RETURNING hATERIALS: 1V1£3I_J P1ace in book drop to LJBRARJES remove this checkout from .--c—. your record. FINES will be charged if book is returned after the date stamped below. NEPHROTOXICITY AND HBPATOTOXICITY OF CHLOROFORM IN MICE By Massumeh Ahmadizadeh A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1982 Copyright By MASSUMEH AHMADIZADEH 1982 ABSTRACT NEPHROTOXICITY AND HEPATOTOXICITY OF CHLOROFORM IN MICE BY Massumeh Ahmadizadeh Administration of deuterium labelled chloroform and chloroform to male ICR and C57 and male and female DBA mice produced dose-dependent damage in the kidney and liver. Deuterium labelled chloroform produced less kidney and liver damage than chloroform. Chloroform caused the same degree of liver damage in male C57 and male and female DBA mice. Nephrotoxicity of chloroform was greater in male DBA than in CS7 mice; female DBA mice failed to develop renal injury following treatment with chloroform. Phenobarbital increased hepatotoxicity but not nephrotoxicity of chloro- form. B-Naphthoflavone enhanced chloroform hepatotoxicity in male CS7 mice but had no effect on nephrotoxicity. Polybrominated biphenyls enhanced hepatotoxicity of chloro- form in both strains but increased renal injury only in male CS7 mice. The lack of correlation between chloroform hepato- toxicity and nephrotoxicity strongly suggests that the kidney is the site of formation of nephrotoxic chloroform metabolite(s). ACKNOWLEDGEMENTS I would like to express appreciation to Dr. Jerry Hook for this opportunity of studying toxicology and his encouragement. I would also like to express sincere gratitude to my other committee members. I thank Martha Thomas for her kind support and academic counseling and Dr. Charles Whitehair for his helpful advice. A special thank you is extended to Dr. Robert Echt for his guidance, encouragement and constructive criticism throughout the morphologic and cytochemical aspects of this investigation, as well as for his moral support during times of frustration. I offer my thanks to Dr. Esther Roege for her technical assistance and to my dear friend Katherine Washko for her literary advice. Finally, I am deeply grateful to my parents, brothers and sisters. Though they are geographically distant from me, they have always been with me in spirit and their moral and financial support has indeed made this entire academic experience possible for me. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 Strain-Related Differences in Nephro- toxicity of CHC13.. 5 Renal Regeneration. . . . . . . 6 Sex- Related Differences in Nephrotoxicity of CHC13 . . . . 8 Morphology of the Kidney in Nephrotox1c1ty of CHC13 . . . . . . . 9 Metabolites of CHC13. . . . . . . . . . . . . . ll Covalent Binding of CHClS . . . . . . . . . . . 13 Depletion of Glutathione. . . . . . . . 15 Classes of Microsomal Enzyme Inducers and Effects of These Agents on CHC13- Induced Toxicity . . . . . . . . . . . 16 PURPOSE. . . . . . . . . . . . . . . . . . . . . . . . 20 MATERIALS AND METHODS. . . . . . . . . . . . . . . . . 21 Materials . . . . . . . . . . . . . . . . . . . 21 Animals . . . . . . . . . . . . . . . . . . . . 21 Treatments. . . . . . . . . . . . . . . . . . 21 Microsomal Enzymes. . . . . . . . . . . . . . . 23 Toxicity Tests. . . . . . . . . . . . . . . . . 23 Light Microscopy. . . . . . . . . . . 25 Transmission Electron Microscopy. . . . . . . . 25 Cytochemistry . . . . . . . . . . . . . . . . 25 Statistical Analyses. . . . . . . . . . . . . . 27 RESULTS. . . . . . . . . . . . . . . . . . . . . . . . 23 Effect of CHC13 or CDC13 on Body Weight, Liver Weight to Body Weight, and Kidney Weight to Body Weight in Male ICR, CS7, Male and Female DBA Mice: Effect of BNF, PB or PBB on These Parameters . . . . . 28 Hepatotoxicity of CHC13 or CDC13 in Male ICR, C57 Male and Female DBA Mice: Effect of BNF, PB or PBB on Hepatotoxicity of CHC13 in Male CS7 and DBA Mice . . . . . . 33 iii Nephrotoxicity of CHC13 or CDC13 in Male ICR, C57 Male and Female DBA Mice: Effect of BNF, PB or PBB on Nephrotoxi— city of CHC13 in Male CS7 and DBA Mice. Effect of BNF, PB or PBB on Hepatic Micro- somal Activities in Male C57 and Male DBA Mice. . . Effect of BNF, PB or PBB on Renal Microsomal Activities in Male C57 and Male DBA Mice. Morphology of Bowman's Capsule in Male CS7 and Male and Female DBA Mice. . . Cytochemistry of the Hepatic and Renal Cytochrome P- 450: Effect of Inhibitors of Microsomal Enzyme Activities on Kidney and Liver. DISCUSSION. . . . . . . . SUMMARY . BIBLIOGRAPHY. iv Page 37 60 64 64 74 81 95 99 Table LIST OF TABLES Effect of a single dose of CHC13 or CDC13 on liver weight to body weight and kidney weight to body weight ratio. . . . Effect of a single dose of CHC13 on ratio of liver weight to body weight (LW/BW) Effect of a single dose of CHC13 on ratio of kidney weight to body weight (KW—BW). Effect of CHC13 and inducers of MFOs on liver weight to body weight (LW/BW) ratio. Effect of CHC13 and inducers of MFOs on kidney weight to body weight (KW/BW) ratio Hepatic and renal AHH activities in adult male C57 and DBA mice. A comparison of parietal epithelium in Bowman's capsule (BC) of male C57 and male and female DBA mice. Page 29 30 31 32 34 61 70 Figure 10 11 LIST OF FIGURES Effect of chloroform (CHClS) and deuterium labelled chloroform (CDC13) on serum glu- tamic pyruvic transaminase (SGPT) activity . Effect of chloroform (CHC13) and deuterium labelled chloroform (CDClS) on p- ammino- hippurate (PAH) accumulation (S/M ratio) by renal cortical slices . . . Effect of chloroform (CHClS) and deuterium labelled chloroform (CDCl3) on tetraethyl- ammonium (TEA) accumulation (S/M) by renal cortical slices. . . . . . . . . . . . Effect of chloroform (CHC13) and deuterium labelled chloroform (CDC13) on blood urea nitrogen (BUN) . . . . Effect of chloroform (CHC13) on serum glu- tamic pyruvate transaminase (SGPT) activity. Effect of chloroform (CHCIS) on -ammino- hippurate (PAH) accumulation (S/M ratio) by renal cortical slices. . . . Effect of chloroform (CHClS) on tetraethyl- ammonium (TEA) accumulation (S/M ratio) by renal cortical slices. . . . . . . Effect of ch10roform (CHC13) on blood urea nitrogen (BUN) . . . . . Effect of chloroform (CHC13) and inducer of MFOs on serum glutamic pyruvic transaminase (SGPT) . . . . . . . . . Effect of chloroform (CHClS) and inducer of MFOs on p-amminohippurate (PAH) accumulation (S/M ratio) by renal cortical slices Effect of chloroform (CHClS) and inducer of MFOs on tetraethylammonium (TEA) accumulation (S/M) by renal cortical slices . . vi Page 36 39 41 43 45 47 49 51 53 SS 57 Figure 12 13 14 15 16 l7 18 19 20 21 22 23 24 25 Effect of chloroform (CHC13) and inducer of MFOs on blood urea nitrogen (BUN). Effect of inducers of MFOs on hydrocarbon hydroxylase (AHH) Effect of inducers of MFOs on hydrocarbon hydroxylase (AHH) Structure of Bowman's capsule Structure of Bowman's capsule mice Structure of Bowman's capsule hepatic aryl- activities renal aryl- activities in male C57 mice in female DBA in male DBA mice Ultrastructure of the cuboidal epithelial cell lining Bowman's capsule in male DBA mice, show- ing the microvilli which project from the apical surface Ultrastructure of the cuboidal epithelial cell lining the proximal convoluted tubule (PCT) in male DBA mice, showing a brush border (BB) which projects from the entire apical surface. Cytochemistry of hepatic cytochrome P- 450 in male C57 mice. Cytochemistry of renal cytochrome P-450 in male DBA mice. Effect of piperonyl butoxide on renal cyto- chrome P-450 in male DBA mice, showing inhibi- tion of microsomal enzyme activities in both proximal convoluted tubular cells and cuboidal epithelial cells of Bowman's capsule (absence of dark areas) Effect of piperonyl butoxide on hepatic cyto- chrome P-450 in male C57 mice, showing inhibi- tion of microsomal enzyme activities by this agent (absence of dark areas). Effect of SKF 525- A on renal cytochrome P- 450 in male DBA mice Effect of SKF SZS-A on hepatic cytochrome P-450 in male C57 mice, showing inhibition of micro- somal enzyme activities by this agent (absence of dark areas) vii Page 59 63 66 67 68 72 73 75 76 77 78 79 80 INTRODUCTION Chloroform (CHClS) was initially used as a medical anesthetic. Later, it was found to cause heart and liver damage (Dipalma, 1971; Goodman and Gilman, 1970). With the discovery of safer agents, CHC13 lost importance as a medical anesthetic. However, CHC13 is still used in industry primarily as a solvent and chemical intermediate in the manufacture of artificial silk and plastic, as a cleaning agent, and as a chemical intermediate for the manufacture of fluorocarbon compounds. Until recently it was also employed as a preservative and/or flavor enhancer in pharmaceutical products such as cough medicine, mouth washes and for extraction and purification of penicillin and other antibiotics. Recently it has been found that chronic oral administra- tion of large doses of CHC13 increased the incidence of carcinoma in mice and rats (Melvin, 1979; Reuber, 1979). A low concentration of CHC13 has been reported in the surface water of industrialized areas of the United States. In addition, CHC13 may arise from the chlorination of organic materials already in the water during chemical sterilization of water supplies (Wade, 1977). These findings have generated widespread concern and caused renewed interest in the study of CHClS toxicity. 