide Dismutase on NADPH- Cytochrome P450 Reductase-Dependent Hydroxyl Radical Formation . . . . . . . . Stimulation of Rat Liver Microsomal NADPH Oxidation by Ferric Chelates . . . . . . . Stimulation of Rat Liver Microsomal NADPH OXLdatiOD by ADP”F€3+ o e e e e o o e 0 Effect of Microsomal Protein Concentration on NADPH-Dependent ADP-Fe3+ Reduction. . Effect of Various ADP:Fe3+ Ratios on the Rate of ADP-Fe3+ Reduction by Microsomes . Reduction of Acetylated Cytochrome c by Xanthine Oxidase . . . . . . . . . . Reduction of Acetylated Cytochrome c by NADPH Cytochrome P450 Reductase . k . . . Stimulation of Acetylated Cytochrome c Reduction by Ferric Chelates . . . . . . Effect of Increasing Cytochrome P450: NADPH-Cytochrome P450 Reductase Ratios on Superoxide Production in the Reconstituted System . ... . . . . . . . . Effect of Increasing NADPH-Cytochrome P450 Reductase: Cytochrome P450 Ratios on Superoxide Production in the Reconstituted System . . . . . . . . . . . Page 22 67 70 72 78 80 83 85 109 110 116 119 121 Figure Page 14 EPR Spectra of Superoxide Produced by Cytochrome P450b in the Reconstituted System . . . . . . . . . . . . . . . . . . . 124 15 EPR Spectra of Superoxide Produced by Cytochrome P450c in the Reconstituted System . . . . . . . . . . . . . . . . . . . 126 16 EPR Spectra of Superoxide Produced by Cytochrome P450d in the Reconstituted System . . . . . . . . . . . . . . . . . . . 128 17 Inhibition of Superoxide-Dependent Cytochrome c Reduction by Microsomes . . . . 133 18 Time Course of Microsomal Hydrogen Peroxide Production . . . . . . . . . . . . 134 19 Effect of Ferric Chelates on Microsomal Hydrogen Peroxide Formation . . . . . . . . 135 20 Effect of Hydrogen Peroxide, Azide, or Hydrogen Peroxide plus Azide on Microsomal Lipid Peroxidation . . . . . . . 164 21 Effect of Azide on Endogenous Catalase Activity in Microsomes and Microsomal Lipid Peroxidation . . . . . . . . . . . . . 166 22 Effect of Hydrogen Peroxide on Microsomal Lipid Peroxidation . . . . . . . 169 23 Effect of Different Commercial Catalase Preparations on Microsomal Lipid Peroxidation . . . . . . . . . . . ; . . . . 171 24 Effect of Benzoate on Microsomal Lipid Peroxidation Dependent Upon Hydroxyl Radical or NADPH . . . . . . . . . . . . . . 175 25 Effect of Mannitol on Microsomal Lipid Peroxidation Dependent Upon Hydroxyl Radical or NADPH . . . ... . . . . . . . . . 177 26 Effect of Superoxide Dismutase on NADPH- Dependent Lipid Peroxidation of Microsomes: A Comparison with Xanthine Oxidase-Dependent Peroxidation . . . . . . . 179 xi Figure 27 28 29 30 31 Effect of Superoxide Dismutase on NADPH- Dependent Lipid Peroxidation of Liposomes: A Comparison with Xanthine Oxidase— Dependent Peroxidation . . . . . . . . . . Elution Profile of NADPH-Cytochrome P450 Reductase and Cytochrome P450-Containing Phospholipid Vesicles Subjected to Gel Filtration . . . . . . . . . . . . . . . . Effect of Vesicle Concentration on the Peroxidation of Phospholipid Vesicles. Effect of ADP—Fe3+ Concentration on the Peroxidation of Phospholipid Vesicles. Effect of Increasing Cytochrome P450 Content on the Peroxidation of Phospholipid Vesicles xii £282 180 203 204 205 207 ADP DLPC DMPO DMPO-OH DMPO-OOH DTPA EDTA EPR GSH GSSG HBB MDA MFO NADH NADPH Reductase SOD 3-MC LIST OF ABBREVIATIONS adenosine diphosphate dilauroylphosphatidylcholine 5,5’-dimethyl-l-pyrroline-N—oxide hydroxyl radical adduct of DMPO superoxide radical adduct of DMPO diethylenetriaminepentaacetate ethylenediaminetetraacetate electron paramagnetic resonance reduced glutathione oxidized glutathione 3,3’,4,4’,5,5’ hexabromobiphenyl malondialdehyde mixed function oxidase or mixed function oxygenase reduced nicotinamide adenine dinucleotide reduced nicotinamide adenine dinucleotide phosphate NADPH-cytochrome P450 reductase superoxide dismutase 3-methylcholanthrene xiii INTRODUCTION Although oxygen is an absolute necessity for aerobes, higher partial pressures of oxygen are frequently toxic to organisms. The phenomenon of oxygen toxicity was described many years ago,1 but the mechanism has remained unresolved at least until the early 1970’s, and some researchers would argue even up to the present time. In the late 1960’s, McCord and Fridovichz discovered that the one electron reduction of ferricytochrome c by milk xanthine oxidase was mediated via 02?. Prior to that time, free radical species of any type and particularly 027 were generally not considered to be produced in biological systems. Concurrently, they described a specific enzymatic activity for the nearly ubiquitous copper-containing protein erythrocuprein that at the time had no known function. They renamed the protein superoxide dismutase, SOD, in recognition of its ability to catalyze the one electron dismutation or disproportionation reaction of 023'.3 Subsequently, it has been “shown that a number of purified enzymes and subcellular organelles generate 02:. Under the assumption that 027 is like other highly reactive free radicals, it has been postulated that organisms must possess sufficient scavenging capacity to protect against the "normal” fluxes of 027 inferred to occur during in vivo metabolism. Accordingly, at least one form of SOD has been identified in nearly all aerobes. However, it is postulated that under certain physiological conditions the normal oxygen radical scavenging system is overwhelmed by increased fluxes of 02? and/or is compromised in some way. This inability to scavenge oxygen radicals has been postulated to result in toxicity or lethality, and this hypothesis has become known as the superoxide theory of oxygen toxicity.‘ There have been a number of studies linking excessive 02? production to toxicity. However, rarely if at all has elevated 02? production in vivo been directly demonstrated. Rather, it has usually been inferred from in vitro data. Thus, some researchers have expressed skepticism over the theory, pointing out that much of the data are _circumstantia1. A few have even maintained that 027 production is unrelated to oxygen toxicity.5'° However, any disagreement over the mechanism of oxygen toxicity does not lessen the significance of McCord and Fridovich’s discoveries, for they have spawned an entirely new area of biological investigation: the role of oxygen radicals in biology and medicine. Over the past decade, 027 and the other reduction products of oxygen have been demonstrated or postulated to be involved in numerous physiological, pathological, and toxicological states including phagocytic killing by leukocytes,’ inflammation,a arthritis,9 mutagenesis,1° tumor promotion,H diabetes,12 ischemia,13 trauma,1‘ spinal cord injury,15 aging,1° inactivation of enzymes and marking them for proteolysis,H peroxidation of membrane 1ipids,1° and the toxicity of numerous xenobiotics.1°'33 This dissertation examines the ability of the NADPH- dependent electron transport in microsomes to generate 02: or reduce iron chelate and the relative importance of these two distinct activities in initiating lipid peroxidation, the oxidative degradation of polyunsaturated fatty acids that is proposed to occur in many of the aforementioned states in which oxygen free radicals are proposed to be involved. Chapter I examines the potential of the microsomal electron transport system to reduce ferric chelates commonly used in lipid peroxidation. Previous work in Dr. Aust’s laboratory has shown that the NADPH-dependent peroxidation of microsomes requires reduced iron, but iron reduction by microsomes did not appear to be dependent upon 02?. The results in this chapter provide evidence for an alternate iron reduction pathway. The potential of rat liver microsomes to generate 027 has been known for over a decade. Yet there is considerable disagreement over the amount of 027 that is produced. In Chapter II, rates' of 027 production by rat liver microsomes, and purified NADPH-cytochrome P450 reductase alone or reconstituted with several purified isozymes of cytochrome P450 are measured using an acetylated cytochrome c reduction assay. The importance of 027 and H202 in the initiation of microsomal lipid peroxidation promoted by ADP-Feat is investigated in Chapter III. According to the iron- catalyzed Haber-Weiss reaction, these partially-reduced oxygen species should be necessary for the generation of -OH, the proposed initiator of lipid peroxidation. Thus various factors that increase or decrease the production of 027 and/or H202 in microsomes have been tested and their effects on the resultant rates of lipid peroxidation have been measured. Lastly, in Chapter IV, the role of cytochrome P450 isozymes in the initiation of microsomal lipid peroxidation is examined. Previous studies have shown cytochrome P460 to function in the decomposition of lipid hydroperoxides, but no studies have shown a role in the initiation of lipid peroxidation. The necessity for cytochrome P450 in lipid peroxidation promoted by ADP-Fe3+ is demonstrated in a model lipid peroxidation system. This dissertation is divided into four chapters, each written in a format similar to that of many scientific Journals. Each chapter contains an abstract of the work, an introduction to the pertinent literature, material and methods section, results, discussion and references. To avoid unnecessary duplication the material and methods sections in subsequent chapters describe only those methods or materials not used in preceding chapters. The chapters are preceded by a broad review of the literature on microsomal electron transport, oxygen radical generation and lipid peroxidation. REVIEW OF LITERATURE Chemistry ofggiogygenggnd Its Reduction Prodggtg Dioxygen - Molecular oxygen or dioxygen has two unpaired electrons in its 7r* antibonding orbital and, therefore, exists in a triplet ground state. As such, kinetic constraints prevent its direct reaction with most organic compounds that are of singlet multiplicity. Before using oxygen as an oxidant, organisms must activate it in order to overcome this spin restriction. Activation can occur via several pathways. Photochemical activation can result in the formation of singlet oxygen, a transient species capable of reacting directly with organic molecules. However, the major means by which organisms activate oxygen involves its reduction and/or complexation with metalloenzymes. Oxygen can be reduced to two molecules of water in four discrete one electron reduction reactions: 02 -t e' ------- > 027 ' (1) 03: + e' ------- > 023‘ (2) H202 + e' ------ > OH + OH‘ (3) -on + e‘ —————— > on- (4) Reduction of oxygen by one, two, or three equivalents results in the formation of 027, H202 or ~OH, respectively. These three species are collectively referred to as the 7 partially reduced oxygen species, oxygen radicals, or oxy radicals. The chemistry of oxygen and its reduction products is complex in part because it can vary drastically with the experimental conditions. As an example, the redox potential of 02/027 varies from -0.58v in aprotic solvents to -0.33v in water.“ The reduction potentials of the other oxygen radical species are all positive, so the overall reduction of oxygen to water is exothermic. Superoxide - The premise of the superoxide theory of oxygen toxicity is that 027 is either intrinsically toxic to organisms or gives rise to other reactive species which subsequently initiate oxidative damage. An examination of the chemistry of 027 reveals that this implication may be a weak link in the theory. In general, despite its free radical nature, 027 is relatively unreactive towards most biological molecules. The chemistry of 027 is, in general, governed by the solvent; it is considerably more reactive in aprotic solvents than in aqueous solutions.2§ In aprotic media it is a good nucleophile and reductant and reacts with many organic compounds. rA matter of 'debate in biology is its purported reactions with biological molecules such as polyunsaturated fatty acidszs, lactate dehydrogenase-bound NADH“ and a( -tocopherol27 since certain cellular microenvironments might be sufficiently aprotic to promote such reactions. In aqueous media 027 is considerably less reactive, its predominant reaction being non-enzymatic dismutation to H202 and oxygen. The rate of dismutation is highly pH dependent, occurring most rapidly at pH 4.8, the pk. for 027. 1102' + 02:41—9H202 + 02 (5) Enzymatic dismutation is catalyzed by a family of SODs. Three types of SOD have been isolated and characterized; each contains a different redox-active metal: manganese, iron or copper. During the catalytic cycle of the enzymatic dismutation, the metal is reduced by the first 0:7 molecule then oxidized by the second. The redox potentials of the 02/02: (-0.33v) and the Oat/H202 (+0.87v)2‘ couples are such that 027 can act as a reductant towards many metal chelates and an oxidant towards the same reduced chelates. Thus, it is not surprising that a dismutase-like activity has also been ascribed to redox-active complexes containing manganese,3° iron,29 and copper.30 However, these complexes do not have the specificity for 027 that characterizes the SODs.“:32 . The reactions of 027 with metal complexes are governed by several factors. First of all the reactions must be sufficiently fast to compete with non-enzymatic dismutation. Second, the redox potential of the metal must be favorable and chelation influences this potential. For example, EDTA chelation of ferrous changes its redox potential from -0.77v to -0.12v.33 The effect of this chelation on ferrous autoxidation is dramatic; ferrous autoxidizes extremely slowly at pH 7, whereas EDTA-Fe2+ autoxidizes extremely rapidly. Chelation can also affect oxygen-metal redox reactions in a kinetic fashion since inner sphere electron transfer is the typical mechanism by which these reactions occur. Chelators like DTPA, desferrioxamine, and phytic acid inhibit these reactions by occupying all coordination sites of the metal.3‘ Hydrogen Peroxide - Like 02?, H202 is relatively unreactive in aqueous solution except in the presence of transition metals. Cleavage of the peroxide bond occurs either homolytically (6) or heterolytically (7), depending upon the oxidation state of the metal: Feat + H202 ------ > 533+ + -OH + OH' (6) Fe3+ + H202 ------ > Feat + H02- + H’ (7) Reaction (6) is known as Fenton’s Reaction.as Originally it was demonstrated to be a redox chain reaction with the substrate radical (formed by the -0H~mediated oxidation of substrate) reducing ferric to ferrous.3° However, in biological systems low concentrations of iron probably preclude such a reaction, so the substrate radical is free to oxidize other cellular constituents. Hydrogen peroxide is scavenged in vivo by two enzymes located at different cellular sites. The hemoprotein catalase is located in the peroxisomes, but due to their 10 fragility, peroxisomes are often ruptured during cell lysis and traces of catalase activity are often found associated with other subcellular fractions. Catalase catalyzes the two-electron dismutation of H202 produced by the peroxisomal oxidases. 28202 ------- > 2820 + 02 (8) This reaction occurs in two distinct steps: Fea+ + 3202 ------- > (Fe0)3+ + H20 (9) (Fe0)3* + R202 ------ > Feat + 02 + H20 (10) where Fe3+ is the heme iron of catalase. The enzyme-oxygen intermediate is known as Compound I and is an intermediate also common to peroxidases, enzymes that are specific for H202 as an oxidant but not as a reductant. Many peroxidases such as that purified from horseradish are relatively non- specific, oxidizing numerous hydrogen donor substrates with many of these reactions forming the basis of assays for H202.37'3° Catalase is more specific than peroxidases, oxidizing predominantly H202, but also short chain alcohols. The selenium-dependent GSH peroxidase is a cytosolic enzyme that catalyzes the reduction of H202 to H20. H202 + ZGSH ------ ) 2H20 + 0886 (ll) Reducing equivalents are supplied by GSH, and the product is 6880. High levels of. GSSG can have adverse effects on cells,3°v‘°' so a: necessary complement to this H202- scavenging system is NADPH-dependent GSH-reductase. 0830 + NADPH ------ > ZGSH + NADP+ (12) 11 Hydroxyl Radical - The -OH is the most potent oxidizing species proposed to be generated in vivo. Its reactions can be characterized into four general types: abstraction, addition, electron transfer, and radical addition or termination.‘1 -0H + Csz ------- > C2H40H~ (13) -OH + Czfls ------- > Csz- + OH ‘ (14) -OH + [Fe(CN)s]" ----- > [Fe(CN)s]3‘ + OH’ (15) -OH + -OH ------- > R202 (16) Many of the reactions of -OH with organic molecules proceed with second order rate constants that approach and occasionally even exceed diffusion-controlled rates.‘1 Thus, the potential of -OH to oxidize biological molecules is unquestioned. Numerous studies have demonstrated the oxidation of proteins, enzymes, nucleic acids, and lipids by -OH generated in vitro. However, there is little evidence for the in vivo generation of -OH or its subsequent oxidation of biological molecules. Measurement of Oxygengggdical Prodgctiog The superoxide theory of oxygen toxicity holds that an overproduction of 027 or a compromised ability to scavenge it renders a cell or organism susceptible to oxidative stress. However, 'to date, many'questions that are raised by this theory have not been answered. ”hat constitutes a damaging flux of 02:? How much scavenging potential does a cell have? How much auxiliary capacity is present? 12 These questions have proven difficult to answer because measurement of oxygen radicals in vivo has not yet been accomplished. In fact, even the measurement of oxygen radicals in vitro has proven to be quite difficult, generally because of several factors. One is the chemical nature of the free radical species themselves. Their intrinsic reactivity usually dictates that the free radicals must be measured indirectly, relying on their reactions with various indicator molecules. Second is the potential for non-specificity in that most of these methods are based on the oxidation or reduction of the indicator molecules, reactions not limited solely to the oxygen radicals of interest. Lastly, there is inherent difficulty in measuring oxygen radical production in a heterogeneous system such as in membranes. Superoxide -Superoxide has proven to be the most difficult of the partially-reduced oxygen species to quantitate because of its unique reactivity. In aqueous solutions its predominant reaction is disproportionation, 'so it does not accumulate in aqueous solutions to a concentration sufficient to directly detect it. Most methods used for detection of 02? rely on its properties as a reductant or oxidant in aqueous solution. Among the most commonly used methods are the reduction of molecules such as cytochrome c,‘3 nitroblue tetrazolium,‘3 tetranitromethane,a and other l3 electron acceptors, or the oxidation of compounds like epinephrine,3 tiron,4‘ hydroxylamine, or sulfite.‘5 By far the most preferable assay is the reduction of cytochrome c and it has been used for the detection of 02: produced by purified enzymes, subcellular fractions and intact cells. One shortcoming of this assay is that ferricytochrome c can be directly reduced by some flavoproteins and cytochromes, including the reductase.‘° With such enzymes, cytochrome c reduction is not suitable as an assay for 027 since it is competing with oxygen for enzymatic reducing equivalents, in addition to scavenging any 02? that might be produced. Several investigators have chemically modified cytochrome c by either acetylation or succinoylation to make its reduction a more broadly applicable assay for 02?.‘5147 Acylation of cytochrome c decreases its tendency for direct reduction, while usually maintaining its ability to react with 027. The suitability of the modified preparations for the measurement of 027 production appears to depend upon the percent modification of cytochrome c. Another commonly used method for 02? production is EPR spin trapping. The detection of 02: or other free radicals is made possible by their reactions with nitrones or nitroso compounds yielding 'more stable “free radicals that can accumulate to concentrations sufficient to detect them directly with EPR spectroscopy.