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Date MSUis an Affirmative Action /Equal Opportunity Institution 0» 12771 lV‘fSI.) RETURNING MATERIALS: Place in book drop to nannies remove this checkout from .—,_. your record. FINES will be charged if book is returned after the date stamped below. RQOM USE oat? MECHANISM OF CHLOROFORM-INDUCED NEPHROTOXICITY By Jacqueline Hagan Smith A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirments for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology and Toxicology Center for Environmental Toxicology 1983 ABSTRACT Mechanism of Chloroform-Induced Nephrotoxicity by Jacqueline Hagan Smith Chloroform (CHCl3) is hepatotoxic and nephrotoxic in most species; in mice only males are susceptible to nephrotoxicity. Hepatotoxicity appears to result from hepatic cytochrome P-4SO-mediated metabolism of CHCl3 to a reactive intermediate, probably phosgene. The purposes of this investigation were two-fold: (l) To test the hypothesis that the kidney metabolizes CHCl3; and (2) To characterize the mechanism of CHCl3-induced nephrotoxicity. Assessment of CHCl3 toxicity in male and female ICR mice sug- gested that nephrotoxicity occurred independent of hepatotoxicity. Nephrotoxicity could be detected 2 hr after CHCl3 administration as decreased accumulation of p-aminohippurate (PAH) and tetraethylammo- nium (TEA) by renal cortical slices from male mice. CHCl3 was not nephrotoxic to females whereas hepatotoxicity was similar in both sexes. .Partial hepatectomy (50-70%) did not alter CHCl3 nephrotoxi- city. Susceptibility to CHCl3 nephrotoxicity was increased in males and females treated with testosterone as were renal mixed function oxidase activities. Both enzyme activities and susceptibility to toxicity were decreased in castrated males. ABSTRACT Mechanism of Chloroform-Induced Nephrotoxicity by Jacqueline Hagan Smith Chloroform (CHCl3) is hepatotoxic and nephrotoxic in most species; in mice only males are susceptible to nephrotoxicity. Hepatotoxicity appears to result from hepatic cytochrome P-450-mediated metabolism of CHCl3 to a reactive intermediate, probably phosgene. The purposes of this investigation were two-fold: (l) To test the hypothesis that the kidney metabolizes CHCl3; and (2) To characterize the mechanism of CHCl3-induced nephrotoxicity. Assessment of CHCl3 toxicity in male and female ICR mice sug- gested that nephrotoxicity occurred independent of hepatotoxicity. Nephrotoxicity could be detected 2 hr after CHCl3 administration as decreased accumulation of p-aminohippurate (PAH) and tetraethylammo- nium (TEA) by renal cortical slices from male mice. CHCl3 was not nephrotoxic to females whereas hepatotoxicity was similar in both sexes. ‘Partial hepatectomy (SO-70%) did not alter CHCl3 nephrotoxi- city. Susceptibility to CHCl3 nephrotoxicity was increased in males and females treated with testosterone as were renal mixed function oxidase activities. Both enzyme activities and susceptibility to toxicity were decreased in castrated males. Jacqueline Hagan Smith Renal cortical slices incubated with CHCl3 jg_vitrg_indicated metabolism was related to toxicity. CHCl3 decreased the ability of slices from male, not female, mice to accumulate PAH and TEA Within a similar time and dose as observed to produce nephrotoxicity ig_ijg, Deuterated-CHCl3 was less nephrotoxic to slices than CHCl3. 14CHCl3 was metabolized to 14 C02, covalently bound radioactivity and aqueous soluble metabolites in greater amounts by male than female renal slices. Metabolism and toxicity were reduced when incubations were conducted under an atmosphere of carbon monoxide. 14CHCl3 was metabolized by male renal cortical microsomes in the presence of NADPH; carbon monoxide inhibited metabolism. CHCl3 pro- duced a type I binding spectrum. Incubation of glutathione with microsomes and 14 CHCl3 increased the amount of aqueous soluble meta- bolites, suggesting the formation of a reactive intermediate, such as phosgene. These investigations support the hypothesis that renal cytochrome P-450 metabolizes CHCl3 to a nephrotoxic intermediate. ACKNOWLEDGEMENTS My deepest thanks go to my mentor, Dr. Jerry B. Hook, for his intellectual guidance, psychological support and high standards of performance throughout my graduate studies at Michigan State Univer- sity. I also would like to thank the members of my dissertation committee, Drs. Theodore M. Brody, Robert A. Roth, w. Emmett Brasel- ton, and Rory Connolly, for their suggestions and assistance in the preparation of this thesis. I wish to acknowledge and thank Drs. Keizo Maita and Stuart D. Sleight for their collaboration in the histopathological aspects of this work. The enthusiastic technical assistance of Ms. Laura Everett, Ms. Gay deShone, Mr. Allen Reynolds, Ms. Julie Eldredge and Ms. Virginia Adler is gratefully acknowledged. Special thanks are due to Diane Hummel for her patience and skillful preparation of this thesis. ii TABLE OF CONTENTS 'Page LIST OF TABLES --------------------------------------------------- vii LIST OF FIGURES -------------------------------------------------- ix INTRODUCTION ----------------------------------------------------- l A. Susceptibility of the Kidney as a Target Organ for Chemicals Requiring Metabolic Activation --------------- 2 B. Relationship Between CHCl3 Metabolism and Toxicity in the Liver ---------------------------------------------- 8 l. Oxidative Dechlorination -------------------------- l4 2. Hydrogen Abstraction ------------------------------ 20 3. Reductive Dechlorination -------------------------- 2l 4. Hydrogen Ion Abstraction -------------------------- 21 C. Characteristics of CHCl3-Induced Nephrotoxicity -------- 24 D. Purpose ------------------------------------------------ 32 METHODS ---------------------------------------------------------- 34 A. Animals ------------------------------------------------ 34 B. Assessment of Renal and Hepatic Toxicity --------------- 34 1. Serum Analysis ------------------------------------ 34 2. Determination of Renal and Hepatic Non-Protein Sulfhydryl Content -------------------------------- 35 3. Histology ----------------------------------------- 35 4. Renal Cortical Slice Accumulation of Organic Ions- 35 5. Definitions of Toxicity --------------------------- 36 C. Analysis of Components of Renal and Hepatic Drug-Meta- bolizing Enzyme Systems -------------------------------- 37 l. Preparation of Subcellular Fractions -------------- 37 2. Determination of Mixed Function Oxidase Activities In Vitro ------------------------------------------ 38 3. ChemiEETs ----------------------------------------- 38 TABLE OF CONTENTS (continued) METHODS (cont'd) D. F. A. Assessment of Mcnizi3 Metabolism _I_r1 Vitro -------------- l. Incubation Proceduaes ----------------------------- 2. Determination of CO Production ................. 3. Determination of Covaiently Bound Radioactivity to Proteins ------------------------------------------ 4. Determination of Aqueous Soluble 14CHCl3 Metabo- lites --------------------------------------------- Individual Experiments --------------------------------- 1. Effect of Dose and Route of Administration on CHCl3 Toxicity in Male and Female ICR Mice -------- 2. Time Course of CHCl3 Toxicity in Male and Female ICR Mice ------------------------------------------ 3. Effect of Decreasing Renal Cortical Non-Protein Sulfhydryl Concentrations on CHCl3 Toxicity in Male and Female ICR Mice -------------------------- 4. Effect of Partial Hepatectomy on CHCl3 Nephrotoxi— city in Male ICR Mice ----------------------------- 5. Effect of Sex Hormone Status on CHCl Toxicity and Renal and Hepatic Mixed Function Oxi ases in ICR Mice .............................................. 6. Assessment of CHCl3 Nephrotoxicity In_Vitro in Mouse Renal Cortical Slices ----------------------- a. Preincubation - In_Vitro CHCl3 Toxicity ------ b. Incubation - Organic Ion Accumulation -------- 7. Assessment of Nephrotoxicity of Hepatic CHCl3 Me- tabolites in Male Mouse Renal Cortical Slices ----- 8. Effect of Mouse Strain Differences on CHCl3 Toxi- city In Vitro and on Mixed Function Oxidases and 4CHCT§ Metabolism by Microsomes ------------------ Statistics ............................................. RESULTS .......................................................... _ Ig_Vivo Studies ........................................ l. Effect of Dose and Route of Administration on CHCl3 Toxicity in Male and Female ICR Mice -------- 2. Time Course of CHCl3 Toxicity in Male and Female ICR Mice .......................................... 3. Effect of Decreasing Renal Cortical Non-Protein Sulfhydryl Concentrations on CHCl3 Toxicity in Male and Female ICR Mice .......................... iv Page 38 38 40 4O 41 4T 4T 42 42 43 43 44 44 45 46 46 47 48 48 48 48 67 TABLE OF CONTENTS (continued) Page RESULTS (cont'd) 4. Effect of Partial Hepatectomy on CHCl3 Nephrotoxi- city in Male ICR Mice ----------------------------- 67 5. Effect of Sex Hormone Status on CHCl3 Toxicity and Renal and Hepatic Mixed Function Oxidases in ICR Mice ------------------------------------------ 72 a. Mixed Function Oxidase Activity -------------- 72 b. CHCl3 Toxicity ------------------------------- 79 c. Histology ------------------------------------ 88 B. In Vitro Studies — Toxicity of CHCl3 in Renal Cortical Slices ------------------------------------------------- 95 1. Assessment of CHCl3 Nephrotoxicity In_Vitro in ICR Mouse Renal Cortical Slices ----------------------- 95 2. Assessment of the Nephrotoxicity of Hepatic CHCl3 Metabolites in Male ICR Mouse Renal Cortical Slices -------------------------------------------- l06 3. Assessment of Mouse Strain Differences on CHCl3 Nephrotoxicity Ig_Vitro --------------------------- l06 c. In Vitro Studies - Metabolism of 14CHCI3 by Renal Cor- tical and Hepatic Slices from ICR Mice ----------------- ll4 D. Ig_Vitro Studies - Microsomal Metabolism of 14CHCl3---- ll8 l. Metabolism of 14CHCl3 Ig_Vitro by Renal Cortical and Hepatic Microsomes - Effect of Time, Microso- mal Protein Concentration and Substrate Concentra- - tion ---------------------------------------------- llB 2. Effefit of NADH and NADPH on Microsomal Metabolism of CHCl3---------------------------------------- 137 3. Subcellular Localization of CHCl3 Metabolism ------ l37 4. Binding Spectra of CHCl3 with Renal Cortical and Hepatic Microsomes -------------------------------- l37 5. Effect of Oxygen Concentration and Carbon Monoxide on l4cnc13 Metabolism ----------------------------- l46 6. ffect of Mixed Function Oxidase Inhibitors on 4CHCl3 Metabolism ----- — -------------------------- l46 . 7. Effect of Strain Differences on Renal Corfiical and Hepatic Mixed Function Oxidases and CHCl3 Metabolism by Microsomes -------------------------- 153 DISCUSSION ------------------------------------------------------- 156 A. Ig_Vivo Evidence for Renal Metabolism of CHCl3 --------- l56 B. I__Vitro Evidence for Renal Metabolism of CHCl3 -------- l6l TABLE OF CONTENTS (continued) Page DISCUSSION (cont'd) C. Evidence for Cytochrome P-450 Mediated CHCl3 Metabo- lism in the Renal Cortex ------------------------------- l67 D. Speculation -------------------------------------------- l7l E. Conclusions -------------------------------------------- l74 SUMMARY ---------------------------------------------------------- l75 BIBLIOGRAPHY ----------------------------------------------------- l79 vi Table 10 ll 12 LIST OF TABLES Page Effect of inducers and inhibitors on mouse renal and hepatic mixed function oxidase activity and the effect on CHCl3 toxicity ------------------------------- 28 Effect of diethyl maleate on renal cortical and hepatic non-protein sulfhydryl concentrations ------------------ 68 Effect of diethyl maleate on CHCl3-induced nephrotoxi- city in male and female mice --------------------------- 69 Effect of testosterone pretreatment on renal and hepa- tic cytochrome content and mixed function oxidases in mice --------------------------------------------------- 77 Effect of castration on renal and hepatic cytochrome content and mixed function oxidases in mice ------------ 78 Effect of testosterone pretreatment and CHCl3 on organ weight/body weight ratios ------------------------------ 80 Effect of castration and CHCl3 on organ weight/body weight ratios ------------------------------------------ 81 Effect of testosterone pretreatment and CHCl on renal and hepatic non-protein sulfhydryl concentra ions ------ 84 Effect of castration and CHCl3 on renal and hepatic non-protein sulfhydryl concentrations ------------------ 