1 2 Physical and chemical properties: Chloroform is a colorless, volatile liquid with a sweetish odor and taste and has a molecular weight of 119.38 daltons. It is insoluble in water but readily soluble in organic solvents such as ether, benzene and alcohols. Chloroform burns with a green flame at high temperatures (Hoover, 1970). Chloroform can be absorbed through the lungs, from the gastrointestinal tract and, to a lesser degree, through the intact skin. In addition, it can penetrate the placental barrier and has been found in fetal liver (Von Oettingen, 1964). After CHC13 is absorbed, it is rapidly distributed to all organs of the body, with the concentration varying according to exposure level and duration. Chloroform is excreted unchanged primarily through the lung. This pul— monary excretion is slower than the absorption process, and CHC13 may persist for several hours or even days after exposure ends. Barret et a1. (1939) showed that a small fraction of the absorbed CHC13 cannot be accounted for by excretory processes, indicating that some degree of decompo- sition must occur within the body after absorption. Neither the precise reaction responsible for CHC13 breakdown in the body nor the metabolic products have been clearly established. Paul and Rubinstein (1963) injected carbon [14] labeled CHC13 into rats and were able to show that 74% of the radioactivity appeared as CHC13 in the exhaled breath; some appeared in the form of carbon dioxide (C02). Reynolds and Yee (1967) hypothesized that the hepatotoxicity of CHC13 is related to the binding of 3 reduction products to the endoplasmic reticulum and to the formation of chloromethylated lipids and proteins in the liver. Most reports of acute CHC13 poisoning in man describe the results of accidental overdose during anesthesia or the characteristics of delayed poisoning following anesthesia. Chloroform also produces a local toxic response when in contact with intact skin because of its strong vesicant action. The symptomatology of CHC13 poisoning varies according to the route of administration and the severity of exposure. Toxicity from ingestion of CHC13 is marked by gastrointestinal disturbances as well as narcosis. Signs of CHC13 toxicity include a characteristic sweetish odor on the breath, cold and clammy skin, and dilated pupils (Kaye, 1970). Ketosis develops as a result of incomplete oxidation of fats as well as increased blood sugar. In addition, ingested CHC13 acts as a strong irritant to the gastrointestinal mucosa. Most CHC13 poisoning results from inhalation of CHC13; it produces strong central nervous system depression and can result in complete narcosis. These poisonings are characterized by loss of sensation and abolition of motor functions. Death may result from respiratory failure attributable to paralysis of the respira- tory center or may result from the reduced blood supply to the brain caused by circulatory failure. The pathological findings associated with CHC13 toxicity are extensive. The outstanding pathological feature is centrolobular necrosis of the liver. The liver is the most 4 notably affected organ, but fatty degeneration of the kidneys and, to a lesser extent, the heart is also charac— teristic. Transient irritation of the kidneys has been observed during CHC13 narcosis; acute exposure may entail damage to the renal tubules. The degenerative changes are most commonly observed in adult or nephropathic animals and are manifested by the accumulation of stainable lipoid material (Von Oettingen, 1964; Casarett, 1975). Although CHC13 has been known to cause renal damage (Von Oettingen, 1964), the precise biochemical mechanisms responsible for nephrotoxicity remain unclear. Increasing evidence indicates that CHC13-induced nephrotoxicity is independent of hepatic metabolism of CHC13; rather, it is the consequence of intrarenal bioactivation (Ilett et al.,. 1978; Kluwe et al., 1978; Kluwe and Hook, 1980). Suscepti- bility of CHC13-induced renal damage is sex-dependent. Female mice of any strain are resistant to nephrotoxicity, while male mice are sensitive to CHC13-induced renal damage (Deringer et al., 1953; Shubik and Ritchie, 1953; Hewitt, 1956; Jacobsen, 1964; Bennet andWhigman, 1964; Zaleska- RutczynskazuuiKrus, 1972; Clemens et al., 1979). However, the susceptibility of male mice to CHC13-induced kidney injury varies markedly among strains. The lack of signifi- cant difference in CHC13 hepatotoxicity in any strain and the difference in observed nephrotoxicity between male and female mice suggest that the mouse is a suitable species for the study and interpretation of renal damage produced by CHC13. 5 Strain-Related Differences in Nephrotoxicity of CHC13 Chloroform has been known to cause liver and kidney damage in humans and experimental animals (Von Oettingen, 1964). In mice, CHC13 produced liver damage in both sexes while causing renal lesions only in males (Eschenbrenner, 1944). Interestingly, the susceptibility of male mice to CHC13-induced renal necrosis varied among different strains. Shunbic and Ritchie (1953) reported a high death rate from kidney necrosis in the DBA strain of male mice following accidental exposure to a low concentration of CHC13 vapor. Deringer et a1. (1953) noted a high death rate in C3H male mice; a smaller portion of the males of strains A and HR also died after exposure to CHC13. In contrast, the kidneys of male C57BL, C57BR/ed, C57L and ST mice were found to be free of any damage following an accidental exposure to CHC13. Hewitt (1956) observed a high death rate in adult male CBA mice; of surviving adult male CBA mice, 72% were found to have severe kidney damage. The renal lesions con- sisted of long areas of complete necrosis confined to the convoluted tubules. No evidence of renal damage was found in any other strain of male mice, although the mice were exposed under the same conditions as the affected mice. Bennet and Whigman (1964) noted that the CBA-P strain of male mice developed renal necrosis. -No lesions were seen in albino ASW male mice following exposure to CHC13. Deringer et a1. (1953) reported that the susceptibility to CHC13 increased with the age of mice; male mice which were immature at the time of the accidental exposure to CHC13 6 were not affected. Hewitt (1956) found that only adult males were susceptible and to varying degrees; he noted no renal damage in females or young males after exposure to CHC13. Such variation with age may reflect possible hormonal influence on CHC13-induced renal necrosis. Hill et a1. (1975) reported that the C57BL/6J strain was about 4 times more resistant to the lethal effects of CHC13 than the male DBA/ZJ mice. Histopathological studies of kidney lesions in male DBA mice have shown calcification in the kidney involving the entire cortex. No signs of damage were observed in other strains of male mice. Damage was localized in particular segments of the tubule; no changes were observed in Bowman's capsule or the first portion of the proximal con- voluted tubule (Eschenbrenner and Miller, 1945; Eschenbrenner, 1944). Several investigators have suggested that strain dif- ferences in CHC13-induced nephrotoxicity are genetically controlled, being transmitted in either a single or multifac- torial manner (Hill et al., 1975; Zaleska-Rutczynska and Krus, 1973). The breeding of resistant C57 mice to sensitive DBA mice produced a generation of males with susceptibility to CHC13 nephrotoxicity intermediate to those of the parental strains. Renal Regeneration Following experiments on strain differences in CHC13- induced lethality, Zaleska-Rutczynska and Krus (1972). 7 reported that all males of the C3H/He strain died following administration of CHC13, while all of the C57/6J strain survived. Hybrid animals also survived, as did their resistant parents. This finding suggested that resistance to lethality is a dominant trait (Zaleska—Rutczynska and Krus, 1973). These investigators stated that all treated males developed renal tubular necrosis after administration of CHC13. Resistant animals, however, were able to regenerate new renal tubular epithelia and survive, whereas the C3H/He animals did not. Treated C3H/He males developed renal cortical calcification,vdfixfllinterfered with tubular regenera- tion, and died. Hill et al. (1975) observed that male C3H/He mice were more susceptible to death because they were unable to repair renal injury. They noted that male DBA mice regenerated renal tubular epithelium, as did the male C57BL mice. Therefore, the sensitivity of male DBA mice to CHC13-induced lethality cannot be attributed to a total failure of repair, as observed in the male C3H/He mice. .Clemens et al. (1979) reported that CHC13 induced renal damage in C57BL as well as DBA/ZJ male mice. However, the male DBA mice underwent regeneration of.tubular epithelium, as did the male C57 strain. Regeneration was characterized by epithelial hyperplasia and increased cellular basophilia. It was con- cluded that strains differ in the capacity for regeneration of renal convoluted tubules. 8 Sex-Related Differences in Nephrotoxicity of CHC13 In contrast to the strain differences observed in male mice, females of all strains of mice are resistant to CHC13 nephrotoxicity. Castrated males, regardless of strain, are also resistant. It appears that testosterone plays a sig- nificant role in sex-related differences in renal toxicity and death produced by CHC13. It may be important in the strain-related differences. In castrated mice, renal toxicity following exposure to CHC13 increases with adminis- tration of increasing doses of testosterone. Analysis of various physiological and behavioral characteristics and determination of plasma testosterone levels suggests that male C57BL/10J mice are relatively testosterone deficient in comparison to male DBA mice (Shire and Bartke, 1972; Bartke and Shire, 1972). If testosterone plays an important role in sensitizing the renal proximal convoluted tubular cells to CHC13, it is important to understand the mechanism of this action. It has been suggested that the hormone enters the cells and binds to a specific cytoplasmic receptor. This is followed by movement of the hormone-receptor complex to the nucleus, where stimulation of nucleic acid and protein synthesis occur (Wilson, 1972). Other mechanisms have been suggested for the initial step in androgen action (Kochakian and Harrison, 1961;_Koth et al., 1971). These include the metabolism of testosterone to another androgen or an estrogen before complexing with a specific receptor and possible testosterone action without mediation of a receptor (Wilson, 1972; Brown et al. , 1976) . 