‘° 14 H O“ H O- , I, ll, H02 + R-C=N-R ------- > R-f-N-R (17) + OOH The intensity of the free radical signal is indicative of the concentration of the radical species, and the hyperfine splitting constants of the radical—spin trap adduct signal can also be used as a means to identify which free radical species has been trapped. However, spin trapping of oxygen radicals, in particular 02?, as a quantitative method has several shortcomings. One of these is the extremely slow reaction of 02? with DMPO, the most commonly used nitrone spin trap for oxygen radical detection. Second-order rate constants on the order of 10 to 20 m'ls”l have been reported,“9 thus large concentrations of DMPO are necessary to compete with the other reactions of 027 that occur with rate constants many orders of magnitude greater than that of the trapping reaction. Secondly, the DMPO-OOH spin-trap adduct is itself not particularly stable having a half-life estimated to be 27—91 sec.50 Moreover, decomposition of DMPO-OOH results in the DMPO OH adduct. Thus spin trapping should really only be considered as a qualitative method for the detection of 02:. Hydrogen Peroxide - Ouantitation of H202 is considerably less difficult than 02?. Nearly all methods rely on the 15 action of catalase or peroxidases in the coupled oxidation of a variety of hydrogen donor substrates.37v3° H202 + Fe3+ ------- > H20 + (FeO)3+ (9) (Fe0)3* + substrate ----- > Feat + product (18) The compound I of catalase or peroxidase can be observed directly51 or oxidizes a number of compounds including ethanol, scopoletin, phenol, o-dianisidine to products detected with uv—visible or fluorescence spectroscopy. However, subcellular fractions typically contain traces of catalase activity, so inhibitors of catalase activity such as azide are normally added to incubations containing these fractions. Hydroxyl Radical - Quantitation of °OH production is nearly as difficult as 027, but for opposite reasons. While the reactivity of 02: is limited in aqueous solutions, the -OH is highly reactive, reacting with most organic compounds. Thus, there is no lack of compounds that can be used to detect -OH, but as with 027 the oxidations of these molecules are not necessarily specific for '-OH. Compounds that have been widely used include methional, dimethyl sulfoxide, benzoate, and salicylate‘5 with the yield of products being indicative of the amount of -OH formed. EPR spin trapping has also been used to detect ~OH formation in vitro.“ However, since 027 is often produced in systems in which the production of -OH is being assayed, it is not always possible to determine the fraction of 16 DMPO-OH signal intensity resulting from the decomposition of the DMPO-OOH. Likewise, there are other factors that may result in a decrease of DMPO-OH signal intensity. Therefore, spin trapping can probably only be considered as a qualitative measure of -OH formation. Cellular Siteg,of Oxygen Rgdical Generation Numerous cellular enzymes utilize oxygen, and those characterized as oxidases reduce oxygen with reducing equivalents derived from the oxidation of their substrates. Although mitochondrial cytochrome oxidase reduces oxygen completely to water without the release of intermediate dioxygen reduction products, most other cellular oxidases produce 027 and H202. It is not known what the consequences of these normal fluxes of 027 and/or H202 are to the cell, but it has been suggested that they might relate to the oxidation of cellular constituents that perhaps is a part of the aging process.15 From the near ubiquitous nature of the scavenging enzymes in aerobes. it is inferred that 02? and H202 generated during normal metabolism could be toxic, and the scavenging enzymes would therefore serve to protect cells from these potentially deleterious oxygen radicals. The scavenging capacity of these enzymes within the cell is not known, but it is proposed that a variety of conditions can result in overproduction of oxygen radicals to levels exceeding the scavenging potential. 17 There are several subcellular locales where 02’ and H202 are produced. Several cellular oxidases are located in the peroxisomes.51 Among them are D—amino acid oxidase, urate oxidase, and fatty acyl CoA oxidase, all which generate H202. Peroxisomes also contain large amounts of catalase, but despite this, some H202 still appears to diffuse from the peroxisomes.51 The toxicity of the hyperlipidemic or peroxisomal proliferating drugs is proposed to be the result of the induction of the peroxisomal H202-producing oxidases and a concomitant increase in peroxisomal H202 production.52 Cytosolic enzymes such as aldehyde oxidase and xanthine oxidase generate 02: and H202 during the oxidation of a variety of substrates. Xanthine oxidase is a popular enzyme for use in in vitro studies of oxygen radicals because it is readily obtainable from biological sources and because it generates both 027 and H202. However, in the liver and in other tissues the physiological form of the enzyme is proposed to be a dehydrogenase, using NAD+ rather than oxygen as the oxidant. During an ischemic insult, Ca2t- dependent proteolysis of the enzyme is proposed to occur, converting the dehydrogenase to an oxidase that upon reperfusion can result in an additional source of 027 and H202.13 Despite the coupled nature of mitochondrial electron transport, a small percent of reducing equivalents appear to "bleed off" the electron transport chain producing 02? via 18 the autoxidation of ubisemiquinone.53 Under conditions such as ischemia or electron transport blockade with azide or cyanide where the mitochondrial electron transport carriers become highly reduced, the concentration of ubisemiquinone increases and its rate of autoxidation increases proportionally. Regardless of the rate of 02? production, it appears that only H202 efflux from mitochondria occurs, presumably because of the efficient scavenging of 027 by the mitochondrial Mn-containing SOD. Polymorphonuclear leukocytes and other phagocytic cells contain a plasma membrane-bound electron transport system consisting of an NADPH dehydrogenase, quinone and a b-type cytochrome.7 Appropriate stimuli trigger the production of 027 by this system, the so-called "oxygen burst." This burst of oxygen radical production contributes to oxidative killing of bacteria, but is also proposed to be involved in the inflammatory response. Another major source of oxygen free radicals is the microsomal electron transport system. Its function is to catalzye the oxidation of numerous substrates both endogenous and exogenous. . NADPH-Ht + 02 + SR ------ > NADPH + H20 + SOH (19) As shown above, it catalyzes the insertion of one atom of oxygen into the 'substrate, thus it has monooxygenase activity. The other atom of oxygen is reduced to water, so the system has both oxygenase and oxidase activity and is often called the MFO system. This electron transport system 19 also generates 027 and H202 as is discussed in a subsequent section. Microsomal Electron Transport Systeg The electron transport system of microsomes is composed of two distinct electron transport chains, differentiated by the pyridine nucleotide cofactors they use as electron donors. One chain is specific for NADH, consists of the flavoprotein NADH-cytochrome bs reductase and cytochrome be, and serves as an electron donor to the stearyl-Co A desaturase system. The other electron transport chain is NADPH—specific, consists of the flavoprotein reductase and the various isozymes of cytochrome P450 and functions in the metabolism of various substrates. While these electron transport chains are separate in terms of function, they do interact. There is a well documented NADH synergism of NADPH-dependent mixed function oxidase activity.“ Others have suggested that the reductase might also reduce cytochrome bs.55 The reductase is an amphipathic molecule. having both a globular hydrophilic domain containing its catalytic sites and a hydrophobic membrane-binding .domain. Its electron transfer properties are quite unique relative to other flavoproteins. It contains 1 mole' FMN and 1 mole FAD per mole of enzyme, distinguishing it from other flavoproteins that usually contain only a single flavin moiety. It utilizes a 2 electron donor in NADPH but reduces cytochrome 20 P450 and other substrates by l electron.5°'57 The variety of substrates it reduces is quite diverse: cytochrome P450 and perhaps cytochrome b5, cytochrome c, ferricyanide, paraquat, 2,6-dichlorophenolindophenol, nitroso compounds, and numerous naphthoquinones and anthracyclines. Early purification procedures relied on the solubilization of the catalytic domain from the membrane by treatment of rat liver microsomes with proteases.93'5° This protease-solubilized reductase retained its ability to reduce numerous exogenously added substrates such as cytochrome c, ferricyanide and 2,6 dichlorophenolindophenol, but its electron acceptor in the microsomal membrane was not identified until later. The reductase has more recently been purified from microsomal membranes following detergent solubilization.°°'°1 Enzyme purified in this manner retains its hydrophobic domain and reduces cytochrome P450 as well as the other electron acceptors. Lu and Coon"2 utilized this detergent-solubilized reductase to reconstitute MFO activity in DLPC micelles also containing cytochrome P450. No activity was observed when the protease-solubilized enzyme was used. Thus, cytochrome P450 isozymes and the reductase incorporated in phospholipid vesicles also form a competent electron transfer chain and exhibit MFO activity. The orientations of the reductase and cytochrome P450 in the microsomal membrane are such that an interaction between them can easily be postulated even though the 21 biochemical mechanism of the interaction is not completely understood. Microsomal cytochrome P450 isozymes are integral membrane proteins, whereas the reductase’s globular region sits above the plane of the membrane perched on its hydrophobic domain anchored in the membrane. There are approximately 20-30 cytochrome P450 molecules per reductase molecule, and there is some controversy over whether the microsomal proteins are present as preformed clusters or laterally diffusing.°3 Clearly, the structure of the reductase suggests its potential to operate within a cluster of cytochrome P450 isozymes reducing some or all of them by pivoting about its hydrophobic domain. Cytochrome P450 isozymes metabolize drug substrates by activating molecular oxygen with reducing equivalents generated by the reductase and catalyzing the insertion of one atom of oxygen into the substrate. It does this by a rather complex series of steps that have not been completely delineated. Figure 1 shows the proposed scheme of the redox cycle of cytochrome P450 isozymes.°‘ The ”resting” state of cytochrome P450 in the membrane is in the ferric oxidation state and mostly low spin (1). However, it exists in an equilibrium between low and high spin and numerous conditions such as temperature, pressure and ionic strength affect this equilibrium.°5‘°7 The most important effector is the binding of substrate which frequently induces a conversion to high spin. The reductase reduces cytochrome P450 by one electron yielding the ferrous form of the Figure 1: Schematic of Cytochroge P450-Dependent Oxidations: The substrate is represented by RH and its oxidation product by ROH. The heme iron of cytochrome P450 is represented by Fe3i. Oxidation states of iron and oxygen are assigned to show stoichiometry only. 23 hemoprotein (II) to which oxygen readily binds (III). The addition of a second electron furnished either by the reductase or cytochrome b: yields a heme iron-oxygen complex reduced by two electrons (IV). These two reducing equivalents reduce one atom of oxygen to water, forming an activated complex of unknown identity but apparently having the oxidation state of Compound I of catalase or peroxidase (V). This complex then oxidizes substrate to generate the product (VI) and the ferric form of cytochrome P450 (VII). Cytochrome P450 can also use reducing equivalents obtained via cleavage of organic hydroperoxides, in much the same way that compound I of catalase is formed with H202.°‘ The activated iron-oxygen intermediate may be the same as that generated by the sequential NADPH-dependent reductions. However, cleavage of the hydroperoxide yields a radical species that can alkylate and inactivate the enzyme. Uncoupling of Microsomal Electron Transport The degree of uncoupling exhibited by the microsomal electron transport system is considerably greater than that of the mitochondria at least in vitro, although oxygen radical production by the endoplasmic reticulum in vivo has not been unequivocally demonstrated. Microsomal uncoupling occurs with the production of 02?, H202, or additional H20 with the following stoichiometries: 24 NADPH-H‘ + 02 ------- > 2 H027 + NADP+ (20) NADPH-H+ + 02 ------- ) H202 + NADP’ (21) 2NADPH-H* + 02 ------ ) 2H20 + 2NADP’ (22) Since microsomal metabolism is often accompanied by greater NADPH and 02 consumption than predicted in (19) and NADPH is consumed in the absence of substrates, the uncoupling reactions listed above appear to occur both in the presence and absence of substrate. It is not clear whether the 02? and H202 produced by microsomes in the absence of substrate is due to the reduction of that fraction of cytochrome P450 present in the high spin conformation. Some have proposed that the reductase generates 027 via autoxidation of a reduced flavin moiety,3°v°° while others have proposed that very little if any is generated.5°-57 However, for several reasons cytochrome P450 is postulated to have a more important role in microsomal 027 and H202 production. Microsomal oxygen radical production is greater in the presence of many substrates than in their absence70-71. Cytochrome P450 inhibitors also inhibit microsomal 02? and H20272'73 production although inhibition is not usually complete. Microsomes isolated from animals treated with inducers of cytochrome P450 exhibit different rates of O2? and H202 production.70 The mechanism by which uncoupling occurs is unclear. It is generally considered that the oxy-ferrous (IV) and ferrous superoxo (V) complexes of cytochrome P450 dissociate to 02? and H202, respectively. It is hypothesized that the 25 bulk of H202 generated by microsomes originates from 0277‘ suggesting that the transfer of the second electron to the oxy-ferrous complex of cytochrome P450 might be a limiting step in drug metabolism and/or oxygen reduction. The observation of a four electron uncoupling reaction75'7° is intriguing because this suggests that the activated cytochrome P450 complex (V) might subsequently be reduced by two electrons to generate additional water. Induction of Cytochrome P450 Another area of active current research in the biochemistry of the cytochrome P450 system that may have significance with respect to oxygen radical production is the inducibility of cytochrome P450 isozymes in response to various xenobiotics.77 Two major classes of inducers have been described. One class of inducers is typified by 3-MC and is termed 3-MC type inducers. Other polyhalogenated aromatic hydrocarbons that can assume planar conformations such as dioxins, naphthalenes, ~¢9 -naphthoflavone, and certain congeners of polychlorinated and 'polybrominated biphenyls, and 3—MC all bind to a cytosolic receptor protein called the Ab receptor. The receptor-ligand complex translocates to the nucleus, binds to chromatin, and induces the transcription of several genes including two isozymes of cytochrome P450. These are termed P450c and d in the nomenclature of Levin et al.*79 The other major class of inducing agent is typified by the barbituate phenobarbital 26 and is characterized by the induction of several forms of cytochrome P450, the major isozyme being cytochrome P450b in the Levin nomenclature.7° Induction of the reductase also occurs upon treatment of animals with phenobarbital. The mechanism by which phenobarbital induction occurs is still not known. Comparatively very little work has been done on the oxidase activities of the various isozymes. The investigations that have been conducted have mostly focused on oxygen radical production by microsomes from phenobarbital-treated animals."°'""‘3°'81 The major phenobarbital-inducible isozyme has been tested for its ability to produce 02? and especially H202 in reconstituted systems.71v75'°1'°2 Other studies have investigated the autoxidation of several purified chemically-reduced cytochrome P450 isozymes, and it appears that ferrocytochrome P450 do autoxidize producing 02: and/or Lipid Peroxidation Biological membranes have a critical role in maintaining cellular integrity and function. This fact, #Other nomenclatures are based on relative mobility of isozymes on SDS slab gels, sequence of elution from DEAE columns or on type of inducing ligand used, but that of Levin is the simplest and will be used throughout this dissertation. 27 when taken with the chemical nature of phospholipids, makes the oxidation of membrane phospholipids a potentially deleterious phenomenon, perhaps a more severe insult to biological organisms than damage to proteins or even to DNA. Due to the free radical nature of the oxidation of phospholipids, initiation of the chain reaction can lead to the oxidation of neighboring phospholipids. Therefore, the formation of a relatively small amount of oxidants can lead to the oxidation of many phospholipids resulting in loss of membrane fluidity, rupture of cells and/or organelles, activation and/or inactivation of membrane-associated enzymes, altered membrane permeability, and ionic gradients, and loss of Ca2+ homeostasis. In addition, by-products of lipid peroxidation such as MDA, 4-hydroxynonena1 and lipid hydroperoxides have been shown to have cytotoxic properties.°5‘°7 The chemistry of lipid peroxidation was detailed in the 1950’s, mostly by oil and food chemists investigating rancidity in fats. The process consists of three distinct phases: initiation, propagation, and termination.°° In initiation reactions, abstraction of allylic or methylene hydrogens from polyunsaturated fatty acids (LH) by an initiating species (I-) results in lipid alkyl radicals (L.). LH + I- ------- > L~ + IH (23) These radicals then enter into propagation reactions where the net number of free radical species is conserved. Alkyl 28 radicals react with dioxygen to form lipid peroxyl radicals (LOO-) that abstract methylene hydrogens from neighboring polyunsaturated fatty acids resulting in lipid hydroperoxides and new alkyl radicals: L- + 02 ------- > LOO- (24) L00- + L’H ----- > LOOH + L'- (25) Secondary initiation reactions involving the cleavage of lipid hydroperoxides by transition metals are also important. These reactions have been termed lipid hydroperoxide dependent initiations°9 and are quite analogous to those described for H202. Feat + LOOH ------- > Fe3+ + LO- + OH‘ (26) Fe3t + LOOH ------- > Fe2t + LOO- + H* (27) Cleavage of lipid hydroperoxides is generally facilitated by acid pH, although heme and certain other ferric chelates rapidly catalyze the decomposition of peroxides at neutral 93.90 Termination reactions result in the net consumption of free radicals. LOO- + L- ------- > LOOL ' (28) From a practical standpoint, termination reactions also occur when free radical species ,abstract hydrogen from compounds that form. more stable free radicals that are unable to participate subsequent propagation reactions. The lipid-soluble antioxidant vitamin E or a(-tocopherol and sulfhydryl compounds are proposed to act in this manner. 