87 Ig_vitro toxicity of CHCl3 and hepatic metabolites in _ male mouse kidney slices ------------------------------- lll Ig_vitro metabolism of 14CHCl3 to 14C02, covalently bound radioactivity and aqueous soluble metabolites by male hepatic and renal cortical slices and female renal cortical slices from ICR mice -------------------------- ll5 14CHCl metabolism by male hepatic and renal cortical, female renal cortical and boiled microsomes from ICR mice --------------------------------------------------- l2l vii LIST OF TABLES (continued) Table l3 l4 l5 l6 17 Page Comparison of renal cortical and hepatic microsomal metabolism of CHCl3 in relation to cytochrome P-450 concentrations ----------------------------------------- l32 Metabolism of 14CHCl to 14C02, covalently bound radio- activity and aqueous soluble metabolites in the pre- sence and absence of glutathione by male renal cortical and hepatic subcellular fractions ---------------------- l42 Strain differences - Renal cortical cytochrome content and metabolism ----------------------------------------- l54 Strain differences - Hepatic cytochrome content and metabolism --------------------------------------------- l55 Assessment of 14C02 incorporation by male hepatic and renal cortical slices and female renal cortical slices from ICR mice ------------------------------------------ l66 viii Figure lO ll LIST OF FIGURES Schematic representation of mechanisms of toxic injury induced by xenobiotics --------------------------------- Hepatic metabolism and activation of cuc13 ............. Possible mechanisms for metabolic activation of CHCl3-- Possible metabolic pathways for metabolism of CHCl3 by (l) oxidative dechlorination, (2) hydrogen abstraction, (3) reductive dechlorination, and (4) hydrogen ion abstraction -------------------------------------------- Effect of route of administration of CHCl3 on hepatic and renal toxicity in male and female ICR mice --------- Time course of non-protein sulfhydryl (NPSH) decrease in liver after 250 pl CHCl3/kg, s.c., in male and female ICR mice ---------------------------------------- Time course of serum glutamic pyruvic transaminase (SGPT) activity after 250 pl CHCl3/kg, s.c., in male and female ICR mice ------------------------------------ Representative hepatocellular morphology after 250 pl CHCl3/kg s.c., in male and female ICR mice ------------- Time course of decrease of renal cortical non-protein sulfhydryl (NPSH) concentration after 250 pl CHCl3/kg, s.c., in male and female ICR mice ---------------------- . Effect of 250 pl CHCl?/kg, s.c., on in vitro renal cortical slice accumu ation of p-amifiohippurate (PAH S/M) and tetraethylammonium (TEA S/M) in male and female ICR mice ---------------------------------------- Time course of blood urea nitrogen (BUN) concentration after 250 pl CHCl3/kg, s.c., in male and female ICR mice ................................................... ix Page 5 I3 15 T6 49 52 54 56 58 61 63 LIST OF FIGURES (continued) Figure 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Page Representative renal tubular morphology after 250 pl CHCl3/kg, s.c., in male and female ICR mice ------------ 65 Effect of partial hepatectomy on CHCl3 (250 ul/kg) nephrotoxicity in male ICR mice ------------------------ 70 Effect of partial hepatectomy on CHCl3 (50 ul/kg) nephrotoxicity in male ICR mice ------------------------ 73 Effect of partial hepatectomy on CHCl3 (10 ul/kg) nephrotoxicity in male ICR mice ------------------------ 75 Effect of testosterone on renal and hepatic toxicity to CHCl3 in male and female ICR mice ---------------------- 82 Effect of castration on renal and hepatic toxicity to CHCl3 in male mice ------------------------------------- 85 Sex differences in ICR mice renal proximal tubule mor- phology ------------------------------------------------ 89 Effect of testosterone on susceptibility of female ICR mice to CHCl3 nephrotoxicity --------------------------- 91 Effect of castration on susceptibility of male ICR mice to CHCl3 nephrotoxicity -------------------------------- 93 Effect of preincubation with 0.5 pl CHCl for 30 to 120 min on PAH and TEA accumulation by renal cortical slices (100110 mg) prepared from male or female ICR mice --------------------------------------------------- 96 Effect of preincubation with CHCl3 concentrations ranging from approximately 0-50 umol (0-4 ul) on PAH and TEA accumulation by renal cortical slices prepared from male or female ICR mice --------------------------- 98 Effect of deuterium on toxicity of CHCl3 jn_vitro in ' male ICR mouse kidney slices --------------------------- 100 Effect of carbon monoxide on toxicity of CHC13'iQ vitro in male ICR mouse kidney slices ------------------ 102 Effect of 0°C incubation temperature on toxicity of CHCl3 in vitro in male ICR mouse kidney slices --------- 104 LIST OF FIGURES (continued) Figure 26 27 28 29 3O 31 32 33 34 35 36 37 38 Page Effect of nitrogen on toxicity of CHCl3 jn_vitro in male ICR mouse kidney slices --------------------------- 107 Effect of diethyl maleate pretreatment in vivo on toxi- city of CHC13_ in vitro in male ICR mouse _kidney slices- 109 Effect of mouse strain differences on CHCl3 neprotoxi- city in vitro ------------------------------------------ 112 Time course of 4C Cl metabolism to covalently bound radioactivity and M8 by male hepatic and renal cor- tical slices from ICR mice ----------------------------- 116 Effect of carbon monoxide or metabolism of 4CHCl3t covalently bound radioactivity and C02 by male hepa- tic and renal cortical slices and female renal cortical slices from ICR mice ----------------------------------- 119 Time course of 14CHC13 metabolism to 14C02 and cova- lently bound radioact1vity by male and female renal cortical microsomes---------------------------------e-- 122 Time course of 14CHC13 metabolism to 14C02 and cova- lently bound radioact1vity by male hepatic microsomes-- 125 Effect of male renal cortical microsomal protein con- centration in 14CHCl3 metabolism to 14C02 and covalent- ly bound radioactivity --------------------------------- 127 Effefit of male hepatic microsomal protein concentration CHCl3 metabolism ---------------------------------- 129 Effegt of substrate concentration on 14CHCl3 metabolism CO2 by male renal cortical microsomes ------------- 133 ||<1neticsto of the renal cortical microsomal metabolism of CHC1314C02 --------------------------------------- 135 ' Effect of4 NADH and NADPH on male renal cortical metabo- lism of14CH013 to covalently bound radioactivity and C02 -------------------------------------------------- 138 Effect of NADH and NADPH on male hepatic microsomal metabglism of1 CHC13 to covalently bound radioactivity and C02 ---------------------------------------------- 140 xi LIST OF FIGURES (continued) Figure 39 4O 41 42 Page Binding spectrum of CHC13 with hepatic and renal corti- cal microsomal cytochrome P- 450 ------------------------ 144 Effect of carbon monoxide and oxygen concentration on male 4renal cortlc cal and hepatic microsomal metabolism MCHCl CO2 and covalently bound radioactivity- 147 Effect of cytochrome P- -420 inhibit? 45 on male renal cortical metabolism of CHCl3 45C02 and covalently bound radioactivity ------------------------------------ 149 Effect of cytoc hrome P- 4504 inhibitors on male hepatic metabolism of? 4CHC13 4C02 and covalently bound radioactivity ------------------------------------------ 151 xii INTRODUCTION Chloroform (CHC13) was introduced as a general anesthetic by Simpson in 1847. Compared to diethyl ether, CHCl3 was less irrita- ting and nonflammable and, thus, became the major anesthetic used during the next several decades (Pohl, 1979; Davidson gt_gl,, 1982). The major disadvantage of CHCl3 use was the potential for hepatic, renal and cardiac toxicity. Thus, the use of CHCl3 as a general anesthetic declined gradually as alternative, less toxic anesthetics were developed. In 1912, the Committee on Anesthesia of the American Medical Association condemned CHCl3 for use in major surgery (Pohl, 1979). ' CHCl3 was used as a preservative and a flavor enhancer in pharma— ceutical products such as cough medicines, mouth washes and tooth- pastes. In 1976, these uses were banned by the Food and Drug Admini- stration following a study by the National Cancer Institute indicating that CHCl3 was a renal and hepatic carcinogen in rodent bioassays (U.S. Food and Drug Administration, 1976; Reuber, 1979). CHCl3 may be found in residual amounts in some drug products from its use as a processing solvent in manufacture or as a by-product in chemical synthesis (IARC, 1979). Currently, CHCl3 is a compound of toxicological concern due to its presence in municipal water supplies. CHCl3 is known to be 1 2 produced environmentally in the chlorination of water where chlorine, added as a disinfectant, interacts with small organic molecules in the water (Bellar £391., l974; Rook, l974; Bunn e_t_§_l_., l975; Deinzer gt gl,, l978). CHCl3 also is used extensively in industry as a solvent and chemical intermediate. Experimentally, CHCl3 is a relatively potent toxicant. The acute oral L050 of CHCl3 for adult rats and mice ranged from 0.8 to 1.3 ml/kg (Klaassen and Plaa, 1967; Kimura g§_al,, 197l; Torkelson §t_al,, l976; Winslow and Gerstner, l978). Administration of CHCl3 by inges- tion, inhalation, injection or percutaneous absorption produced hepa- tic and renal damage in all species studied, including man (Davidson gt_al,, 1982). Other toxic effects of CHCl3 included eye irritation, cardiovascular effects and depression of the respiratory and central nervous systems (Torkelson et_al,, 1976; Winslow and Gerstner, l978). A. Susceptibility of the Kidney as a Target Organ for Chemicals RequiringiMetabolic Activation Xenobiotics are eliminated primarily by the kidney, lung and/or liver. Consequently, these organs are frequently targets for toxicity produced by a variety of chemicals. The role of metabolic activation of xenobiotics in the liver in relation to the occurrence of hepato- toxicity has been studied extensively in the past 15 years with such model hepatotoxicants as acetaminophen, bromobenzene, carbon tetra- chloride and chloroform. In contrast to the liver, very little infor- mation is available on biochemical mechanisms of metabolic activation within the kidney as a prerequisite for the manifestation of nephro- toxicity. Fortunately, the extensive information on CHCl3-induced 3 hepatotoxicity provides a background for an evaluation of the mecha- nisms of metabolism and nephrotoxicity of this compound. The kidney, and particularly the proximal tubular cells, may be much more susceptible than other organs to the toxic effects of a variety of chemicals for a number of reasons. The kidneys comprise only 0.4% of the body weight in most mammals, but receive 20% of the cardiac output (Maher, 1976). This high blood flow dictates that large quantities of xenobiotics in the systemic circulation will be delivered to the kidneys, especially to the renal cortex, which re- ceives over 90% of the renal blood flow (Maher, 1976). Furthermore, the ability of the kidney to concentrate tubular fluid may enhance toxicity due to increased xenobiotic concentrations. Additionally, specialized functions of the proximal tubular cells may enhance toxicity in several ways which may contribute to high intracellular concentrations of potentially toxic xenobiotics. For example, solutes may be reabsorbed by passive or active mechanisms by the tubular cells. Additionally, many organic compounds are secreted into the tubular lumen by organic acid or base transport mechanisms, hence passing through or accumulating within the proximal tubular cells, exposing those cells to very high concentrations. These secre- tory processes can transport protein-bound xenobiotics as well as those in free solution. Anatomically, the renal cortex contains glomeruli, proximal and distal tubules, is richly vascular, and receives the majority of renal blood flow. Functionally, the renal cortex contains many active 4 transport processes, is highly aerobic, is metabolically very active and is exquisitely sensitive to oxygen deprivation. Once in the kidney, a chemical may act directly or indirectly to produce a toxic response. In general, three independent mechanisms can be envisioned for chemicals that result in renal damage (Figure l). First, a chemical may enter renal cells and interfere directly with an essential metabolic or functional process resulting in cellu- lar damage (Scheme I). Second, a chemical may be metabolized in the kidney to a highly reactive