9 Flutamide is a nonsteroidal antiandrogen that inhibits testosterone action by competing with the hormone for its cytoplasmic receptor (Wilson, 1972; Brown et al., 1976)- Kidneys of all CS7BL female mice were sensitized by testos- terone to the toxic effects of a subsequent dose of CHC13; kidneys of all animals treated with both testosterone and flutamide failed to show damage from CHC13 (Clemens et al., 1979). These experiments suggested that testosterone sensi- tization in the mouse kidneys acts through the testosterone receptor mechanism. The protective role of castration in mice and the production of kidney lesions in testosterone- treated female mice appears to indicate that that or a metabolite of testosterone in the kidney may be responsible for development of CHC13-induced renal damage in the mouse. The lack of sensitivity of female mice to CHC13-induced nephrotoxicity could be due to the possible protective effect of female hormones. However, oophorectomized females are also insensitive to CHC13-induced renal injury. There- fore, it is unlikely that these hormones are involved as necrosis-preventing agents (Krus et al., 1974). Morphology of the Kidney in Nephrotoxicity of CHC13 Sex-related differences in kidney morphology have been reported by Crabtree (1941), who found that the parietal epithelium of Bowman's capsule in male mice was composed of cuboidal cells similar to those lining the proximal convoluted tubules. Later, she observed brush borders lining the cuboidal type cells in Bowman's capsule (Crabtree, 1941). Eschenbrenner and Miller (1945) confirmed this finding. In 10 addition, they noted renal lesions following exposure to CHC13 in male mice. In contrast, female mice were not affected. These investigators also reported that castrated mice failed to develop renal necrosis following exposure to CHC13. However, female mice treated with testosterone developed renal tubular necrosis following CHC13 administra- tion. Selye (1939) stated that testosterone-treated female mice showed increased kidney weight due to hypertrophy of the epithelium of the proximal and distal convoluted tubules and of the epithelium lining Bowman's capsule. Pfeiffer et a1. (1940) reported that in the kidney of mice receiving testosterone propionate, a portion of the normal squamous epithelium of Bowman's capsule was replaced in many instances by cuboidal cells, cytologically indistinguishable from those of the proximal tubules. However, the source of the thickened cells of Bowman's capsule was not clear. Pfeiffer et a1. (1940) suggested that either the epithelial cells of the proximal tubule entered the capsule by migration and gradually replaced the squamous cells, which degenerated and dropped out, or the parietal cells themselves underwent metaplasia and converted to a type indistinguishable from those of the tubules. Crabtree (1940) noted that castration produces in the kidney of male mice a return to the immature or female type cell morphology but does not result in a complete layer of cuboidal cells in the capsule. However, castration affects the mouse kidney by causing a slight decrease in the kidney ratio of kidney weight to body weight 11 and a fall in the percentage of cuboidal cells in the capsule to approximately the female level for mice of the same age. The administration of testosterone propionate to castrated mice results in marked hypertrophy of all parts of the kidney and an increase in the percentage of cuboidal cells in Bowman's capsule to nearly the normal value for males of the same age (Selye, 1939; Pfeiffer, 1940). These observations indicate that testosterone appears to play a significant role in sex-related differences in kidney morphology as well as renal toxicity of CHC13. However, these authors did not indicate a cause and effect relationship between the two phenomena. Since hepatotoxicity of CHC13 is not related to changes in hormone activities (Culliford and Hewitt, 1957), the data suggest that either the nephrotoxicity and hepato- toxicity of CHC13 occur by different mechanisms or that the formation of a CHC13 metabolite is stimulated by testosterone in the kidney of the mouse. Metabolites of CHC13 The toxicity of many drugs is due to the formation of toxic metabolites. This also appears to be true for CHC13; reactive metabolite(s) of CHC13 are responsible for toxicity. Several investigators have suggested that the initial step leading to CHC13-induced tissue injury is biotransformation of CHC13 to the reactive intermediate phosgene (COCL2) by enzymes of the mixed-function oxygenase system (MFO) (Pohl et al., 1977; Mansuy et al., 1977; Sipes et al., 1977). Formation of COCL2 was postulated to proceed through an oxidative dechlorination mechanism which involved oxidation 12 of the C-H bond of CHC13 to produce the trichlomethanol (CC13-OH) derivative. This unstable derivative would then spontaneously dehydrochlorinate to COC12. The electrophilic COC12 could react with water to form carbon dioxide (C02), a known metabolite of CHC13 in vitro (Paul and Rubinstein, 1963) and in vivo (Pohl and Krishna, 1978), or with micro- somal enzyme to yield a covalently-bound product (Ilett et al., 1973; Brown et al., 1974; Uehleke and Werner, 1975; Gillette and Pohl, 1977; Sipes et al., 1977; Uehleke et al., 1977). To support the hypothesis that intermediate product of CHC13 is toxic and causes cell injury, the relative hepato- toxicity of CHC13 and deuterium-labeled chloroform (CDC13) was compared. The approach was based upon the fact that an isotope effect was observed in the in Vitro formation of COC12 by liver microsomes at approximately half the rate of CHC13. In vivo studies also indicated that CDC13 was less hepatotoxic than CHC13 (Pohl and Krishna, 1978). These observations suggested that the cleavage of the C-H bond is the rate-limiting step in the activation of CHC13. The observation was made that, when rats or mice are treated with [14C1CHC13, the extent of hepatic necrosis parallels the amount of [14C] label bound irreversibly to liver protein (Ilett et al., 1973). This supported the concept that a reactive metabolite of CHC13 is responsible for its toxicity. Although CHC13 has been known to cause renal damage (Von Oettingen, 1964), the precise biochemical mechanism responsible for nephrotoxicity remains unclear. 13 The strain and sex differences in susceptibility to CHC1- induced renal necrosis raise the possibility that the nephrotoxicity is caused by a metabolite of CHC13. However, increasing evidence indicates that CHC13-induced nephro- toxicity is independent of hepatic metabolism of CHC13; rather, it is the consequence of intrarenal bioactivation (Ilett et al., 1973; Kluwe et al., 1978; Kluwe and Hook, 1980). In summary, drug metabolizing enzyme systems in the liver and kidney convert CHC13 to one or more chemically reactive metabolites. Cellular necrosis caused by this toxic metabolite(s) appears to be related to the alkylation of macromolecules in the target cells. However, the bio- logical process by which alkylation of macromolecules may lead to cell necrosis is not completely understood. Covalent Bindingof CHC13 The covalent binding of CHC13 metabolite(s) to liver and kidney protein was observed by Ilett et al. (1973), who showed that the livers of male and female CS7BL/6J mice not only developed similar degrees of hepatic necrosis but also covalently bound a similar amount of radioactivity to hepatic protein following administration of [14C]CHC13. In contrast, more radioactivity was bound to renal protein by male than female mice. In addition, they noted that female mice were relatively resistant to CHCL3-induced renal injury. Taylor et al. (1974) found that in the kidneys of male mice the concentration of [14C] was much higher than in female CBA mice. Testosterone treatment of female mice resulted in a 14 greater concentration of radioactivity in the cortex and castration reduced the amount of renal cortical radioactivity in males (Taylor et al., 1974) and decreased the renal damage produced by CHC13. Clemens et al. (1979) reported that radioactivity from [14C]CHC13 bound to renal protein appeared to be greater in male DBA/ZJ mice which are sensi- tive to CHC13-induced nephrotoxicity than in relatively resistant male C57/6J mice. Ilett et a1. (1973) suggested that a cause and effect relationship may exist between the covalent binding of a CHC13 metabolite and acute renal and hepatic necrosis. Pretreatment of mice with phenobarbital (PB) increased both the degree of liver damage and the level of radioactivity bound to protein in this tissue. In con- trast, pretreatment of mice with an inhibitor of MFO activities such as piperonyl butoxide decreased the hepato- toxicity and covalent binding of [14C]CHC13. Thus, consider— able evidence indicates that the extent of renal and hepatic necrosis in mice parallels the amount of CHC13 covalently bound to protein in these tissues (Ilett et al., 1973). The metabolic activation of CHC13 by liver microsomes appears to be catalyzed by cytochrome P-450, because the binding to microsomal protein is NADPH-dependent and inhibited by carbon monoxide, SKF SZS-A and piperonyl butoxide (Sipes et al., 1977). In contrast, omission of NADPH from mouse kidney microscomal preparation caused only a slight decrease in the amount of covalent binding (Sipes et al., 1977). In addition, carbon monoxide and SKF 525-A failed to inhibit the irreversible binding of CHC13 to renal microsomes (Pohl 15 and Krishna, 1978). Thus, the failure of SKF 525-A and carbon monoxide to protect the kidney from CHC13 toxicity indicates that CHC13 may be metabolized to a nephrotoxic product by an enzyme system sensitive to inhibition by piperonyl butoxide but not blocked by SKF SZS-A or carbon monoxide. Depletion of Glutathione Glutathione (GSH), a sulfhydryl tripeptide, found in high concentration in the liver, may play a key role in protecting the liver from the toxic effects of CHC13 (Brown et al., 1974). It has been found that one of the physio- logical functions of GSH is to protect cellular components from attack by electrophilic chemicals or metabolites which may cause tissue damage by reaction with macromolecules (Jakoby, 1977). Results of several observations suggested that treatment of rats with CHC13 depelets hepatic GSH (Brown et al., 1974; Johnson, 1965), but acute liver damage occurs only with doses of CHC13 sufficient to reduce the. concentration of hepatic GSH below a critical level. It has been suggested that toxic, electrophilic metabolites of CHC13 are detoxified by conjugation with GSH. However, when hepatic GSH has been depleted below a critical level, the electrophilic metabolites can attack essential cellular components. Pretreatment of mice with a liver microsomal inducer such as phenobarbital markedly increased the depletion of liver GSH by CHC13; no corresponding depletion of GSH was found in the kidney (Dock, 1976). In addition, although 16 massive liver necrosis was observed in mice treated with phenobarbital, no kidney damage was noted in these mice. This finding further supports the concept that GSH protects the kidney from the injurious effects of CHC13. A protective role of GSH is supported by the observation that the hepato- toxicity of CHC13 is potentiated when liver GSH is depleted by pretreatment of animals with diethyl maleate (Brown et al., 1974). Since CDC13 is not nearly as potent as CHC13 in deplet- ing GSH, it would indicate that the hydrogen in CHC13 may be involved in the formation of the reactive intermediate that might deplete GSH conjugate (Docks and Krishna, 1976). The finding that CDC13 was also less hepatotoxic than CHC13 (Docks and Krishna, 1976; Pohl and Krishna, 1978) suggests that CHC13 is presumably metabolized in the liver to an electrophilic molecule, possibly phosgen, which depeletes GSH by formation of GSH conjugates (Docks et al., 1974). Classes of Microsomal Enzyme Inducers and Effects of These Agents on CHC13- Induced ToXICity The mechanism by which CHC13-induced nephrotoxicity and hepatotoxicity develops is not completely understood. However, the initial step in the pathway leading to CHC13 toxicity is generally believed to be biotransformation of CHC13 to a reactive intermediate, possibly phosgen (COC12) by a mixed-function oxygenase (MFO) present in the endo- plasmic reticulum of susceptible tissue (Ilett et al., 1973; Pohl et al., 1977; Manusy et al., 1977; Sipes et al., 1977; Brown et al., 1974). The presence of MFO systems in both 17 the kidney and liver of various animal species, including mice, has been reported by many investigators (Kuntzman, 1969; Gillette, 1963; Conney, 1967; Parke, 1968; Brodie, 1958; Goldstein et al., 1968). A number of compounds have been reported to induce microsomal enzymes. Based on differences in the profile of biological effects, most of these agents can be placed into one of three general groups. 