29 The degree or extent of lipid peroxidation can be assayed in a number of ways. One of the most popular methods involves the condensation of MBA with thiobarbituric acid to form a Schiff base with an absorbence maximum at 536 nm. Other methods of assessing lipid peroxidation include assays for lipid hydroperoxides, disappearance of polyunsaturated fatty acids, conjugated diene formation, direct HPLC assays for malondialdehyde, release of 510r, lactate dehydrogenase and other cytosolic enzymes from cells in culture, and ethane and pentane expiration in animals.”1 The choice of assay methodology is usually based on the particular system of study. Cellular Antioxidgpt Defenses As previously mentioned cells contain SOD, catalase, and GSH peroxidase that scavenge the oxygen radicals produced during cellular metabolism. In addition to reducing H202, GSH peroxidase also catalyzes the reduction of lipid hydroperoxides to the corresponding alcohols. However, besides these enzymes, cells. have several additional antioxidant defense mechanisms. Perhaps the most studied of these is 0(-tocopherol or vitamin E. Vitamin E is a Ilipid-soluble antioxidant present at different concentrations in various tissues. It is a component of membranes and is proposed to terminate the free radicals chain reaction of lipid peroxidation. The importance of vitamin E is clearly evident from the 30 nutritional studies that demonstrate the protective effects that vitamin E supplementation or the deleterious effects vitamin E deficiency has on organisms subjected to oxidative stress."2 Other species like GSH, and protein or non-protein sulfhydryls may act in a similar manner to vitamin E by reacting with lipid peroxyl radicals to yield less reactive radicals that can not abstract -H from neighboring polyunsaturated fatty acids. Whether in this capacity or as a cofactor for GSH peroxidase, GSH-S-transferase or in some other role, GSH has been shown to be a critical factor in maintaining cellular antioxidant defenses.°3'°‘ Phospholipase A2 is also proposed to have a function in protecting membranes against lipid peroxidation. The enzyme has enhanced activities towards oxidized fatty acids esterified to phospholipids.°5 By hydrolyzing the ester bond linking the fatty acid to the phospholipid, the oxidized fatty acid is removed from the membrane and prevented from participating in further propagation reactions within the membrane bilayer. Initiator Forggtion Once initiated, A lipid peroxidation is a self— propagating event; Thus, the bulk of scientific investigations in this field have dealt with the factors important to the initiation process, the identities of the initiating species, and means to inhibit their formation. 31 There are two major pathways by which oxidizing agents capable of initiating lipid peroxidation are proposed to be formed. The first of these involves the metabolism of certain xenobiotics to free radical species that directly abstract methylene hydrogens and initiate lipid peroxidation. The classical example of such a compound is carbon tetrachloride;°° it is metabolized by the hepatic microsomal MFO system to the trichoromethyl radical (-CCla). This radical abstracts hydrogen from a polyunsaturated fatty acid to initiate lipid peroxidation. Of particular relevance to oxygen toxicity is the other major route for the initiation of lipid peroxidation and involves the participation of 02? and H202 and their subsequent redox reactions. Haber-Weiss Reaction - Superoxide and H202 are usually considered to be toxic by virtue of their reaction to form more reactive species capable of oxidizing cellular macromolecules such as polyunsaturated fatty acids. The most often proposed initiator of lipid peroxidation is -OH formed by the Haber-Weiss reaction:97 I 027 + H202 ------ > 02 + OHT + -OH (29) This reaction clearly provides a mechanism for the proposed toxicity of excessive 02' and H202 production. However, what was initially overlooked by the first investigators who proposed it was the requirement for metal ion catalysis. It was not until 1978 when several groups of scientists 32 demonstrated that the rate of the uncatalyzed Haber-Weiss reaction was extremely slow, probably too slow to account for the biological damage attributed to 02: and H202 production. Since then, the Haber-Weiss reaction has become known as the iron-catalyzed Haber—Weiss reaction, not because catalysis of this reaction is a property exclusive to iron, but rather in recognition of the biological abundance of iron and its propensity to undergo one electron oxidation-reduction reactions. 027 + Fe3t ------- > Fe2* + 02 (30) 02? + H02 ------- > H202 + 02 (5) Fe2+ + H202 ------ > Fe3+ + OH' + -0H (6) This reaction sequence has been most often invoked as the causative factor in oxygen toxicity and most of the physiological, toxicological and pathological states mentioned previously. It is generally difficult to determine the participation of 02? and H202 in the reactions leading to the formation of an initiating species. The most widely used method for determining 027 and H202 participation is the addition of SOD or catalase, respectively. In many cases in vitro lipid peroxidation is inhibited by SOD or catalase, but rarely) is inhibition complete. Thus, one cannot discount the possibility that other substances can substitute for 02? or H202 and perhaps produce other initiating species besides -OH. Likewise, it has proven extremely difficult to ascertain to what extent and when 33 -OH-mediated oxidation of biological molecules has occurred. This can be attributed at least in part to the intrinsic reactivity of -OH which readily reacts with most molecules. Thus, it may often be difficult to achieve sufficient concentrations of detector molecules to compete with the spectrum of organic molecules with which -OH can react, especially in vivo. Objections to the Haber-Weiss Reaction - A closer consideration of the iron-catalyzed Haber-Weiss reaction has led some investigators to question its nearly universal acceptance in explaining oxidative damage to biomolecules. The purpose of 027 in the iron-catalyzed Haber—Weiss reaction is two-fold: reduction of ferric (30) and a source of H202 via dismutation (5). Thus, the requirement for 02? can be met by supplying alternate reductants and sources of H202. Fee has recognized this and has termed the iron- catalyzed Haber-Weiss reaction "superoxide-driven Fenton’s chemistry."°° His contention is that 027 is only one of numerous cellular reductants and is present at miniscule concentrations relative to other reducing agents such as ascorbate or glutathione. Numerous in vitro studies have demonstrated that these and other reducing agents can support lipid peroxidation, and' in these systems lipid peroxidation is not inhibited by SOD. Other objections to the Haber-Weiss reaction are based on a different premise, that being the chemistry of iron- 34 oxygen reactions. The iron-catalyzed Haber-Weiss reaction is merely a reduction of ferric followed by the oxidation of the ferrous. As such, the tendency of the iron chelates to undergo reduction and/or oxidation is determined by the redox potential of the chelate, not by the presence of 02? or H202. Thus, there are examples of ferrous chelates that rapidly autoxidize such as EDTA, citrate, pyrophosphate” and ones that are not oxidized by either oxygen or H202 such as o-phenanthroline, dipyridyl, and bathophenanthroline sulfonate.1°° Therefore, it will be important to identify biologically-relevant iron chelates and characterize their redox chemistry to determine their tendency to catalyze the Haber-Weiss reaction or other reactions leading to strong oxidizing species in order to ascertain the extent to which the iron-catalyzed Haber-Weiss reaction might occur in vivo. A chelator of special note is ADP. It was the chelator used by Hochstein and Ernster”1 when they observed microsomal lipid peroxidation. A comparison of the data in the literature indicates that ADP-iron promotes greater rates of lipid peroxidation than other. iron chelates, especially when phospholipids are present as bilayers (i.e., liposomes, microsomes, vesicles). Moreover, although ADP- iron can catalyze -OH' formation, the yield of -OH is lower than with other iron'chelates.”2 'In fact, when ADP-Fe3+ is used to promote lipid peroxidation, it appears that production of -OH and initiation of lipid peroxidation may be inversely related.103 35 A final consideration in the debate over the significance of the Haber-Weiss reaction in vivo relates to the bioavailability of iron. While iron is an abundant element in biology, it is not readily available; most iron is present in high molecular weight proteins.1°‘ Very low amounts of what is termed low molecular weight iron has been isolated from cells,104 and recent studies have begun to assess the conditions under which the high molecular weight iron proteins will release iron.1°3‘1°7 Thus, the metal catalyst for the Haber-Weiss reaction or other oxygen radical reactions may well be the limiting factor in the formation of strong oxidizing species in vivo. Therefore, perhaps the rigid control of iron metabolism by the cell should also be considered as a major antioxidant defense mechanism. Alternate Initiatora - A growing number of investigations have demonstrated that SOD and catalase did not always inhibit lipid peroxidation or other oxidative damage. This is an indication that the 'OH may not. be the only initiations species. Some investigators have referred to a crypto--OH as being the initiating species.”8 This species is proposed to possess equal or nearly equal reactivity to free -OH but be kinetically distinct from -OH. Others have suggested various iron-oxygen complexes as being responsible for initiation including perferryl ion,1°' an adriamycin complex with ADP—Fe3*,11° ferryl iron,111 an activated 36 methemoglobin or metmyogoblin complex,112 and a ferrous- dioxygen-ferric complex.113 Lipid peroxidation initiated by these proposed initiators was not inhibited by SOD, catalase, or -OH scavengers. Just as the ability of SOD, catalase, or -0H traps to inhibit lipid peroxidation is not definitive evidence for -OH participation, the inability of these scavengers to inhibit does not discount the possibility that 02?, H202, or -OH were involved. In fact, the studies in which alternate initiators were proposed have been criticized on the grounds that the data can be explained by an inaccessibility of the scavengers to the site(s) of oxygen radical generation.““»115 To settle this question it will be necessary to characterize these alternative initiating species. _ADPH—Dependent Lipid Peroxigation Hochstein and Ernster were the first to report that rat liver microsomes incubated with NADPH underwent peroxidation.”1 They characterized the process, finding a requirement for ADP-Fe“.116 Lipid peroxidation was not inhibited by SOD, catalase, or ~OH traps and a perferryl ion (Fe3*02) was proposed to be the initiating species.109 However, perhaps the most thorough study on microsomal lipid peroxidation was done by McCay and his associates. They characterized the process with regard to the alterations of phospholipid, oxygen uptake, polyunsaturated 37 fatty acid loss, MDA formation, phospholipid peroxides as intermediates and the enzymatic nature of the process.117' 131 Pederson and Aust were the first to demonstrate that the enzyme responsible for catalyzing NADPH oxidation as a precedent to lipid peroxidation was the reductase.122 They did this by inhibiting microsomal lipid peroxidation with Fab fragments prepared from rabbit IgG raised against purified reductase and by developing a model lipid peroxidation system containing the purified enzyme, microsomal phospholipid liposomes and iron chelates. Whereas ADP-Fe3+ was the only iron chelate needed to promote lipid peroxidation in the intact microsomal system, Pederson and Aust noted an additional requirement for EDTA- Fe3+ in their reconstituted system. A previous study had indicated that the direct addition of ferrous to microsomes resulted in peroxidation even in the absence of NADPH,123 so these data taken together suggested that the role of NADPH and the reductase in the microsomal system was to reduce ADP-Fe3+ in the microsomal system. In addition there appeared to be some substrate specificity to the reduction in that the purified enzyme seemed to reduce EDTA-Feat but not ADP-Fe3t. Rolaa of Cytochroae P450 in LipiaiPeroxidation Cytochrome P450 is a group of isozymes having broad and overlapping substrate specificities. To date, the relative ability of individual forms of cytochrome P450 or the 38 complement of isozymes induced by xenobiotics to promote the initiation of lipid peroxidation has not been demonstrated. Perhaps one of the reasons for this can be attributed to the controversy over how microsomal lipid peroxidation is initiated and what factors contribute to the formation of an initiating species. Previous results have shown that xanthine oxidase could promote the ADP-Fe3i-dependent peroxidation of microsomal phospholipids where 027 was required for iron reduction.12‘ Therefore, the reconstitution of the reductase with cytochrome P450, that has been shown to generate 02?, would be predicted to promote peroxidation also. However, in only one instance has a reconstituted MFO system been shown to initiate lipid peroxidation.125 In this case, there was no apparent requirement for iron, although iron Chelators did inhibit peroxidation. Whereas the role of cytochrome P450 in initiation of peroxidation is questionable, it can apparently function in lipid hydroperoxide-dependent initiation reactions. Cytochromes P450 can utilize lipid hydroperoxides or other organic hydroperoxides as a means to produce activated oxygen capable of oxidizing MFO substrates.°‘ They catalyze a homolytic cleavage of the peroxide bond generating an alkoxyl radical and an activated heme iron intermediate that may be related to the Compound I intermediate of catalase or species (VI) of cytochrome P450 (Figure l). Fe3+ + LOOH ------- > (FeO)3+ + L0- (27) 39 The alkoxyl radical is proposed to be capable of abstracting hydrogens from neighboring fatty acids to continue the free radical chain reaction of lipid peroxidation. Svingen et al.89 have shown that cytochrome P450 could promote the peroxidation of phospholipids liposomes containing lipid hydroperoxides in the absence of additional iron or reducing equivalents. 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Stimulation was dependent upon iron, since neither DTPA nor EDTA affected rates of NADPH oxidation. Microsomal NADPH oxidation was also stimulated by EDTA-Fe3+ and DTPA-Feat, but to a lesser extent by ADP-Fe3t. Increasing concentrations of EDTA-Feat and DTPA-Fe3+ resulted in increasing rates of NADPH oxidation by microsomes or the purified reductase. These results suggested that EDTA-Fe3t and DTPA-Fe3+ were substrates for the reductase. This was demonstrated with EPR spin trapping experiments in which NADPH-dependent reduction of EDTA-Fe3+ and subsequent -OH formation was not inhibited by son. That EDTA-Fe3t and DTPA-Fe3t were indeed substrates for the reductase was confirmed by monitoring the NADPH-dependent reduction of these chelates in anaerobic incubations containing microsomes, purified reductase, or reductase reconstituted with cytochrome P450 in DLPC micelles. These results are consistent with the NADPH oxidation data in that EDTA-Feat and DTPA-Fe3+ are directly 55 56 reduced primarily by the reductase and perhaps also by cytochrome P450 but to a much lesser extent. The purified reductase was unable to reduce ADP-Fe3+ but microsomes or the reconstituted electron transport system reduced ADP-Fe3+ in addition to EDTA-Fe3+ and DTPA-Feai. INTRODUCTION The NADPH-dependent peroxidation of microsomal lipids was first described by Hochstein et a1.1 They characterized this process with respect to its requirements for NADPH and ADP-Fe3t.2 Subsequent studies indicated that the addition of ferrous to microsomes resulted in peroxidation in the absence of NADPH.3 Thus, it was inferred that NADPH was the source of reducing equivalents for the reduction of iron and subsequently the reductase was shown to be (one of) the microsomal enzyme(s) necessary for iron reduction.‘ That iron must be first reduced is implicit in virtually all mechanisms proposed for the initiation of lipid peroxidation.5-1° However, the mechanism by which iron is reduced by the purified reductase or microsomes has remained a subject of controversy. Researchers have theorized that the reductase generates 02: as the first step towards the formation of -OH by a Ozt-driven iron-catalyzed Haber-Weiss reaction.7v°-1° 02: + Fe3+ -—-—> re2+ + 02 (1) 202: + 23+ ----> 8202 (2) Fez* + H202 ----> Fe"+ + -OH + 0H“ (3) Superoxide dismutase should scavenge 02? and prevent its reduction of ferric (1). However, SOD has not consistently been reported to inhibit NADPH-dependent lipid peroxidation. One group of investigators has postulated 57 58 that although SOD was not inhibitory in their system, reduction of ferric was still via 02?;7 others have postulated that the reason that SOD failed to inhibit lipid peroxidation might be that it is inaccessible to the site(s) of 02: generation.1°'11 This is clearly a difficult argument to disprove, or for that matter to prove. Other investigators have hypothesized that the inability of SOD to inhibit lipid peroxidation is because the mechanism of iron reduction is not dependent upon 02:; they postulated that iron reduction occurs via a direct transfer of an electron to ferric from the NADPH-dependent electron transport systemflnfiv12 Microsomes have been shown to possess both NADH- and NADPH-dependent ferricyanide reductase activities catalyzed by cytochrome b5 reductase13 and the reductase,14 respectively, so there is a precedent for such an activity. Inherent in this direct reduction hypothesis is that the ferric chelate is in fact a substrate for an electron transport protein and presumably its binding to the protein .should stimulate NADPH oxidation by the reductase. This is what occurs following the addition of ferricyanide to microsomes. There may be a second consideration that could be inferred from the existence of a direct iron reduction pathway in microsomes. One might predict some specificity for particular ferric chelates that would not be exhibited by Oat-dependent reduction systems. This has not been directly observed, but some indirect evidence that this is 59 indeed the case is based on the ability of ADP-Fe3* to promote the NADPH-dependent lipid peroxidation in a reconstituted lipid peroxidation system in the presence of EDTA-Fe3+ but not in its absence.12 It was postulated that EDTA-Fe"+ but not ADP-Fe3+ was reduced by the reductase (in a Ozr-independent process). More recent results seem to support this interpretation since ADP-Fe3t has been shown to promote xanthine oxidase-dependent peroxidation of phospholipid liposomes in the absence of EDTA-Fe3+ and in a process completely inhibited by SOD.15 Previous studies had shown that EDTA-Fe3+ stimulated NADPH oxidation by the purified reductase,°'15 however the ability of ferric chelates commonly used in the study of oxygen radical-mediated oxidations to act as electron acceptors in NADPH-dependent electron transport has not been extensively studied. Therefore, we have investigated the ability of microsomal MFO components to reduce several ferric chelates and concomitantly oxidize NADPH. MATERIALS AND METHODS Chemicals: Cytochrome c (Type VI), SOD, bromelain, xanthine oxidase, NADPH, ADP, DTPA, DLPC, o-phenanthroline, and xanthine were all obtained from Sigma. Desferrioxamine under the proprietary name desferal was obtained from CIBA- Geigy and Aldrich was the source for DMPO which was vacuum distilled before use. Catalase was a product of Millipore and EDTA was purchased from Mallinckrodt Chemical Company. All other reagents were of analytical grade and used without further purification. Solutions intended for use in EPR, NADPH oxidation, or iron reduction assays were treated with Chelex 100 (Bio Had) to remove contaminating transition metal ions. Preparation of Microaaaaa: Rat liver microsomes were isolated from male Sprague-Dawley rats (250-300g) (Charles River) by the method of Pederson and Aust.17 Microsomal pellets were resuspended in 10 mM EDTA, 1.158 HCl pH 7.0 and centrifuged at 105,000 xg for 60 min. Pellets were resuspended in 50 mM NaCl previously passed through a Chelex 100 and washed twice “to remove residual EDTA. Microsomes were used immediately or stored at -20°C in 50 mM Tris pH 7.5 containing 503 glycerol. 