Microsomal Mixed-function Oxidase Research Articles

Chlordecone‐induced hepatic dysfunction

Chlordecone (Kepone) is a decachloroketone analog of the dodecachlorohydrocarbon mirex and is used as a stomach poison insecticide. Despite the structural similarity to mirex, chlordecone is unlike mirex in general organ-specific toxic properties. Chlordecone is primarily accumulated in the liver, where it causes a variety of morphological and biochemical alterations. Although less effective than mirex as a hepatotoxin, it causes liver enlargement, focal necrosis, mitochondrial changes, fatty infiltration of hepatocytes, and proliferation of endoplasmic reticulum. Chlordecone accumulation and morphological alterations in the liver were also observed in occupationally exposed human patients. Induction of hepatic microsomal mixed-function oxidases (MFOs) and impaired production and utilization of hepatocellular energy are the principal biochemical aberrations produced by chlordecone. Chronic exposure causes carcinogenesis in mice and rats. Hyperplastic nodules, which progress to hepatocellular carcinomas, are the principal pathological lesions. Acute and chronic exposures to chlordecone result in hepatobiliary dysfunction manifested as impaired excretion of anionic compounds accompanied by choleresis. Exposure to chlordecone results of greatly potentiated haloalkane hepatotoxicity, representing a most potent toxic interaction at otherwise individually nontoxic levels. In view of the demonstrated carcinogenic effect of chlordecone, such interactions at very low levels assume extraordinary significance in terms of chronic toxicological and pathological manifestations induced by combinations of toxic chemicals.

The influence of disulfiram and other inhibitors of oxidative metabolism on the formation of 2-hydroxyethyl-mercapturic acid from 1,2-dibromoethane by the rat

The mercapturic acid derivative, N- acetyl-S-2- hydroxyethyl- l - cysteine , is a major metabolite of 1,2-dibromoethane in vivo. This compound can be formed via two pathways, both involving a potentially dangerous reactive intermediate. One way involves the intermediacy of bromoacetaldehyde, formed by microsomal oxidation, followed by loss of hydrogen bromide. The second pathway, direct conjugation of 1,2-dibromoethane with glutathione, gives rise to S-2-bromoethyl glutathione. Using several inhibitors of microsomal mixed function oxidases, it was found that under these conditions about 10% of the mercapturic acid derivative formed via direct conjugation. Disulfiram, an inhibitor of aldehyde dehydrogenases, but also of microsomal oxidation, also markedly inhibits the excretion of the mercapturic acid, after administration of a single high dose (1 g/kg) or upon chronic treatment with a low dose (50 mg/kg). The inhibitory effect is maximal after 10 days of chronic treatment. Administration of large amounts of 1,2-dibromoethane (>0.20 nmole/rat) following a single lower dose of disulfiram (125 mg/kg) also leads to a lower excretion of mercapturic acid metabolite a phenomenon associated with a decrease in cytochrome P-450 levels. From these results it is concluded that the enhanced carcinogenic effect of the combination disulfiram (chronic)/1,2-dibromoethane is not caused by bromoacetaldehyde, since its formation is completely inhibited under these conditions, but by S-2-bromoethyl-glutathione, although a role for 1,2-dibromoethane itself cannot be excluded.

Studies on the mechanism of denaturation of cytochrome P-450 by cyclophosphamide and its metabolites.

Several lines of investigation were pursued to understand mechanisms involved in the in vivo depression of rat hepatic microsomal mixed function oxidase by cyclophosphamide, an important anti-cancer and immunosuppressive agent. Essentially exclusive metabolism-dependent binding to microsomal proteins of 14C from [4-14C]cyclophosphamide, compared with 3H from [chloroethyl-3H]cyclophosphamide, suggests the binding of the metabolite acrolein. Of the various metabolites and analogs of cyclophosphamide tested (which did not contain a peroxy or a hydroperoxy group), only acrolein and 4-hydroxycyclophosphamide (which releases acrolein in solution) caused denaturation of microsomal cytochrome P-450 in vitro; this denaturation was identical with that produced by sulfhydryl reagents. Of the various chemicals tested, only those which contained either a free amino group (except lysine) and/or a free sulfhydryl group (e.g. semicarbazide, cysteine, glycine, glucosamine) effectively blocked (40-80%) the binding of 14C as well as protected against acrolein-induced denaturation of cytochrome P-450. These data further suggested interaction of cyclophosphamide metabolite with free amino and/or free sulfhydryl groups in proteins. However, comparison with [3H]aflatoxin B2a which interacts with free protein amino groups via the formation of Schiff bases, clearly attributed the preferential binding of 14C to cysteine sulfhydryl groups in these proteins. Studies on chemical models derived from reaction between acrolein and cysteine also supported this suggestion. When microsomes isolated from incubations metabolizing [4-14C]cyclophosphamide were subjected to gel electrophoresis, the major radioactive band detected by autoradiography was associated with a cytochrome P-450 band at 55,000 daltons, the major band induced by phenobarbital in the rat. All these results taken together strongly point to the possibility that acrolein is the cyclophosphamide metabolite responsible for the depression of the mixed function oxidase activities. Acrolein most likely produces this effect by alkylation of the sulfhydryl group(s) in the active site of cytochrome P-450.