1. Phenobarbital type: Phenobarbital (PB) administra- tion enhances the metabolism of drugs by microsomal enzymes by increasing total liver mass, microsomal protein and enzyme-specific activity. It also increases the amount of both NADPH and cytochrome P—450. 2. Polycyclic hydrocarbon types: Two of the most common examples of this group are 3-methylcholanthrene (3MC) and 3,4-benzo(a)pyrene (BP). Although many compounds in this group are carcinogens, there is no relation between their carcinogenic capacity and enzyme induction. There are 3 general differences between the induction effect of PB and 3MC: a. 3-Methylcholanthrene produces more selective stimulation of hepatic enzymes; i.e., it increases the metabolism of fewer substrates. . b. 3-Methylcholanthrene increases the amount of cytochrome P-450 but does not alter the amount of NADPH cytochrome c reductase. c. The delay between exposure and maximum enzyme induction is in the order of hours for 3MC and days for PB. 18 3. Anabolic steroids: Comparatively little is known about the mechanism of induction of microsomal drug metabolism by anabolic steroids. Administration of tes- tosterone or methyltestosterone to female or castrated male rats increases drug metabolism by hepatic microsomes. How- ever,co-administrationcfi?anabolic steroids and phenobarbital results in a summation of their inductive effects, suggesting that they act through different mechanisms. The time required for maximal enzyme induction by the anabolic steroids differs from both other groups. Whereas 3MC produces maximum effects within 12 to 24 hours and PB within 2 to 3 days, the anabolic steroids require 2 to 4 weeks. Their administra- tion is not accompanied by significant effect on liver weight or microsomal protein content. Moreover, the steroids do not greatly increase the amount of cytochrome P-450 in liver microsomes. Stimulation of hepatic MFO activity by PB not only increased hepatic MFO activities but also increased hepatic damage following CHC13 administration in mice (Burger and Herdson, 1966; Kluwe et al., 1978). In contrast, PB did not affect renal MFO activity or alter the sensitivity of mice to CHC13-induced nephrotoxicity. Pretreatment of mice with 3MC enhanced MFO activities in both liver and kidney. This agent did not alter the liver damage produced by CHC13 but markedly decreased the nephrotoxic effect of CHC13 (Kluwe et al., 1978). Polybrominated biphenyls (PBB) have been found to enhance enzyme sensitive to both PB and 3MC (Dent et al., 1976). Pretreatment of mice with PBB both increased 19 MPG activities in liver and kidney and enhanced the nephro- toxicity and hepatotoxicity of CHC13 (McCormack et al., 1978; Kluwe et al., 1978). PURPOSE The overall purpose of these studies was to test the hypothesis that chloroform-induced nephrotoxicity is due to biotransformation of chloroform into one or more toxic metabolites intrarenally and not by translocation of the toxic agent from the liver. These investigations were divided into 3 segments: 1. Determination of nephrotoxicity and hepatotoxicity of chloroform in male ICR mice and determination of the effect of deuterium substitution on toxicity. 2. Study of induction of drug metabolizing enzymes in kidney and liver and their effects on renal and hepatic toxicity of chloroform in inducible and noninducible strains of male mice. 3. Comparison of renal and hepatic injury produced by chloroform in male and female mice. 20 MATERIALS AND METHODS Materials Chloroform (CHC13) and phenobarbital (PB) were obtained from Mallinckrodt (St. Louis, MO); deuterium-labelled chloroform (CDC13) and B-naphthoflavone (BNF) were purchased from Sigma Chemical Company (St. Louis, MO). Polybrominated biphenyls (PBB) were received from the Michigan Chemical Company (St. Louis, MI). Animals Male ICR mice (25-30 g) were purchased from Spartan Farms (Haslett, MI); male C57BL/BJ (C57) and male and female DBA/ZJ (DBA) strain mice (25-30 g) were obtained from Jackson Laboratory (Bar Harbor, MO) and housed in groups of 5 in plastic shoebox cages in temperature and humidity controlled rooms with a light cycle of 12 hours light and 12 hours dark. Treatments Chloroform (CHC13) and deuterium-labelled chloroform (CDC13) were dissolved in corn oil and administered intra- peritoneally (ip) at doses of 0.1, 0.25, 0.5, 0.75, and 1.0 ml/kg into male ICR mice. Control mice were given 21 22 corn oil only. B-Naphthoflavone (BNF) was dissolved in acetone and mixed slowly and evenly into the ground rodent diet (Wayne Lab Blox, Chicago, IL) to produce a final concentration of 1 g/kg BNF diet. Male C57 and male DBA mice were maintained on a 1 g/kg BNF diet for 5 days before exposure to CHC13. Control mice were fed a regular rodent diet. Polybrominated biphenyl (PBB) was dissolved in acetone and mixed slowly and evenly into ground diet (Wayne Lab Blox, Chicago, IL) to produce a final concentration of 100 parts per million (ppm). Male C57 and male DBA mice treated with PBB were placed on 0 or 100 ppm of PBB for 28 days before exposure to CHC13. In another series of experiments, phenobarbital (PB) was dissolved in saline (13.3 mg/ml saline) and administered ip into mice (both male C57 and male DBA mice) at a dose of 80 mg/kg once daily for 3 consecutive days. Twenty-four hours after the last injec- tion, animals were exposed to CHC13. Following pretreatment with BNF, PBB or PB, mice were administered ip 0.025, 0.05, 0.1 or 0.25 ml/kg of CHC13 (in peanut oil). Control mice received peanut oil alone. In another series of experiments, male C57 and male and female DBA mice received CHC13 (in peanut oil) ip at doses of 0.05, 0.1, 0.25 or 0.5 ml/kg. All animals were killed 24 hours after administration of CHC13. 23 Microsomal Enzymes Some control and inducer-treated animals were killed prior to exposure to CHC13 and livers and kidneys were quickly removed, weighed, minced and homogenized in 3 volumes of ice-cold 1.15 KCl (livers) or 66 mM Tris-HCl buffer, pH 7.4 (kidneys). The homogenates were centrifuged at 10,000 x g for 20 minutes. All assays were performed on the day of supernatant preparation; protein was measured by the method of Lowry et al. (1951), using bovine serum albumin as a standard. Arylhydrocarbon hydroxylase (AHH) activity was determined by the method of Nebert and Gelboin (1968). Toxicity Tests Male ICR, male C57 and male and female DBA mice and male C57 and male DBA mice pretreated with BNF, PB or PBB with controls (each set had separate controls) were chal- lenged with a single injection of CDC13 (male ICR mice) or CHC13 and killed 24 hours later. Blood was collected and livers and kidneys were quickly removed and weighed. The blood sample was allowed to clot for 1 hour at room tempera- ture and serum was prepared. HepatotoxicitygTests Serum glutamic pyruvic transaminase (SGPT) activity, which has been suggeSted to be a sensitive index for hepatotoxicity (Balazs et al., 1962), was determined by the method of Reitman and Frankel (1957). A Sigma Chemical 24 Company reagent kit was used. The results were expressed as SF units/m1 serum. Nephrotoxicity Tests Blood urea nitrogen (BUN) was determined as described in the Sigma Technical Bulletin No. 640 using Sigma Chemical Company reagents. Renal cortical slice transport capacity for organic anions (p-aminohippurate, PAH) and organic cations (tetraethylammonium, TEA) was determined. For estimation of PAH and TEA uptake, renal cortical slices were prepared (30-100 mg) and placed in 4.0 ml of phosphate buffered (pH 7.4) incubation medium (Cross and Targart, 5 5 M [14C]TEA. 1950) containing 7.4 x 10' M PAH and 1.0 x 10’ Renal cortical slices were incubated for 90 minutes at 25 C under an atmosphere of 100% oxygen in a Dubnoff metabolic shaker. Following incubation, slices were removed, blotted, weighed and homogenized with 3% trichloroacetic acid (TCA) in a final volume of 10 m1. Two milliliters of medium was treated in a similar manner. After centrifugation, the supernatant was assayed for PAH and TEA. p-Aminohippuric acid (PAH) was determined spectrophotometrically by the method of Smith et al. (1945). Radioactivity of TEA in tissue and medium supernatant was determined by liquid scintillation spectrometry using 1.0 m1 aliquots of tissue or medium following addition of 10 ml aqueous counting scintillant (ACS). Accumulation of PAH or TEA was expressed as the slice-to-medium (S/M) ratio, where 8 represents mg of PAH or TEA per g of tissue and M represents mg of PAH or TEA per ml of medium. 25 Light Microscgpy In another series of experiments, untreated male C57 and male and female DBA mice were killed by decapitation. Kidneys were quickly removed. For light microscopy, the tissue was sectioned and several samples were immersed in 10% buffered formalin fixative, conventionally processed, and embedded in paraffin. Sections were cut at 5 pm and stained by either hematoxylin and eosin (HEB) or periodic acid-Schiff (PAS) stain. Observations were recorded for at least 5 animals in each group. All glomeruli in 1 section of each kidney were counted and the epithelial cells of the parietal layer of Bowman's capsule were classified as either squamous or cuboidal as described by Crabtree (1941). Ten to fifteen micro- scopic fields for each kidney were observed, and the per- centage of squamous and cuboidal cells was determined. Transmission Electron Microscopy For ultrastructural studies, kidneys from each animal were excised. The cortex was cut into pieces of approxi- mately 1 mm3 and immersed in 4% glutaraldehyde in 0.1 M phosphate buffer. Following a 24-hour fixation period, they were washed in Zetterqvist's wash solution and then postfixed for 2 hours in Zetterqvist's 1% osmium fixative. The tissue was dehydrated in ascending grades of alcohol and cleared in propylene oxide for 2 hours. Tissues were embedded in a mixture of Epon-Araldite. One micron sections were cut; selected areas for tissue orientation by light 26 microscopy were stained with toluidine blue. Selected areas for further examination were sectioned (approximately 900 A). Thin sections were stained with uranyl acetate and lead citrate and examined with a Zeiss EM 9 electron microscope. Cytochemistry For study of cytochrome P-450 in microsomal reactions, fresh cryostat sections of unfixed liver and kidney tissue of male C57 and male DBA mice were prepared and incubated in the reaction medium for 10 minutes at 37 C. Following incubation, slides were washed with distilled water and mounted in Farran's medium (Chayen et al., 1972). To determine the effect of the inhibition of microsomal enzyme activities, piperonyl butoxide or SKF 525-A was added to the reaction medium. The reaction medium included the following: Tris buffer (pH 7.8) was prepared; 0.3 g of nitroblue tetra- zolium (NBT) was added to 100 m1 of the buffer. Twenty grams of polyvinyl alcohol (PVC) was also added to the Tris buffer to inhibit diffusion of reaction products. NADPHZ (5 mg/ml) was added to the PVC-buffered solution. To test the effect of piperonyl butoxide or SKF SZS-A on hepatic and renal microsomal activities, these compounds were added into the incubation medium (pH 7.8). 