intermediate that may covalently bind to protein or initiate lipid peroxidation resulting in cellular damage (Scheme II). Finally, a chemical may be metabolized by extrarenal enzymes to a stable metabolite that may enter the systemic circulation (Scheme III). In the kidney, this metabolite may result in toxicity in a manner similar to Scheme I or II. . Drug metabolizing enzymes, including cytochrome P-450 dependent mixed function oxidases, are present in the kidney, as well as in other extrahepatic organs, though the specific activities of enzymes present are typically much less than found in the liver (Litterst et- al,, 1975, 1977; Fry gt_al,, 1978). Due to the heterogeneity of cell types in this organ, homogenates of whole kidney or kidney cortex contain diluted concentrations of these enzymes. Thus, use of kidney hombgenates may dramatically underestimate the metabolic activity of certain regions of the kidney. The subcellular locations and actions of renal drug metabolizing enzymes are generally analogous to those described for the liver and other extrahepatic tissues. In contrast to the liver, there are regional differences in the relative amounts Figure 1. Schematic representation of mechanisms of toxic injury induced by xenobiotics. CHEMICAL ? e CHEMlCAl —""lAl‘—" Reactive “Detoxification (A) —-(A) DAMAGE (A) Protein renal cell Metabolite Covalent DAMAGE Binding renal cell lll CHEMICAL 7 Stable (A) —'(A)_’Metabolite (X) hepatic cell l (X) 7 Prolain Reactive Metabolite DAMAGE Detoxification *— Binding renal cell Figure 1 7 of certain enzymes due to the greater cellular heterogeneity of the kidney. Renal mixed function oxidases are not uniformly distributed within the kidney, but exhibit a cortico-papillary gradient with activity being highest in the cortex (Oees gt_§l,, 1982; Rush gt 21,, 1983). Very little information is available on the direct metabolic activation of nephrotoxicants by renal cytochrome P-450. However, due to the similarities of the renal mixed function oxidases to those in the liver the potential for activation of chemicals by this mechanism in the kidney, as has been shown in the liver, must be considered. Furthermore, while cytochrome P-450-type reactions have been most extensively investigated in the kidney to date, there are other pos- sible biochemical mechanisms of intrarenal metabolic activation. Considering the unstable nature of putative reactive interme- diates, it seems likely that the site of toxicity within a tissue would be in close proximity to the site of activation, which is con- sistent with ig_§jtu_metabolism of nephrotoxicants within the kidney. This may be reflected by the discrete regions of renal damage produced by a variety of nephrotoxic agents believed to require some type of metabolic activation. Thus, the kidney is a target organ for chemicals requiring meta- bolic activation. The extent of jfl_§jtu_metabolism and the suscepti- bility to toxicity may be greatly exaggerated by the achievement of much greater concentrations of a toxic chemical within certain regions of the nephron. Additionally, it is quite possible for complex inter- actions of these various mechanisms to produce a nephrotoxic response that might not be seen with any of the events occurring individually. 8 8. Relationship Between CHCl, Metabolism and Toxicity;in the Liver The concept that metabolism of CHCl3 was required for toxicity and the molecular basis for the toxicity of CHCl3 has been a subject of investigation for many years. In 1883, Zeller observed an increase in chloride ion excretion after CHCl3 administration to dogs (Zeller, 1883). Phosgene (COClz) was reported to be a component of CHCl3 in 1894 (Hofmeister and Lenz, 1894). As early as 1912, it was known that CHCl3 could be oxidized to phosgene and hydrochloric acid in the presence of sunlight at room temperature (Baskerville and Hamor, 1912). Furthermore, phosgene was known to form carbon dioxide and hydrochloric acid in the presence of water. The net chemical reaction was expressed as: CHCl3 + 0=C0c12 + HCl COCl +Ho=co 2 2 2 + 2HC1 (Graham, 1915) Therefore, it was reasoned that since one molecule of CHCl3 could produce 3 molecules of hydrochloric acid and since the liver was known to be a metabolically active organ, the most favored mechanism pro- posed for CHCl3 toxicity was the formation of hydrochloric acid re- sulting in tissue necrosis (Graham, 1915). The earlier hypothesis of Mfiller (1911) that phosgene might be the agent responsible for the tissue necrosis produced by CHCl3 was discounted until recently. Research on the mechanism of CHCl3 toxicity during the next 60 years concentrated primarily on documenting the metabolism of CHCl3 to hydrochloric acid (Lucas, 1928; Heppel and Porterfield, 1948; Bray gt_ 21,, 1952) and to carbon dioxide (Paul and Rubinstein, 1963; Van Dyke 9 gt__l,, 1964; Fry gt_al,, 1972; Brown gt_al,, 1974b; Lavigne and Marchand, 1974). However, as research in the area of chemical carci- nogenesis indicated the role of metabolism to reactive electrophilic metabolites and covalent binding to tissue macromolecules was related to tissue damage (Miller and Miller, 1947; 1966), emphasis on the mechanism of toxicity of drugs and chemicals also was shifted toward the concept of reactive intermediates (Gillette gt_gl,, 1974; Gil- lette, 1974). Evidence had accumulated that a reactive intermediate was formed during CHCl3 metabolism. Whole body autoradiography experiments in mice 2 hr after inhalation of 14CHCl3 indicated the presence of non- volatile radioactivity in the liver and duodenum, suggesting the 14 14 presence of a compound other than CHCl3 or CO2 (Cohen and Hood, 1969). Autoradiograms of liver and kidney of male mice killed 30 hr after 14 CHCl3 administration revealed localized accumulation of radio- activity in necrotic centrilobular hepatocytes and in necrotic proxi- mal convoluted tubular cells, further suggesting a relationship be- tween covalent binding and necrosis (Ilett gt_al,, 1973). A similar correlation between covalently bound radioactivity and the degree and site of hepatotoxicity had been reported for other model hepatoxicants believed to require metabolic activation, such as acetaminophen (Mitchell gt_al,, 1973; Jollow gt_al,, 1973) and bromobenzene (Reid g3_ 21,, 1971; Brodie §t_al,, 1971). Evidence that radioactivity asso- ciated with necrotic tissue observed jn_yjyg_occurred as a result of covalent binding of a reactive metabolite(s), and not merely the 14 incorporation of a CHCl3 metabolite such as CO2 into normal cellular lO macromolecules, was provided by experiments with hepatic microsomes. Covalent binding to protein and lipid was observed after incubation of 14CHCl3 with liver microsomes prepared from rats (Reynolds and Yee, 1967; Brown gt_gl,, 1974a; Uehleke and Werner, 1975; Sipes gt_al,, 1977), mice (Ilett gt_gl,, 1973; Uehleke and Werner, 1975) or rabbits (Uehleke and Werner, 1975). In a microsomal reaction, the enzymes necessary for incorporation of C02 into cellular macromolecules, such as lipids or proteins, are not available. This would indicate that a metabolite of CHCl3 could be bound covalently to tissue macromolecules 12m. The metabolism of CHCl3 by hepatic microsomes suggested that CHCl3 was metabolized by a cytochrome P-450-dependent mechanism. In_ gjtrg_activation of CHCl3 by liver appeared to be catalyzed by a phenobarbital-inducible form of cytochrome P-450 and required oxygen (Ilett gt_gl,, 1973; Uehleke and Werner, 1975; Sipes gt_al,, 1977; Pohl gt_§l,, 1980), required NADPH (Rubinstein and Kanics, 1964; Ilett gt_al,, 1973; Sipes gt_al,, 1977), was inhibited by carbon monoxide (Ilett gt_gl,, 1973; Sipes gt 91,, 1977), and was inhibited by the cytochrome P-450 inhibitor SKF 525-A (Sipes gt_§l,, 1977). Further evidence for the role of cytochrome P-450 was the obser- vation that there were parallel alterations in the hepatotoxicity and metabolism of CHCl3 in rats and mice pretreated with inducers and inhibitors of hepatic microsomal enzymes. Inducing agents which increased CHC13-induced hepatotoxicity included phenobarbital (Schol- 1er, 1970; Ilett gt_al,, 1973; Brown gt_al,, 1974a; Lavigne and Mar- chand, 1974; Gopinath and Ford, 1975; Uehleke and Werner, 1975; Docks 11 and Krishna, 1976; Cascorbi 21_21,, 1976; Pohl and Krishna, 1977; Sipes 22_21,, 1977; Pohl §£”_l-’ 1980; McMartin 22_21,, 1981), ketones and ketogenic chemicals (Hewitt 2__21,, 1980a,b), DDT (Gopinath and Ford, 1975), ethanol (Klaassen and Plaa, 1966), polybrominated bi- phenyls (Kluwe and Hook, 1978) and phenylbutazone (Gopinath and Ford, 1975). Not all hepatic microsomal inducers increased hepatotoxicity; inactive agents included 3-methylcholanthrene (Kluwe 21_21,, 1978; Pohl 22_21,, 1980), polychlorinated biphenyls and 2,3,7,8-tetrachloro- dibenzo-p-dioxin (Kluwe 21_21,, 1978). Hepatotoxicity was decreased when animals were pretreated with inhibitors of cytochrome P-450 such as SKF 525-A (Gopinath and Ford, 1975), piperonyl butoxide (Ilett 22_ 21,, 1973; Kluwe 21_21,, 1978) or carbon disulfide (Watrous and Plaa, 1972; Gopinath and Ford, 1975). These data were consistent with the 12_11222 observations that a phenobarbital-inducible form of cyto— chrome P-450 mediated the metabolism of CHCl3. Additional evidence that CHCl3 hepatotoxicity was associated with the formation of a reactive metabolite was the observation that hepatic glutathione concentrations were decreased in phenobarbital pretreated rats that were subsequently treated with CHCl3 (Brown 22_21,, 1974a; Docks and Krishna, 1976). Glutathione, a nucleophilic tripeptide containing cysteine, was believed to play a role in protecting the liver from electrophilic metabolites of other hepatotoxicants such as bromobenzene (Reid 21_21,, 1971) and acetaminophen (Mitchell 22_21,, 1973). In 1977, two laboratories independently reported the identifica- tion of COCl2 as a hepatic metabolite of CHCl3 (Mansuy 21_21,, 1977; 12 Pohl 22_21,, 1977). In these studies, 14 CHCl3 was incubated with cysteine, a nucleophilic trapping agent, in the presence of liver microsomes prepared from phenobarbital pretreated rats. The metabo- lite identified from the incubation was 2-oxothiazolidine-4-carboxylic acid (OTZ). An earlier report had indicated that phosgene reacted spontaneously with cysteine to form OTZ (Kaneko 22_21,, 1964). Pohl and coworkers have investigated extensively the hepatic metabolism of CHCl3 and the role of phosgene in CHCl3-mediated toxicity. Phosgene has been trapped as a metabolite of CHCl3 12_1112_with cysteine (Pohl 22_21,, 1979) and 12_1112_and 12_21122_with glutathione to form diglutathionyl dithiocarbonate (Pohl 21_21,, 1981). Figure 2 illu- strates the metabolic pathway of CHCl3 proposed by Pohl and coworkers on the basis of these experiments (Pohl, 1979; Pohl 2§_21,, 1981). The chemical properties of phosgene make this compound a likely candidate for a role in the toxicity produced by CHC13. Phosgene is extremely electrophilic and reacts readily with various nucleophiles; the half-life of phosgene in water at 37°C was calculated to be less than 0.04 sec (Nash and Pattle, 1971). For example, phosgene reacts with water to produce CO2 and HCl (Babad and Zeiler, 1973), a reaction which would be consistent with the metabolism of CHCl3 12“!1§§g_and 12_ 11!9_to these products. Phosgene was predicted to react rapidly with various endogenous nucleophiles such as thiols, hydroxyls, and amino groups in proteins and lipids (Nash and Pattle, 1971; Babad and Zeiler, 1973; Pohl, 1979), a reaction that would be consistent with the metabolism of CHCl3 12_vitro and 12_vivo to covalently bound radioactivity. The conclusion is supported further by the observed 13 H l Cl—(‘i—Cl Cl Microsomes P450. 02 OH I Ct—C -- CI l Cl HCI O HE:—§_z-o.. 0 HS NH2 H20 II Cysteine ZHCI+002 ‘— Cl—C—Cl : ' 2 HCl ‘ 2 GSH ' 2 HCI Cavalent Binding 0 H 68- C—SG Duglutathionyl Duthiacavbonate H H 0 H?——c-3—ow s\ NH C/ 2- Clothiatolidine ~ 4 - COIbOIleC and l Oil 1 Figure 2. Hepatic metabolism and activation of chloroform. 14 interactions of phosgene with protein 12_vjtgg_(Cessi 2§_21,, 1966) and 12_1112_(Reynolds, 1967), with glutathione 12_yjtgg_(Pohl 22 21., 1981) and with cysteine 12_!11§9_(Kaneko 21_21,, 1964). The inter- action of phosgene with glutathione probably was responsible for the CHCl3-mediated depletion of hepatic glutathione 12_yjyg_(Johnson, 1965; Brown 21_21,, 1974a; Docks and Krishna, 1976). Pohl and coworkers have reviewed several possible metabolic pathways for the metabolism of CHCl3 (Pohl, 1979; Pohl 2§_21,, 1980). These include metabolism of CHCl3 by (1) oxidative dechlorination, (2) hydrogen abstraction, (3) reductive dechlorination, and (4) hydrogen ion abstraction. These pathways are illustrated in Figures 3 and 4 and are discussed below. 