60 61 Purification of Enzymes: Cytochrome P450b and the reductase were isolated from liver microsomes of rats pretreated with 0.1% phenobarbital in their drinking water for 10 days prior to sacrifice. Protease-solubilized reductase was isolated using the procedure of Pederson a2 a2.17 with minor modifications. Fractions from the Sephadex G-100 column (Pharmacia) were pooled on the basis of cytochrome c reductase activity, and instead of ion exchange chromatography, the pooled fractions were subjected to affinity chromatography on 2’-5’ ADP Agarose18 (PL Biochemicals) using buffers containing no dithiothreitol nor detergent. Specific activities of purified preparations ranged between 40 and 66 units/mg. Detergent-solubilized reductase was solubilized from microsomes by treating them with Emulgen-Qll (Kao Atlas, Japan) to 1.5% final concentration and chromatographing on DEAR-Sephadex A-25 (Pharmacia) by the method of Dignam and Strobe1.1° The fractions having cytochrome c reductase activity were pooled and diluted two-fold with 20* glycerol and 0.18 Emulgen-911 and subjected to affinity chromatography on 2’-5’ ADP-Agarose as per Yasukochi and Masters.18 Specific activities ranged from 40-60 units/mg protein. Cytochrome P450b was purified from the flow-through of the DEAE Sephadex A-25 column using the procedure of Waxman and Walsh.20 Briefly this consists of DEAR-Cellulose 62 chromatography (Whatman) as described by West and Lu21 with subsequent chromatography on hydroxyapatite and CM-Sepharose (Pharmacia). Purified preparations had specific contents of 10-14 nmol/mg protein. Assays: Purified preparations of reductase and commercial xanthine oxidase were chromatographed on Sephadex G-25 (Pharmacia) to remove buffer salts and contaminating metal ions, and assayed for their ability to reduce cytochrome c. Cytochrome c reduction was monitored spectro- photometrically at 550 nm with 1 unit of enzyme activity defined as the amount of enzyme reducing l/pmol cytochrome c/min/ml at 25°C using an extinction coefficient of 21 x 103 M'1 sec‘l- Cytochrome P450 was assayed by the procedure of Omura and Sato.22 NADPH oxidation was monitored at 340 nm using an extinction coefficient of 6.22 x 103 M'1 sec-1. Catalase activity was assayed using the method of Bears and Sizer,23 and SOD was assayed as per McCord and Fridovich.3‘ Protein was assayed either by the method of Lowryzs or the bicinchoninic acid method26 adapted for use with microtiter plates.27 EPR Measurements: Electron paramagnetic resonance spin trapping experiments were performed using a Varian Century- 112 EPR spectrometer at 20°C. Spectrometer settings were 3320 G magnetic field, 9.412 GHz, lOOOKHz modulation 63 frequency, l5mW microwave power, 0.63 modulation amplitude, 1 sec time constant and 8 min scan time. Iron Reduction Assays: Iron reduction in incubations containing microsomes or purified enzymes was monitored spectrophotometrically at 510 nm, observing the absorbsnce of the o-phenanthroline-Fe2* complex. Aliquots of anaerobic incubations (0.5 or 1.0 ml) were quenched in 0.2% o-phenanthroline (1 m1) and the protein precipitated by the addition of 0.5 ml of 20% TCA. The o-phenanthroline-Fe2+ chelate was extracted from the acidified mixture with 2 ml of n-amyl alcohol and the absorbence of the organic phase was measured. Standard curves were prepared using known ferric chelate concentrations reduced with excess thioglycolate. RESULTS Stimulation of NADPH-Cytochroae P450 Ageductaae-Depenaent NADPH Oxidation by Ferric Chelates: The rate of NADPH oxidation catalyzed by the reductase in aerobic solution is shown in Table 1. The ability of EDTA-Fe3+ and DTPA-Feat to markedly stimulate basal NADPH oxidation rates is also shown. The stimulation by these chelates was dependent upon iron as evidenced by the inability of the chelating agents to stimulate NADPH oxidation in the absence of added iron. Neither ADP-Fe3+ nor desferrioxamine-Fe3t stimulated NADPH oxidation by the reductase to any significant extent. From these data it could not be determined whether EDTA-Fe3+ and DTPA-Fe3+ were actually substrates for the flavoprotein reductase or simply acted in a catalytic fashion to induce the reductase to generate 02? via autoxidation of reduced flavins. Therefore, the effect of varying concentrations of DTPA-Fe3+ and EDTA-Fe3+ on NADPH oxidation was investigated and the results are shown in Figure 2. Both DTPA-Fe3+ and EDTA-Fe3* appeared to act as substrates for the reductase having apparent Km’s of 540 f" and 170 PM respectively. EPR Spin Trapping: Evidence suggesting the direct reduction of EDTA-Fe3t by the reductase was also obtained with EPR spin trapping. The ability of a xanthine-xanthine oxidase 64 65 Table 1. Effect of Ferric Chelates on the Rate of NADPH Oxidation by NADPH-Cytochrome P450 Reductase NADPH oxidation (nmollunit) Complete System 1 +EDTA l +EDTA-Fe3+ 130 +DTPA 2 +DTPA-Fe3+ 220 +ADP 2 +ADP-Fe3t 1 +desferrioxamine l +desferrioxamine-Fe3* l The complete system contained NADPH (0.1mM), purified reductase (0-007u/ml) and where indicated, chelator or ferric chelates in 0.3M NaCl pH 7.0 at 37°C. The concentration of Chelators and chelates was 0.11 mM chelator and 0.1 mM FeCla except for ADP which was 0.5 mM. NADPH oxidation was expressed per unit of cytochrome c reductase activity. 66 .macwuflhun—OOOOO OudHOAO Owhhvh On» gaseous vouuoHa one: moflummusoosoo oumHoao amazon some as Aaa\swm\fiossv newumvwxo mmnwu an: we newumamawum "m ossmfim 78 ..EE .nedasm \ _ ON 0. b d L h J P h d n .225 £398: u in ON 00 d d (D 0 0 8 o' o 3, o l_(|u1/ugui/|ouJU) 000.0 0.0.0 sill/low") M I K) 5, O _uw/u All 79 .toom1m9< mo modumuuaoosoo on» unsfisus vouuoam one: sowusuusoosoo +nomlma< some as newusvwxo mmnsmn< .+nvhlmn< hp nodumvwxo mae gum: A23 Hv mmo H202 ' (2) EDTA-Fe2* + H202 ----- > EDTA-F83+ + 'OH + OH‘ (3) Superoxide dismutase readily inhibits iron reduction by 02: (1). Therefore, in the xanthine oxidase system that serves as a control, SOD completely inhibits the DMPO-OH signal intensity, whereas when EDTA-Fe3+ reduction is via NADPH and the purified reductase, SOD has no apparent effect on the DMPO-OH signal intensity. 89 The direct reduction of ferric chelates was demonstrated by incubating the reductase, rat liver microsomes or a reconstituted MFO system with NADPH under anaerobic conditions. Under these conditions, it is unlikely the observed rate of iron reduction is in fact the actual rate, since it is extremely difficult to completely prevent ferrous chelate oxidation during the incubation and handling of the sample. Nevertheless, ferrous chelate formation under these conditions did occur, demonstrating the potential of microsomes to directly reduce several iron chelates commonly used in lipid peroxidation. From a thermodynamic standpoint reduction of most ferric chelates by 02? should be a favorable process since the redox potential of the 02/02? couple is -0.33v31 in aqueous solution. Most iron chelates have negative redox potentials. The redox potential of hydrated ferrous is- 0.77v,32 but it is difficult to test the reduction of ferric since it is highly insoluble at neutral pH. Chelators maintain ferric in a soluble form, permitting iron reduction to occur, but alter the redox potential of the iron. However, from a kinetic standpoint, chelators can also affect iron redox activity. In general oxygen radical redox reactions tend to ' be inner-sphere electron transfer processes, so chelators that hinder the access of 02: to the metal can inhibit its reduction. This was indeed the case with desferrioxamine as well as several other ferric chelators tested by Graf t 1.33 In agreement with their 90 results, desferrioxamine was not reduced by the microsomal electron transport system in this investigation. In contrast to 02?-dependent reduction, the direct reduction of ferric chelates might be expected to exhibit some substrate specificity, and indeed this is what is observed in the present study. The purified reductase reduces both EDTA-Fe3+ and DTPA-Fe3t but not ADP-Fe3+ whereas rat liver microsomes reduce ADP-Fe3+ as well as the other two ferric chelates. The reduction of EDTA-Feat and DTPA-Fe3+ by microsomes would appear to occur at the flavoprotein reductase, but the site of ADP-Fe3t reduction is not clear. The reduction of ADP-Fe3+ by a reconstituted MFO system occurs as well, so this would suggest that cytochrome P450 might be the microsomal enzyme ultimately responsible for reducing ADP-Fe3t. In addition, per unit of reductase activity, microsomes oxidize more NADPH (in the presence of EDTA-Fe3+ or DTPA-Fe3t) than the purified reductase, suggesting that EDTA-Fe3+ and DTPA-Feat may also be reduced at other sites on the electron transport chain, perhaps at cytochrome P450. LIST OF REFERENCES P. Hochstein and L. Ernster. ADP-activated lipid peroxidation coupled to the TPNH oxidase system of microsomes. Biochaa, Biophys. Bes. 00222 22: 388-394 (1963). P. Hochstein, K. Nordenbrand, and L. Ernster. Evidence for the involvement of iron in the ADP-activated peroxidation of lipids in microsomes and membranes. 2iochea2 Biophya. Rea. 00522 22: 323-328 (1964). A. Beloff-Chain, G. Serlupi-Cresenzi, R. Cantanzaro, D. Venettacci, and M. Balliano. Influence of iron on oxidation of NADPH in rat liver microsomes. Biochim. Biophys. Acta 21: 416-421 (1965). T.C. Pederson and S.D. Aust. NADPH-dependent lipid peroxidation catalyzed by purified NADPH-cytochrome c reductase from rat liver microsomes. Biochea. Biophya2 Res. Comm. 22: 789-795 (1972). D.J. Kornbrust and R.D. Mavis. Microsomal lipid peroxidation. I. Characterization of the role of iron and NADPH. Mol. Pharmacol. 21: 400-407 (1980). T. Noguchi and M. Nakano. Effect of ferrous ions on microsomal phospholipid peroxidation and related light emission. Biochem. Biophys. Acta 368: 446-455 (1974). R.L. Fong, P.B. McCay, J.L. Poyer, B.B. Eeele, and H. Misra. Evidence that peroxidation_ of lysosomal membranes is initiated by hydroxyl free radicals produced during flavin enzyme activity. J. Biol. Chem. 248: 7792-7797 (1973). C.-S. Lai and L.H. Piette. Spin-trapping studies of hydroxyl radical production involved in lipid peroxidation. Ayah. Biochea. Biophys. 222: 27-38 (1978). J.R. Bucher, M. Tien, and S.D. Aust. The requirement for ferric in the initiation of lipid peroxidation by chelated ferrous iron. Biochea. Biophys. Res. Comm. 222: 777-784 (1983). 91 I | 19 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 92 J.F. Roster and R.G. Slee. Lipid peroxidation of rat liver microsomes. Biochia2 Biophys Acta. 620: 489-499 (1980). R.G. Mimnaugh and M.A. Trush. Superoxide anion- dependency of NADPH-dependent rat liver microsomal lipid peroxidation as demonstrated by the inhibition of peroxidation by superoxide dismutase. In: Oxy Radica2a and Their Scavenger Systems. Volume I: Molecular Aspects (G. Cohen and R.A. Greenwald, eds.) pp. 300- 303, Elsevier, NY (1983). T.C. Pederson, J.A. Buege, and S.D. Aust. Microsomal electron transport: The role of NADPH-cytochrome c reductase in liver microsomal lipid peroxidation. 12 Biol. Chea2 222: 7134-7141 (1973). P. Strittmatter and S.F. Velick. A microsomal cytochrome reductase specific for NADH. J. Biol. Chea. 221: 277-286 (1956). C.H. Williams, Jr. and H. Kamin. Microsomal triphosphopyridine nucleotide-cytochrome c reductase of liver. J. Biol. Chem. 237: 587-595 (1962). M. Tien, B.A. Svingen, and S.D. Aust. An investigation into the role of hydroxyl radical in xanthine oxidase- dependent lipid peroxidation. Arch. Biochem. Biophys. 222: 142-151 (1982). M.H. Bilimoria and H. Kamin. The effect of high salt concentrations upon cytochrome c, cytochrome be and iron-EDTA reductase activities of liver microsomal NADPH-cytochrome c reductase. Ann. N.Y. Acad. Sci. 222: 428-448 (1973). T.C. Pederson and S.D. Aust. Aminopyrine demethylase: Kinetic evidence for multiple microsomal activities. Biochea. Pharmacol. 22: 2221-2230 (1970). Y. Yasukochi and B.S.S. Masters. Some properties of a detergent-solubilized NADPH-cytochrome c (cytochrome P450) reductase, purified by biospecific affinity chromatography. J. Biol. Chaa2 222: 5337-5344 (1976). J.D. Dignam ande.W. Strobel: NADPH-cytochrome P450 reductase from rat liver: Purification by affinity chromatography and characterization. Biochemistry 22: 1116-1123 (1977). 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 93 D.J. Waxman and C. Walsh. Phenobarbital-induced rat liver cytochrome P450: Purification and characterization of two closely related isozymic forms. J. Biol. Chea2 221: 10446-10457 (1982). S.B. West, M.-T. Huang, G.T. Miwa, and A.Y.H. Lu. A simple and rapid procedure for the purification of phenobarbital-inducible cytochrome P450 from rat liver microsomes. Arch. Biochea, Biophya2 222: 42-50 (1979). T. Omura and R. Sato. The carbon monoxide-binding pigment of liver microsomes. II. Evidence for its heme protein nature. J. Biol. Chea2 222: 2370- 2378 (1964). R.F. Beers, Jr. and I.W. Sizer. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Che22 195: 133-140 (1952). J.M. McCord and I. Fridovich. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J. Biol. Chea. 244: 6049-6055 (1969). O.H. Lowry, N.J. Rosebrough, A.L. Farr and H.J. Randall. Protein measurement with the folin phenol reagent. J. Biol. Chea. 193: 265-275 (1951). P.E. Smith, R.I. Krohn, G.T. Hermanson, A.H. Mallia, F.H. Gartner, M.D. Provenzano, R.E. Fujimoto, N.M.Goeke, B.J. Olson, and D.C. Klenk. Measurement of protein using bicinchoninic acid. Anal. Biochea2 222: 76-85 (1985). M.G. Redinbauch and R.B. Turley. Adaptation of the bicinchoninic acid protein assay for use with microtiter plates and sucrose gradient fractions, Anal. Biochem. 153: 267-271 (1986). E. Finkelstein, G.M. Rosen, and E.J. Rauckman. Spin trapping of superoxide and hydroxyl radicals: Practical aspects. Arch. Biocaaa. Biophya2 222: 1-16 (1980). Y.A. Ilan and G.' Czapski. The rection of superoxide radical with iron complexes of EDTA studied by pulse radiolysis. Biochia. BiophyaagActa2 498: 386-394 (1977). 30. 31. 32. 33. 94 V. Massey, S. Strickland, S.G. Mayhew, L.G. Howell, P.C. Engel, R.G. Matthews, 8. Schuman, and P.A. Sullivan. The production of superoxide anion radicals in the reaction of reduced flavins and flavoproteins with molecular oxygen. Biochem. biophya. res. c0222 22: 891-897 (1969). J.A. Fee and J.S. Valentine. Chemical and physical properties of superoxide. In: Superoxide and Superoxide Disaatases (A.M. Michelson, J.M. McCord, and I. Fridovich, eds.), pp. 19-60, Acadaaic Presa, New York (1977). W.M. Latimer. Oxidative Potentials, Prentice-Hall, Englewood Cliffs, NJ (1952). E. Graf, J.R. Mahoney, R.G. Bryant, and J.W. Eaton. Iron-catalyzed hydroxyl radical formation: Stringent requirement for free iron coordination site. J. Biol. Chem. 259: 3620-3624 (1984). CHAPTER II Superoxide Production by Mixed-Function Oxidase Components 95 ABSTRACT Superoxide production by purified reductase, rat liver microsomes, and reconstituted MFO system containing reductase, DLPC, and one of several purified cytochrome P450 isozymes, was determined using an acetylated cytochrome c reduction assay. The reductase generated only a very low rate of 02? production (0.2% of its reduction capacity towards cytochrome c) but 02: production was stimulated by EDTA-Fe3+ and DTPA-Fe3t, the ferric chelates that were previously shown to stimulate NADPH oxidation and be anaerobically reduced by the reductase. Microsomes generated considerably more 02: than the reductase alone, presumably due to the presence of cytochrome P450 isozymes. Several cytochrome P450 isozymes were tested for their potential to reduce oxygen to 02? in the presence of the reductase and DLPC. Cytochrome P450d generated slightly more 02: than did cytochrome P450b and both generated more than cytochrome P450c. Superoxide production required the same components necessary for successful reconstitution of MFO activity, detergent-solubilized reductase, DLPC and cytochrome P450. Superoxide production in the reconstituted system was confirmed using EPR spin trapping with DMPO. The ability of microsomes isolated from rats pretreated with various inducers of the MFO system to oxidize NADPH and produce 02? and H202 was also examined. In agreement with 96 97 the literature, microsomes from rats induced with phenobarbital generated more 02? and H202 than microsomes from untreated or HBB-treated animals. Despite the ability of HBB to induce cytochrome P450d the isozyme which stimulated 02? production in the reconstituted system, microsomes from HBB-induced animals did not generate appreciably more 02? and H202 than microsomes from phenobarbital-induced animals. INTRODUCTION There are numerous enzyme and enzyme systems that have been shown to generate 02? and H202.“2 However, as of yet it has not been possible to determine the significance of the production of oxygen radicals and in most situations an accurate quantitation of oxygen radical production 2a y2ya has not been made. Thus, the quantitation of oxygen radical production 2a y22;a is a crucial first step towards the eventual understanding of the potential significance of oxygen radical production 2a y2ya. One of the enzyme systems that has received a great deal of attention with respect to oxygen radical generation is the microsomal MFO system. This electron transport chain catalyzes the monooxygenation of numerous substrates, both endogenous and exogenous. During oxidation of drug substrates and even in the absence of substrates, this electron transport system has been shown to produce 02? and H202. Based on initial studies where SOD, and copper complexes having dismutase activity were purported to inhibit MFO activity,3'4 it was suggested that the reduction of cytochrome P450 might be via 02? generated by the reductase.° However, more recent.studies have demonstrated that the reductase reduces cytochrome P450 directly. Significant disagreement still exists over the ability of the reductase to generate 02: 2a vitro.5‘° The previous 98 99 results in Chapter I indicated that EDTA-Fe3t, DTPA-Fe3+ and perhaps other iron chelates may significantly enhance NADPH oxidation by the reductase, and this may be a factor in rates of 02: measured in various systems. Moreover, previous reports in which the epinephrine assay was used to quantitate 02? production have since been shown to be suspectfl"11 Regardless of the ability of the reductase to generate 02?, several lines of evidence indicate that the isozymes of cytochrome P450 are directly responsible for the bulk of microsomal oxygen radical generation. Higher rates of H202 production have been observed with microsomes isolated from rats pretreated with cytochrome P450 inducers.12 Many chemicals that are metabolized by the hepatic MFO system stimulate H202 production.12 Inhibitors of cytochrome P450 such as carbon monoxide13 and metyrapone,1‘ also inhibit microsomal oxygen radical production, and purified oxyferrous complexes of cytochrome P450 isozymes have also been shown to autoxidize or dissociate generating 02? and H202.15'17 There have been several studies in which the production of 02: and H202 by microsomes have been studied. However, it is difficult to make many generalizations about the results for a number of reasons. First of all, the measurement of H202 and especially 02? is inherently difficult due to the transient nature of these partially ,reduced oxygen species. 100 As previously mentioned, the type of assay can dramatically affect the results that are obtained. Cytochrome c reduction is usually considered as the most reliable assay for 02? production, but it cannot be used for quantitation of microsomal 02? production since it is an excellent substrate for the reductase. Thus, investigators have modified cytochrome c by acetylation or succinoylation to reduce its tendency for direct reduction by the reductase but yet maintain its potential for reduction by reductants such as 02?.”