The comparison of in vivo plasma radioactivity clearance and 14CO 2 breath elimination of model drugs in the rat: A study in regional hepatocyte function

We systematically related the in vivo plasma radioactivity clearance and the 14CO 2 breath elimination of two drugs ([4- dimethyl- 14C]aminopyrine and [1- methyl- 14C]caffeine), whose major sites of hepatic metabolism correspond to the microsomal mixed-function oxidase system (cytochromes P-450 and P-448, respectively), and two compounds ([1- 14C]ethanol and [ 14C]methanol], whose major sites of hepatic metabolism correspond to the cytosol. All studies were carried out in the freely moving rat. Measurements of plasma radioactivity clearance and 14CO 2 breath elimination were done with normal animals as well as with animals pretreated with agents that either stimulate or inhibit the model drugs' in vivo metabolism. Following pretreatment with phenobarbital, the aminopyrine plasma radioactivity clearance was significantly increased over normal, and this was paralleled by a significant increase of the 14CO 2 breath elimination as well. The ratio (plasma elimination/breath elimination) was unchanged between the normal and phenobarbital-pretreated animals and approached unity. by contrast, neither the aminopyrine plasma radioactivity clearance nor the 14CO 2 breath elimination was significantly altered by pretreatment with 3-methylcholanthrene. When the plasma radioactivity clearance and the 14CO 2 breath elimination were studied after administration of [ 14C]caffeine, both the plasma radioactivity clearance and 14CO 2 breath elimination showed a significant increase in the 3-methylcholanthrene-treated animals as compared to normal. The ratio (plasma elimination/breath elimination) was unchanged, however, between the normal and 3-methylcho-lanthrene-pretreated animals. Neither the caffeine plasma radioactivity clearance nor 14CO 2 breath elimination was significantly altered by pretreatment with phenobarbital. In animals pretreated with pyrazole, the ethanol plasma radioactivity clearance and 14CO 2 breath elimination were both significantly decreased. However, neither measurement was significantly altered after pretreatment with 3-amino-1,2,4-triazole. By contrast, the methanol plasma radioactivity clearance and 14CO 2 breath elimination were significantly decreased in both the pyrazole and 3-aminio-1,2,4-triazole-pretreated animals. These studies illustrate that for the model compounds selected and the dosages utilized, the 14CO 2 breath elimination rate closely reflected the plasma radioactivity elimination rate.

Cholesterol 7 alpha-hydroxylase of rat liver. Studies on the solubilisation, resolution and reconstitution of the enzyme complex.

1. Cytochrome P-450 was prepared from the liver microsomes of cholestyramine-fed rats by solubilisation with the non-ionic detergent Nonidet P42 followed by chromatography on a DEAE-cellulose column and on hydroxyapatite. NADPH--cytochrome P-450 reductase was prepared by a technique of affinity column chromatography using 2',5'ADP Sepharose. 2. The activity of cholesterol 7 alpha-hydroxylase was measured in a reconstituted system of microsomal mixed-function oxidase containing cytochrome P-450 and NADPH--cytochrome P-450 reductase from rat liver plus cholesterol and NADPH. Endogenous cholesterol was largely depleted from the enzyme preparations by the treatment of the microsomes with cold n-butanol/acetone. 3. The reconstituted system of mixed-function oxidase catalysed a highly effective and specific 7 alpha-hydroxylation of cholesterol. The reconstituted system showed a higher activity of cholesterol 7 alpha-hydroxylase than was observed in native liver microsomes. The reconstituted system had an absolute requirement for cytochrome P-450, NADPH--cytochrome P-450 reductase and NADPH. 4. The apparent Km for cholesterol in the reconstituted system was 15 microM and the V was 1.4 nmol 7 alpha-hydroxycholesterol formed min-1 (nmol cytochrome P-450)-1. 5. The reconstituted system also catalysed the 7 alpha-hydroxylation of taurodeoxycholic acid, the 7 alpha-hydroxylation of 26-norcholesterol and to a limited degree the 12 alpha-hydroxylation of cholest-4-ene-3-one-7 alpha-ol. The ability of this reconstituted system to effect these two 7 alpha-hydroxylation reactions and the 12 alpha-hydroxylation reaction was significantly less than the ability of the system to 7 alpha-hydroxylate cholesterol.