1. Oxidative Dechlorination The most likely pathway for CHCl3 metabolism is an oxidative dechlorination in which an activated oxygen would be inserted directly across the C-H bond of CHCl3 to form trichloromethanol (C13C-0H). The reaction is depicted in Figures 2-4. Trichloromethanol has not been identified directly as a metabolite; it is believed to be very un- stable such that it would rapidly and spontaneously dehydrochlorinate to phosgene (COClz) (Pohl, 1979). The trihalomethanol derivative, trifluoromethanol (F3C-OH), has been synthesized and observed to dehydrofluorinate spontaneously to carbonyl fluoride (COFZ) at tempera- tures above -20°C (Seppelt, 1977; Kloter and Seppelt, 1979). The potential role of phosgene in mediating the toxicity of CHCl3 has been discussed previously. 15 CHLOROFORM METABOLISM l. Oxidative Dechlorination CHC|3 —e CI3COH 2. Hydrogen Abstraction CHCI3 —-> C136 + H 3. Reductive Dechlorination c1103 —> 0ch + C1 4. Hydrogen Ion Abstraction CHC|3 —-> 038' + H"‘ Figure 3. Possible mechanisms for metabolic activation of chloroform. 16 .cowpomcumnm cow cmmoguxc -wao_;umu m>wbmuwxo AFV V can .cowwmcwco_;umu w>wpozvmc Amv .cowuuwcumnm cmmocuzz Amy .covpmc q Ma Ecowoco—go mo Emwponmpms Loy mxmzsuma owponmpms m—nwmmoa .e wcsmwm 17 c mczmwm U: + 3 U: u + + 83 «3 a O I .u Imwfi .5 fl OmOUmO Vll .UOUmO VI «.900 l' 551:3 .:e_a>ou r50 .30 # T35”— a «o «0 do 9 o 535 l .un_u+ .: lll £qu ll 2. + mono *@ -_u .u. are I + cot—ugxeec E 3 NGIU. ON... 0.52.3 .ce.a>ou on}. tees—vex .x/b “.3032; sort 3 + .u: u 18 Carbon monoxide has been detected as a very minor metabolite of CHC13, though the structural analogs iodoform (CHIB) and bromoform (CHBr3) yield greater amounts of C0 12_11122_(Ahmed 22_21,, 1977) and 12_yjyg.(5tevens and Anders, 1981). From these data, Anders and coworkers have proposed that sulfhydryl compounds, primarily gluta- thione (GSH), may mediate the conversion of phosgene to C0 (Figure 4) (Ahmed 22_21,, 1980). This could occur, for example, via the attack of glutathione on phosgene to yield glutathione-S-formyl chloride (650001), and subsequent attack by a second glutathione on the sulfur of the first to result in formation of carbon monoxide (CO) and oxi- dized glutathione (6556) (Stevens and Anders, 1979, 1980; Ahmed 21_ 21,, 1980). This pathway has been developed primarily using bromoform as a model (Ahmed 22 21,, 1977; Anders 22_21,, 1978; Stevens and Anders, 1979). In these studies, it was observed that CO produced in the presence of 1802 was enriched in 0180 while no C180 was seen when H2180 was used in the incubation mixtures (Stevens and Anders, 1979). These data indicate that the oxygen atom in CO was derived from mole- cular oxygen, and thus are consistent with the oxidative dechlorina- tion pathway. Evidence that molecular oxygen is the source of oxygen in the chloroform metabolite was provided by incubation of rat liver microsomes with CHCl3 in the presence of cysteine under an atmosphere of 18O2 (Pohl 21_21,, 1977). In these incubations, 18O was incor- porated into the 2-oxo position of 2-oxothiazolidine-4-carboxy1ic acid. It was estimated that nearly 100% of 2-oxo oxygen was derived 19 from molecular oxygen based on the isotope purity of 18 02 used in these experiments (Pohl 22_21,, 1977). Evidence for the direct involvement of cytochrome P-450 as the enzyme catalyzing the metabolism of CHCl3 to phosgene 12 11222 was provided by the observations that molecular oxygen was the source of oxygen in phosgene (Pohl 22_21,, 1977); that production of phosgene was inhibited by SKF 525-A and carbon monoxide (Pohl and Krishna, 1978) and was stimulated by phenobarbital pretreatment of rats (Pohl 22_21,, 1981). The importance of the metabolism of the C-H bond in the metabolic activation and toxicity of CHCl3 has been demonstrated in experiments using deuterium-labelled CHCl3 (CDC13). The C-D bond has a higher chemical energy than the C-H bond; thus, metabolism and toxicity should be reduced if cleavage of the bond is a requisite step in CHCl3 metabolism and toxicity. The formation of 2-oxothiazolidine- 4-carboxylic acid from CDCl3 was 50% of that formed from an equimolar concentration of CHCl3 (Pohl and Krishna, 1978). This indicated that less COCl2 was formed from CDCl3 than from CHClB. Likewise, 00013 was significantly less hepatotoxic than CHCl3 in rats 24 hr after admini- stration (Pohl and Krishna, 1978). These results indicated that cleavage of the C-H bond was a requisite step in the hepatotoxicity of CHCl3 and that COCl2 probably was produced 12_11!2_in the liver. Subsequent experiments have revealed there is less 12_!1!2_formation of phosgene from CDCl3 than from CHCl3 (measured as formation of 2- oxothiazolidine-4-carboxy1ic acid in the livers of rats pretreated with cysteine and phenobarbital) (Pohl 22_21,, 1979). Likewise, less 20 diglutathionyl dithiocarbonate was formed from CDCl3 than from CHCl3 in rat liver microsomes incubated in the presence of glutathione (Pohl 22_21,, 1981). 2. Hydrogen Abstraction Another possible pathway for CHCl3 metabolism involves the abstraction of a hydrogen atom resulting in the formation of a tri- chloromethyl radical (C13C-, Figure 3) (Pohl 22_21,, 1980). Tri- chloromethyl radical is a reactive metabolite produced during the reductive metabolism of carbon tetrachloride (CC14), another exten- sively studied model hepatotoxicant (Butler, 1961; Reynolds, 1967, 1977; Recknagel and Glende, 1973; Recknagel 21_21,, 1977). Formation of this intermediate should produce a pattern of metabolites and characteristics of toxicity similar to those observed for CC14. The characteristics of hepatotoxicity produced by CCl4 are markedly different from those produced by CHC13; these differences will be discussed below. The trichloromethyl radical has several fates, which have been summarized in a recent review by Hanzlik (1981). Both the trichloromethyl radical and chlorine radical could bind irreversibly to protein and lipid (Butler, 1961; Reynolds, 1967, 1977) or could abstract hydrogen atoms from lipids or other potential sources of hydrogen atoms to form CHCl3 (Butler, 1961; Paul and Rubinstein, 1963; Rubinstein and Kanics, 1964; Uehleke 2§_21,, 1973; Shah gt_21,, 1979; Kubic and Anders, 1981). Additionally, the trichloromethyl radical could react with molecular oxygen to form phosgene (Shah 22_21,, 1979; Kubic and Anders, 1980); or it could react with another 21 trichloromethyl radical to form hexachloroethane (Fowler, 1969; Uehleke 22_21,, 1973). The reactions of the trichloromethyl radical are depicted in Figure 4. An additional reactive metabolite which may be formed is dichloromethyl carbene (C12C:), which would result from the further reductive metabolism via chloride elimination of the trichloromethyl radical (Wolf 21_21,, 1977). The metabolic fate of dichloromethyl carbene (C12C:) will be discussed with (4) Hydrogen Ion Abstraction. 3. Reductive Dechlorination The reductive dechlorination of CHCl3 would produce a di- chloromethyl radical (CleC-) which would undergo reactions similar to the trichloromethyl radical (Figure 3) (Pohl 21_21,, 1980). Thus, as illustrated in Figure 4, this radical intermediate could bind irre- versibly to tissue macromolecules, or abstract hydrogen atoms from lipids or other potential sources of hydrogen atoms to form dichloro- methane (CHZClz). The toxicity produced by this intermediate should be similar to that produced by other free radical intermediates, such as trichloromethyl radical. 4. Hydrogen Ion Abstraction The final alternative pathway of CHCl3 metabolism involves the abstraction of a hydrogen ion to produce trichloromethyl carbanion (C13C:') (Figure 4) (Pohl, 1979; Pohl 22_21,, 1980). This interme- diate could be hydroxylated to produce phosgene (C0012), or could abstract a hydrogen ion (H+) to form CHClB, or could spontaneously eliminate a chloride ion (Cl') to form the reactive electrophile 22 dichloromethyl carbene (C120:). Dichloromethyl carbene may bind irreversibly to tissue macromolecules or may be further metabolized, under anaerobic conditions in the presence of water, to form carbon monoxide (CO) and HCl (Wolf 21_21,, 1977; Ahmed 22_21,, 1977; Anders 22_21,, 1978; Ahr 21 21,, 1980; deGroot and Haas, 1981). Although there is evidence for a small amount of carbon monoxide formation during CHCl3 metabolism, this appears to occur under aerobic condi- tions with the incorporation of molecular oxygen as discussed above (Stevens and Anders, 1979). Thus, extensive studies of hepatic metabolism of CHCl3 and its trihalomethane analogs indicate that oxidative dechlorination is the primary metabolic pathway. There is little evidence to support the formation of CHCl3 metabolites that would be produced by the alternative metabolic pathways of hydrogen abstraction, reductive dechlorination, or hydrogen ion abstraction. The main evidence against most of these alternative pathways comes from the differences observed between CHCl3 and CCl4 metabolites f 14c and manifestations of toxicity. For example, the pattern 0 incorporation into constituents of liver 12_vjyg_and liver microsomes 12_!1222_is markedly different for CCl4 and CHCl3. A larger portion of 14CHCl3 is bound to protein than with 14CCl4 (Reynolds and Yee, 1967; Uehleke and Werner, 1975). Likewise, CCl4 produces extensive lipid peroxidation in livers of untreated rats (Recknagel 21_21,, 1977) whereas CHCl3 does not (Klaassen and Plaa, 1969; Reynolds, 1972; Brown 22_21,, 1974a) except when hepatic mixed function oxidases have been induced (Brown 22_21,, 1974a). CCl4 will rapidly decrease 23 hepatic cytochrome P-450 concentrations (Recknagel and Glende, 1973) whereas CHCl3 does not in non-induced rats (Brown 22_21,, 1974a). Additionally, CHCl3 depletes hepatic glutathione concentrations where- as CCl4 does not (Johnson, 1965; Brown 22_21,, 1974a; Docks and Krishna, 1976). Furthermore, covalent binding of radiolabelled CCl4 indicates 14C and 36Cl (Reynolds, 1967) suggesting the 3 incorporation of both generation of free radicals. Negligible amounts of H or 36Cl incor- poration have been detected after incubation of CHCl3 with rat liver microsomes under various incubation conditions (Pohl 22_21,, 1980). Electrophilic chlorine has been trapped with 2,6-dimethylphenol after incubation of CC14, but not CHC13, with rat liver microsomes (Mico 22_ 21,, 1982). These data indicate that trichloromethyl radical (C13C-), dichloromethyl carbene (012C:), or dichloromethyl radical (ClZHC-) are not major reactive metabolites of CHCl3 (Pohl 2§_21,, 1980). Dehalogenation, CCl4 metabolism and irreversible binding are favored when C014 is incubated anaerobically with rat liver microsomes whereas the metabolism and binding of CHCl3 requires oxygen (Uehleke and Werner, 1975; Sipes 23_21,, 1977). Finally, when CDCl3 was administered to mice, no exchange of the deuterium label to form CHCl3 was observed (Krantz 21_21,, 1967). CHCl3 would be expected to form if the alternate metabolic pathways of hydrogen abstraction, reductive dechlorination, or hydrogen ion abstraction were of significance. 24 C. Characteristics of CHCl3-Induced Nephrotoxicity Susceptibility to CHCl3 nephrotoxicity varies between species. The renal lesion induced by CHCl3 was localized primarily to the proximal tubule and was characterized by increased kidney weight, cloudy swelling of tubular epithelium, fatty degeneration, tubular casts and/or marked necrosis of proximal tubular epithelium with a lesser involvement of the distal tubules (Eschenbrenner, 1944; Derin- ger 21_21,, 1953; Plaa and Larson, 1965; Thompson 23_21,, 1974; Torkel- son 22_21,, 1976; Clemens 22_21,, 1979). Renal functional changes include proteinuria and glucosuria, decreased excretion of exogenously administered phenolsulfonphthalein and increased blood urea nitrogen (Plaa and Larson, 1965; Klaassen and Plaa, 1967; Kluwe and Hook, 1978). 