?20 Secondly, SOD and catalase frequently contaminate microsomal preparations and scavenge 02? and H202, yielding erroneous results.21'22 Many investigators have added EDTA, or DTPA-""3””,23 which (if iron was present) could have lead to artificially high rates of NADPH oxidation as was shown in Chapter I and 02? production as will be demonstrated in this chapter. Also, recent results have shown that the production of H202 varies as the ratio of NADPH to substrate is altered.24 Thus, the method of preparation of microsomes and purified enzymes as well as reaction conditions may also affect the observed rates of 02? production. One aspect of particular interest and one in which very little information is available is the potentials or tendencies of the ,various cytochrome P450 isozymes to generate 02?. Cytochrome P450 isozymes exist in equilibrium .mixtures containing both high and low spin forms, and Several isozymes are isolated from microsomal membranes 101 predominantly in the high spin state,25'2° the spin state induced by the binding of many substrates29 and the conformation favored for the reduction of heme iron by the reductase.3° Reduction of heme iron by the reductase precedes the binding of molecular oxygen and its subsequent activation, so greater rates of oxygen activation could be predicted to occur under conditions where more high spin cytochrome P450 is present. The treatment of animals with various xenobiotics results in the induction of different complements of cytochrome P450, isozymes. The toxicity exhibited by these xenobiotics could conceivably be due, at least in part, to their induction of isozymes with increased tendencies for uncoupled reduction of oxygen. However, it is not clear whether the rate of 02? production by microsomes correlates with the presence of particular isozymes or with the fraction of isozymes in the high spin conformation. Relatively few studies have addressed the oxidase activities of purified cytochrome P450 isozymes. A few studies have investigated 02? production by partially purified preparations of MFO components.31'32 A greater number of investigators have been. concerned with oxygen radical production by microsomes and the overall percentage of NADPH oxidation that results in the uncoupled reduction of oxygen to H202 or water in microsomal or reconstituted MFO assays.2°-33‘35 Thus, the following studies were initiated to better characterize the uncoupling of the 102 microsomal MFO system and the experimental factors that influence the measurement of oxygen radical production by microsomes or the reconstituted system. MATERIALS AND METHODS flarification of Cytochroae P450c and P450d: Male Sprague Dawley rats (150—250 g) were pretreated with HBB (10 Pmol/kg) given by gavage and sacrificed 3 days later. Liver microsomes were isolated as previously described in Chapter I and solubilized with sodium cholate added to a final concentration of 0.83 (w/v). Solubilized microsomal protein was subjected to affinity chromatography on N-octylamino Sepharose.3° The cytochrome P450 fraction eluting at 0.33 sodium cholate, 0.13 Lubrol PX (Sigma) was dialyzed and then applied to a DEAE-Sepharose column and chromatographed by the method of Kstrom and DePierre.3° The pools of fractions containing cytochrome P4500 and P450d were each subjected to hydroxyapatite chromatography, washing with 2 column volumes of 30, 90, and 180 mM phosphate buffer. Purified cytochrome P450c eluted with 90 mM phosphate whereas cytochrome P450d eluted with 180 mM phosphate. Preparations of these cytochrome P450 isozymes were homogeneous on SDS gel electrophoresis and had specific contents of 12-18 nmol/mg protein. Acetylation of Cytochroae c: Cytochrome c (Sigma) was acetylated using the procedure of Wade and Okunuki.37 A 1 mM cytochrome c solution in half-saturated sodium acetate on ice was treated with a 20-fold molar excess of acetic 103 104 anhydride over the constitutive lysine residues. The constantly stirred solution was maintained at pH 7.0 over the course of reaction through the dropwise addition of l N NaOH. After 30 min, the reaction mixture was dialyzed against 200 volumes of distilled H20 at 4°C (three changes). The dialyzed protein solution was centrifuged at 10,000xg for 20 min to remove any insoluble material. The resulting supernatant was adjusted to pH 7.5 and applied to a l x 20- cm column of DEAR-cellulose (Whatman, Clifton, N.J.) previously equilibrated with 5 mM HHzPOc, pH 7.0. The column was first washed with 150 ml of equilibration buffer before the bulk of the acetylated cytochrome c was eluted with 0.2 M KHzPOa, pH 7.0. Acetylated cytochrome c was dialyzed against distilled H20 before lyophilization. Prior to use, the lyophilized preparation was dissolved in 0.3 M NaCl, and EDTA was added to 10 mM final concentration. The use of EDTA was intended to chelate any divalent cations loosely associated with the acetylated protein. This solution was incubated on ice for 1 hr. The solution was centrifuged at low speed to remove any insoluble material prior to chromatography on Sephadex G-25. The percentage acetylation of cytochrome c ranged from 95-993 using picrylsulfonic acid assay.19 Acetylated Cytochroae c Redaction. The reduction of acetylated cytochrome c was monitored spectrophotometrically at 550 nm with a Cary 219 recording spectrophotometer. The 105 cuvette chamber was maintained at 37°C. The initial rate of acetylated cytochrome c reduction was monitored in each instance, and a value of 21 mM'1cm‘1 for the extinction coefficient of acetylated cytochrome c was used. When 02? production by a reconstituted system was monitored, cytochrome P450 isozymes were preincubated with the reductase and DLPC for 5 min prior to the addition of the other reagents. When no preincubation was performed, initial rates of NADPH oxidation and 02: production were not maximal indicating that efficient complex formation had not occurred in the diluted solution. Hydrogan Peroxide Prodagtion: Hydrogen peroxide was measured by a modification of the procedure employed by Hildebrandt and Roots.12 Microsomes (0.2 mg/ml) were incubated with NADPH (0.5 mM) in the presence of azide (3mM) and desferrioxamine (1 mM) at 37° C in a shaking water bath. Azide was added to prevent catalatic decomposition of H202 and desferrioxamine was used to inhibit non-heme iron- catalyzed decomposition of H202. Aliquots from the incubation mixture were quenched in ice cold 33 TCA and allowed to stand on ice for 20' min before they were centrifuged at low speed for 10 min to pellet precipitated material. Supernatants (0.66 ml) were neutralized with 2 M triethanolamine (0.06 ml). Finally, o-dianisidine and horseradish peroxidase (l‘pg/ml) were added and the H202- dependent oxidation of o-dianisidine38 was monitored at 106 460 nm and compared to a standard curve prepared with known concentrations of H202. it Fe 0m de Cy' RESULTS Redaction of Acetylated Cytochroae c by Xanthine Oxidaaegaa_ NADPH Cytochroae P450 Reductaaa; The reduction of acetylated cytochrome c by 02? produced by xanthine oxidase is shown in Figure 9. The rate of reduction was linear for at least 5 min when catalase was included in the incubation mixture. No reduction of acetylated cytochrome c was observed when SOD was added. Purified reductase also catalyzed acetylated cytochrome c reduction (Figure 10). However, the rate of reduction was linear either in the presence or absence of catalase and was inhibited only slightly by SOD. The difference between the rate of acetylated cytochrome c reduction in the presence and in the absence of SOD was used as the measure of 02? generation. Superoxide Generation by Xanthine Oxidase: Effect of Iron Chelates: The effects of several chelators and ferric chelates on the production of 02? by xanthine oxidase are shown in Table 6. Superoxide production was calculated as the fraction of the acetylated cytochrome c reduction rate inhibited by SOD per unit of unmodified cytochrome c reduction. Using. this assay,. approximately 663 (658 nmol/unit) of the 02? produced by xanthine oxidase could be detected, although by either increasing the acetylated cytochrome c concentration or decreasing the xanthine 107 108 .Asa\= ewes uneseoeo to Ase\e emmv eom enemaseoo ease meeseeneoea «new .e.e we .Hoez z m.e es AHa\ua m.HV o usesn00u>o oeuoamuoos use .Aza bfi.ov onwaussx .AH3\= No.ov onmvfixo sawdusnx monwsusoo anewumnsosH mmmmwmm ocwnusmx up u oaosnooumo voumahuoo< mo seguosvom "m shaman 109 efiamzsk 0 n N _ l n e - - w, - o dom+ no.0 03—0500 I..O_.O 1.9.0 loud fl u .0 3228... mm (““1 099) A.LISNBO 'lVOlldO 110 0.25 " .. p 8 l 1 + Col\ol.ose Complete E I: O U) E! t 0.15- - B I! DU 0 0.10 .. .J 4 C) § 0.05 .. 0. O 0 ~l L 1 J l___1 O l 2 3 4 TIME (min) Figure 10: Reduction of Acetylated Cytochrome c by NADPH-Cytochrome P450 Reductase Incubations contained purified reductase (0.015 u/ml), NADPH (0.1 mM), and acetylated cytochrome c (1.6 mg/ml) in 0.3 M NaCl, pH 7.0. Some incubations also contained SOD (230 u/ml) or catalase (460 u/ml). 111 Table 6. Superoxide Production by Xanthine Oxidase Acetylated Cytochrome c Superoxide Reduction Production (nmollunit) (nmol/unit) Complete system 660 660 (100) +800 0 +EDTA 580 580 (88) +EDTA + SOD 0 +EDTA-Fe3+ 530 530 (80) +EDTA-Fe3t + SOD 0 +ADP 570 570 (86) +ADP + SOD 0 +ADP-Fe3+ 550 550 (83) +ADP-Fe3+ + SOD 0 +DTPA 600 600 (91) +DTPA + SOD 0 +DTPA-Feat 620 620 (94) +DTPA-Fe3t + SOD 0 +Desferrioxamine 650 650 (98) +Desferrioxamine + SOD 0 +Desferrioxamine-Fe3+ 620 620 (94) +Desferrioxamine—Fe3t+SOD 0 Incubations contained xanthine oxidase (0.001 u/ml), xanthine (0.17 mM), acetylated cytochrome c (1.6 mg/ml), and catalase (460 u/ml) in 0.3 M NaCl, pH 7.0. Incubations also included SOD (230 u/ml), chelator (0.11 mM), or ferric chelate (0.11 mM chelator, 0.1 mM FeCla) as indicated. ADP and ADP-Fe3+ concentrations were 0.5 mM and 0.5 mM; 0.1 mM FeCla, respectively. Values in parentheses refer to the percentage of 02? production in the complete system. 112 oxidase concentration, greater percentages of 02? were detected. Any one of the chelators or ferric chelates .tested caused a modest inhibition of apparent 02? production by xanthine oxidase, but under no circumstances did acetylated cytochrome c reduction occur in the presence of SOD. 2aperoxide Prodaction by Purified NADPH Cytochroae P450 Reductase: Effect of Iron Chelatea. Acetylated cytochrome c reduction and 02? production by the purified reductase are shown in Table 7. The significant differences between xanthine oxidase and reductase-dependent reduction of acetylated cytochrome c apparent in Figures 9 and 10 are also evident from the data comprising Table 7. The addition of SOD to the incubations containing the reductase did not completely inhibit acetylated cytochrome c reduction. Superoxide production by the reductase was 2.3 nmol/unit cytochrome c reductase activity (0.23 of its ability to reduce unmodified cytochrome c). In contrast to their inhibition of xanthine oxidase-dependent acetylated cytochrome c reduction, all iron chelates that were tested enhanced reductase-dependent acetylated cytochrome c reduction. Ferric chelates enhanced both 02?-dependent and 02?-independent rates of acetylated cytochrome c reduction. Particularly noteworthy was the stimulation of apparent 02: production by the reductase when either EDTA-Fe3+ or DTPA- Fe3* was included in the assay, for these iron chelates are 113 Table 7. Superoxide Production by Purified NADPH-Cytochrome P450 Reductase Acetylated Cytochrome c Superoxide Reduction Production (napllanit), (nmol/unit) Complete system 9.7 2.3 (100) +SOD 7.4 +EDTA 13 4.8 (209) +EDTA + SOD 8.2 +EDTA-Fe3t 93 42 (1826) +EDTA-Fe3t + SOD 51 +ADP 16 7.1 (309) +ADP + SOD 8.9 +ADP-Fe3t 24 12 (522) +ADP-Fe3+ + SOD 12 +DTPA 9.4 1.3 (56) +DTPA + SOD 8.1 +DTPA-Fe3+ 140 20 (870) +DTPA-Fe3+ + SOD 120 +Desferrioxamine 13 3.1 (135) +Desferrioxamine + SOD 9.9 +Desferrioxamine-Fe3+ 11 3.0 (130) +Desferrioxamine-Fe3+ + SOD ' 8.0 Incubations contained NADPH (0.1 mM), purified reductase (0.014/ml) and acetylated cytochrome c (1.6 mg/ml) in 0.3 M NaCl pH 7.0 with SOD (230 u/ml), chelator (0.11 mM) or ferric chelate (0.11 mM:0.l mM FeCla) added as indicated. The concentration of ADP was 0.5 mM. Values in parentheses refer to the percentage of 02? production in the complete system. 114 often used in EPR spin trapping, lipid peroxidation, and reconstituted drug metabolism systems. These chelates were shown to be substrates for the reductase in the previous chapter. In the absence of reductase, ferrous chelates were shown to reduce unmodified cytochrome c as well as acetylated cytochrome c (data not shown). It was also observed that EDTA-Fe3t or DTPA-Fe3+ did not affect the rate of unmodified cytochrome c reduction. The stimulation by EDTA-Fe3+ or DTPA-Fe3+ of acetylated cytochrome c reduction by the purified reductase was concentration dependent in that increasing concentrations of ferric present as either the DTPA or EDTA chelate resulted in increasing rates of acetylated cytochrome c reduction (Figure 11). Relatively little stimulation of acetylated cytochrome c reduction in the presence of ADP-Fe3+ or desferrioxamine-Fe3t was observed. 2aperoxide Generation by Rat Liver Microaomes: Afiffect of Iron Chelates: NADPH-dependent acetylated cytochrome c reduction and 02: production by rat liver' microsomes is shown in Table 8. The rate of 02? production was 15.6 nmol/unit unmodified cytochrome c reductase activity. Therefore, 02? production accounted for only about 1.53 of the total NADPH-dependent reduction capacity of microsomes. Chelating agents (without added iron) had little effect on microsomal acetylated cytochrome c reduction or 02. production. However, ferric chelates, especially EDTA-Fe3+ 115 Figure 11: Stiaulation of Acetylated Cytochraaa c Redaction by Ferric Chelates Incubations contained purified reductase (0.01 u/ml), NADPH (0.1 mM), acetylated cytochrome c (1.6 mg/ml) and varying concentrations of ferric chelates in 0.3 M NaCl pH 7.0. The chelator: iron ratios were all 1.1:1 except ADP-Feat which was 5:1. The chelates used were: (0 ), DTPA-Fe3t; ( I ), EDTA-Feat; (' ), ADP-Fe3’; ((3 ), desferrioxamine-Fe3t. 116 3700* 200T 1700- I300- o p 0 mw 2 22:8 es 8532 o 052828 86334 3300* 2900' 250 200 50 FeCI3()1M) 117 Table 8. Superoxide Production by Rat Liver Microsomes Acetylated Cytochrome c Superoxide Reduction Production (naal/unit) Ajnmol/aait) Complete system 28 15 (100) +SOD l3 +EDTA 30 15 (100) +EDTA + SOD 15 +EDTA-Fe3+ 140 70 (467) +EDTA-Fe3+ + SOD 70 +ADP 26 9.0 (60) +ADP + SOD l7 +ADP-Fe3+ 61 33 (220) +ADP-Fe3+ + SOD 28 +DTPA 27 ll (73) +DTPA + SOD 16 +DTPA-Fe3+ 260 30 (200) +DTPA-Fe3+ + SOD 230 +Desferrioxamine 24 10 (67) +Desferrioxamine + SOD 14 +Desferrioxamine-Fe3+ 28 10 (67) +Desferrioxamine-Fe3t + SOD 18 Incubations contained NADPH (0.1 mM), rat liver microsomes (0.05 mg/ml) and acetylated cytochrome c (1.6 mg/ml) in 0.3 M NaCl pH 7.0 with SOD (230 u/ml), chelator (0.11 mM), or ferric chelate (0.11 mM; 0.1 mM FeCla) added as indicated. The concentration of ADP was 0.5 mM and ADP-Fe3+ was 0.5 mM ADP, 0.1 mM Fe013. Values in parentheses refer to the percentages of 02? production in the complete system. 118 and DTPA-Feat, stimulated the rate of acetylated cytochrome c reduction measured either in the presence or absence of SOD. Thus, ferric chelates appeared to enhance both 02?- dependent and 02?-independent pathways for acetylated cytochrome c reduction. Superoaide Production by the Reconatituted Mixed Function Oxidase System: The previous results demonstrated that microsomes generated more 02? than did the purified reductase when normalized to equal reductase activities. This, along with reports from the literature indicated that increased 02? production by microsomes was probably due to the presence of cytochrome P450. This was confirmed by the results of experiments shown in Figure 12 in which purified cytochrome P450 isozymes were reconstituted with detergent- solubilized reductase in DLPC micelles, the standard reconstituted system shown by researchers to form a competent electron transport chain capable of oxidizing various MFO substrates. The addition of increasing cYtochrome P450 isozyme concentrations resulted in increased o2=iproduction. Cytochrome P450d produced slightly more 02? than did cytochrome P450b and either produced more than QYtochrome P450c. Superoxide production by all isozymes increased until the molar ratio reached about 4-5 nmol cYtOChrome P450/nmol reductase. Beyond that ratio the rate 0f 02? production remained the same suggesting that the 119 I I I I I I I ‘ P4 d 018- 50 " 75' 1 "\g 015- ‘9 P450b E E 012- P450c . J5 ll c: .9 .. *5 0.09 3 “U 9 9- 0.06 ‘ l-N C) 0.03 “ 0' L l L A l I l l I 0 l0 2.0 3.0 4.0 P450 / Reductase Figure 12: 2ffect of Increasing;Cytochroae P450: NADPH Cytochrome P450 Reductase Ratios on Supapoxide Prodaag1aa in the Reconatituted System: Incubations contained (0.012 nmol reductase), NADPH (0.1 mM), acetylated cytochrome c (1.6 mg/ml), catalase (500 u/ml), DLPC (20 ‘pg/ml), desferrioxamine (0.5 mM) and varying amounts of cytochrome P450 isozymes in 0.05M Tris pH 7.5. Duplicate incubations containing SOD (150 u/ml) were also monitored and 02? production was calculated as before. The cytochrome P450 isozymes used were: ( I ), cytochrome P450d: ( O ), cytochrome P450b; (‘ ), cytochrome P450c. 120 reduction of cytochrome P450 by the reductase was becoming rate-limiting. However, increasing the concentration of reductase relative to cytochrome P450 isozymes resulted in a continual increase in 02? production (Figure 13), presumably due to the potential of the reductase to generate 02?, regardless of the presence of cytochrome P450 isozymes. The effects of omitting various reagents from the acetylated cytochrome c reduction assay on the observed rate of 02? production is shown in Table 9. Conditions necessary to reconstitute MFO activity (cytochrome P450, detergent- solubilized reductase and DLPC) are also necessary for 02? production. On the other hand, the omission of desferrioxamine or catalase, added as precautionary measures to prevent iron-stimulated 02? production or H202-dependent ferroacetylated cytochrome c oxidation respectively, generally had lesser effects on the observed rates of 02: production. These results were confirmed using EPR spin trapping (Figure 14-16). The DMPO-OOH adduct was observed in incubations containing purified cytochrome P450 isozyme, detergent-solubilized, and DLPC. However, the adduct signal was not as unequivocal as that observed in the xanthine oxidase system (Chapter I, Figure 3); one can clearly observe both DMPO-OOH and the appearance of the DMPO-OH adduct over the time of the scan. That the formation of DMPO-OH was primarily due to the decomposition 121 I ' I ' i I I I l P450d 0.24 r - 0.2l - ' ' 5 £5. E P450b 5 015 - - ' I: .9 g O.l2 - . “U 9 0L .. N0.09 - O a 0.06 . 0’ 0.03 . O l I l l I l I I I 0 10 2.0 3.0 4.0 Reductase/ P 450 Figure 13: §2ject of Increasing NADPH-CyAaahroae P450 Reductaae: Cytochrome P450 EatioaA on Saperoxide Production in the fieconatituted System: Incubations contained cytochrome P450 isozymes: ( I), cytochrome P450d; ( Q ), cytochrome P450b, and ( A ), cytochrome P450c, 0.012 nmol/m1, NADPH, acetylated cytochrome c, catalase, DLPC, desferrioxamine and SOD at the concentrations specified in Figure 12. The concentration of reductase was adjusted to obtain the indicated ratios. 122 Table 9. Requirements for Superoxide Production by the Reconstituted Mixed Function Oxidase System P450b P450c P450d Complete system 1003 1003 1003 -P450 isozyme 113 253 83 -DLPC 183 513 373 -Catalase 1303 1003 933 -Desferrioxamine 583 973 753 +Protease-solubilized reductase 63 223 33 The complete system contained DLPC (20 jug/m1), desferrioxamine (0.5 mM), acetylated cytochrome c (1.6 mg/ml), catalase (500 u/ml) NADPH (0.1 mM), cytochrome P450 isozymes (0.1 nmol/m1) and reductase (0.025 nmol/m1) in 50 mM NaCl pH 7.0 at 37°C. Duplicate incubations with SOD (150 u/ml) were also monitored. Superoxide production was calculated from the differences in the rate of acetylated cytochrome c reduction in incubations with and without SOD and expressed as a percent of the complete system. 123 .:m cod no: moses o>mzoso«m on» uaooxo H moussso aw vosflfiuso no use» on» one: nusauuue souoaouuoomm .6 mm Homz 23 cm cw A23 000 case was AHB\= ombv oanouso .Aza m>.oV omfiamxowuuownov .23»... one 0.3: .3535. 398 33268 AHE\Hoam bH.oV somem oSOLAUOumo posmmusoo nmowuonsomn Amusemm sensuaumeooem one an comes unwanooumo an mooomvosm ovwxosoasm mo suuooam- mum “on ossmwh 124 25%;: 13>»: 00. 22:3 22.32% 388$ + 058220.23me 1 .3238 - 05o . 8m + 539m 3238 125 .vovvs as: A~m\aoms 6H.ov oomvm osounUOuho awn» newuaooxo on» new: ea ousuwh aw veawsuop no omen on» one anewunnsosm a eeeefifleeoom of 5 domed emeteoeilo an voosvosm ovflxouomwm mo ospoomm mmm "ma ousmwm 126 $2263”. g 8938 + $69.20 12;)? mEonoEfimmo I iii/gig mmO_O«UU I... 00. E396 Emano 127 .vomvs as: AHB\Hoas 5H.ov venom omosn00umo ass» seauaooxo on» new: vH ousuwm a“ voHMauov no name on» use asowumnsosH snowman eummssemeoomm we» es eemee emoteoeeso an wwwmvoam ovwxozommm Mo mauoomm mum "0H ossmfim 128 :15: 00. 