27 Statistical Analyses Data were expressed as mean i standard error. The results were analyzed by analysis of variance in a com- pletely randomized design. Treatment differences were identified by the method of least significant difference (Steel and Torrie, 1965). The 0.05 level of probability was used as the criterion of significance. RESULTS Effect of CHC13 or CDC13 on Bodngeight, Liver Weight to Body Weight, and KidneykWeight to Body Weight in Male ICR, C57, Male and Female DBA’Mice: Effect of BNF, PB or PBB on These Parameters Body weight (BW) was not affected by treatment of animals with CHC13 or CDC13 (male ICR mice). Similarly, BW did not change in male C57 or male DBA mice treated with BNF, PB or PBB alone or following administration of CHC13. Liver weight to body weight ratio (LW/BW) was not signifi- cantly affected by treatment of male ICR mice with CHC13 or CDC13 (Table 1). Treatment of male C57 and male and female DBA mice with CHC13 did not alter LW/BW ratio (Tables 2 and 4). However, these ratios were significantly increased in PB- and PBB-treated animals. Liver weight to body weight ratio increased in male C57 but not in male DBA mice following BNF treatment (Table 4). Administration of CHC13 following these inducer treatments did not further increase LW/BW ratios (Table 4). The kidney weight to body weight ratio (KW/BW) increased in male ICR mice after administration of CHC13 or CDC13 (Table l). The ratio of kidney weight to body weight was not affected in male C57 and female DBA mice following exposure to CHC13 (Table 3). In contrast, these ratios 28 29 Table 1. Effect of a single dose of CHC13 or CDC13 on liver weight to body weight and kidney weight to body weight ratio3 Dosage Liver Weightxloo Kidney WGightxloo Solvent (ml/kg) Body Weight Body Weight --- 0(corn oil) 6.14 i 0.18 1.44 i 0.01 00013 0.10 5.37 i 0.29 1.53 : 0.04b 00013 0.10 6.23 i 0.13 1.61 i 0.04 00013 0.25 6.25 i 0.16 1.48 : 0.03b 00013 0.25 5.88 i 0.11 1.62 i 0.04 00013 0.50 5.88 i 0.14 1.90 i 0.08::C 00013 0.50 5.72 t 0.19 2.32 i 0.14 00013 0.75 5.99 t 0.30 1.80 i 0.08: CHC13 0.75 6.74 i 0.22 1.87 i 0.14 00013 1.00 6.21 i 0.42 2.26 i 0.13: 00013 1.00 6.33 x 0.30 2.34 i 0.12 aMice received a single injection of various doses of CHC13 or CDC13 dissolved in corn oil and were killed 24 hours later. Control animals received corn oil alone. Each number represents the mean i S.E. of five or more animals. bSignificantly different from mice receiving corn oil only (P<0.05). cSignificantly different from mice receiving the same dose of CHC13 (P<0.05). 3O whoa HO m>Mm MOM .m.m mHmEMam HOMMOOU .Hmo.ovmv HHOO HMO pscmom mcM>MOOOM OOME EOMM MGOMOMMMO xHuchMchme 6.. came 23 mucomoumOM osHm> comm K .mHmEMcm .OCOHO HMO panama wo>MOOOM .MOMOH mpsoc em OOHHMH OM63 van HMO Manama :M wo>HOmmMO mHumu MO mmmow mOOMMm> HO :OMOOOMOM onan m OO>MOOOM OOME MH M6 oMpmM co mHOmO mo mmop OchMm O HO puomwm Hzm\zqu .N ofinaM 31 .Hmo.ovmv >H:O HMO panama mcM>MOOOH OOME EOMM MOOMoMMMO >HucmOMmManm an OM05 .MO o>MM MOM .m.m H H82: on“ mucomopamfi osHm> comm mHmEMam HOMucou .mHmEMcm .anHm HMO unseen O0>MOOOH .MoumH meson em OOHHMH OM63 was HMO panama :M Oo>HOmmMO mHuzu MO momow mOOMMm> MO :OMMOOmcM OchMm m OO>MOOOH OOME MOOOM OOME OOMOOMucs EOMM McOMOMwMO xHucmOMMMcmMm K -OMQOM ODHO> :uwm .meEwa mHOE HO ®>Hm m0 .MOMOH mMson 4N OOHHMH OMO3 OOMZ .m.m H wOchHHmsu OMOB mmm MO mm .mzm :MMS OOMOOMMOMQ mHmEMcm MO MH no mo»: O6 muousecM 4:4 mHOmO O6 OOOOOO .4 mHan 33 significantly increased in male DBA mice following CHC13 administration (Table 5). Pretreatment with BNF, PB or PBB had no effect on KW/BW ratio following CHC13 administration (Table 5). Hepatotoxicity of CHC13 or CDC13 in Male ICR, C57 Male and Female DBA Mice: Effect OfIBNF, PB or PBB on Hepatotoxicity of CHC13 in Male C57 and DBA MiCe A dose-related increase in SGPT activity was observed in male ICR mice following the exposure of animals to CHC13 or CDC13 (Figure l). Chloroform elevated SGPT more than CDC13. For instance, at 0.75 and 1.0 ml/kg, CHC13 increased SGPT activity 28- and 84-fold above the control, respective1y3 whiler CDC13 increased SGPT only 14- and 35~ fold (Figure 1). A dose-dependent relation in SGPT activity was also observed in male C57 and male and female DBA mice after treatment with CHC13 (Figures 5 and 9). Interestingly, CHC13 produced the same degree of liver damage in male C57 'and male and female DBAmice (Figures 5 and 9) . Treatment with BNF, PB or PBB alone did not affect SGPT activity in either strain of male mice. However, an eleva- tion of SGPT was observed in BNF-treated male C57 mice following administration of CHC13 (Figure 9). In contrast, administration of CHC13 did not alter SGPT activity in BNF-treated male DBA mice (Figure 9). Administration of CHC13 in PB- or PBB-treated male CS7 and male DBA mice markedly increased SGPT activity (Figure 9). However, SGPT activity was considerably higher in PBB-treated male C57 mice compared to SGPT activity in PBB-treated male DBA mice following exposure to CHC13 (Figure 9). 34 .Hm6.ovav mHO2O MO OmOw OEOm Onp wa>MOOOM OOME OOMOOMMOO Onu EOMM McOMOMMMO MszmOMMMamMm .mHOEMam OMOE MO O>MM MO .m.m M :mOE Onu mucOmOMaOM O=Hm> comm .MOMOH mMsoc 4N OOflHMH OMOz OOH: .MHuzu MO mOmOO mOOMMO> MO chMMOOncM OH OchMm :MMB OOchHHmnu OMO3 OEMM MO mOOMMOO mcM -sz> MOM mmm MO mm .mzm £MM3 mHOEMcm MGOEMOOMMOMQ MO 36 O CORN Oll. lltcocu, 7F cucu, <11 025 DOSE (ml/ kg) Figure 1 of 5 0.75 1.0 37 Nephrotoxicity of CHC13 or CDC13 in Male ICR, C57 Male and Female DBA Mice: Effect Of BNF, PB or PBB on Nephrotoxicity of CHC13 in Male C57 and DBA Mice Twenty-four hours after a single administration of CHC13 or CDC13, accumulation of PAH and TEA into renal cortical slices in vitro was depressed in a dose-dependent manner (Figures 2, 3, 6, 7, 10 and 11). The effect was considerably greater in kidneys from male ICR mice treated with CHC13 than in those treated with CDC13 (Figures 2 and 3). Administration of an equivalent dose of CHC13 reduced PAH and TEA S/M ratios to a greater extent in male DBA mice than in male CS7 mice (Figures 6, 7, 10 and 11). In contrast, administration of CHC13 in female DBA mice had no effect on PAH and TEA S/M ratios (Figures 6 and 7). Treatment of male C57 and male DBA mice with BNF, PB or PBB alone did not affect these ratios (Figures 10 and 11). Neither BNF nor PB affected PAH and TEA S/M ratios following CHC13 administration in male C57 or male DBA mice (Figures 10 and 11). However, dietary PBB caused considerable depression in PAH and TEA S/M ratios following CHC13 administration in male CS7 mice. In contrast, male DBA mice receiving PBB failed to alter PAH and TEA S/M ratio following exposure to CHC13 (Figures 10 and 11). A dose-dependent increase in BUN was found after exposure of male ICR, C57 and DBA mice to CHC13 or CDC13 (male ICR mice). In contrast, administration of CHC13 in female DBA mice had no effect on BUN content (Figures 4, 8 and 12). Blood urea nitrogen increased following treatment 38 Figure 2. Effect of chloroform (CHC13) and deuterium labelled chloroform (CDC13) on p-amminohippurate (PAH) accumulation (S/M ratio) by renal cortical slices. Male ICR mice were challenged with a single IP injection of CHC13, CDC13 or corn oil. Animals were killed 24 hours later. Each point and vertical bar represents the mean 2 S.E. of five or more animals. *Significantly different from mice receiving the same dose of CDC13 (P<0.05). PAH (S/M) 20 I O CORN Oll. I 000:, O O chu, ‘5 b g i .1 IO - " C: 5 .— 0 co 7" 03 0.25 0.5 0.75 1.0 DOSE (ml/kg) Figure 2 40 Figure 3. Effect ofchlorof0r01(CHCl3) and deuterium labelledchlorofonn(CDC13) on tetraethylammonium (TEA) accumulation (S/M) by renal cortical slices. Male ICR mice were challenged with a single IP injection of CHC13, CDC13 or corn Oil. Animals were killed 24 hours later. Each point and vertical bar represents the mean i S.E. of five or more animals. *Significantly different from mice receiving the same dose of CDC13 (P<0.05). 4O 3O TEA (S/M) h) C 10 41 O CORN OII. I CDCI3 O CHCI3 O O O CO ” .10 0.25 0.50 0.75 1.0 DOSE (ml ”(9) Figure 3 y 42 Figure 4. Effect of chloroform (CHC13) and deuterium labelled chloroform (CDC13) on blood urea nitrogen (BUN). Male ICR mice were challenged with a single IP injection of CHC13, CDC13 or corn oil. Animals were killed 24 hours later. Each point and vertical bar represents the mean 2 S.E. of five or more animals. *Significantly different from mice receiving the same dose of CDC13 (P<0.05). 