12_11322_accumulation of organic ions by renal cortical slices also was decreased by 12_yjyg_CHC13 administration (Watrous and Plaa, 1972; Kluwe and Hook, 1978; Kluwe, 1981). In mice, CHCl3 did not produce glomerular damage and the renal lesion was sex-dependent; only males were susceptible (Eschenbrenner, 1944; Deringer 2;_21,, 1953). The mechanism of CHCl3-induced nephrotoxicity is unknown but may be the result of 1) direct CHCl3 nephrotoxicity, 2) a hepatic metabo- lite that is directly nephrotoxic; 3) a hepatic metabolite that is further metabolized by the kidney to a nephrotoxic compound, or 4) the renal metabolism of CHCl3 to a nephrotoxic compound. Most of the experimental evidence supports either the 12_§112_metabolic activation of CHCl3 or a hepatic metabolite of CHCl3 as the mechanism for nephro- toxicity. For example, autoradiography studies indicated that the location and distribution of radioactivity to the kidney after 25 administration of ‘4 CHCl3 was proportional to the extent of nephro- toxicity (Ilett 21_21,, 1973; Taylor 22_21,, 1974). Furthermore, the radioactivity was localized to the necrotic proximal tubular cells and centrilobular hepatocytes, the regions of the highest mixed function oxidase activity in these organs (Ilett 21_21,, 1973). Since CHCl3 does not bind readily to macromolecules without metabolism, renal metabolism of CHCl3 to some reactive intermediate is probably required to elicit nephrotoxicity. CHCl3 also produced a dose-dependent reduc- tion in renal glutathione concentrations in male ICR mice similar to that found in the liver which may result from an electrophilic inter- mediate of CHC13. Glutathione depletion alone did not produce nephro- toxicity. However, depletors of glutathione content such as diethyl maleate potentiated the extent of nephrotoxicity induced by CHCl3 (Kluwe and Hook, 1981). In the liver, cleavage of the C-H bond in CHCl3 was a rate-limiting step in CHCl3 metabolism as 00013 was less hepatotoxic than CHCl3 (Pohl and Krishna, 1978; Pohl gt_21,, 1979). Likewise, the nephrotoxicity in mice was less in animals receiving CDCl3 than in those receiving an equal dose of CHCl3 (Ahmadizadeh 22_ 21,, 1981). Thus, it appears that metabolism of CHCl3 is a requisite step in CHCl3 nephrotoxicity. However, whether this metabolism occurs in the kidney or liver is unknown. Several experimental approaches utilizing different sexes and strains of mice, as well as dose-response rela- tionships, indicated that the hepatic and renal toxicity of CHCl3 were independent events. Eschenbrenner (1944) originally observed renal necrosis in the absence of liver necrosis following low concentrations 26 of CHCl3 in male mice. Watrous and Plaa (1972) determined that the minimum subcutaneous dose of CHCl3 effective in producing nephrotoxi- city in mice was 0.005 m1/kg with a near maximal effect at 0.025 m1/kg, as determined by the decreased ability of renal cortical slices to accumulate p-aminohippurate (PAH). These doses produced little or no hepatotoxicity. Functional studies also have detected renal toxi- city in ICR mice at doses of CHCl3 showing no evidence of hepato- toxicity (Kluwe and Hook, 1978). In contrast, to other animal species, in mice only males were susceptible to CHClB-induced nephrotoxicity, whereas hepatotoxicity occurred in both sexes (Eschenbrenner, 1944; Eschenbrenner and Miller, 1945; Shubik and Ritchie, 1953; Hewitt, 1956; Culliford and Hewitt, 1957; Klaassen and Plaa, 1967; Ilett 21_21,, 1973; Taylor 22_21,, 1974; Clemens 21_21,, 1979). This sex difference in CHCl3 nephro- toxicity in mice appeared to be an effect of testosterone. Immature or castrated male mice were not susceptible to CHCl3 nephrotoxicity (Eschenbrenner and Miller, 1945; Deringer 21_21,, 1953; Culliford and Hewitt, 1957); female mice or castrated male mice treated with testo- sterone were susceptible to CHCl3 nephrotoxicity (Eschenbrenner and Miller, 1945; Culliford and Hewitt, 1957; Taylor 22_21,, 1974). The lack of a testosterone effect on CHCl3 nephrotoxicity in female mice treated concomitantly with testosterone and the antiandrogen fluta- mide, or in androgen unresponsive Tfm/Y mice, suggested that an andro- gen receptor may mediate the effect of testosterone on CHCl3 nephro- toxicity (Clemens 2t_21,, 1979). In fact, it has been suggested that the dramatic strain differences in mice with respect to the degree of 27 CHCl3 nephrotoxicity may by related to strain differences in androgen production (Hill 22_21,, 1975; Clemens 21_21,, 1979). Alteration of sex-hormone status had no effect on CHCl3 hepatotoxicity (Ilett 21_ 21,, 1973; Taylor 22_21,, 1974). Testosterone induces dramatic morphological and biochemical alterations in the proximal tubules of mouse kidney. These changes include renal hypertrophy (Selye, 1939), enhanced RNA and protein synthesis (Kochakian 22_21,, 1963; Koths 22_21,, 1972), larger and more extensively developed organelles such as mitochondria, lysosomes, smooth and rough endoplasmic reticulum, and Golgi apparatus (Koenig 22 21,, 1980) and increased activity of many renal enzymes. The effect of testosterone on renal mixed function oxidases has not been quanti- fied. The inability of female mouse kidney to activate dimethyl- nitrosamine to a mutagen in contrast to male mice suggests there may be sex-related differences in mouse renal cytochrome P-450 concentra- tions (Weekes and Brusick, 1975). Morphologically, male mice have a greater amount of smooth endoplasmic reticulum than female mice (Koenig 21_21,, 1980), also suggesting there may be sex-related differ- ences in renal cytochrome P-450 concentrations. Therefore, sex- related differences in the susceptibility to CHCl3-induced nephro- toxicity may be explained by alterations in the ability to generate a nephrotoxic metabolite within the kidney. Further evidence for intrarenal metabolic activation of CHCl3 comes from studies employing inducers and inhibitors of drug metabo- lizing enzymes (Table 1). However, these results are complicated by the simultaneous induction of hepatic as well as extrahepatic enzymes, 28 mmcmzu o: u o mmmwcomu u . mmmmgucw n + pwmp .xooz can mzsz + . u + . mummpme fixcpwwo PmmF .xooz ecu oza—x + u + . wuu< on: humorxop mmwpw>muu< on: mUCmLmkmm IOLSQwZ chwm IOHGQGI UwHmeI momuexoe m_u=o co poaccm ago new abw>woo< Aoazv amaewxo cowpocsd umxwz ovumam: ucm chmm mmzoz co mcopwnwch vcm memosucH mo muummmm — m4m

wucw 00 use mppwo mo mcwcooFPmn .m:w_~ozm wuxooueam; umxeme .m—uzu Levee L; em .mpmz on .Umuocuwc mew mop»00pmam; msom mmmpxoopeam; mo mchszm empznopvwe use uwcpcmu mmpuzu cmuem e; m .m—memu on .cowuomnzw mpuzo Loewe L; m op e pcmgmaam pmcwm we: mesh .mmpxu -opmam; mo mcwp_mzm Lepaaopwepcmu Amparo cmpwm L: m .mpmz Amv .ew>wp Focucou .msz Awuepcmmmeamm .m mcamwd 57 z “vein“... I. (figure. V A (. ”3&5...“ w .4» .. . s Q _v4' _ 4““ < I r 58 Figure 9. Time course of decrease of renal cortical non-protein sulfhydryl (NPSH) concentration after 250 pl CHCl3/kg, s.c., in male and female ICR mice. Values are mean : S.E.M., n=4. 59 KIDNEY Control pmal NPSH lg tissue A Female 4.9 “30.4 0 Male 5.3 ‘1' 0.3 140 l. 120 N30 80 x C antral 401 2O Figure 9 60 control values during the 24 hr period. Decreases of the steady-state accumulation of PAH and TEA could be detected 2 hr after CHCl3 admini- stration in male mice, and were maximal by 5 hr (Figure 10). PAH and TEA S/M ratios remained decreased during the 24 hr time course. These functional changes paralleled the alterations detected in renal corti- cal non-protein sulfhydryl concentrations in male mice. In female mice, the accumulation of PAH and TEA was variable at the early time points, but there were no significant decreases of the S/M ratios during the 24 hr period to indicate nephrotoxicity. Slight increases of BUN concentration occurred in male mice by 5 hr after CHCl3 admini- stration (Figure 11). By 24 hr, the increase in BUN was more drama- tic. There was no increase of BUN at any time point in female mice, again indicating a lack of susceptibility to CHCl3 nephrotoxicity. Histological comparison of kidneys from male and female mice showed equally profound results -- no lesions were observed in female mouse kidneys at any time (Figure 12). In male mice, the earliest evidence of necrosis as detected by light microscopy was in proximal tubular cells 5 hr after CHCl3 aministration. Nuclei were pyknotic and there was loss of reticular cytoplasmic structure (Figure 12A). The lesions progressed to frank necrosis by 8 hr and increased in distribution and severity with time in the proximal tubular region only. By 12 hr, the tubular lumens were occluded with hyaline casts (Figure 128). Epithelial cells of Bowman's capsules and distal tubules still appeared normal at 24 hr (Figure 12C). 61 Figure 10. Effect of 250 p1 CHCl3/kg, s.c., on in vitro renal cortical slice accumulation of p-aminohippurate (PAH 37M) and tetraethylammonium (TEA S/M) in male and female ICR mice. Values are mean :_S.E.M., n=4. PAH SIM TEA SIM 25 N O 5 62 A Female 0 Male Figure 10 63 Figure 11. Time course of blood urea nitrogen (BUN) concentration after 250 p1 CHCl3/kg, s.c., in male and female ICR mice. Values are mean :_S.E.M., n=4. BUNlmg/dl) 64 A Female ID Aflcfle 140 [— 120 - 100 - Figure 11 65 .oopx .cwm m :wmom use cwpxxoumem: .cowpeeummcwsue mpuzu empwm umeesouo mcowpmcwppm Fmmeopocpmaopmw; o: m _uzu gmu+m g; cm .mFmEme mov .uumucw smegma mpsmamo m.:mszom Co mppmo meFmguwam "memocow: LmFanzu Fmewxocq m>wmcmpxw egos m Puzo genes as em .mpez Auv .mwmocumc cmpsnap _mswxoca m>wmmme mmpuxu Levee a; w .mpmz Amv .mFPwu cmpznau PeEFxocg Co census Fpesm m cw cmueon cmsgn mo mmop use wwpozc owpocxxa mm_u:o emumm a; m .mpmz Awumucmmwcamm .NF oczmwu 66 ~_ mesmee 67 3. Effect of Decreasing_Renal Cortical Non-Protein Sulfhydryl Concentration on CHClQ Toxicity in Male and Female ICR Mice The role of non-protein sulfhydryl concentrations in the susceptibility of male and female mice to CHCl3 nephrotoxicity was evaluated in mice pretreated with diethyl maleate to reduce renal glutathione concentrations. Male and female renal cortical non-protein sulfhydryl concentrations were reduced by 66 and 80%, respectively, and by approximately 90% in the liver of both sexes 30 min after an i.p. injection of 0.6 m1 diethyl maleate/kg (Table 2). Administration of diethyl maleate 30 min prior to CHCl3 (50 or 500 p1/kg, s.c.) did not alter the susceptibility of female mice to nephrotoxicity but did potentiate nephrotoxicity in male mice (Table 3). 4. Effect of Partial Hepatectomy on CHCl, Nephrotoxicity in Male ICR Mice ” Partial hepatectomy of male mice did not alter the nephro- toxic response to CHCl3 compared to control or sham-operated mice (Figures 13-15). Four hours after the s.c. injection of 250 pl CHClB/kg, accumulation of PAH and TEA and renal cortical non-protein sulfhydryl concentrations were significantly decreased in all three groups (control, sham-operated and partial hepatectomized) (Figure 13A-C). There was no difference in the nephrotoxic response to CHCl3 of partial hepatectomized mice compared to control. Since 250 pl CHCl3/kg, s.c., produced a maximal nephrotoxic response in the intact mouse (Figure 5), the effect of partial hepatectomy on the nephro- toxic effect of two lower doses of CHCl3 was assessed. Four hours after 50 pl CHCl3/kg, s.c., accumulation of PAH and TEA was signifi- cantly decreased in all three groups, but again there was no 68 .eue ..z.e.m.H gees eee meepe> .msozeez ee umsmsommu me umsmeseamu wee: msomuesusmosoo Azmszv pxsuaswpam symposssos owuesms use Feuwpsoo pesos use sompomwsw smaee see om uePFPx use: mow: .eoms muH mFesmm use mpes on Amx\_E mv sompomnsw .s.w >3 umsmumwswe -ue we: pmo ussems sw ue>pommwu mpempee Fxsumwu mx\PE 0.0 so Pwo pasemse Noe e~.sm~e.o om.QMem.e eFeEee see m_.o+ee._ me.o+e_.m e_ez eeeees ems ee.gMee.o em.smM_.e eFeEee see em.o+eo._ om._+ee.m epez ee>_s mammmu m\_oEs .sowuesusmusou :msz Peeeeou sees eeeepes ssseeeo Fee eeeeea xem cease mmemsumo & psmEpemspess emsowpespsmusou pxsuasmpum sweeosssoz uwpesm: use peowpsoo Pesem so muempez pxspmws mo aummem N mgm