82633”. 2.2.9.8 + 30882 «5682:3me l .3228 I 00.5 I oow + nomad I 88.0.3 829:8 129 of the DMPO-OOH was demonstrated by showing that the addition of SOD inhibited both the DMPO-OOH and DMPO-OH signals. The inability of desferrioxamine, which inhibits iron-catalyzed -OH formation39 to inhibit the DMPO-OH signal intensity is further indication that DMPO-OH arose mainly from DMPO-OOH decomposition. Pitfalls in Measuring Oxygen Radical Production by Microsomes: Although it is of interest to determine 02? production by individual forms of cytochrome P450, of perhaps greater importance is the production of 02? by the complement of cytochrome P450 isozymes present in hepatic microsomes. However, oxygen radical quantitation is inherently difficult, but in reconstituted MFO systems, there is usually no problem with inadvertent scavenging of 02? or H202. Ouantitation of oxygen radical production by microsomes is considerably more difficult however, and depending upon the isolation procedure, microsomes may contain varying amounts endogenous SOD activity, catalase activity and even ferritin which also has' been shown to scavenge 02?.22 Some investigators have attempted to account for microsomal scavenging of 02? by measuring the decrease in 02? production by the xanthine-xanthine oxidase system in the presence of microsomes and obtaining a correction factor which they applied to NADPH-dependent 02? production to obtain the "true” value for 02‘ production.30 130 A similar approach was used to examine several microsomal preparations for their ability to inhibit 02?- dependent cytochrome c reduction by xanthine oxidase (Figure 17). Rat liver microsomes isolated by the routine differential centrifugation procedure dramatically inhibited cytochrome c reduction. Further washing of the microsomes with 1.153 HCl resulted in a preparation that inhibited cytochrome c reduction to a considerably lesser extent. Subsequent chromatography of the KCl-washed microsomes on Sepharose 2B resulted in microsomes having essentially no inhibitory effect on cytochrome c reduction. Similar effects of washing and chromatography on the catalase activity in microsomes were also noted (See Chapter III). However, even chromatography was not sufficient to remove the last traces of catalatic activity from microsomes. Therefore, azide, a potent catalase inhibitor, has been used to inhibit the remaining activity present in microsomes.12 The effect of azide on microsomal H202 production is shown in Figure 18. In the initial stages of the incubations, H202 was formed. However, in incubations without azide the final amount of H202 recovered from the incubations was not indicative of the total amount of H202 produced due to the Catalase activity in microsomes. This was true either for ,basal rates of H202 production, or in incubations where H202 production was stimulated by the redox cycling agent paraquat. In the presence of azide, catalatic decomposition of H202 was prevented, and H202 131 .A O v nan—H00 mm onossaaom s so vomamuuoumsosao 3:323: e228: .3 A I V Sm «24 it. venues essences“. .A ‘0 soflumuswwuusoo stusouommfiv hp vousaomw aolonosOMH so>wa use no anewumuusoosoo anewus> covsaosw omHs nmowumnsosfl .sOwuflvvs sH .m.> mm .nwua 2.. on S .22... 2: o 282033 can :5: 3.8 «2.3.8 sawdusmx .Aza mm.ov osflnusmx poswsusoo anewumasomfi HH< moaomosowz an sofiuomfiom o oaosnooufio usovsomoauuwwxosoamw mo cofiuwpwgsa "5H oaswflm 132 I I I I V U to b ‘9 " d ‘ A E \ . .8 0’ if 3 .E . Q) ' E . 0'6 ‘(D E O (I) .. .. g .9 2 . .o q. .. .0 N m d O a I J l s O a: :2 g o 0' d 9 (layman/og vv) uogpnpea 0 ewmuoouig 133 ' I I I I I 300*- 0 - a Q 200- o . E 75 E _ . 5 (£0 N 100 ~ I: A O _ 1 n l 0 8 l0 l2 Time (min) Figure 18: Time Courae of Microsomal Hydrogengaroxiaa Formation ~ Incubations contained microsomes (0.25 mg/ml), and NADPH (0.5 mM) in 0.05 M NaCl pH 7. Additions to the incubations were as follows: none, ( ‘ ); azide, (3 mM), ( A ); paraquat, (0.1 mM), ( Q ); paraquat plus azide, ( C) ). Hydrogen peroxide was assayed as detailed in Material and Methods. 134 accumulated over the entire time course of the of the experiment. The effect of several ferric chelates on H202 production by microsomes was also investigated. Most ferric chelates that were tested had an inhibitory effect on microsomal H202 production (Figure 19). This was attributed to iron-catalyzed decomposition of H202, a property characteristic of numerous ferrous or ferric chelates. Of the iron chelates tested, only desferrioxamine-Fe3+ had no effect on the accumulation of H202 relative to control incubations. Therefore, desferrioxamine was added to all subsequent assays to prevent the decomposition of H202 during the incubation. Stoichioaetry of Microsoaal Oxyggn Radical Production: The rates of NADPH oxidation and 02? production by liver microsomes isolated from untreated rats or rats pretreated with phenobarbital or HBB are compared in Table 10. Significantly higher rates of NADPH oxidation and 02? occurred in liver microsomes from phenobarbital- and HBB- pretreated animals than in untreated animals. This corresponded to an increase in cytochrome P450 content in these microsomes relative to control. Hydrogen peroxide production was assayed and the results are also shown in Figure 10. Again rates of H202 were greater in microsomes isolated from phenobarbital- or HBB—pretreatd rats than in those of control animals. 135 r d 80 - J . J ‘5: L d E 60 ‘x '5 E " d 5 N C%u‘"3” ' I 20 - .. 1- cl -—W C’0 5 o :5 20 25 Time (min) Figure 19: Effect of Ferric Chelates on Microaoma2 Hydrogen Peroxide Foraation: All incubations contained rat liver microsomes (0.26 mg/ml), and NADPH (0.5 mM) in 50 mM NaCl, pH 7.0 at 37°C. The following iron chelates were added (0.5 mM chelator: 0.1.mM FeCla) as- indicated: none, ( Q); desferrioxamine-Feat, ( II ); DTPA-Fe3t, ( )( ); EDTA- Fe‘”, ( O ); ADP-Feat, (‘ ). Hydrogen peroxide was assayed as detailed in Materials and Methods. 136 Table 10. Stoichiometry of Oxygen Reduction by the Microsomal Mixed Function Oxidase System Hydrogen NADPH Superoxide Peroxide Pretreatment Oxidation Production Production None 30 3.4 20 Phenobarbital 40 8.1 28 HBB 43 9.4 25 Microsomes were isolated from untreated rats or rats treated with phenobarbital or HBB (10 mg/kg in corn oil given by gavage). NADPH oxidation was monitored at 340 nm in incubations containing 0.1 mM NADPH in 0.05 M Tris pH 7.5. Hydrogen peroxide was measured as outlined in Material and Methods and 02? was quantitated as described in' Table 8. Values are in nmol/min/mg protein. 137 Even though cytochrome P450 activates molecular oxygen and is postulated to produce 02? and H202 via uncoupled reduction of oxygen, the possibility exists that the either in microsomes or the reconstituted system, the presence of cytochrome P450 isozymes may alter the electron transfer properties of the reductase such that generates 02? and/or H202.33 This hypothesis can be tested by examining the rates of NADPH oxidation and 02? production from liver microsomes isolated from rats treated with isosafrole. Isosafrole pretreatment results in the induction of cytochrome P450d, and its 2a y2ya metabolism results in the formation of a metabolite which binds tightly to the heme iron of cytochrome P450d, preventing the binding of CO or oxygen and the metabolism of substrates.°° Thus, these microsomes contain a specific isozyme previously shown to produce significant amounts of 02?, but that is specifically blocked by the isosafrole metabolite from activating oxygen. The metabolite can be removed by treating microsomes with butanol,“1 restoring its CO and oxygen binding capacities and its MFO activity. Comparison of the rates of NADPH oxidation and 02? production by microsomes with or without the metabolite allows an assessment of the contribution of P450d to the rates of oxygen activation in these microsomes. ThSB data are shown in Figure 11. .Butanol treatment of the ni<3rosomes restored CO binding capacity and resulted in a near 6-fold increase in 02? production. This was 138 Table 11. Effect of Butanol Pretreatment on NADPH Oxidation and Superoxide Production from Liver Microsomes Isolated from Isosafrole-Induced Rats Total NADPH Superoxide Cytochrome Cytochrome Oxidation Production P450 P450 Microsomes 36 5.2 1.45 2.75 Microsomes 52 31 2.25 2.50 + Butanol Microsomes were isolated from rats pretreated with isosafrole (150 mg/kg isosafrole in corn oil given by gavage daily for 4 days) by differential centrifugation and washed with 1.153 KCl, 10 mM EDTA pH 7.0 and then chromatographed on Sepharose 2B as in Figure 17. The isosafrole metabolite was displaced from microsomes by treating them with butanol (100 mM), for 1 hr. at room temperature prior to chromatography. Cytochrome P450 was determined by a carbon monoxide difference spectrum“2 and total cytochrome P450 was the sum of that determined with carbon monoxide and that of the isosafrole metabolite peak having A455-A490 of approximately 75 mM‘1.°3 Rates of NADPH oxidation and 02? production are in nmol/min/mg protein and cytochrome P450 contents are in nmol/mg protein. accompanied by oxidation. near 139 stoichiometric increase in NADPH DISCUSSION It was originally reported that the reductase produced 02?, however, the epinephrine oxidation assay used to detect 02? formation has since been demonstrated to be rather poor for quantitating 02?.“:11 The reduction of cytochrome c is perhaps the assay of choice for the measurement of 02? under many circumstances, but microsomes and the reductase catalyze the direct reduction of cytochrome c. Acetylation (or succinoylation) of ferricytochrome c limits its potential for direct enzymatic reduction. Therefore, the reduction of acetylated cytochrome c is a more specific measure of 02? production than the reduction of unmodified cytochrome c. However, modification of cytochrome c does not totally eliminate the possibility of its direct reduction. Therefore, a highly modified cytochrome c preparation with a minimal tendency for direct enzymatic reduction has been used in these investigations. The differences between xanthine ‘ oxidase- and reductase-dependent reduction of a highly acetylated cytochrome c preparation were readily apparent. Superoxide dismutase completely “inhibited xanthine oxidase-dependent acetylated cytochrome c reduction, but only partially inhibited reductase-dependent reduction. Also, the xanthine oxidase-dependent reduction of acetylated cytochrome c was not linear; a net oxidation of acetylated ferrocytochrome c 140 141 became evident after several minutes of reaction. Minakami had previously reported that the susceptibility to oxidation of acetylated cytochrome c was enhanced as its percent modification was increased.H Because xanthine oxidase may generate H202 directly or by 02? dismutation, the net oxidation of acetylated ferrocytochrome c that was observed was attributed to an accumulation of H202 in the incubation. The addition of catalase to the incubation prevented the oxidation of ferrocytochrome c, presumably by catalyzing the dismutation of H202 thus preventing its accumulation. Catalase was also added 3-4 min after the addition of xanthine oxidase (when a net oxidation of acetylated cytochrome c was occurring). In this case, oxidation of acetylated ferrocytochrome c was also prevented, and the rate of reduction of acetylated cytochrome c was equal to the rate of reduction in incubations to which catalase was added prior to xanthine oxidase. In contrast, the rate of reductase-dependent reduction of acetylated cytochrome c was linear in the absence of catalase and not affected by the presence of catalase. Superoxide dismutase did not greatly inhibit the rate 0f reduction. Therefore, these results are consistent with the reductase generating only small amounts of 02?. Efficient scavenging of 02? by acetylated .cytochrome c prevents a BiSnificant accumulation of H202, and prevents the oxidation 0f acetylated cytochrome that was observed with xanthine oxidase. These results would also suggest that the 142 reductase does not produce H202 by direct 2 electron reduction of oxygen in agreement with previous studies of its electron transfer properties.‘5 The rate of reduction of acetylated cytochrome c which was inhibited by SOD was used as the measure of 02' production. This technique capitalizes on the known specificity of SOD for 02?‘° and is, therefore, a more reliable method for quantitating 02? production. In some instances the rate of 02? production was expressed as a function of the unacetylated cytochrome c reductase activity of the purified reductase or intact microsomes. In this way the amount of 02? relative to the total NADPH-dependent reduction capacity of the reductase (or microsomes) became apparent. The reductase generated 2.3 nmol 02?/unit cytochrome c reductase activity, a rate which was approximately 0.23 of its NADPH-dependent reduction capacity. Because certain ferric chelates will react with 02?,‘7' ‘“ they may compete with acetylated cytochrome c and inhibit its rate of reduction. This was apparently the case with xanthine oxidase-dependent acetylated cytochrome ' c reduction, as added chelators or ferric chelates inhibited the reduction of acetylated cytochrome c by as much as 203. However, SOD still completely inhibited the reduction. In contrast, certain ferric chelates stimulated acetylated cytochrome c reduction by the purified reductase or intact microsomes. Both 02?-independent and 02:- 143 dependent rates of reduction were enhanced. The ferric chelates that markedly stimulated reductase-dependent acetylated cytochrome c reduction, and 02: production (EDTA- Feat and DTPA-Fe3t) also stimulated NADPH oxidation in the absence of acetylated cytochrome c (Chapter I). An explanation consistent with the data can be proposed. Oxygen must be considered as a poor substrate for the reductase relative to unmodified cytochrome c or cytochrome P450, its physiological electron acceptor. Therefore, 02? production by the reductase in the absence of more suitable electron acceptors appears to be a slow process, accounting for only a minor percentage of its potential reduction capacity. The addition of EDTA-Fe3+ or DTPA-Feat (better substrates than oxygen) results in an increased rate of NADPH oxidation with concomitant direct reduction of the ferric chelate (Chapter I). The resultant ferrous chelate could then reduce acetylated cytochrome c directly, accounting for the 02?-independent component of this reduction. The 02?-dependent component of reduction is probably a result of ferrous chelate oxidation. Ferrous- EDTA autoxidizes quite rapidly in contrast to DTPA-Feat, and therefore, the 02?-dependent component of acetylated cytochrome c reduction in the presence of EDTA-Fe3+ was much greater than that observed in the presence of DTPA-Feat. This was despite the fact that the total rate of acetylated cytochrome c reduction in the presence of DTPA-Fe3+ was greater. However, the mechanism(s) of autoxidation of 144 ferrous chelates has (have) not been elucidated, so it is not know whether the 02?-dependent fraction of acetylated cytochrome c reduction is due only to autoxidation. Conceivably, ferric chelates could also enhance 02: production by the reductase by a different but unknown mechanism. As with the purified reductase, microsomal 02? production was low (15.6 nmol 02?/unit cytochrome c reductase activity), accounting for only a minor percentage of its total reduction potential (about 1.53). Data in the literature as well as the results in this chapter undoubtedly indicate that the difference in 02? production between the purified reductase and microsomes is due at least in part to cytochrome P450s. Ferric chelates, especially EDTA-Fe3+ and DTPA-Fe3+ stimulated microsomal 02: production, although the degree of stimulation was less than with the purified reductase. This result brings into question the results of previous experiments in which EDTA was added to microsomal incubations in which NADPH oxidation, and 02: and H202 production were measured. It is not clear exactly what effects trace EDTA-Fe3+ concentrations might have had on the observed stoichiometry of microsomal oxygen radical formation. Results in this Chapter and Chapter I would suggest a stimulation of NADPH oxidation and 02? production, perhaps yielding a stoichiometry in which a greater percentage of NADPH oxidized resulted in 02? production, a conclusion that 145 Huthan a2 a22 reached.20 The stoichiometry obtained in this chapter agrees more closely with that of Zhukov and Archakov,34 but is debatable whether the excess NADPH oxidation (over H202 formation) shown in this chapter is truly indicative of direct 4 electron reduction of oxygen to water as previously suggested.33'34 Previous results obtained using a reconstituted system33 would appear to be better evidence for a 4-e1ectron oxidase activity than results obtained with microsomes, due to the problem of catalatic activity in microsomes. The ability of cytochrome P450 isozymes to enhance 02? production over that generated by the reductase alone was also demonstrated. 4 Increased rates of 02? production were observed as the molar ratio of cytochrome P450/reductase was increased, until the ratio reached approximately 4 or 5 to 1. This is consistent with previous studies that had shown that the rate of metabolism of MFO substrates in a reconstituted system increased until the cytochrome P450/reductase ratio approached 5.50 The production of 02? by the reconstituted system followed previous observations of the requirements for obtaining drug metabolism in a reconstituted MFO system, i.e. the presence 0f DLPC, the detergent-solubilized reductase, and cytochrome P450 .isozymesJ‘1 Use of the protease-solubilized enzyme or the exclusion of DLPC resulted in a marked inhibition in the rates of 02? production. 146 Superoxide production by the reconstituted MFO system was confirmed using EPR spin trapping. However, because of the slow rate of reaction of 02? with DMPO, the signal observed was apparently a combination of DMPO-OOH and DMPO-OH adducts. The direct production of -OH as a source of DMPO'OH was prevented by the addition of desferrioxamine, which inhibits the iron-catalyzed Haber-Weiss reaction.39 The addition of SOD to the incubation prevented both DMPO-OOH and DMPO-OH adduct signals, so the DMPO-OH most probably arose from DMPO-OOH decomposition. A difference in the rates of 02? production was observed depending upon the isozyme of cytochrome P450 that was reconstituted with the reductase. Cytochrome P450d, the isozyme isolated in the high spin conformation from rats treated with 3-MC type inducers produced slightly more 02? than did cytochrome P450b, the major phenobarbital-inducible isozyme. Cytochrome P450c, the other major isozyme induced by 3-MC produced somewhat less 02?. A recent report has indicated that the reductase binds more tightly to high spin cytochrome P450.52 This is consistent with the data illustrated in Figure 12; the slope of the curve with cytochrome P450d is considerably greater than those of the other two isozymes, 'indicating that high spin cytochrome P450d is able to , saturate the reductase at lower concentrations than either cytochromes P460b or P450c. Treatment of rats with isosafrole results in increased levels of hepatic microsomal cytochrome P450d. However, the 147 binding of an isosafrole metabolite to cytochrome P450d prevents the determination of cytochrome P460 using a CO difference spectra and inhibits 02? production presumably by preventing the coordination of oxygen to the heme iron. Nevertheless, these microsomes still catalyze appreciable rates of NADPH oxidation and 02? production. Removal of the isosafrole metabolite by preincubating with butanol resulted in microsomes having increased capacity for 02? production as well as a near stoichiometric increase in the rate of NADPH oxidation. These results would suggest that 02? production by cytochrome P450d is prevented when the heme iron of cytochrome P450d is occupied with other ligands. This would tend to discount the hypothesis of Gorsky a2 a2_.