43 120 - 1000 o CORN on I CDCI3 O CHCI3 G O r_1 BUN (mg 7:) 8 4o 6 O 2!,- 0 607’ 01.1- 0.25 0.5 0.7510 DOSE (ml/kg) Figure 4 44 Figure 5. Effect of chloroform (CHC13) on serum glu- tamic pyruvate transaminase (SGPT) activity. Male C57 and male and female DBA mice were challenged with a single IP injection of various doses of CHC13 dissolved in peanut oil and were killed 24 hours later. Each point and vertical bar represents the mean i S.E. of five or more animals. *Significantly different from mice receiving peanut 011 only (P<0.05). SGPT (units/ml ) 600 500 400 300 200 100 45 ll 1 J 1 I ” 0.05 0.1 0.25 0.5 Dose ( mI/kg) Figure 5 46 Figure 6. Effect of chloroform (CHC13) on p-ammino- hippurate (PAH) accumulation (S/M ratio) by renal cortical slices. Male C57 and male and female DBA mice were challenged with a single IP injection of various doses of CHC13 dissolved in peanut oil and were killed 24 hours later. Control animals received peanut oil alone. Each point and vertical bar represents the mean : S.E. of five or more animals. *Significantly different from mice receiving peanut oil only (P<0.05). PAH(S/M) 25 20 15 1O o 6‘ 057 El JDBA O 9 DBA CO 47 0.05 Dose( mI/kg) Figure 6 48 Figure 7. Effect of chloroform (CHC13) on tetraethyl- ammonium (TEA) accumulation (S/M ratio) by renal cortical slices. Male C57 and male and female DBA mice were challenged with a single IP injection of various doses of CHC13 dissolved in peanut oil and were killed 24 hours later. Control animals received peanut oil alone. Each point and vertical bar represents the mean i S.E. of five ore more animals. *Significantly different from mice receiving peanut oil only (P<0.05). TEA (S/M) 49 35 - o 6'057 DO'DBA o 9 DBA 30 - O 253" O 20 - * 15 - 1O " 5 - o L 1 1 4 l 1 co + 0.05 0.1 0.25 0.5 Dose(ml/kg) Figure 7 50 Figure 8. Effect of chloroform (CHC13) on blood urea nitrogen (BUN). Male CS7 and male and female DBA mice were challenged with a single IP injection of various doses of CHC13 dissolved in peanut oil and were killed 24 hours later. Control animals received peanut oil alone. Each point and vertical bar represents the mean i S.E. of five or more animals. *Significantly different from mice receiving peanut oil only (P<0.05). BUN(mg%) 200 160 120 80 4O 51 o 6'057 J [I l J J 00 ” 0.05 0.1 0.25 0.5 Dose (ml/kg) Figure 8 52 Figure 9. Effect of chloroform (CHC13) and inducer of MFOs on serum glutamic pyruvic transaminase (SGPT). Control male C57 and DBA mice or pretreated animals with BNF, PB or PBB for varying periods of time were chal- lenged with a single IP injection of CHC13. Mice were killed 24 hours later. Each point and vertical bar represents the mean i S.E. of five or more animals; SGPT activity was expressed as percentage of control male C57 or DBA mice. *Significantly different from mice receiving the same dose of CHC13 (P<0.05). C). and H6 mod .4.qu u m OM:w_; 3.5.538 6.3 3 36 Rome 36 3 36 6.qu u I I I I~\J I I I I:\ I I I I IZ>EU< II< KIDNEY Figure 14 67 Figure 15. Structure of Bowman's capsule in male C57 mice. Male C57 mice were killed by decapitation. Kidneys were removed, fixed with 10% buffered formalin, stained with HGE or PAS. Bowman's capsules were analyzed with light microscopy. (X400) 68 Figure 16. Structure of Bowman's capsule in female DBA mice. Female DBA mice were killed by decapitation. Kidneys were removed, fixed with 10% buffered formalin, stained with HGE or PAS. Bowman's capsules were analyzed with light microsc0py. (X400) 69 Figure 17. Structure of Bowman's capsule in male DBA mice. Male DBA mice were killed by decapitation. Kidneys were removed, fixed with 10% buffered formalin, stained with HGE or PAS. Bowman's capsules were analyzed with light microscopy. (X400) 70 Table 7. A comparison of parietal epithelium in Bowman's capsule (BC) of male C57 and male and female DBA mice No. of Cu- Percent Cu- No. of boidal or Percent boidal or Squamous Cuboidal G Squamous Cuboidal G Sex Species BC Squamous BC BC Squamous BC M C57 56 16 71.43 28.57 M C57 64 25 71.80 28.10 M CS7 46 12 79.30 20.70 M C57 50 12 80.65 19.35 M C57 53 14 79.11 20.89 M DBA 20 64 15.79 84.21 M DBA 10 45 18.18 81.82 M DBA 10 50 16.67 83.33 M DBA 16 52 23.53 76.47 M DBA 17 58 22.67 77.33 F DBA 60 8 88.24 11.76 F DBA 57 3 95.00 5.00 F DBA 63 4 94.33 5.97 F DBA 70 9 88.61 11.39 F DBA 61 8 89.33 10.67 Male C57 and male and female DBA mice were killed by decapitation. Kidneys were removed, fixed with 10% buf- fered formalin, stained with HaE or PAS. Bowman's capsules were analyzed with light microscopy. 71 Ultrastructural features of the parietal cuboidal epi- thelial cells of Bowman's capsule in male C57 and male and female DBA mice were generally similar to those of the epithelium lining proximal convoluted tubules (PCT) (Figures 18 and 19). The cuboidal cells lining Bowman's capsule contain microvilli constituting a brush border at the apical cell surface. However, this brush border does not project from the entire cell surface and microvilli appear shorter and not as dense numerically when compared to PCT epithelium (Figures 18 and 19). The lateral and basal plasma membranes of the cuboidal capsule cells exhibit infolding, which appears to be more extensive in the cuboidal capsule than that observed in PCT epithelial cells. Although cuboidal cells of Bowman's capsule and PCT both contain numerous mitochondria as well as ribosomes, PCT epithelial cells appeared to have more mitochondria per unit of cytoplasm and fewer ribosomes associated with endoplasmic reticulum than Bowman's capsule cells. The morphology of the lyso- somes and vacuoles associated with the endocytotic apparatus was similar in PCT and Bowman's capsule cuboidal epithelium. Bowman's capsule cells contain a round to oval nucleus with a prominent nucleolus and extensive peripheral hetero- chromatin. The Golgi apparatus was not Observed in Bowman's capsule (Figures 18 and 19). 72 Figure 18. Ultrastructure of the cuboidal epi- thelial cell lining Bowman's capsule in male DBA mice, showing the microvilli which project from the apical surface. Note that the brush border (BB) does not project from the entire surface and that there are fewer mitochondria (M) per unit area of cytoplasm. (X6077.5) 73 Figure 19. Ultrastructure of the cuboidal epi- thelial cell lining the proximal convoluted tubule (PCT) in male DBA mice, showing a brush border (BB) which projects from the entire apical surface. There appears to be more mitochondria (M) per unit area of cytoplasm but fewer ribosomes (arrow) associated with endoplasmic reticulum when compared to cells lining Bowman's capsule (Figure 18). (X7350) 74 Cytochemistry of the Hepatic and Renal Cytochrome PFZSO: Effect of Inhibitors of Microsomal Enzyme Activities on Kidney and Liver The presence of cytochrome P-450 in kidney as well as in liver of male C57 and male DBA mice was noted (a deposit of the colored formazan) (Chayen et al., 1972) (Figures 20 and 21). However, the activity of renal and hepatic microsomal enzymes was markedly reduced when piperonyl butoxide was added into the reaction medium (Figures 22 and 23). Interestingly, SKF 5256A only inhibited hepatic micro- somal activity; this agent failed to reduce renal microsomal activity in either male C57 or male DBA mice (Figures 24 and 25). The presence of microsomal enzyme activity in the cuboidal epithelium of Bowman's capsule was observed. The activity of the microsomal enzyme in the cuboidal cells was reduced by piperonyl butoxide but not by SKF 525-A (Figures 22 and 24). 75 . 91 r... ”’4 9 The dark area indicates the Cytochemistry of hepatic cytochrome presence of microsomal enzyme activities in the centre— (X400) Figure 20. 450 in male C57 m1ce. lobular region. P 76 Figure 21. P-450 in male DBA mice. presence of microsomal enzyme activities in proximal convoluted tubular cells as well as the cuboidal epithelial cells of Bowman's capsule. (X250) Cytochemistry of renal cytochrome The dark areas indicate the 77 Figure 22. Effect of piperonyl butoxide on renal cytochrome P-450 in male DBA mice, showing inhibition of microsomal enzyme activities in both proximal con- voluted tubular cells and cuboidal epithelial cells of Bowman's capsule (absence of dark areas). (X400) 78 Figure 23. Effect of piperonyl butoxide on hepatic cytochrome P-450 in male C57 mice, showing inhibition of microsomal enzyme activities by this agent (absence of dark areas). (X400) 79 Figure 24. Effect of SKF 525-A on renal cyto- chrome P-450 in male DBA mice. Dark, cytochemically stained areas reflect the presence of microsomal enzyme activity in the cuboidal epithelial cells lining proximal convoluted tubules and Bowman's capsules. (X250) 80 Figure 25. Effect of SKF 525-A on hepatic cyto- chrome P-450 in male C57 mice, showing inhibition of microsomal enzyme activities by this agent (absence of dark areas). (X400) DISCUSSION The mechanism by which CHC13 produces toxicity is not completely understood. The initial step in the pathway leading to CHC13 toxicity is generally assumed to be the biotransformation of CHC13 to a reactive intermediate, possibly phosgene (COC12) by mixed-function oxygenases (MFO) present in the endoplasmic reticulum of susceptible tissue (Ilett, 1973; Brown et al., 1974; Sipes et al., 1977; Pohl et al., 1977; Mansuy et al., 1977). The MFO system is a heterogeneous group of enzymes responsible for oxidative metabolism of polycyclic hydrocarbons and drugs and for the manifestation of the nephrotoxic and hepato- toxic effects Of a variety of compounds including CHC13. The presence of the MFO system in kidney as well as liver microscomes in various animal species has been described by many investigators (Ichihara, 1969; Levis, 1970; Ichihara et al., 1971; Ellin et al., 1972; Jakobsson et al., 1970; Jakobsson and Cinti, 1973; Ichikawa, 1975). An important function of these enzymes is the metabolism of various exogenous and endogenous compounds. Broide et al. (1958) introduced the term "drug metabolizing enzyme." The reactions involved in the biotransformation include oxidation, reduction, demethylation and hydroxylation. 81 82 Biotransformation of xenobiotics does not necessarily result in substrate detoxification. It has been shown that the resulting metabolite can be as toxic as the foreign chemical itself, or more toxic. This mechanism is at least partly responsible for the hepatotoxic and nephro- toxic effect of compounds Such as carbon tetrachloride and chloroform (McLean, 1970; Scholler, 1970; Hewitt et al., 1979). Phosgen (COC12) has been postulated to be a possible reactive intermediate(s) formed upon CHC13 oxidation (Pohl et al., 1977; Mansuy et al., 1977; Sipes et al., 1977). Phosgen is a very reactive electrophilic compound and reacts with tissue macromolecules; formation of irreversible covalent binding occurs and causes tissue necrosis (Ilett et al., 1973; Pohl and Krishna, 1978). Increased serum glutamic pyruvic transaminiase (SGPT) activity has been suggested as a sensitive index of hepatic injury (Balazs et al., 1962). By this criterion, CDC13 produced less liver damage in mice than the same dose of CHC13 (Figure 1), consistent with findings in rats (Pohl and Krishna, 1978). Pohl and Krishna (1978) reported that 2.49 mmOl/kg CDC13 produced only minor histological changes and no elevation of SGPT relative to a sesame oil control; at this dose CHC13 produced considerable necrosis of the centrolobular region of the liver and elevation of SGPT. Thus, a deuterium isotope effect appears to influence hepatotoxicity of CDC13 in rats and mice, suggesting that 83 similar pathways and mechanisms of CHC13 toxicity exist in the liver in both species. Accumulation of p-aminohippuric acid (PAH) or tetra- ethylammonium (TEA) into renal cortical slices in Vitro is an energy-dependent active transport process. Altered