wmomc wows pocpcou soc» pcmgmccwu appcmurmwzmwm4" .mo.ova Amparo mcw>wmumc Ho: aaoco “cmEummLu wswm as» cw wows soc» ucmcmmwwc xpucmowwwcmwmuw .mu: ..z.m.m + some mcm mmapm> .cowpumncw mpuzu cmgmm c; w cmcsmmms «cm: mcowumcgcmucou :maz uwpmaw; Adv ucm .muw>wuom Ahmwmv mmmcwsmmcmcu o_>:c>a owEwgzpm Escmm Amy .Azamv cmmocpwxlmmc: noopn ADV .cowpmcucmucou Azmmzv ~>cu>;$_zm ammuocnucoc quwpcou rmmmc Auv .Az\m cw cowpm~353uum ancoEEmpxzummcpmg qumugou Paco; Amy .Az\m z cm cowme253oom mpmcsaamgocwsmua Fmowpgou pmcmm A Lo Amx\~n ommv Puzu .mums mum mFms cw xumuwxopocgam: mpuzu co xsouumumaw: pampcma we pummmm .m_ mczmwd anu HEPAIECIOMY 250 pl CHCl3/hg a Conno' s Sham - Ooomnd . Pomol Hepolouovny 71 U ___my,;,l, A 'C’ " Jig/,2 5'1 0 OJ {111111- muguuuuu#1111911 l l l J ‘4 V M N "' o (0058;. Shown) HSdN '0)gu°) 10003 ....................... m ae‘ W42 g ( mmmmumm V :n Z42; : 1 "a ,. ~ " I1 2 .:umnuuuunmnmmuuuu1. l 1 4 A _l O o O O O V H N " wls v31 4 L l I I O O O O M (N "' wls ma ."‘ u I u u. 01 a 1 1 1 n I l 9 o o v a o (snug; 6 I|Owfl) HSdN anode” _n v I u w . 4 O 5' 1 1 J 8 o o o 8 N ‘— (1w In) mos _M U I U D O c 1 O O O O o v m (IF/5W) mm Figure 13 72 difference in the degree of nephrotoxicity in partially hepatectomized mice (Figure l4). Decreasing the CHCl dose to 10 ul/kg did not pro- 3 duce nephrotoxicity within the 4 hr period (Figure 15). There was no increase of BUN by 4 hr after CHCl3 administra- tion, consistent with the data from the time course study (Figure ll). SGPT was increased after 4 hr in the sham-operated and partially hepa- tectomized mice. This increase probably reflects damage to the liver during the surgical procedure since there were increases of SGPT in mice not receiving CHCl3. The decrease of hepatic non-protein sulfhy- dryl concentrations in mice receiving 250 pl CHCl3/kg probably is the result of CHCl3 administration. 5. Effect of Sex Hormone Status on CHCl Toxicity and Renal and Hepatic Mixed Function Oxidases in ICR’Mice a. Mixed function oxidase activity Concentrations of cytochromes P-450 and b5 and ethoxy- coumarin-O-deethylase activity were greater in male than in female mouse kidney microsomes (Table 4). Treatment of male and female mice with testosterone increased these mixed function oxidase components to similar activities; whereas castration of male mice reduced renal mixed function oxidase activity to that observed in untreated female mouse kidneys (Tables 4 and 5). Altering testosterone concentrations in mice did not affect renal NADPH cytochrome-c reductase or ethoxy- coumarinFO-deethylase activities, nor were there any alterations of hepatic cytochrome content or hepatic mixed function oxidase activi- ties (Tables 4 and 5). 73 .mo.ovg .mpozu mo mmov msmm any mcw>wmomc wows Focpcou soc» ucmcmmwwu >chmuwwwcmwmxn .mo.ova .mpuzu mcw>wmomg yo: azocm acmEpmmgu mamm msu cw wows soc» pcmcmwmwc >_u:quwmcmwmnw .vu: ..z.m.m.H cums mew mm=Fm> .cowpumncw mpuzu cmpcm c; e uwcsmmme mgmz mcowumcycmocou :maz ovumam; Adv can .xpw>wpum Apmwmv mmmcwsmmcmcu uw>zc>a umEmuapm Eacmm Amv .Azzmv :mmocuwc1mmc: coopn on .cowumcucmucou Armazv chuxsmpzm cwmuocauco: Pmuwucou rmmmc on .Az\m cw cowumpzszoum Ezwcoesmpzsammcpmu Fmowpcou chmc Amv .Az\m :«av ocpw> cw cov9m93530um muwczanwsocwsmua quwpcoo chmm A__mwggma co .cmumcmaousmgm .Focucou op Amx\FE NV .u.m umcwumwcwEum mm: A.o.a .Fwo “seawav mpumgm> so Am¥\~n omv mpuzu .muwe mum opus cw >HWwaouoggamc mpuzo co xeouuoumam; Powucma we uumwmm .ep mcamwu PAlllAl HEPATECTOMY so ul c1403] kg 3 Control Sham - Operated \V L; V I Partial Hapanuarny ;::r225::aaazzaaaaaasaazazzzz ZZZ;33333ZZ22252!?!iiifiiZZZZiaafififiifi III12:llllllllllllllllllllllllllllIlllllllllllllll 74 (1403 P.O n N '- O L 1 4 I o o o o n N ‘- wls 1131 _t") -x- gm 5; U 0 a; L 1 l J O O O O n N "' wls HVd l_ l l l 0 V N 3’me ::::311111111111111111111111111111 2333?¥333¥33822 iiiilllllllllllllllll“ (:1 (anuu B’gaum) HS‘N :ggadaH [1111] 000 822§3° Ifl'MNQ (aw/n) 1495 ZWW 'Iiiilllllllll|l|llllllll O l 21 .21 n 1:) (I) ¢:> 4:) -GD 'I' “' (191”) m ("(13 RC. c1103 P.O. (1103 RC Figure 14 75 .me.eva .m_e:e we emee esem ecu mcw>weeeg mews wecpeee Eecw peecewwwe z—uceewwwcmwmxn .mo.ove .mwozu mew>weeec we: eeeem useEeeeLu eEem ecu cw mews Eecw peegewwwe xpaceewwwemwmxn .muc ..z.m.m.H some wee meewe> .eewpeenew mwuxu Leuwe c; e emcememe wee: meewuecuemeeee :mez owpeee; Amy ece .xuw>wuee Awewmv emeewaemeecu ew>eexe ewEeuewm Eecem ADV .Azsmv :emecupc ewwm eeepe Auv .eewuecuceecee Armezv wxcexz -wwem cweeeceueee Peewucee Page; Amv .Az\m z cw eewpe_:seeee eueceeewgeewEeue weewugee weeem A we Amx\wn owv MFUIU .eewe muH eFes cw xpwewxepecseee mwuzo :e zsepeepeee; Fewucee we ueewwm .mp egemwd PARTIAL HEPATECTOMY 10 pl CHCl3/kg 76 ................ ................ ................. CHC|3 22illlllllllllllflllIlllllllll IIIWWWMW 1IIIIllIllllllllflmmmlllllllIll L I 1 1 .0 Q N O (enssg. 6’ lawn) HSdN puma: Iauaa PO. > E .0 2 2 3 E ‘6 O O. Q 0 o I 3 u -. z E .2 C O 5 O .5 O U m & II I :':*~';-.-:3:':-:-: .n W ‘1’ U < :W 2 Zillllllllllllllll L L l J O O O O M N "’ wfs mu 7:;1WIWW ::1|IllllllllllIllllmlIllllllllllllllllllllllmllll1 IttllllllllllIllllllllllllllllllllllIllllllllllllllllllll"‘ 1 L 4 1 1 +_1 9 an o v n o ( onssu Snow“) HSdN sand.” I 1 l l l _J 2 8 8 8 8 ° 2 2 .9. 2 2 (1min) 1:195 le/fl/flz ' ‘.‘.’.'. .'.'. ............. .................... L L 1 _.l O O O O 0 Q N (nu/5w) won 010;; P.O. cuc13 P. O CHCI3 P.O. 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L l 1 0 so 500 D 10000 - E 1000 h '3 E 100 - o tit 10 - L 1 1 0 so 500 pl CHClalkg Figure l7 87 TABLE 9 Effect of Castration and CHCl3 on Renal and Non-Protein Sulfhydryl Concentrations aHepat1c Non-Protein Sulfhydryl Concentration‘ (nmol/g tissue) Treatment CHCl3 (pl/kg) 0 so 500 RENAL Control 6.63:0.050 3.93:0.193 2.43:0.19g Sham 7.16:9.062 3.65:0.28C 2.33:0.07c Castrate 5.95:9.068 5.72:1.12 6.39:9.62 HEPATIC Control 14.3o:2.78 9.l7+l.64 9.68+l.38 Sham 14.11:2.06 lO.28E].77 8.29:).59 Castrate 8.45:2.61 14.41:].36 12.94:2.28 aMale ICR mice were castrated, or underwent sham surgery, or were un- treated (control) as described in METHODS. CHCl3 (so or 500 ul/kg) or vehicle (peanut oil) was administered s.c. (2 ml/kg) after a 3 week surgical recovery period. 24 hr after CHCl3 Values are mean :_S.E.M., n=3. NPSH concentrations were determined injection in hepatic and renal cortical tissue. bSignificantly different from mice within the same pretreatment group receiving 0 ul CHCl3/kg, p<0.05. cSignificantly different from control mice, p<0.05. 88 where there was a trend for SGPT to be increased to a greater extent than in peanut oil-treated male or female mice (Figure 160) and for hepatic non-protein sulfhydryl concentrations to remain decreased 24 hr after exposure to CHCl3 (Table 8). c. Histology Testosterone treatment of female mice produced dramatic morphological changes in the kidney and in the renal response to CHCl3. Testosterone increased the size of proximal tubular cells in the outer cortex but not in the cortico-medullary region (Figure l8). After testosterone treatment of female mice the extent and degree of renal cortical necrosis produced by CHCl3 was similar in severity to lesions in male mice (Figure l9). The marked hypertrophy of renal cortical cells and the increased severity of histological lesions were not detected in kidneys of male mice treated with testosterone, though KN/Bw and the biochemical indices of nephrotoxicity suggested there may have been some hypertrophy of male mouse kidney after testosterone treatment (Figure 16, Tables 6 and 8). No renal lesions were detected in castrated male mice at either dose of CHCl3 (Figure 20). No reduc- tion of proximal tubular cell size was observed in castrated male mice. Castration of male mice did not affect the hepatic lesions produced by CHCl3. The degree of hepatic lesions was greater in untreated female than in male mice or in testosterone-treated female mice. 89 Figure 18. Sex differences in ICR mice renal proximal tubule morphology. Male and female mice were injected s.c. for 3 weeks on alternate days with O.l ml of testosterone (1 mg) or peanut oil. (A) Male. (B) Female. (C) Female treated with testoster- one; increased size of proximal tubular cells in outer cortex. Hematoxylin and eosin stain, xlOO. 90 Figure l8 — 91 Figure l9. Effect of testosterone on susceptibility of female ICR mice to CHCl3 nephrotoxicity. CHCl (500 pl/kg) or vehicle (peanut oil) was administered s.c. (2 ml/kg to pretreated female mice in- jected s.c. for 3 weeks on alternate days with O.l ml of testoster- one (1 mg) or peanut oil. Mice were killed 24 hr after CHCl injec- tions for histological examination. (A) Female pretreated w1th peanut oil 24 hr after 500 pl CHCl3/kg, no histopathological altera- tions occurred after CHClg. (B) Female pretreated with testosterone 24 hr after 500 pl CHCl /kg; extensive proximal tubular necrosis. Hematoxylin and eosin sEain, xlOO. 92 . . _'~‘ I:.p;:'"v, (‘ 7: .'{TN‘ r 1 V‘ . b 1570 saver: .1 » ' - 1. _ . . 2‘ 3, . . . u ‘1'“ (1‘th «*Afmimga ‘2. ' “‘3... #84519.“ a“; _ ‘ “ Figure 19 93 Figure 20. Effect of castration on susceptibility of male ICR mice to CHCl3 nephrotoxicity. CHCl (500 pl/kg) or vehicle (peanut oil) was adm1nistered s.c. (2 ml/kg to male mice which were castrated, or underwent sham surgery, or were untreated (control) as described in METHODS. Mice were killed 24 hr after CHCl3 injections for histological examination. (A) Control male 24 hr after 500 pl CHCl3/kg; extensive proximal tubular necrosis. (8) Castrated male 24 hr after 500 pl CHCl3/kg; no histopathological alterations occurred after CHC13. Hematoxylin and eosin stain, xlOO. 94 e\B.le. V. ow. .. 1‘1. \‘0. e . . . .. n .. . J. A.“ .x v 1. “may“! “in“. . 4.1., .s. 4.0”»! , ». O .‘ lax .. “we . . are: i A. , . .MwfimL . . . N .mc‘.gn p nypmw.w_ ; 3...... h&uW.J.\..1z- . r not? . .ex. 1..., I. q. .92. a... loam. “_‘...“.x . \_ u. .‘u .. \\ A 4 . ’e.~‘.w ¢ ...o e .. I .. Qua»... Figure 20 95 B. In Vitro Studies - Toxicity of CHCl3 in Renal Cortical Slices 1. Assessment of CHCl, Nephrotoxicity In Vitro in ICR Mouse Renal Cortical Slices Preincubation of renal cortical slices from male and female mice with 0.5 pl (26.25 pmol) CHCl3 for up to 2 hr produced decreases in organic ion accumulation in male kidney slices only (Figure 21). TEA S/M ratios were decreased after 60 min of preincubation while the decrease of PAH S/M ratios was not observed until 90 min. The effect on organic ion accumulation was near maximal after a 2 hr incubation at this concentration of CHCl3. Preincubation with up to 4 pl (250 pmol) CHCl3 for 90 min in_ vitrg_produced a concentration-related decrease in the ability of slices from males to accumulate PAH and TEA, while slices from female mice were only affected at the highest CHCl3 concentrations (Figure 22). CHCl3 (4 pl/ 250 pmol) reduced the PAH and TEA S/M ratios in slices from female mice less than the reduction in slices from male mice incubated with only 0.25 pl (23.1 pmol) CHCl3. Organic ion accumulation was decreased less after preincuba- tion of male kidney slices with CDCl3 (1 pl; 212.5 pmol) than with an equimolar concentration of CHCl3 (Figure 23). Preincubation of slices with CHCl3 under an atmosphere of carbon monoxidezoxygen (80:20) or at 0°C prevented the CHCl3-induced decrease of PAH and TEA accumulation (Figures 24 and 25). Carbon monoxide alone decreased the PAH S/M (15.1 versus 10.3), but there was no further decrease after the addition of CHC13. In general, the accumulation of TEA appeared to be less sensitive to alterations of 96 .eue ..z.m.m.H sees ace mmepe> .moozwmz cw eeewcemee me eeueeeeee ewe: :ewpew3530ee .mooxwmz cw eweweemwe we ewpeeeeee me: :ewpe_:E=0ee cw mwozu we zuwewxeu :e Aouv wewxeees :eeeew we pewwwm .em weemww 103 we weaeee Sue-10.00 - 00 :2... 3: 3e: - 30:0 30:0 30:0 +00 30:0 00 62200 +00 H J O W 1 2 "III I. * * w e. 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Assessment of the Nephrotoxicity of Hepatic CHCl Metabolites in Male ICR Mouse Renal Cortical Slices Preincubation of male mouse renal cortical slices with 6.25 or 12.5 pmol CHCl3 for 90 min at 37°C decreased the ability of slices to accumulate PAH and TEA (Table 10). Preincubation of slices with 6.25 pmol 2-oxothiazolidine-4-carboxy1ic acid (OTZ) or diglutathionyl dithiocarbonate (GSCOSG) under the same conditions had no effect on PAH and TEA S/M ratios. 3. Effect of Mouse Strain Differences on CHC13T0xicityiln Vitro CHCl3 toxicity was assessed in renal cortical slices pre- pared from three different mouse strains reported to exhibit different susceptibilities to CHCl3 nephrotoxicity jn_vivg_(Figure 28). Previous investigations have indicated that male mice of the C5781/6 strain are relatively resistant and those of the DBA/Z strain are relatively sensitive to the nephrotoxic effects of CHCl3 (Hill et_al,, 1975; Hill, 1977; Clemens gt_gl,, 1979). Based on the jn_vit§9_effect of CHCl3 on the ability of renal cortical slices to accumulate PAH and TEA, male ICR mice were most susceptible to nephrotoxicity. Male C57Bl/6 and DBA/2 mice showed similar susceptibilities to CHClB-induced nephrotoxicity, slightly less than that observed in male ICR mice. 107 .85; $55 :5: 228.2% 328:??? .mo.ovq .Fogucou Eocm acmcmmwwv xpuchwacmwmxn.¢uc ..z.m.m.H come mgm mmzpm> .moozhmz cm cwnwgummu mm cmpuzucoo we; cowpm_:E:uum cw mpozu mo xpwuwxou co :mmocpw: mo pumwmm .mm mesmvu 108 * u. n65 +N2 n .UIU N2 .0528 E * all om aczmwa Nz x oo— - «z 2253: 3 3 - mazu fin! IO— JON Len W/S V31 ”55 +~z n55 Nz ‘01»ZOU Q * * LII-III‘II ii [HH- *1 * Z 0— ON on WIS HVd 109 .mo.ova .mFozu EOLF ucmngFFv FFchoFFchFm+ .mo. ovm .2mo EogF ucmcmFFFo prcmuFFchFme .mo.ova Focpcou EoLF acmLmFFFc xFucmuFFchFman .