__33 who suggested that the presence of cytochrome P450 isozymes might stimulate the reductase to produce more 02? or other partially reduced oxygen species. However, from the data on the rates of NADPH oxidation and 02? production from microsomes isolated from rats pretreated with MFO inducers it was apparent that the presence of particular isozymes of cytochrome P450 was not the only factor influencing oxygen radical production by microsomes. While induction with phenobarbital resulted in microsomes having greater basal rates of NADPH oxidation and 02? and H202 production, induction of cytochrome P460d by HBB did not result in much of an increase in microsomal uncoupling (compared to microsomes from phenobarbital- treated rats) despite the fact that cytochrome P450d 148 stimulated 02? production in the reconstituted system to a greater extent than cytochrome P450b. In fact, if rates of 02? production were expressed per unit of reductase activity, 02? production was comparable (11.0, 13.6 and 17.9 nmol/unit for microsomes from control, phenobarbital-, and HBB-treated rats, respectively). This result would suggest that under certain conditions, the level of reductase may limit 02? production by microsomes in much the same fashion as it has been proposed to limit MFO activity.3°'53 Finally, the potential of the MFO system to generate partially reduced oxygen species 2a y2yg should be addressed. From one perspective, uncoupling represents a futile cycle with reducing equivalents from NADPH eventuating in the net reduction of oxygen to water without substrate oxidation. Futile cycles are certainly not the rule in biological systems and the cell usually prevents such unnecessary consumption of energy by inhibiting the competing enzyme or pathway. However, in at least one respect the microsomal electron transport system represents an atypical enzyme system. It appears to be ideally suited to handle a wide variety of structurally-diverse substrates. As such, a limited amount of uncoupling of MFO activity may be the biological price to be paid for the low degree of specificity exhibited by the MEG system relative to more conventional enzymes or enzyme systems. on the other hand, oxygen radical production by microsomes may be an artifact of isolation and, therefore, 149 essentially a property of microsomes but not the intact endoplasmic reticulum. There is evidence for an asymmetric distribution of MFO components within the endoplasmic reticulum, and the normal homogenization procedure might disrupt the organization of the electron transport system and promote oxygen radical formation. A limited amount of data would suggest that MFO- dependent oxygen radical production does indeed occur 2a y2ya. However, that data had been obtained using indirect determinations, for example, monitoring the alteration of metabolism of methanol during the 2a y2yp metabolism of ethylene g1ycol.54 A recent report details the decrease of H202 production by the MFO system upon the treatment with allylisopropylacetamide,55 a cytochrome P460 inhibitor, but this study used as a measure of H202 production the residual catalase activity remaining in liver following aminotriazole treatment. Thus, the evidence for 2a y2ya formation of H202 can not be considered as particularly strong at the present time. 1. 10. LIST OF REFERENCES B.A. Freeman and J.D. 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Evidence that isoniazid and ethanol induce the same microsomal cytochrome P450 in rat liver, an isozyme homologous to rabbit liver cytochrome P450 isozyme 3a. Arch. Biochea. Biophys. 222: 633-644 (1986). D.R. Hoop, E.T. Morgan, C.E. Tarr and M.J. Coon. Purification and characterization of a unique isozyme of cytochrome P450 from liver microsomes of ethanol- treated rabbits. J. Biol. Chem. 257: 8472-8480 (1982). D.A. Haugen and M.J. Coon. Properties of electrophoretically homogeneous phenobarbital-inducible and ‘y9 -naphthoflavone-inducible forms of liver microsomal cytochrome P450. J. Biol. Che22 261: 7929- 7939 (1976). J.D. Schenkman, S.G. Sligar and D.L. Cinti. Substrate interaction with cytochrome P450. In: Hepatic Cytochrome P450 Monooxygenase Systea. (J.B. Schenkman and D. Kupfer, eds.) pp. 587-615, Pergamon Press, NY (1982). 30. 31. 32. 33. 34. 35. 136. :37 38. 39. 153 I. Bjorkhem. Rate limiting step in microsomal cytochrome P450 catalyzed hydroxylations. In: Hepatic Cytochrome P450 Monooxygenase Sysgaa. (J.B. Schenkman and D. Kupfer, eds.) pp. 645-666, Pergamon Press, NY (1982). S.K. Soodaera, E.D. Skotzelyas, A.A. Zhukov and A.I. Archakov. Comparative studies of superoxide radical generation in microsomes and reconstituted monooxygenase systems. In: Cytochroae P450, Biochemiatry, Biophysics, and Environaental 2ap1ications (E. Hietanen, M. Laitinen and O.Hanninen, eds.) pp. 615-48, Elgevier, Amsterdam. H. Huthan, H. Tsuji, H. Graf, V. Ullrich, J. Werringloer and R.W. Estabrook. Generation of superoxide anion as a source of hydrogen peroxide in a reconstituted monooxygenase system. FEBS Lett. 22: 343-345 (1978). L.D. Gorsky, D.R. Hoop and M.J. Coon. On the stoichiometry of the oxidase and monooxygenase reactions catalyzed by liver microsomal cytochrome P450. J. Biol. Chaa2 259: 6812-6817 (1984). A.A. Zhukov and A.I. Archakov. Complete stoichiometry of free NADPH oxidatin in liver microsomes. Biochem. Biophys. Res. Comm. 109: 813-818 (1982). G.D. Nordblom and M.J. Coon. Hydrogen peroxide formation and stoichiometry of hydroxylation reactions catalyzed by highly purified lier microsomal cytochrome P450. Arch. Biochem. Biophys. 180: 343-347 (1977). A. Xstrom and J.W. DePierre. Identification of the forms of cytochrome P450 induced in rat liver by 2- acetylaminofluorene using immunoblotting and partial purificaiton. Biochem. Biophys. Res. Comm. 222: 578- 585, (1985). K. Wada and K. Okunuki. Studies on chemically modified cytochrome c. I. The acetylated cytochrome c. 12 Biochem. 22: 667-681 (1968). Worthington Enzymes, Enzyme Reagents and Relajed Biochemicals pp. 43-45, Worthington Biochemical Corporation, Freehold, NJ (1972). E. Graf, J.R. Mahoney, R.G. Bryant and J.W. Eaton. Iron-catalyzed hydroxyl radical formation: Stringent requirement for free iron coordination site. J. Biol. Chem. 259: 3620-3624 (1984). 40. 41. 42. 43. 44. 45. 46. 473 48. ‘19. £50. 51. 154 G.J. Fisher, H. Fukushima and J.L. Gaylor. Isolation, purification and properties of a unique form of cytochrome P450 in microsomes of isosafrole-treated rats. J. Biol. Chem. 256: 4388-4394 (1981). M. Dickins, C.H. Elcombe, S.J. Moloney, H.J. Netter and J.W. Bridges. Further studies on the dissociation of the isosafrole metabolite-cytochrome P450 complex. Biochaa. Pharmacol. 22: 231-238 (1979). T. Omura and R. Sato. The carbon monoxide-binding pigment of liver microsomes. II. Evidence for its heme protein nature. J. Biol. Chea2 239: 2370-2378 (1964). M. Murray, H. Hetnarski and C.F. Wilkinson. Selective inhibitory interactions of alkoxymethylene dioxybenzenes towards monooxygenase activity in rat hepatic microsomes. Xenobiotica 22: 369-379 (1985). S. Minakami, H. Titani and H. Ishikura. The structure of cytochrome c. II. Properties of acetylated cytochrome c. J. Biochem. 22: 341-348 (1958). B.S.S. Masters, M.H. Bilimoria, H. Hamin and C.H. Gibson. The mechanism of 1- and 2-e1ectron transfers catalyzed by reduced triphosphopyridine nucleotide- cytochrome c reductase. J. Biol. Chea2 222: 4081-4088 (1965). P. Wardman. Specificity of superoxide dismutase in catalyzing redox reactions: A pulse radiolysis study. Stud. Phys. Theor. Chim. 2: 189-196 (1979). B. Halliwell. The superoxide dismutase activity of iron complexes. FEBS Lett. 22: 34-38 (1975). J.M. McCord and E.D. Day. Superoxide-dependent production of hydroxyl radical catalyzed by the iron- EDTA complex. FEBS Lett. 22: 139-142 (1978). J. Butler and B. Halliwell. Reaction of iron-EDTA chelates with the superoxide radical. Arch. Biochea2 Biophys. 218: 174-178 (1982). E.T. Morgan, D.R. Hoop and M.J. Coon. Catalytic activity of cytochrome P450 isozyme 3a isolated from liver microsomes of ethanol-treated rabbits. J. Biol. Chem. 221: 13961-13957 (1982). A.Y.H.Lu, R.W. Strobel and M.J. Coon. 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FEBS Lett. 199: 19-22 (1986). CHAPTER III The Roles of Superoxide and Hydrogen Peroxide in NADPH-Dependent Microsomal Lipid Peroxidation 156 ABSTRACT The requirement for 02? and H202 in the initiation of NADPH-dependent microsomal lipid peroxidation has been investigated. To assess the potential involvement of H202 in the generation of -OH, the concentrations of microsomal H202 have been altered and resulting rates of lipid peroxidation have been measured. Hydrogen peroxide concentration in microsomes was altered by washing to remove endogenous catalatic activity, by adding exogenous catalase, adding exogenous H202 and/or by inhibiting endogenous catalase activity with azide. In only one instance was the rate of lipid peroxidation altered; exogenous H202 added to azide-treated microsomes inhibited lipid peroxidation, the opposite effect from that predicted if -OH generated from H202 is actually the major initiating species. These results do not support the role of free H202 in the initiation of microsomal lipid peroxidation. The Participation of 02? generated by the reductase in NADPH- dePendent lipid peroxidation of microsomes was also investigated and compared with results obtained witha xanthine oxidase-dependent lipid peroxidation system. PerOxidation of phospholipid liposomes or rat liver microsomes in the presence of ADP-Fe3+ was demonstrated to be independent of 02: generation by the reductase. 157 INTRODUCTION Lai et al.,1‘3 Fong et a1.,4 and Roster and Slee5 have all suggested that the -OH is the initiating species in reductase-dependent peroxidation of microsomal lipids. They propose that, during oxidation of NADPH by microsomes, reduced iron and H202, formed by a 02?-dependent mechanism, react to form -OH. This series of reactions is known as the iron-catalyzed Haber-Weiss reaction: 02? + F83+ -------- > Fe2+ + 02 202? + 2H+ -------- > H2 02 + 02 Fe2+ + H202 ------- > Fe3+ + -OH + OH'. It has been questioned whether this mechanism represents the predominant means by which the initiator of microsomal lipid peroxidation is generated.6 The endogenous catalase activity present in microsomes7 should scavenge H202, thereby limiting the amount available to react with reduced iron to generate -OH. Thurman 22 122.3 have shown ‘that, an inhibitor of catalase must be added to microsomes ill order to quantitate H202 production, indicating that the 8teady-state concentration of H202 must be very low in -i43rosomes incubated with NADPH. - Furthermore, Cohen and Cederbaum" have shown that azide-treated microsomes produce lore -OH than do microsomes incubated in the absence of aziJie. Therefore, addition of exogenous H202 and/0r the 158 159 inhibition of endogenous catalase activity should cause an increase both in -OH production and in subsequent rates of microsomal lipid peroxidation if it is indeed initiated by the mechanism shown above. However, there are no reports that thoroughly investigate the effects of H202, azide, and catalase on microsomal lipid peroxidation. There have been several reports claiming that catalase inhibited NADPH-dependent lipid peroxidation,2'5 whereas other investigators have reported no effect.1°'11 Since catalase is available from several sources and because antioxidants are sometimes included in the preparations, the effect of several different catalase preparations on lipid peroxidation was examined. Likewise, there is controversy as to whether in NADPH- dependent lipid peroxidation reduction of ferric is via 02: (1) or not.3“3:'12 Previous studies presented in Chapter I indicated that microsomes possess a NADPH-dependent ferric reductase activity, that might eliminate a requirement for 02? for example as is proposed in the Haber-Weiss reaction. Thus, relative to xanthine oxidase, microsomes or the purified reductase should promote greater rates of lipid peroxidation. Moreover, lipid peroxidation promoted by xanthine oxidase should be inhibited by SOD whereas NADPH- dependent peroxidation should not. MATERIALS AND METHODS Chemicals: Butylated hydroxytolulene, 2-thiobarbituric acid, glutathione reductase and sodium azide were all purchased from Sigma Chemical Company. Malondialdehyde bis (dimethyl acetal) was obtained from Aldrich Chemical. Catalase preparations were from Sigma, Boehringer-Mannheim and Millipore (3 preparations). Other chemicals were of reagent grade and used without further purification. Solutions used in lipid peroxidation assays were treated with Chelex 100 to remove contaminating transition metal ions. Preparation of Phospholipid Liposomes: Microsomes were prepared as described in Chapter I and microsomal phospholipids were extracted by the procedure of Folch 22 a2.,13 and stored at -20°C in CHCla:CHaOH (2:1). Phosphate was assayed by the procedure of Bartlett.14 Liposomes were prepared by taking an aliquot of the CHC13:CH20H extract and drying it under a stream of Argon, diluting it with 50 mM NaCl and sonicating until all lipid was resuspended. Lipid Peroxidation Assays: Stock solutions of microsomes or phospholipid liposomes, ADP-Fe3t, NADPH, purified reductase, or xanthine oxidase prepared in 30 mM NaCl (pH 7.0) were used to constitute lipid peroxidation mixtures that were 160 161 incubated in a shaking water bath maintained at 37°C. Final concentrations of the solutions are given in the figure and table legends. Lipid peroxidation was initiated by the addition of NADPH or xanthine oxidase. When both azide and H202 were added to microsomes, azide was always added prior to H202 to inhibit the endogenous microsomal catalatic activity. When only H202 was added, it was added just before the initiation of peroxidation with NADPH. At specific times aliquots (0.5 ml or 1 ml) were removed and assayed for MDA by the thiobarbituric acid assay.15 The procedure of Paglia and Valentine was used for the determination of H202-dependent glutathione oxidase activity.16 RESULTS Role of Hydrogen Peroxide in Microsoaal Lipid Peroxidation: Figure 20 is time course of microsomal NADPH-dependent lipid peroxidation and shows the effect of H202 added either without or with azide to inhibit endogenous catalase activity. The addition of either azide (0.4 mM) or H202 (0.1 mM) to microsomal incubation mixtures containing both NADPH and ADP-Fe3+ had no effect on the rate of lipid peroxidation. However, upon the addition of both H202 and azide, the initial rate of lipid peroxidation was decreased markedly. The simultaneous addition of H202 (0.1 mM), NADPH (0.1 mM) and azide (0.4 mM) to microsomes in the absence of ADP-Fe3+ did not result in lipid peroxidation (Figure 20), indicating that neither endogenous heme iron, such as cytochrome P-450 or cytochrome b5, nor non-heme iron was able to decompose H202 at least not to an extent sufficient to observe lipid peroxidation. The ability of azide to inhibit the endogenous catalase activity of microsomes was demonstrated by the results shown in Figure 21. In the absence of azide, the catalase activity of microsomes was approximately 140 units/mg protein. Fifty percent inhibition of this activity was obtained by the addition of 0.025 mM azide (Figure 21, inset), and 0.4 mM azide caused greater than 97 percent inhibition of the microsomal catalase activity. Although 162 163 .Azs e.ov ovwws was .Azs “.00 new: .Azm ~.ov mmnuuano and» Mo noususoouum no vonnouaxo use sowusluom <9! 00 neusm .ovwws leaves no encapsuusoosoo escape» @0000 use: dawn: on how ossuwm aw monflsoeovv mouamn ouoamaoo on» moswsuaoo anewusnmoaw sofluovwxouoa magma Assamese“: .uvwsm asfivon mo anewusuusoosoo anewum> so“: Am.emav .oaumu z me.e e2 «cum 2: eN eea .Ha\eseeoee Helonouowm mm H.o unmeasumoo ouao>so am a a mu essences no: nomenoaOMI a“ huw>wuos onsamuso nmosouovsm .sOMHMfiwXOLom vflmwu Hsaonosowz cam. moaonosoflz cw huw>wuo< ons~oumo umosuwovsm so ovww< mo uooumm .HN shaman 166 MDA FORMATION (H) (zcoumou -100 "' 50 0 d '5 . _s 4: . 0 lg - ( a“! o I a 8 ° - |* mmov «010100 A 0.4 0.2 M: g 0 I00 ("1 (101111100 .7.) All/\IIOV asvmyo AZlDE (mM) 167 inhibition of catalase activity by azide should increase the H202 concentration in microsomes, NADPH and ADP-Fe3*- dependent lipid peroxidation was clearly not inhibited by azide concentrations up to 1 mM (Figure 21). The effect of H202 on microsomal lipid peroxidation was also studied by adding exogenous H202 to microsomes (Figure 22). In the absence of azide, H202 concentrations up to 150 uM were without effect on the rate of MDA formation. However, in agreement with the results shown in Figure 20, the addition of H202 in the presence of azide (0.4 mM) caused a significant decrease in the rate of lipid peroxidation. Fifty percent inhibition occurred in the presence of approximately 20 juM H202. The results of control experiments indicated that neither H202 nor azide had an effect on the thiobarbituric acid assay for MDA. Effect of Catalase on Lipid Peroxidation: The involvement of H202 in microsomal lipid peroxidation has been studied previously by adding exogenous catalase. However, the results of these studies are contradi'ctoryfl-l'n1°!11 Accordingly, five different commercial catalase preparations were tested for their effects on this process. The results are shown in Figure, 23. Three of the five catalase preparations had no.effect on microsomal lipid peroxidation (Figure 23A). However, two catalase preparations significantly inhibited the rate of MDA formation (Figure 233). Pederson and Aust suggested that. the conflicting 168 .Ioumhm ouoHafloo 0:0 as vo>somn0 some so nousueoosoa no vonmosaxo use sowumasom n vuvamloo on» :w vo>uonno pug» we nuuauaoouoa nu vouoounxo one newuuuuom <9: mo «can: .aaowuauomoum voaaahwouoaouAOIno: 0» Lemma uHonlha venoHo “unawuuuaauum voanouuouoIOhno a» young uHonahu ammo .Ama\nuw:: bmo.¢¢v 83:3: A oiv E; “Au-<32: 2363 2&3. A AYCV .aouuhu oumanaoo an» 0» vovvu awn» van .huw>waoo you vohwnuo .mmlo xovunmvm co vonnouUOuuHOLno one: ouons .aowuavwxouom vwawa Halonoqua vouwnwnnw amaaauoo mo anewuouoaoun 039 "m gonna .AvONMHMnmo>H vonuwaham 6532: «3;: 82:2: A 4v 23 "Awake“? «863 9832:. A I V 22:32: 23.2: 322.3: Iuouafiuavom ADV u< fiocam "anewuauomoun onoawuwo ucwzofiaom on» no mausolu m:o«u¢> vuvva «no: soda: cu Aom ousmmuv acumhm mumHmaoo an» vocfiauaoo maowuwnnoaw HH< .aoAuvaxouom vwmfia Hoaomouofiz no anewuwuw ”hm wmwfiwuoo waouvmmoo unvumMMwn mo uummkm .MN ouamwh 171 on 00. a 3: £5.90. xv >.E>_._.o N I d A2; 30.3 38.2.; o . 0 AS; 833 an; 0.4 «3.2. 38 voznfmouogucuc: 4 Q 0 0 ‘ ‘ 32:38 vocamgmouoggfi < $448.4 mm" m c N O a . . . . l AS; «8.5 932:; 4 I A8; .833 28:2: I Awe? 0863 SucccuzLoucIcoom O ( I01W00 7.). NOLLVWBOA VOW 00. 172 reports of the effect of exogenous catalase on microsomal lipid peroxidation could be due to antioxidants present in several commercial enzyme preparations. When the catalase preparations which inhibited lipid peroxidation [Sigma (30,000 units/mg) and Millipore (44,087 units/mg)] were chromatographed on Sephadex G-25 to remove low molecular weight contaminants, no inhibition of MDA formation could be observed upon their addition to the complete system (Figure 238). Previously, it was demonstrated that endogenous catalase activity could be significantly removed from microsomes by washing.17 In this study, washed microsomes were found to contain both lower catalase activity and lower H202-dependent, glutathione oxidase activity than was found in unwashed microsomes, yet rates of microsomal lipid peroxidation were essentially unaffected (Table 12). Washed microsomes to which exogenous catalase was added to an activity equal to that of unwashed microsomes also showed rates of lipid peroxidation similar to those seen with unwashed control microsomes (Table 12). Chromatography of microsomes on Sepharose ZB reduced catalase activity to approximately 10 units/mg. The ability of .the -0H trapping agents benzoate and lannitol to inhibit.NADPH-dependent lipid peroxidation was investigated and compared to their effect on ~0H-dependent liDid peroxidation promoted by Fenton’s reagent (Figures 24 and 25, respectively). Whereas either mannitol or benzoate 173 .moaomouoHa omAmmsa: nufia vm>uomno umzu mo mmwmucmuumm mm vmmmmuaxo mum :0Humahom <9: wo mmumm .mwsomouoHB oonmmacs mo umnu 0» Homes huH>Huum onHooam m ou mmaomOHUHa vwnmms ou xomn woven mos ommHmumo msocwwoxm mGOHumnsucH meow CH .O.n ma Homz xa 0m cH A28 H.Ov mmnuH>Huum mmmHmumo AHouucoo NV cowumEMOO <02 COHumvonumm OHOHH Hmsomouowz co mmmHmumo wo COHqum< mnu was mcHSmmB mo uummwm .NH mHan 174 .Aza A.ov «Cum coo Hz: N.OV «Hook AHa\us 0.00 monouosoHl uo>HH was vomHmuaoo mooHuonsoaH ucovcouovnHonvah HuxOMvhm .ON moame mH vonHuomov ops aeoHuonsocH uoovoooovlmm0Huom ommuosvom so.