uptake of these ions is a sensitive index of nephrotoxicity, especially injury of proximal tubular cells (Hirsch, 1976), and has been used in quantifying CHC13-induced nephrotoxicity (Watrous and Plaa, 1972; Kluwe et al., 1978; Hewitt et al., 1979). Treatment with CHC13 depressed accumulation of PAH and TEA by rat and mouse renal cortical slices in Vitro. Deuterium-labelled chloroform appeared to be less nephro- toxic than CHC13 (Figures 2 through 4). Since kidneys usually have low drug-metabolizing enzyme activities (Litterst et al., 1975), chemically induced nephrotoxicity has been assumed to be produced by toxic intermediate(s) generated in the liver and transported to the kidney. If this were the case, phosgene could be pro- duced from CHC13 in the liver and transported to the kidneys to produce nephrotoxicity. Male C57 and male and female DBA mice which developed the same degree of liver damage following exposure to CHC13 (Figures 5 and 9) should show the same degree of kidney damage as well. However, results of renal function tests indicated that male DBA mice were more sensitive to CHC13-induced nephrotoxicity than male C57 mice. In contrast, female DBA mice failed to develop renal damage after administration of CHC13 (Figures 6 through 8). This observation was consistent with Clemmens et al. 84 (1979) and Hill et al. (1975), who reported that covalent bind- ing of [14C]CHC13 to renal microsomes was greater in male DBA mice than in male C57 mice. Female DBA mice failed both to develop tubular lesions and to accumulate [14C]CHC13 bound to renal microsomal protein. Since renal injury does not parallel hepatotoxicity, it is unlikely that CHC13 nephrotoxicity is due to intermedi- ate(s) formed in the liver. Thus, kidney damage following administration of CHC13 appears to be caused by toxic reactive intermediate(s) generated within the kidney. Strain-dependent nephrotoxicity following CHC13 admin- istration to male mice, in the absence of significant dif- ferences in hepatic injury, strongly suggests that CHC13 is metabolized intrarenally and that generation of toxic metabolites in the kidney is responsible for renal injury. Male C57 mice have lower renal MFO activity than male DBA mice (Table 6); they also are relatively resistant to CHC13-induced nephrotoxicity. It seems reasonable to attribute reduced susceptibility to CHC13-induced renal necrosis to lower renal microsomal drug metabolizing enzyme activity. The activity of microsomal enzymes can be altered by administration of various agents such as phenobarbital (PB), 3-methylcholanthrene (3MC), tetrachlorodibenzo-p- dioxane (TCDD), polybrominated biphenyls (PBB), poly- chlorinated biphenyls (PCB) and B-naphtheflavone (BNF). Both liver enlargement and increased hepatic activity of drug metabolizing enzymes with PB-induced proliferation of 85 smooth endOplasmic reticulum in the hepatic cells in rats have been reported (Gopinath and Ford, 1975; Burger and Herdson, 1966). Induction of the MFO system in male C57 and DBA mice and CHC13-induced hepatotoxicity were observed following treatment of animals with PB (Figures 9 and 13). In contrast, PB did not alter renal MFO activity, nor did it affect CHC13-induced nephrotoxicity. Similar observations were made by Kluwe et al. (1978), who reported that treat- ment with PB increased the activity of drug metabolizing enzyme in the liver but not in the kidney of male ICR mice. Since PB only enhances hepatotoxicity and not nephrotoxicity of CHC13, it is unlikely that CHC13 nephrotoxicity is due to intermediate(s) formed in the liver. 3-Methylcholanthrene appears to be less effective than PB in promoting the hepatotoxicity of CHC13. B-Naphthoflavone (BNF), which is a 3MC type inducer (Boobis et al., 1976), significantly induced both renal and hepatic MFO activity in liver and kidney in male CS7 mice (Figures 13 and 14). These figures are consistent with the observation of Nebert et al. (1972). Nephrotoxicity of CHC13 was not altered in either strain of male mice treated with BNF (Figures 10 through 12). Pre- treatment of animals with BNF only enhanced liver injury by CHC13, further supporting the idea that liver is not the source of nephrotoxic CHC13 metabolites. Polybrominated biphenyls (PBB) were used commercially as fire retardants. Recently these were accidentally mixed into animal feed, resulting in heavy exposure of domestic farm animals to dietary PBB. Increased MFO activities in 86 kidney as well as liver following PBB administration have been reported (McCormack et al., 1980). A similar class of compounds, the polychlorinated biphenyls (PCB), have been extensively studied. Polybrominated biphenyls (PBB) may share many of the biological and toxic properties of PCB. In laboratory animals, PCB and PBB cause an altera- tion of hepatic function (Johnstone et al., 1974) and induce both hepatic and renal microsomal enzyme activities (Alvores et al., 1973; Kluwe and Hook, 1978; McCormack et al., 1979; McCormack et al., 1980). Although PCB is similar to PBB in many respects, they differ in their toxic effects. Polychlorinated biphenyls have been reported to protect against CHC13 administration (Kluwe and Hook, 1978). The differences between the effects of PBB and PCB on CHC13 toxicity suggest that these agents may have different effects on the overall metabolism of CHC13 (Kluwe et al., 1978). The effect of PBB on hepatic microsomal activities was compared with the effect produced by PB and 3MC, agents which represent two distinct classes of inducing agents. The PBB are unique in that they stimulate hepatic enzymes sensitive to both PB and 3MC (Dent et al., 1976). Poly- brominated biphenyls induce hepatotoxicity as well as nephro- toxicity of CHC13 in mice (Kluwe et al., 1978). These results suggest that PBB ingestion, in subtoxic amounts, stimulates the metabolism of CHC13 to hepatotoxic and nephrotoxic agents and enhances the sensitivity of the liver and kidney to CHC13 injury (Kluwe and Hook, 1978). Hepatic microsomal enzyme activities, as well as 87 CHC13-induced hepatotoxicity, have also been studied in male C57 and DBA mice (Figures 9 and 13). The degree of MFO inducibility and the toxic effect of CHC13 are con- siderably higher in male C57 than in male DBA mice. In male DBA mice, PBB did not affect renal MFO activities or alter the CHC13-induced renal injury. In contrast, pre- treatment of male C57 mice with PBB resulted in markedly increased renal MFO activity as well as enhanced CHC13 nephrotoxicity (Figures 14 and]I)through 12). These find- ings further support the idea that nephrotoxicity of CHC13 is likely to be caused by CHC13 metabolites formed intrarenally. If the liver were the site of production of nephrotoxic CHC13 metabolites, pretreatment with PBB should induce renal damage in male DBA mice as well as hepatic injury. Since this was not the case, it is unlikely that the liver is responsible for the generation of nephrotoxic CHC13 metabolites. Arylhydrocarbon hydroxylase (AHH) is an example of a mixed-function oxidase (MFO) (Nebert et al., 1972). The in vitro enzyme assay was used as an index for enzymatic conversion of CHC13 both in the liver and kidney. Follow- ing treatment of male C57 and DBA mice with PBB, hepatic AHH activity was significantly higher in male C57 than in male DBA mice (Figure 13). Similarly, SGPT activity, which is a sensitive index of hepatic injury (Balazs et al., 1962), was considerably higher in PBB treated male C57 mice than in male DBA mice after CHC13 (Figure 9). Hepatic AHH activity and CHC13 hepatotoxicity were also 88 enhanced in male C57 and male DBA mice following administra- tion of PB (Figures 13 and 9). Stimulation of hepatic AHH activity and CHC13 hepatotoxicity was noted in BNF- treated male C57 mice (Figures 13 and 9). Interestingly, BNF did not affect hepatic AHH activity or CHC13-induced hepatic injury in male DBA mice (Figures 13 and 9). These findings may suggest that hepatic AHH activity may parallel the activity of enzyme(s) responsible for the metabolite of CHC13. Untreated male C57 mice had higher hepatic AHH activity in comparison to the hepatic AHH activity in male DBA mice (Table 6). However, when both strains of male mice were challenged with the same dose of CHC13, hepatic injury pro- duced by CHC13 was the same (Figure 9). Similar observations on male ICR mice were carried out by Kluwe et al. (1978), who reported that hepatic AHH activity increased following treatment of male ICR mice with PCB, 3MC or TCDD, but hepato- toxicity of CHC13 was not affected by these agents. These findings, which are contrary to the previous observations, indicate that determination of AHH activity alone is not a suitable index for interpretation of the MFO activity responsible for metabolism of CHC13 in the liver. In kidney, AHH activity was higher in untreated male DBA mice than in untrated male C57 mice (Table 6). Similarly, CHC13-induced renal injury was greater in male DBA than in male C57 mice (Hill et al., 1975; Clemens et al., 1979) (Figures 14 and 10-12). In addition, when both male C57 and male DBA mice were treated with PBB, nephrotoxicity of 89 CHC13 was enhanced as well as renal AHH activity in male C57 mice (Figures 14 and 10-12). In contrast, PBB did not alter nephrotoxicity or renal AHH activity in male DBA mice (Figures 14 and 10-12). Renal AHH was not altered by treatment of male C57 and DBA mice with PB. Similarly, PB did not affect nephrotOxicity of CHC13 in either strain of male mice. However, treatment of male C57 mice with BNF induced renal AHH activity, but there were no significant effects by this agent on nephrotoxicity of CHC13 (Figures 10-12). In addition, Hook et al. (1978) and Kluwe et al. (1978) found that TCDD, 3MC or PCB induced renal AHH; in contrast, nephrotoxicity of CHC13 was reduced following treatment of male ICR mice with these agents. From these observations, it was also concluded that determination of renal AHH activity alone also is not a suitable index for the detection of renal MFO activity responsible for CHC13 metabolism. In conclusion, failure to correlate the induction of renal and hepatic AHH activities and nephrotoxicity and hepatotoxicity of CHC13 suggests that CHC13-induced renal and hepatic toxicity may not parallel AHH activity. As a result, enzymes responsible for metabolism of CHC13 comprise a subpopulation of the MFO system, the activity of which does not correlate with the degree of inducibility of AHH activity. Inhibition, as well as stimulation, of drug metaboliz- ing enzyme systems can alter the nephrotoxicity and hepato- toxicity of CHC13. Piperonyl butoxide is an inhibitor of 90 the microsomal drug metabolizing enzyme system in both liver and kidney (Figures 22 and 23). Ilett et