«u c ..z. m. m + cmme mgm mmaFm> .mooszz :F umnFLummc mm umuuzucou mm: coFFszsaoum :F mFuzu Fo proFxou :o o>F> :F FamEpmogpmga mummFmE Fxcmeu Fo pumFFm .FN mesmFm 110 «65 F +23 520 {HHHHHHHHIIHHH IH {*- FN atamFu 9:356 - sac :25 22 .a and .326 55 23 .953 , o 111 j J 9000. 1 o. . n H... v F H w I ON L 8 I -HHHIHHHHHHHHHHHHH +23 «55 .23 6523 O— ON on WIS HVd 111 TABLE 10 lg_Vitro Toxicity of CHCl and Hepatic Metabolites in Male Mouse Eidney Slicesa PAH S/M TEA S/M Control l5.5:p.l 24.2:9.8 6.25 pmol CHCl3 (0.5 ul) 9.8:9.8* 14.7:9.9* l2.5 pmol CHCl3 (1.0 pl) 7.2:].4* l3.6jj.5* 6.25 pmol OTZ 16.0:p.8 24.6:l.l 6.25 pmol GSCOSG 18.9:J.6 26.7:J.8 aMale ICR mouse renal cortical slices (1001J0 mg) were incubated in 2 ml of an isotonic phosphate-buffered medium (pH 7.4) with CHCl3 or its metabolites, 2- oxothiazolidine-4-carboxylic acid (OTZ) or digluta- thionyl dithiocarbonate (GSCOSG), for 90 min at 37°C in an oxygenated, sealed 25 ml Erlenmeyer flask. After preincubation, slices were assessed for their ability to accumulate PAH and TEA as described in METHODS. Values are mean :_S.E.M., n=3. *Significantly different from control, p<0.05. 112 Figure 28. Effect of mouse strain differences on CHCl3 nephrotoxi- city jn_vitro. Renal cortical slices were prepared from male or female ICR, male C57BL/6 and male DBA/Z mice. Slices (100110 mg) were placed in a 25 ml Erlenmeyer flask containing 4 ml of medium and gassed for 5 min with oxygen. CHCl3 was added with a 5 pl micro- syringe. The flasks were stoppered and incubated at 37°C for 90 min in a Dubnoff metabolic shaker. After preincubation, the assessment of PAH and TEA accumulation was conducted as described in METHODS. Values are expressed as percent decrease of S/M compared to slices of the same strain with no CHCl3. Values are mean :_S.E.M., n=4. S Decrease of PAH SIM S Deacon oi TEA SIM +20P 20- 4o- 60- 801- .001 +20r- 20- 40F oo- 80*- loot 113 A Female ICR A Male ICR 0 Male C57 Bllb 0 Male DBAI2 O— l 2 ‘3 4 pl 1 1 1 1 1 #1 0 lo 20 30 40 50 nmol 1 1 1 1 J O 1 2 3 4 ul 1 1 1 1 1 J 0 10 20 3O 40 50 pmol CHCl3 I Incubation Figure 28 114 Renal cortical slices prepared from female ICR mice did not appear to be susceptible to CHCl3-induced nephrotoxicity. C. In Vitro Studies - Metabolism of ‘4 Hepatic Slices from ICR Mice CHClgiby_Rena1 Cortical and 14CHCl3 was metabolized to 14C02, covalently bound radioactivity and aqueous soluble metabolites by slices prepared from male liver and renal cortex and female renal cortex of ICR mice (Table 11). A con- centration of ‘4 CHCl3 was used that reduced PAH and TEA accumulation near maximally (Figures 22 and 28). Following a 2 hr incubation at 37°C, the production of 14C02 was greatest in slices from liver, with smaller amounts formed in renal cortical slices from male and female mice. The extent of covalent binding to liver and kidney did not parallel the evolution of 14 C02. Covalent binding was greatest in male kidney slices followed by liver slices and then female kidney slices. The production of aqueous soluble metabolites paralleled the evolution of 14C02, with liver producing more than male and female kidney. The extent of 14 CD2 production and covalent binding was deter- mined over a 3 hr incubation period in male kidney and liver slices incubated with 3.12 umol CHCl3 (Figure 29). Again, the amount of covalent binding was greater in male kidney slices than in liver slices at all incubation times (Figure 29). The degree of covalent binding to both liver and kidney was linear for approximately 1 hr. 14CO2 production in the 100 mg of slices was much greater in the liver 115 TABLE 11 lg_Vitro Metabolism of 14CHCl3 to 14CD , Covalently Bound Radioactivity and Aqueous Soluble Metabolites by Male Hepatic and Renal Cortical Slices and Female Renal Cortical Slices from ICR Micea Tissue Male Liver Male Kidney Female Kidney Detected 65.46:_5.04 14.36:D.95 8.30:9.54 (nmo1/100 mg tissue) Covalent Binding 1.84: 0.30 3.04:0.20 0.51:0.12 (nmol/mg protein) Aqueous 59.63120.00 11.94:].01 10.02:D.76 (nmol/100 mg tissue) aReactmn vessels contained 100 mg of tissue slices and 6. 25 pmol CHCl3 (specific activity ~D. 5 uCi/umol added in a volume of 5 ul dimethyl formamide) in 2. 0 m1 of phosphate buffer, pH 7. 4, composed of 96. 7 mM NaCl, 7. 4 mM sodium phosphate buffer, 40 mM KCl, and 0. 74 mM CaClg. Reaction vessels were gassed with 100% 02 for 5 min prior to adding 14CHCl3 and incubations were conducted at 37°C in an oscillating water bath (100 cycles/min). Incubations were terminated by the injection of 2. 0 ml 10% TCA. Values have been corrected for non- enzymatic metabolism of 14CHC13 using boiled slices. Values are mean + S. E. M. , n= -4. 116 .4": 22.5 H =85 a: 832, SF F2 E 3 .5 8.50% .:F ecu Fa mEFF umpmuFucF mg» pm umumcFELmF mcmz mconmasucF .AcFE\mmFuxu oon span Lopez mcFumFFFomo an :F ooFm um omuusvcou ago: mcoFumnzucF ucm mFuzucF mchum op goFLa :FE m LOF No FooF :FFz ummmmm mgmz mmemm> coFuommm .NFomu ze em.o can Fox :5 cc .LmFan mumcamosa Eanom :5 ¢.F .Fumz :2 F.mm Fo vmmoaeoo .¢.F In .LwFan mpmcamosa Fe Fe o.m :F quFEoELoFszpmch F: m.~ Fo mazFo> a :F umuum Foe:\Fu: m.ou qu>FFum uFFFumamv mFuzue Foe: NF.m can mmuFFm mammFu Fo as ooF umcqucou mmemm> :onummm .moFE «UH EoLF mmuFFm meFuLou chmc ucm uFFmam; «Fae >2 NouwF can AFF>FpomoFumc uczon szchm>oo on EmFFonmumE nFozqu Fo mmczou mEFF .mm mcamFm 117 C KIDNEY IOOr- A [IVER I BOILED L l P fl’ 5! :3 ° (onssga B111 om Haunt) P0130600 z03,, b O N -—-o > a I” 111 53-.” . :ES 0‘- l 1 l I 1'2 9 "1 ° (0mm 6111001] pwu) Bugpugg iuo'anog Incubation Time (hr) Incubation Tim (In) Figure 29 118 than the kidney at all time points (Figure 29). 14C02 production continued to increase in liver slices up to 3 hr, while a plateau was reached in kidney slices after incubation for 30 min. The metabolism of 14CHCl3 to covalently bound radioactivity and 14CO2 was reduced by carbon monoxide in liver slices and in male and female renal cortical slices (Figure 30). A 4:1 mixture of CO and D2 reduced covalent binding by 93% in male and by 82% in female kidney slices. The reduction of oxygen concentration in the incubation vessel could only account for a 40-50% reduction of covalent binding in all of the tissues, as indicated by the incubations conducted under a 4:1 mixture of N2 and 02. Likewise, incubation of slices under an atmosphere of carbon monoxide reduced the metabolism of 14 14 CHCl3 to C02 in all tissues similar to the reduction of covalent binding. 14 D. In Vitro Studies - Microsomal Metabolism of CHCl3 l. Metabolism of 14CHCl-4 In Vitro by Renal Cortical and Hepatic Microsomes - Effect of Time, Microsomal Protein Concentra- tion and Substrate Concentration 14 14 CHCl3 was metabolized to C02 and covalently bound meta- bolites by microsomes prepared from male mouse kidney cortex and liver (Table 12). Metabolism of 14CHCl3 by microsomes prepared from female kidney cortex was similar to non-enzymatic 14CHCl3 decomposition in heat-denatured (boiled) microsomes. There was little or no metabolism of 14CHCl3 to 14CD2 or covalently bound radioactivity by female renal cortical microsomes up to 2 hr incubation (Figure 31). 14CHCl3 meta- 14 bolism by male renal cortical microsomes to CO2 was linear with time for approximately 15 min; metabolism to covalently bound radioactivity 119 .mu: ..z.u.m + cams wee mwsFm> . :onummm .NFumu :5 4F.o eew Fox :5 oe .meczn mascamoga schom :2 ¢.F .Fumz :3 F.o¢ mchFmacou .4.F Ia .LmFan apmgamoga co Fe o.N CF AmnFsmecochgpmch F: m.N to ms=Fo> F cF cause Foe:\Fun m.ou FpF>FFom uFFFumamv mFuzu Foe: NF.m ncm mmoFFm mammFu Fe 95 ooF umcFmpcou mmemm> :onomwm .moFE moH soLF mmuFFm quFwwou Foams mFmeF new mmuFFm quFFgou chmc vcm uFFmam; «Fae Fa Noqu vcm auF> inomochL uczon aFucmFm>ou op mFuzqu Fo EmFFoanmE :o mchocoE concwu Fo uuwFFm .om mcsmFu 120 on mgamFu 8:8 go 2.0 .b o 0v LJ 0 IQE :33. 0E Do: .065 3833 N3: 3... gm 73.: a... oo: .95.. 02.023 p2ma<>0u :3; «0" NZ :3. No.8 N0.2:: 121 .en: ..z.m.m + cams mam mmaFm> . :onommm .FE m.F Fo wsaFo> Fmpop m =F .¢.F In .cmFan mgmzamoga EzFuom z F.o cF NFumz Foss mm.o .mmmcmmoguzgmu oumcamosa -o-mmou=Fm mFch e .mpmgamoga-o-mmou=Fm Foen wF .=o m F.F umocm FoEn\Fun m.on auF> -Fuom uFFFumamv mFuzo Foe: NF.m .cFmpoLa FmsomoguFE as N umcqucoo mmemm> coFFuommm «F FNF.Qw©~F.F nmo.gwwNN.F ,OFF.QwF~m.oF mFF.mmFmo.om om coo.owmom.o FmF.oHNmF.F Fem.owomo.m Fow.NHFFm.o~ mF mmo.o+~mm.o ooo.o+mFm.o mmm.o+Fom.o oFm.o+wNm.m m Amema> coFFumma\Fos=V QNFumFmo NouaF mFo.QmFFF.o mmo.ng-.o FFN.omFmo.F mmF.memm.m om eFo.oHFmF.o Fmo.ouomF.o Fem.onmm~.F Fom.owomo.m mF mmo.o+oo~.o eFo.o+omF.o Foo.o+omm.o omN.o+me.F m Acquogn mE\FoE:v wzHosz Fzm4<>ou FcFEv mEFF cmFFom xmchx mFmsmm choFx msz Lm>F4 msz :oFumnzucH mmqu «UH EocF mmsomogqu vaFom new FmoFugou chmm mesmm .quFuLou chmm new uFumam: mez Fa EmFFoamumz mFuzuqF NF m4m .mmsomochE cmFFon mcFm: mFuzu Fo EmFFoanmE oFFmstcmico: LoF umuumccou :mmn m>mc wmaFm> . :oFFummm .FE m.F Fo agaFo> quou w cF .¢.F :a .ngFan mpmsamoga Echom z F.o :F NFumz Foes mo.o cam mmmcwmogcxcmc mumsamogaioimmousz mcha 4 .mbmgamoga-o-amou:Fm Foe: mF .Io a :F cmuum .353anm m.on FFF>Fpum oFFFomamv mFoxueF Foe: NF.m .cquoLa FmaomoLqu as N umcFmacou mmemm> coFFummm .mmEomoguFE FmoFuLou chmg mFmEmF ucm mFmE an FFF>Fuumonmc uczon FFucmFm>ou new N8S oF EmFFonmpwE mFuzueF Fo mmgsou mEFF .Fm mgszm 123 1 1 1 1 1 *4 in o «'1 q A 1Q 0 oi N "' '- O KIDNEY Q Mala I Fomalo (uguoad Bunowu) Bugpugg wo'aAo) 1 1 an o in O a- a— (goqu uogpooanowu) popoioq lo)" 60 90 120 30 Incubation Time (min) Figure 31 60 90 120 30 Incubation Timo (min) 124 was linear with time from 5 to 30 min (Figure 31). Hepatic microsomal 14 metabolism of CHCl3 to 14CO2 was linear with time for 15 min and metabolism to covalently bound radioactivity was linear for 30 min (Figure 32). In contrast to 14 CHCl3 metabolism by male hepatic and renal cortical slices (Table 11), 14CHCl3 metabolism to MC02 ang_ covalently bound radioactivity always was greater by hepatic than by renal cortical microsomes from male mice (Table 12, Figures 31 and 32). Furthermore, there was an increase in the magnitude of difference between hepatic and renal cortical metabolism as incubation time in- creased. 14cum to ‘4 3 and covalently bound radioactivity was linear with protein concentra- Male renal cortical microsomal metabolism of C0 2 tion to at least 2 mg of microsomal protein during a 10 min incubation 1 14 14 with 3.12 nmo cum3 (Figure 33). Metabolism of CHCl to 14co2 3 and covalently bound radioactivity by hepatic microsomes appeared linear up to 8 mg of microsomal protein, the highest concentration used in these studies (Figure 34). On the basis of these data, an incubation period of 10 min with a microsomal protein concentration of 2 mg per 1.5 ml reaction was used for subsequent experiments. The greater degree of hepatic microsomal metabolism of CHCl3 relative to that of renal cortical microsomal metabolism was similar to the results observed for metabolism of 14CHCl3 to 14CO2 by hepatic and renal cortical slices. The cytochrome P-450 content in microsomes per mg microsomal protein was approximately four times greater in 125 .eu: ..z.u.m.H came mew mszm> .mmEomoLoFE umFFon mcFm: mFuzu Fo EmFFonmuws oFamE>~cmucoc coF umpumccoo :mmn m>mc mmsFm> . :oFuomwm .FE m.F Fo wasFo> quow w cF .¢.F In .LwFan mumcamoga EsFuom z F.o :F NFumz Foe; mo.o vcm mmmcmmoguzcmv manganesa-o-emou=Fa mFch e .eeeeamoca-o-amou=Fm Foe: mF .Io4z Foe: N.F .+ao a :F uwvvm .FoE:\Fu: m.on FFF>Fpum uFFFumamv mFuzu F Foe: NF.m .cFmFoca FmsomoLuFE as N nmcFmpcou mmemm> :onomwm .mmsomoLqu qumam; mFme an WFF>FFumoFumL canon FFucmFm>ou ucm NouwF op EmFFoampme mFozueF Fo mmgaoo mEFF .Nm mcszm 126 1 1 1 1 1 #1 t" O “I o. "I o oi (5 N In 1 N (ugoimd 8111 Noam) Bugpugg tuolaaog 8 g 1 : l L 1 J l 1 l o o o a o a o 0 ID V M N '- (pcsoa uogpaunowu) poiaoioa to)" 60 90 120 30 60 90 120 30 Incubation Timo (min) Incubation limo (min) Figure 32 127 .FcFoa mumu :umm LoF m ow mu: ..z.m.m.H :mme mcm mmaFm> .memm> :oFuumm: emu umELoF mFFFoanmE Fo mmFoE: op umFmFoamcpxm mam cam mFuzueF Fo EmFFoanme qumEFNcmico: :oF umuumgcou cmmn m>ms mszm> .u oon sump cmpm: mcFumF -FFUmo :m :F QOFm um vmuozucou mam: mcoFumnsocF cam .mFozo F mcFunm op :oFca cFE m :oF Nouc FooF :FF: ummmmm mgm: mmemm> :oFuummm .FE m.F Fo mstow Fmpop m :F .