“ communes vac vouooHHoo one: AH: HO mcoHuowum .sasHoo mleo enoumsmom so omxH a so commonwOHmsonao mm: moHone> mo uosuHHo Ha H c .soHumouHHm How on covoewnsm moHonos OHNHHonmmonm mcHsHmucoouomem omounoouho vac emeuosvom omen emotnooumoummaHuom eumuosvon use .n.b mm mHma an on mH Hza N.OV mmama mooHuemmaomo oHoHuo> ddoHOHmW> OHNHHonomoam mo noHumexonom 0:» so unoucoo omen omosmooumo wwwmwooosm mo voomkm .Am .....2 207 NHV . 00m+ 25:35 8.3 395er H mqu _ :u _ ("lulQI/IUJ/IOHJU) vow 208 incubations resulted in 30-SOX inhibition, with higher degrees of inhibition occurring at lower cytochrome P450 contents. DISCUSSION The reactions leading to the formation of an initiating species of lipid peroxidation are thought to involve ferrous iron and oxygen (or perhaps partially—reduced forms of oxygen). The function of the microsomal MFO is usually considered to the reduction of ferric chelates. Microsomal lipid peroxidation is promoted by ADP-Fe3+ and NADPH, but a reconstituted lipid peroxidation system that included the purified reductase could not support peroxidation unless EDTA-Fe3+ was also included.‘ Thus it was postulated that EDTA-Fe3+ replaced an endogenous component of microsomes permitting the reduction of ADP-Fe3+ and the subsequent initiation of peroxidation. If this hypothesis is indeed correct, cytochrome P450 isozymes are probably the most likely microsomal components necessary for ADP—Fe3+ reduction. In Chapter I, cytochrome P450 was shown to reduce ADP-Feat under anaerobic conditions when incubated with NADPH and the reductase. Thus, what remained to be shown was that the addition of cytochrome P450 to a reconstituted lipid peroxidation system already containing the reductase, ADP-Feat, and NADPH could result in peroxidation, without the additibn of EDTA-Feat. However, the incubation of cytochrome P450 with the reductase in DLPC micelles did not result in ADP-Fe3*- dependent peroxidation, unless EDTA-Feat was also added. 209 210 This is somewhat surprising in that this reconstitution yields an active MFO system that is capable of reducing ADP- Fe3+ (Chapter I), but it is essentially no different from reconstituted system of Pederson and Aust with respect to its ferric chelate requirements. The reductase and cytochrome P450 were incorporated in asolectin vesicles using a sonication procedure. Incubation of these vesicles with NADPH and ADP-Fe3* resulted in peroxidation as evidenced by the formation of MDA. Essentially no peroxidation was observed in the absence of NADPH or ADP-Fe3+, or when EDTA-Fe3+ was substituted for ADP-Fe3t. Increasing the concentration of vesicles in the incubation resulted in increased MDA formation, as did increasing the concentration of ADP-Fe3t. Moreover, vesicles containing increasing cytochrome P450 contents exhibited increased rates of lipid peroxidation. Thus, it appears that the incorporation of cytochrome P450 and the reductase in phospholipid vesicles results in the peroxidation of the vesicles incubated with NADPH and ADP- Fe3*. . The addition of SOD to these incubations resulted in a moderate inhibition of the rates of ,MDA formation, just as was the case with intact microsomes. However, as the cytochrome P450 content of the' vesicles increased, MDA formation was inhibited by SOD to lesser extents. It has been a subject of debate as to whether or not NADPH- dependent microsomal lipid peroxidation is dependent upon 211 023.12'15 Whereas the addition of cytochrome P450 to the reductase resulted in increased rates of 027 production (Chapter II) it also resulted in the direct anaerobic reduction of ADP-Fe3+ (Chapter I). Thus, regardless of the mechanism of iron reduction, the data in this thesis would support the role of cytochrome P450 in the initiation of microsomal lipid peroxidation. Inhibition by SOD was less than 50%, so the major pathway for ADP-Fe3* reduction in this reconstituted vesicular system would appear to be direct. In a previous communication, Elkstrom and Ingelman- Sundberg had reached a similar conclusion.8 However, they did not demonstrate a requirement for low molecular weight chelates, despite that they reported an inhibition by iron chelators desferrioxamine, EDTA, and DTPA. Thus, it could not be ascertained whether cytochrome P450 was promoting lipid peroxidation via enhancing initiation reactions by reducing low molecular weight iron chelates or via participating in the decomposition of lipid hydroperoxides as had previously been demonstrated.7 The data in this chapter supports a role for cytochrome P450 in the reduction of iron in the promotion of lipid peroxidation. It should be indicated that if the asolectin used as a phospholipid source in these studies had contained a significant amount of lipid hydroperoxides, peroxidation of the vesicles would have been promoted by EDTA-Fe3+ and reductase alone, since the reductase readily reduces EDTA-Fe3+ and EDTA-Fe3+ 212 readily decomposes lipid hydroperoxides.7 In this case, no requirement for ADP-Fe3* or for cytochrome P450 would have been evident. It is also interesting to speculate on the apparent necessity for cytochrome P450 to be incorporated into the phospholipid vesicles in order to promote lipid peroxidation. The MFO reconstituted system customarily used in drug metabolism investigations was not able to promote lipid peroxidation despite being capable of reducing ADP- Fe3+ (Chapter I). This might indicate a different type of "site specific" mechanism, that is the reduction of ADP-Fe3+ must occur close to or at the membrane surface in order to .efficiently promote lipid peroxidation. This differs from other "site specific" hypotheses in that it has been postulated that the iron that acts as a catalyst for potentially deleterious redox reactions is bound at critical cellular sites or chelated by chemicals having selective cellular binding sites.17'1° Relatedly, in Chapter I it was shown that microsomes or the reconstituted MFO system reduced ADP-Fe3+ at a slow rate relative to EDTA-Fe3+. This would indicate that in order to efficiently promote lipid peroxidation the low level of ADP- Fe3+ formed must be able to: successfully interact with the lipid bilayer, a more likely prospect when cytochrome P450 is incorporated into vesicles. 10. LIST OF REFERENCES P. Hochstein and L. Ernster. ADP-activated lipid peroxidation coupled to the TPNH oxidase system of microsomes. Biochem. Biophys. Res. Comm. lg: 388-394 (1963). P. Hochstein, K. Nordenbrand and L. Ernster. Evidence for the involvement of iron in the ADP-activated peroxidation of lipid in microsomes and membranes. Biochem. Biophys. Res. Comm. l1: 323-328 (1964). A. Beloff-Chain, G. Serlupi-Cresenzi, R. Cantanzaro, D. Venettacci and M. Balliano. Influence of iron on oxidation of NADPH in rat liver microsomes. Biochim. Biophys. Acta 21: 416-421 (1965). T.C. Pederson and S.D. Aust. NADPH-dependent lipid peroxidation catalyzed by purified NADPH-cytochrome c reductase from rat liver microsomes. Biochem: Biophys. Res. Comm. 4E: 789-795 (1972). Y. Hirokata, A. Shigematsu and T. Omura. Immunochemical study on the pathway of electron flow in reduced nicotinamide adenine dinucleotide-dependent microsomal lipid peroxidation. J. Biochemm EE: 431-440 (1978). S. Orrenius, G. Dallner, and L. Ernster. Inhibition of the TPNH-linked lipid peroxidation of liver microsomes by drugs undergoing oxidative demethylation. Biochem. Biophyg. Res. Comm; mg: 329-334 (1964). B.A. Svingen, J.A. Buege, F.O. O’Neal, and S.D. Aust. The mechanism of NADPH-dependent lipid peroxidation. 1; Biol. Chem. E54: 5892-5899 (1979). G. Elkstrom and M. Ingelman-Sundberg. Cytochrome P450- dependent lipid peroxidation in reconstituted membrane vesicles. Biochem. Pharmacol. EE: 2523-2525 (1984). J.D. Dignam and R.W. Strobel. NADPH-cytochrome P450 reductase from‘ rat liver: .Purification by affinity chromatography and characterization. Biochemistry lg: 1116-1123 (1977). E. Racker. A new procedure for the reconstitution of biologically active phospholipid vesicles. Biochem. Biophyg. Res. Comm. §§: 224-230 (1973). 213 ll. 12. 13. 14. 15. 16. 17. 18. 214 G.M. Keller and C.R. Jefcoate. Benzo(a)pyrene activation to 7, 8-dihydrodiol 9, lO-oxide by rat liver microsomes. Control by selective product inhibition. J. Biol. Chem; EEE: 13370-13776 (1984). E.G. Mimnaugh and M.A. Trush. Superoxide anion- dependency of NADPH-dependent rat liver microsomal lipid peroxidation as demonstrated by the inhibition of peroxidation by superoxide dismutase. In: Oxy Radicals and Their Scavemger Systems. Volume I: Molecmlar Aspects. (G. Cohen and R.A. Greenwald, eds.). pp. 300- 303, Elsevier, NY (1983). C.-S. Lai, T.A. Grover and L.H. Piette. Hydroxyl radical production in a purified NADPH-cytochrome c (P450) reductase system. Arch. Biochem. Biophys. 193: 373-378 (1979). R.L. Fong, P.B. McCay, J.L. Poyer, B.B. Keele and H. Misra. Evidence that peroxidation of lysosomal membranes is initiated by hydroxyl free radicals produced during flavin enzyme activity. J. Biol. Chem; EEE: 7792—7797 (1973). J.F. Roster and R.G. Slee. Lipid peroxidation of rat liver microsomes. Biochim. Biophym. Acta 620: 489-499 (1980). M. Tien, B.A. Svingen and S.D. Aust. Superoxide- dependent lipid peroxidation. Fed. Proc. gm; 179-182 (1981). A. Samuni, M. Chevion and G. Czapski. Unusual copper- induced sensitization of the biological damages due to superoxide radicals. J. Biol. Chem. 256: 12632-12635 (1981). N.R. Bachur, S.L. Gordon and M.V. Gee. A general mechanism for microsomal activation of quinone anticancer agents to free radicals. gmncer Res. EE: 1745-1750 (1978). SUMMARY Several mechanisms for the formation of an initiating species of microsomal lipid peroxidation have been proposed, but one feature common to all of these proposals is the reduction of ferric iron. Iron reduction has been proposed to be either 023-dependent or direct (Ozi-independent). In this dissertation, two activities of the microsomal MFO system (027 production and direct iron reduction) have been examined and their relative significance in the initiation of lipid peroxidation of phospholipid membranes has been assessed. A summary of the data is presented in Table 17. Rat liver microsomes incubated with NADPH produced 02: and reduced the ferric chelates ADP-Fe3+ and EDTA-Fe3t. The NADPH-dependent peroxidation of microsomes required only the presence of ADP-Fe3t, in agreement with earlier work. Despite the greater rates of EDTA-Fe3+ reduction exhibited by microsomes, no lipid peroxidation promoted by EDTA-Fe3+ was observed. This emphasizes that the reduction of any ferric chelate is not necessarily sufficient to result in the initiation of lipid peroxidation. However, the reduction of ADP-Fe3+ does result. in the peroxidation of membrane phospholipids. Peroxidation was not inhibited by SOD, indicating that the direct iron reduction mechanism has 215 216 Table 17. Summary of Results Superoxide Direct Iron Lipid Production Reduction Peroxidation Reductase +/- EDTA-Fe3+ EDTA-Fe3* ADP-Fe3+ Microsomes + EDTA-Fe3* ADP-Fe3+ ADP-Fe3+ Reconstituted MFO + EDTA-Fe3+ ADP-Fe3+ ADP-Fe3* 217 greater significance in the initiation of lipid peroxidation than does 02? production. Purified reductase generated very low rates of 02: production under aerobic conditions, but catalyzed the reduction of EDTA-Fe3+ under anaerobic conditions. Lipid peroxidation of phospholipid liposomes promoted by the reductase could not be initiated in the presence of ADP-Fe3+ alone, consistent with the inability of the enzyme to reduce ADP-Fe3*. However, the reductase did reduce EDTA-Fe3+ and the incubation of EDTA-Fe3+ and ADP-Fe3* with liposomes resulted in NADPH-dependent peroxidation. The small amount of 027 produced by the reductase could not promote ADP-Fe3+ reduction or subsequent lipid peroxidation. Peroxidation promoted by EDTA-Fe3+ and ADP-Fe3+ was not inhibited by SOD, indicating that the direct reduction of EDTA-Fe3* was of utmost importance in the initiation of lipid peroxidation. The addition of cytochrome P450 isozymes to the purified reductase resulted in increased rates of 027 production, indicating that cytochrome P450 is the microsomal component mainly responsible for 02? generation. Furthermore, this reconstituted MFO system exhibited ADP- Fe3+ and EDTA-Pea+ reduction activities. When incorporated into phospholipid vesicles, the reconstituted MFO system promoted NADPH-dependent peroxidation of the vesicles incubated with ADP-Fe3t. Peroxidation of vesicles containing increased cytochrome P450 contents was inhibited to decreasing extents by SOD. 218 In conclusion, these data indicate that direct iron reduction by the microsomal MFO system or its purified components is of greater importance in the initiation of lipid peroxidation than reduction of iron dependent upon 02?. APPENDIX A 219 220 Appendix A PUBLICATIONS Tien, M., Morehouse, L.A., Bucher, J.R. and Aust, S.D. (1982) "The Multiple Effects of EDTA in Several Model Lipid Peroxidation Systems," ArchivegmBiochemistry Biophysics 218, 450. Morehouse, L.A., Tien, M., Bucher, J.R. and Aust, S.D. (1983) ”Effect of Hydrogen Peroxide on the Initiation of Microsomal Lipid Peroxidation," Biochemical Pharmacology EE, 123. Bucher, J.R., Tien, M., Morehouse, L.A. and Aust, S.D. (1983) "An Investigation into the Involvement of Superoxide in Thiol-Dependent Lipid Peroxidation,” In: Oxy Radicals and Their Scavenger System; (Cohen, G. and Greenwald, R.A., eds.) pp. 360, Elsevier, New York. Bucher, J.R., Tien, M., Morehouse, L.A. and Aust, S.D. (1983) "Three Mechanisms for the Formation of an Initiator of Lipid Peroxidation by Xanthine Oxidase," In: Oxy Radicals and Their Scavenger System; (Cohen, G. and Greenwald, R.A., eds.) pp. 296, Elsevier, New York. Morehouse, L.A., Bucher, J.R., Tien, M. and Aust, S.D. (1983) "The Promotion of 'Fenton’s Chemistry’ by EDTA-Iron,” In: Oxy Radicalg and Their Scavenger Eystems (Cohen G. and Greenwald, R.A., eds.) pp. 288, Elsevier, New York. Bucher, J.R., Tien, M., Morehouse, L.A. and Aust, S.D. (1983) "Influence of Superoxide Dismutase and Catalase on Strong Oxidant Formation During Autoxidation of Ferrous Chelates," In: Oxy Radicals and Their Scavenger Systems (Cohen, G. and Greenwald, R.A., eds.) pp. 292, Elsevier, New York. Bucher, J.R., Tien, M., Morehouse, L.A. and Aust, S.D. (1983) "Redox Cycling and Lipid Peroxidation: The Central Role of Iron Chelates," Fundamental and Applied Toxicology E, 222. Morehouse, L.A., Thomas, C.E. and Aust, S.D. (1984) "Superoxide Generation by NADPH-Cytochrome P450 Reductase: The Effect of Iron Chelators and the Role of Superoxide in Microsomal Lipid Peroxidation," Archives of Biochemistry and Biophysicg EEE, 366.’ ' Thomas, C.E., Morehouse, L.A., and Aust, S.D. (1985) ”Ferritin and Superoxide-Dependent Lipid Peroxidation," Journal of Biological Chemistry 260, 3275. 221 Appendix A Aust, S.D., Morehouse L.A. and Thomas, C.E. (1985) ”Role of Metals in Oxygen Radical Reactions," Journal of Free Radicals in Biolggy and Medicine A, 3. Aust, S.D., Thomas, C.E., Morehouse, L.A, Saito, M. and Bucher, J.R. ”Active Oxygen and Toxicity," In: Biological Reaction Intermediates III (In Press), Plenum Press, NY. Aust, S.D., Thomas, C.E., Morehouse, L.A. and Saito, M. (1985) "Membrane Oxygen Radical Formation and Lipid Peroxidation," In: Cell Membranes and Cancer, (Galeotti, T., Cittadini, A., Neri, 6., Papa, S. and Smets, L.A., eds.) pp. 235, Elsevier, Amsterdam. Morehouse, L.A. and Aust, S.D. (In Press) "Oxygen Radical Produciton by the Microsomal Mixed Function Oxidase System," In: Cellular Antioxidant Defense Mechanisms, (Chow, 0., ed.) CRC Press, Boca Raton, FL. Morehouse, L.A. and Aust. S.D. (In Preparation) "Superoxide Production by Microsomes and Reconstituted Mixed Function Oxidase Systems.” Morehouse, L.A. and Aust, S.D. (In Preparation) ”Cytochrome P450 Isozymes are Required for the ADP-Fe3* Dependent Peroxidation in Phospholipid Vesicles.” Saito, M., Morehouse L.A., and Aust, S.D. (In Press) "Transferrin-Dependent Lipid Peroxidation," Journgl of Free Radicals in Biolpgy and Medicine. ABSTRACTS Morehouse, L.A., Tien, M. and Aust, S.D. ”The Effect of Hydrogen Peroxide on Microsomal Lipid Peroxidation," Michigan Regional Society of Toxicology Annual Meeting, May 1981. Tien, M., Bucher, J.R., Morehouse, L.A. and Aust, S.D. ”Spin Trapping of Radicals Involved in Lipid Peroxidation,” International Symposium on Spin Trapping and Nitroxyl Radical Chemistry, July 1981. Aust, S.D., Tien, M., Morehouse, L.A. and Bucher, J.R. ”Mechanisms of Microsomal Lipid Peroxidation,” International Conference on Peroxides in Biological Systems," Otzenhausen, Saar, West Germany, September 1981. 222 Appendix A Morehouse, L.A., Tien, M., Bucher, J.R. and Aust, S.D. "The Role of Hydrogen Peroxide in Microsomal Lipid Peroxidation," Society of Toxicology, February 1982. Bucher, J.R., Tien, M., Morehouse, L.A. and Aust, S.D. ”Effect of Iron Chelators on the Mechanism of NADPH- Dependent Lipid Peroxidation," Society of Toxicology, February, 1982. Bucher, J.R., Tien, M., Morehouse, L.A. and Aust, S.D. "Redox Cycling and Lipid Peroxidation: The Central Role of Iron Chelates," Michigan Regional Society of Toxicology Annual Meeting, June 1982. Morehouse, L.A., Bucher, J.R., Tien, M. and Aust, S.D. ”EDTA and Lipid Peroxidation," American Society of Pharmacology and Experimental Therapeutics/Society of Toxicology, August 1982. Aust, S.D., Bucher, J.R., Tien, M. and Morehouse L.A. "The Central Role of Iron Reduction in Lipid Peroxidation,” American Society of Pharmacology and Experimental Therapeutics/Society of Toxicology, August 1982. Bucher, J.R., Tien, M., Morehouse, L.A. and Aust, S.D. ”Effect of Superoxide Dismutase and Catalase on Lipid Peroxidation Initiated by Ferrous Chelate Autoxidation," American Society of Pharmacology and Experimental Therapeutics/Society of Toxicology, August 1982. Morehouse, LA. and Aust, S.D. ”The Significance of Superoxide (02’) in NADPH-Dependent Lipid Peroxidation,” Michigan Regional Society of Toxicology Annual Meeting, May 1983. Aust, S.D., Bucher, J.R., Morehhouse, L.A. and Tien, M. "Mechanisms of Iron-Catalyzed Lipid Peroxidation,” Michigan Regional Society of Toxicology Annual Meeting, May 1983. Morehouse, L.A., Thomas, C.E. and Aust, S.D. "The Effect of EDTA-Fe on NADPH Cytochrome P450 Reductase- Dependent Superoxide Generation and Lipid Peroxidation," American Society of Biological Chemists, June 1983. Voorman, R., Mills, R.A., Bumpus, J.A., Morehouse, L.A. and Aust, S.D. "Metabolism of PBB Congeners by Reconstituted Monooxygenase Systems,” Society of Toxicology, February 1984. Morehouse, L.A., Bumpus, J.A., Voorman, R., Thomas, C.E. and Aust, S.D. ”Superoxide Production by Reconstituted Cytochrome P450-Dependent Mixed Function Oxidases," Michigan Regional Society of Toxicology Annual Meeting, May 1984. 223 Thomas, C.E., Morehouse, L.A., and Aust, S.D. "Superoxide- Dependent Ferritin Catalyzed Lipid Peroxidation," Michigan Regional Society of Toxicology Annual Meeting, May 1984. Appendix A Voorman, H.V., Mills, R.A., Bumpus, J.A., Morehouse, L.A. and Aust, S.D. "Metabolism of PBB Congeners by Reconstituted Monooxygenase Systems,” Michigan Regional Society of Toxicology Annual Meeting, May 1984. Thomas, C.E., Morehosue, L.A. and Aust, S.D. "Ferritin- Dependent Microsomal and Superoxide-Catalyzed Lipid Peroxidation,” American Society of Biological Chemists, June 1984. Great Lakes Workshop on Oxygen Radicals in Biology and Medicine, November 1984. Morehouse, L.A., Bumpus, J.A., Voorman, H.V. and Aust, S.D. "Superoxide Production by Reconstituted Cytochrome P450- Dependent Mixed Function Oxidases," American Society of Biological Chemists, June 1984. Great Lakes Workshop on Oxygen Radicals in Biology and Medicine, November 1984. Leedle, R., Morehouse, L.A., and Aust, S.D. "Oxygen Radical Production by the Rat Lung Mixed Function Oxidase (MFO) System: Comparison to Liver." Gordon Research Conference on Toxicology, July 1986.