a1. (1973) reported that pretreatment of male C57 mice with piperonyl butoxide reduced the hepatotoxicity and nephrotoxicity of CHC13. Interestingly, SKF SZS-A inhibited the activity of the MFO system in liver but not in kidney of male C57 or male DBA mice (Figures 24 and 25). Similarly, decreased CHC13-induced hepatotoxicity is found when mice were pre- treated with SKF 525-A. This compound, however, failed to protect the kidney from CHC13 injury. The precise effects of these agents on CHC13 metabolism are not completely understood. However, these two inhibi- tors are believed to affect different subpopulations of the MFO enzyme systems in the kidney. Decreased CHC13 nephro- toxicity in animals pretreated with piperonyl butoxide and the failure of SKF SZS-A to block renal injury produced by CHC13 suggest that CHC13 is metabolized in the kidney by enzyme system(s) sensitive to inhibition by piperonyl butoxide but not by SKF 525-A. The mechanism by which CHC13 produces renal necrosis in male but not in female mice is not clear. Sex-related differences in kidney morphology of mice have been reported by Crabtree (1940). She observed that most cells in the parietal epithelium of Bowman's capsule in male albino mice was composed of cuboidal cells resembling those which line the proximal convoluted tubule cells. In female albino mice the parietal epithelium of Bowman's capsule is composed of squamous cells. The observation of Eshenbrenner and Miller 91 (1945) on strain A mice confirmed these findings. In addi- tion, they noted renal lesions occurred in males following exposure to CHC13 but not in similarly treated female mice. Therefore, they suggested that there is a correlation between the structure of Bowman's capsule in male and female mice and sex dependent nephrotoxicity of CHC13. Male DBA mice have a higher percentage of cuboidal epithelial cells in the parietal layer of the Bowman's capsule. Male C57 mice, which are relatively resistant to CHC13 nephrotoxicity (Figures 6-8, 10-12), have a higher percentage of squamous epithelial cells (Table 7). However, female DBA mice which are resistant to renal damage following exposure to CHC13 (Figures 6-8) have the lowest percentage of cuboidal cells in the pareital epithelium of Bowman's capsule (Table 7). These studies also demonstrated that the ultrastructural features of cuboidal cells in Bowman's capsule of male C57 or DBA mice are generally similar to the epithelial Cells lining proximal convoluted tubules (Figures 18 and 19). The finding that the effect of CHC13- induced renal damage parallels the percentage of the cuboidal cells in the pareital epithelium of Bowman's capsule (Table 7 and Figures 6-8) suggests a correlation between the structure of Bowman's capsule in male and female mice and susceptibility to nephrotoxicity of CHC13 (Eshenbrenner and Miller, 1945). These correlations may exist among dif- ferent strains of male mice. However, the data do not indicate a cause and effect relationship between the 92 morphology of Bowman's capsule and susceptibility of CHC13- induced renal necrosis. The role of testosterone in developing cuboidal epi- thelial cells in Bowman's capsule has been reported. Crabtree (1941) noted that castrated male mice failed to develop cuboidal cells. Setye (1939) found development of cuboidal cells in the parietal epithelium of castrated mice treated with testosterone and in testosterone-treated female mice. Furthermore, Crabtree (1941) observed that the euboidal cells do not increase in number in male mice until sexual maturity. Testosterone also plays a significant role in CHC13- induced nephrotoxicity. Renal tubular necrosis in testosterone-treated female mice following exposure to CHC13 has been noted (Krus et al., 1974). Renal injury with CHC13 exposure has been found also in castrated mice treated with testosterone (Eshenbrenner and Miller, 1945). The role of testosterone may also be important in strain-related differences in renal injury produced by CHC13 (Shire and Bartke, 1972). Interestingly, testosterone is not affected in hepatotoxicity of CHC13 (Culliford et al., 1957). Results clearly establish that kidney of mice is par- ticularly sensitive to testosterone and under the influence of the hormone undergoes histological changes in Bowman's capsule, as well as testosterone-induced CHC13 toxicity in the kidney but not in the liver. It should be emphasized that an explanation of these two phenomena is not clear. However, a lack of correlation between nephrotoxicity and 93 hepatotoxicity of CHC13 strongly suggests that the kidney is the site of formation of CHC13 metabolite(s). There is considerable evidence to support a cause and effect relationship between [14C]CHC13 covalent binding and tissue necrosis. In kidney, the covalent binding of [14C]- CHC13 and CHC13-induced renal necrosis was carried out with male and female C57 mice by Ilett et al. (1973). They observed that the amount of covalent binding and severity of necrosis were significantly higher in male C57 mice when compared to female C57 mice. Similarly, Clemens et al. (1979) observed that covelant binding of CHC13 by renal microsomal protein was greater in male DBA than in male C57 mice, and male DBA mice were more sensitive to CHC13- induced renal injury than male C57 mice. Taylor et a1. (1974) reported that the amount of radioactivity of [14C]- CHC13 in the kidneys of male CF/LP, CBA and C57BL mice was significantly greater when compared to the female kidneys of each strain of mice. These observations support the idea that there is a cause and effect relationship between [14C]CHC13 covalent binding and kidney necrosis. Pretreatment of male C57 and male DBA mice with PB enhanced covalent binding of [14 C]CHC13 by renal microsomes in male DBA but not in male C57 mice (Clemens et al., 1979). In addition, Ilett et a1. (1973) reported that pretreatment of male C57 mice with PB decreased covalent binding of [14C]CHC13 in the kidney. Interestingly, administration of CHC13 to male C57 and male DBA mice pretreated with PB did not alter nephrotoxicity of CHC13 in either strain of 94 male mice (Figures 10-12). This finding, which is in con- trast to the previous suggestion of cause and effect rela- tionship between covalent binding and kidney necrosis, suggests that covalent binding of radioactivity of CHC13 by kidney microsomes.may not parallel the nephrotoxicity of CHC13. SUMMARY Administration of deuterium-labelled chloroform (CHC13) to male ICR mice and chloroform (CHC13) to male ICR, C57 and male and female DBA mice produced dose-dependent damage in the liver (elevated SGPT activity) and kidney (increased BUN, decreased accumulation of PAH and TEA). Deuterium- labelled chloroform produced less kidney and liver damage than CHC13 in male ICR mice, suggesting that the kidney may metabolize CHC13 in the same manner as the liver. Chloro- form caused the same degree of liver damage in male C57 and male and female DBA mice. However, nephrotoxicity of CHC13 was greater in male DBA than in male C57 mice. Female DBA mice failed to develop renal injury following treatment with CHC13. Pretreatment of male C57 and DBA mice with phenobarbital (PB) markedly increased hepatotoxicity of CHC13 but did not affect nephrotoxicity of CHC13. B-Naphtho- flavone enhanced CHC13 hepatotoxicity in male C57 mice but had little effect on nephrotoxicity. Polybrominated biphenyl (PBB) enhanced hepatotoxicity of CHC13 in both strains. In contrast, PBB increased renal injury in male CS7 mice but not in male DBA mice. Therefore, the lack of correlation between hepatotoxicity and nephrotoxicity produced by CHC13 strongly suggests that the liver is not the site of formation of nephrotoxic CHC13 metabolite(s). 95 96 Arylhydrocarbon hydroxylase (AHH) is an example of a mixed-function oxygenase. Following treatment of male C57 and DBA mice with PBB, hepatic AHH activity was signifi- cantly higher in male C57 than in male DBA mice. Similarly, hepatotoxicity of CHC13 was considerably higher in PBB treated male C57 than in male DBA mice pretreated with PBB. Hepatic AHH activity and CHC13 hepatotoxicity were also enhanced in male C57 and DBA mice following administration of PB. Stimulation of hepatic AHH activity and hepato- toxicity of CHC13 were noted in BNF-treated male C57 mice. Interestingly, BNF did not affect hepatic AHH or CHC13-induced hepatic injury in male DBA mice. However, untreated male C57 mice had higher hepatic AHH activity in comparison to the hepatic AHH activity in male DBA mice. But when both strains of male mice were challenged with the same doses of CHC13, hepatic injury produced by CHC13 was the same. In kidney, AHH activity was higher in untreated male DBA than in untreated male C57 mice. Similarly, CHC13- induced renal injury was greater in male DBA than in male CS7 mice. In addition, when both male C57 and male DBA mice were treated with PBB, nephrotoxicity of CHC13 and renal AHH activity were increased in male CS7 mice. In contrast, PBB did not alter nephrotoxicity or renal AHH activity in male DBA mice. Renal AHH was not altered by treatment of male C57 and DBA mice with PB. Similarly, PB did not affect nephrotoxicity of CHC13 in either strain of male mice. However, treatment of male CS7 mice with BNF induced renal AHH activity, but there were no significant 97 effects by this agent on nephrotoxicity of CHC13 in male C57 mice. In conclusion, renal and hepatic AHH activities may not parallel the CHC13-induced renal and hepatic injury. Enzymes reSponsible for metabolism of CHC13 comprise a sub- population of the MFO system and their activity does not correlate with the degree of inducibility of AHH activity in the target organ. Inhibition as well as stimulation can alter nephro- toxicity and hepatotoxicity of CHC13. Piperonyl butoxide, an inhibitor of microsomal drug metabolizing enzymes, inhibits both liver and kidney microsomal enzyme activities. Similarly, it has been reported that nephrotoxicity and hepatotoxicity of CHC13 are markedly reduced following pre- treatment of mice with piperonyl butoxide. However, SKF 525-A only inhibits hepatic microsomal enzyme activity and has little effect on renal microsomal enzyme activity. In addition, it has been found that hepatotoxicity of CHC13 decreased in mice pretreated with SKF 525-A. In contrast, this agent failed to protect the kidney from the toxicity of CHC13, suggesting that the kidney may metabolize CHC13 by a subpopulation of the MFO system which is sensitive to inhibition by piperonyl butoxide but not by SKF SZS-A. Male DBA mice had the highest percentage of cuboidal epithelial cells in Bowman's capsule in comparison to male C57 and female DBA mice. 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