¢.F 2g .cmFan mums? -moga Echom z F.o cF NFumz Foes mo.o cam mmmcmmocvzcmu mamcamogaioummouaFm mcha e .mumgamoca toimmousz Foe: mF .:o m :F umnum .FoEn\Fu: m.on FFF>FFum uFFFumamv mFuzucF Foe: NF.m .cFmpoga FmEomoLoFe as m o» m.o cmcqucou mmemm> conommm .FFF>FFumonmg meson FFucmFm>ou ucm NouaF ow EmFFon imam: mFuzqu :o conmcpcmucou :quoga FmeomochE quFpgou chm: mFmE Fo FumFFm .mm mgsmFm MAlE KIDNEY 128 (tosson uogpaullomu) Bugpugg 111010110) 1 l l l 1 l __J C: O Q 0 ¢ N O — (tosson uogtaaoanouJU) popotaq 303 91 Mic rosomal Protein (m9) Microsomal Protein (mg) Figure 33 129 .choo momo :omm :oF m cm mu: ..z.m.m.H cmms mom mszm> .memm> coFuommL 2mm omELoF mpFFoomoms Fo mmFoE: op omomFoomcoxm mom ocm mFuzqu Fo EmFFoomomE oFFmsxmcmico: :oF omuomccoo :mmo m>mg mmoFm> . coFoommm .FE m.F Fo me:Fo> Fmpop m cF .o.F :o .LmFFso momgomosm EzFoom z F.o :F NFumz Foss mo.o mom mmmcmmogoxcmo mpmgamozoioummoosz moch e .momgomosoimimmoosFm Foe: mF .zoqz Foe: N.F .+mo m :F omoom .FoE:\Fu: m.on FFF>Foom oFFFomomv mFuzue Foe: NF.m .cFmooco FmeomoLoFE as w op m.o omcFmocoo mmemm> :oFoommm .FFF>FFomoFomL oczoo wchmFm>oo ocm Noqu op EmFFoomame mFozqu co :onmcocmocoo :quooo FmsomoLoFE oFumom; mFmE Fo uomFFm .om mcszm 130 TO do «V ~01 -O l l l l l J I I I o 10 o in 0 1n 0 1n 0 V M M N N '- '- (pssen uogpaoauomu) Bugpugg tuolanog TO “'0 3 Z "t .1 ‘N ‘0 1__ 1 1 1 1 l O O O O O 0 m V‘ M N '- ('OSSOA uogpaotnowu) P0130800 £0391 Microsomal Protein (mg) Microsomal Protein (mg) Figure 34 131 liver than male kidney cortex (Table 13). Microsomal metabolism of 14CHCl3 to covalently bound radioactivity and 14CO2 was approximately two times greater by liver than by kidney after a 10 min incubation of 3.12 um01 ‘4 CHCl3 with 2 mg microsomal protein. However, when micro- somal metabolism was expressed in terms of nmol of metabolites per nmol cytochrome P-450, CHCl3 metabolism was approximately two times greater in male mouse renal cortical microsomes than in hepatic micro- somes (Table 13). Male mouse renal cortical metabolism of 14CHCl3 to 14C02 increased linearly with increasing substrate concentration up to 5.0 pmol of CHCl3 (Figure 35). An Eadie-Hofstee plot was constructed to estimate the kinetic parameters for the renal cortical microsomal metabolism of CHCl3 (Figure 36) (Eadie, 1952; Hofstee, 1952). The Michaelis constant (KM) for the reaction was 2.78 pmol and the Vmax was 0.782 nmol/2 mg microsomal protein/min. 0n the basis of these data, the substrate concentration was increased in subsequent reac- tions to decrease the potential that CHCl3 concentrations would become rate-limiting. The substrate concentrations used in these experiments (1.25 to 12.5 pmol CHCl3) were not sufficiently high to estimate the kinetic parameters for hepatic microsomal metabolism of CHCl3; a plot of velocity versus substrate concentration was parallel to the abscissa indicating the Vmax had been reached (data not shown). 132 TABLE 13 Comparison of Renal Cortical and Hepatic Microsomal Metabolism of P-450 Concentrationsa CHCl3 in Relation to Cytochrome Male Kidney Cortex Liver Cytochrome P-450 nmol/mg protein 0.453:0.029 1.627:D.065 Covalent Binding nmol/mg protein l.086:0.167 2.092:0.264 nmol/nmol cytochrome P-450 2.435:0.410 1.237:0.ll4 14002 Detected nmol/mg protein 3.468:0.360 6.681:0.476 nmol/nmol cytochrome P-450 7.803:l.024 4.103:0.245 aReaction vessels contained 2 mg microsomal protein, 3.12 pmol 14CHClg (specific activity 20.5 uCi/umol added in a volume of 2.5 ul dimethylformamide), 0.4 pmol NADPH, 1.2 pmol NADP+, 1.2 pmol NADH, 18 pmol glucose-6-phosphate, 4 units glucose- 6-phosphate dehydrogenase and 0.65 mmol MgC12 in 0.1 M sodium phosphate buffer, pH 7.4, in a total volume of 1.5 ml. Reaction vessels were gassed with 100% 0 for 5 min prior to adding 14CHCl3, and incubations were con ucted at 37°C in an oscillating water bath (100 cycles/min) for 10 min. Incu- bations were terminated by the injection of 1.5 ml of 10% TCA. Values have been corrected for nan-enzymatic metabolism of 14CHCl3 and are expressed as nmol mg microsomal protein gr_per nmol cytochrome P-450. are mean :_S.E.M., n=5. CHCl3 metabolized per Values 133 .mn: ..z.u.m.H :mms mom mszm> .mmeomocoFe omFFoo mcFms mFozuo Fo EmFFoomoms oFomschmico: :oF omoomggoo :mmo m>mc mmoFm> . :oFoommm .FE m.F Fo mooFo> Fmooo m :F .o.F In .cmFF3o mumzmmoga EzFoom z F.o :F NFumz Foss mm.o ocm mmmcmmocoxgmo momsomocoimimmoosz mchz o .mpmsomocoimimmoooFm FoE3 mF .:o z FoE1 N.F .+mooca om Fozo m>Feemomeceoe Foe: mN.FF om mN.o sza FFoe:\Fo: m.ou FeF>Feum eFFFumemv mFozue Foe: mN.F .cquoco FmeomoooFE as N omcqucoo mmemm> coFoommm .mmeomocoFE FmoFucoo chme me5 Fa NoooF op EmFFoomume mFozooF :o :oFchocmocoo mumgomozm Fo uomFFm .mm mgszm 134 I4 1 12 IO l I J I I I I N. ‘0. “l. V. “'2 N, "- o o o o o o o (aim/1355911 uogpaeqpopetep 303“ lomu) AIIDO'IBA CHCl3 (pmol) Figure 35 135 .mnc .coFomozocF moo op omoom mFozu Foe: n m ocm :FE\memm> :oFoomm:\omoomomo Noqu Foe: n > .FNmmF .mmFmFo: mNmmF .mFomuv mmomFo: ocm mFomm Fo oocpms moo an omopoFo mom: mama .coFFFocoo :oFomooocF moo Fo :oFooFLommo m coF mm moomFm mmm .FoE: m.NF op mN.F EooF ommcm: mcoFumLucmocoo momcom imam .NouoF op mFuzoeF Fo EmFFoomomE FmaomocoFE FmoFoooo chm: ms» Fo moFomch .mm moomFm 136 mm 953... F3 2 > no No F.o m .05: on .N :_E F 5205 35033.5 mEN F .95. «mud ad o.F 137 14 2. Effect of NADH and NADPH on Microsomal Metabolism of CHCl3 Metabolism of MCHCl3 by renal cortical and hepatic micro- somes required the presence of a NADPH regenerating system (Figures 37 and 38). The extent of 14CHCl3 metabolism to 14C02 and covalently bound radioactivity was similar in incubations with heat-denatured microsomes or with microsomes in the absence of a NADPH regenerating system. Enzymatic metabolism of 14CHCl3 by renal cortical microsomes in the presence of NADH alone was only about 5% of that observed in the presence of NADPH (Figure 37). In contrast, hepatic microsomal MCHCl3 metabolism in the presence of NADH alone was approximately 40% of that observed in the presence of NADPH alone (Figure 38). Addition of both NADH and NADPH to the incubation mixture increased the meta- bolism of 14CHCl3 in an additive manner. f 14 3. Subcellular Localization o CHC13_Metabolism Hepatic and renal cortical 9000 g supernatant and the micro- somal fraction from male ICR mice metabolized 14CHCl3 to 14CO2 and covalently bound radioactivity (Table 14). Metabolism of 14 CHCl3 by the cytosol fraction was very low. There was little or no conversion to aqueous soluble metabolites by the microsomal fraction alone, however when glutathione was added, the amount of aqueous soluble counts was increased. There was no metabolism of 14 CHCl3 by any of these fractions in the absence of a NADPH regenerating system (data not shown). 4. Binding Spectra of CHCl, with Renal Cortical and Hepatic Microsomes “ The binding of CHCl3 to oxidized cytochrome P-450 from male renal cortical and hepatic microsomes produced a typical type I binding spectrum (Figure 39). 138 .on: ..z.m.m.H cmme mom mmsFm> . :oFoommm .cFE m LoF omFFoo mom: poo .mxmmFF Imo :oFuommL omFFom .mmmcmmoeoxgmo mumgomozouoummoooFm mFch m.F ocm mumcomogoioimmoooFm :5 m .NFomz :5 m sza .mmmmoFmeF mm ano coFuommm .Fzmo Fmooo m :F .¢.F Io .cmFFoo momsomogo EoFoom z F.o :F FmoFEmseoF -cmomEFo F: m.N Fo mazFo> m :F omoom Fosn\Fua m.ou zuF>Foom oFFFomomv mFuzum Foe: NF.m mom :Fmpoeo FmeomocoFE as F omcqucoo mmemm> coFFomm: FF< .Noqu ocm FFF>FpomoFowc ocooo szcmF im>oo o» mFozqu Fo EmFFoomme FmoFFLoo chm.F mFmE co Imo c2309. 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F .95... 86:3 F8.. 0.— On On Go. Qm md QF m.— Ga $2. 529.. 2.1.0.8 E 261012 a 58: Fe m 149 Figure 41. Effect of cytochromeC P- 450 inhibitors on male renal cor— tical metabolism of 14CHC13 to1 and covalently bound radioacti- vity. Reactions vessels contained WI mg microsomal protein, 3.12 pmol 14CHCl3 (specific activity ~0. 5 uCi/umol added in a volume of 2. 5 ul dimethylformamide), 0.1 pmol NADPH, 0. 3 pmol NADP+, 0. 3 pmol NADH, 4. 5 pmol glucose- 6- -phosphate, 1 unit glucose- -6- -phosphate dehydrogenase and 0.l6 mmol MgClzi n0. 1 M sodium phosphate buffer, pH 7. 4, in a total volume of 0.75 ml. Stock solutions of inhibitors or vehicle (H20 or ethanol) were added as 1% of the total reaction volume to achieve the indicated inhibitor concentrations. SKF 525-A and metyrapone were formulated in H20; piperonyl butoxide and a-naphthoflavone were for- mulated in ethanol. Reaction vessels were gassed with 100% 02 for 5 min prior to adding1 4CHCl and incubations were conducted at 37°C in an oscillating water bath (100 cycles/min) for 10 min. Incubations were terminated by the injection of 0. 75 ml of 12% TCA. Values have been4 corrected for non- enzymatic metabolism of CHCl3 Metabolism 4CHCl3 by microsomes plus vehicle (Control + H2 0 and Control + Ethanol) was expressed as nmol CHCl metabolized to 1 C02 and cova- lently bound radioactivity. Metabo ism of CHCl by microsomes in the presence of inhibitors was expressed as % of Ehe appropriate vehicle control. Values are mean + S. E. M. ., n= 5. Statistical signi- ficance was determined by calculat1ng the 95% confidence interval for microsomal reactions containing inhibitors. *Significantly different from Control + appropriate vehicle, p<0.05. X oI Control 1 ol Control 100 80 60 4O 20 I20 I00 80 60 4O 20 .- ISO KIDNEY “co, omcno (nmol! roouion vossol) (onnol ‘ H20 3 5063 I 349 SKF 525’A Mooycopono Connol 9 Ethanol l 603 2 05M 5 F...) ||||||Illlllllllllllllllllllllllllll 10“ lo" 10" 10" M Piporonyl c-Nophthollovono loco-id. KIDNEY COVAlENl BINDING (nmol I rng protein) (onOroI 0 H20 I 259 3 0 CIO Control 0 Ethanol 0 03I 9 0 I69 l J. 10" 1o“ 10“ 10" SK. 575" Molyropone 10“ lo" 10“ 10'5 M 'lfiOIOH'I c-Nophrhollovono .U.°I Id. Figure 4l 151 Figure 42. Effect of cytochrome P-450 inhibitors on male hepatic metabolism of 14CHCl3 to 14C02 and covalently bound radioactivity. Rfiactions vessels contained l mg microsomal protein, 3.l2 pmol CHCl3 (specific activity =0.5 uCi/pmol added in a volume of 2.5 ul dimethylformamide), 0.1 pmol NADPH, 0.3 pmol NADP , 0.3 pmol NADH, 4.5 pmol glucose-6-phosphate, l unit glucose-G-phosphate dehydroge- nase and 0.l6 mmol MgClZ in O.l M sodium phosphate buffer, pH 7.4, in a total volume of 0.75 ml. Stock solutions of inhibitors or vehicle (H20 or ethanol) were added as l% of the total reaction volume to achieve the indicated inhibitor concentrations. SKF 525-A and metyrapone were formulated in H20; piperonyl butoxide and a-naphtho- flavone were formulated in ethanol Reaction vessels were gassed with l00% 02 for 5 min prior to adding 14CHCl3, and incubations were con- ducted at 37°C in an oscillating water bath (lOO cycles/min) for l0 min. Incubations were terminated by the injection of 0.75 ml of l % TCA. Values haxe been corrected for non-enzymatic metabolism of I CHCl3. Metabolism of CHCl3 by microsomes plus vehicle (Control + H2 and Control + Ethanol) was expressed as nmol CHCl3 metabolized to 4C0 and covalently bound radioactivity. Metabolism of 14CHCl3 by micro- somes in the presence of inhibitors was expressed as % of the appro- priate vehicle control. Values are mean :_S.E.M., n=5. Statistical significance was determined by calculating the 95% confidence interval for microsomal reactions containing inhibitors. *Significantly differ- ent from Control + appropriate vehicle, p<0.05. 3 ol Control % oI Control 152 uvea "‘co2 omcuo lnmol / reaction vessel) (mmol 0 H2O Control 0 ("nmol '20 ’ 4645 9 0657 i Jill 3 0437 ‘00? §\‘ E .. g Fh . a g *“ § 2 E °°- § 2 'g‘ M a E 40» § : E \ s 2 cl .\ i ‘2‘ 1o“3 10“ 10" 1o" 10" 10" 1o" 10" M SK! 525 A Motyropono Pup-nonyl c Nophthotlovono Onto-.60 [IVER COVAlENI BINDING (nmol I mg protoin) Control 0 H20 2 290 t 0 30. Control 9 Ethanol [539 t 0 30l I I I nor 1 * Jfl l :—: g -_ E too > g ":—_= E a E E g +l so » . g 3% g l 1E _E_ l i E 60 . g E g- I g E I E E 40* : E: E: 1 E E l E E 20 * : E E 1 g g 0 l. I 7— 7... 10" 1o" 10" 10” M SKF 525‘ Metyrapone Pipotonyl o-Nophthotlovono auto-id! Figure 42 153 4 and lO'SM) nor significant degree. Neither piperonyl butoxide (10' a-naphthoflavone (10'4 and lO'SM) decreased covalent binding, though there were similar decreases in 14CO2 production. 7. Effect of Strain Differences on Renal Cortical and Hepatic Mixed Function Oxidases and TZICHCl.2 Metabolism by_Micro- somes “ Renal cortical cytochrome content and mixed function oxidase activities (ethoxycoumarin-O-deethylase and 14CHCl3 metabolism) were similar statistically in C57Bl/6, DBA/2 and ICR strains of male mice (Table 15). Female ICR mouse renal cortical cytochrome content and mixed function oxidase activities were significantly less than male mice of the three strains; there was a trend for increasing activi- ties from C57Bl/6J to DBA/6 to male ICR mice. 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