Differences in the nature of induction of mixed-function oxidase systems of the rat liver among phenobarbital, DDT, 3-methylcholanthrene, and TCDD

A comparative study of the induction patterns of rat hepatic microsomal mixed-function oxidases was carried out in male Sprague-Dawley rats. The pattern of induction by DDT was different from that of phenobarbital. First, DDT induction was not acompanied by an increase in δ-aminolevulinic acid synthetase, and second, it was not affected by prior treatment with actinomycin D or α-amanitin in vivo. Otherwise, induction by these two chemicals as determined by differential increases in various drug-metabolizing activities and cytochrome P-450 was indistinguishable, indicating the basic similarities of these two induction patterns. Both TCDD and 3-methylcholanthrene (3-MC) induced a similar type of change in the cytochrome as judged by ethyl isocyanide- and CO-binding spectra. However, induction by TCDD and 3-MC was also different in two important areas: first, induction by 3-MC was inhibited by a prior or simultaneous treatment with actinomycin D, while that by TCDD was not. Second, the pH response of the induced cytochrome P-448 in terms of ethyl isocyanide-binding spectral changes was different in the two cases.

Studies on the effects of l-ascorbic acid on acetaminophen-induced hepatotoxicity : 1. Inhibition of the covalent binding of acetaminophen metabolites to hepatic microsomes in vitro

The covalent binding of [ 14C]acetaminophen metabolites to male mouse hepatic microsomes was inhibited by the sulfhydryl compounds, reduced glutathione, cysteamine, and l-cysteine, and also by l-ascorbic acid (vitamin C, LAA). Although the sulfhydryl compounds were more effective inhibitors of macromolecular binding than LAA, the combination of LAA with any of the thiol agents resulted in additive inhibition of covalent binding of [ 14C]acetaminophen metabolites. Similar results were obtained in studies with hepatic microsomes from female mice and male hamsters. Investigations into the mechanism of inhibition of covalent binding of [ 14C]acetaminophen metabolites indicated that LAA probably acts by scavenging the reactive intermediates generated by the microsomal mixed-function oxidase enzymes rather than by the inhibition of their formation. The results suggest that LAA, at concentrations found in rodent and human liver, may supplement the endogenous protective mechanisms (such as reduced glutathione) which operate in vivo to prevent the covalent binding of reactive acetaminophen metabolites and hence hepatic necrosis. The possible application of this study to the use of LAA in the prevention and treatment of acetaminophen-induced hepatotoxicity in man is discussed.

Effects of dietary pectin and cellulose on hepatic and intestinal mixed-function oxidations and hepatic 3-hydroxy-3-methylglutaryl-coenzyme a reductase in the rat

The aim of this study was to determine if feeding dietary fiber (cellulose or pectin) to male rats could influence hepatic and intestinal mixed-function oxidation. We simultaneously compared hepatic drug-oxidizing activity with the activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme for cholesterol biosynthesis. Three groups of six animals were fed a purified diet containing by weight either 10.4% cellulose or 10.4% pectin, or a standard cereal-based diet containing 4.5% crude fiber; the caloric contributions by carbohydrate, protein and fat in the three diets were similar. In the cellulose-fed rats, the hepatic microsomal cytochrome P-450 content and the activities of ethylmorphine N-demethylase and aniline hydroxylase were significantly lower when compared with those of rats fed pectin or the cereal-based diet. The hepatic microsomal cytochrome P-450 content and the activities of ethylmorphine N-demethylase and aniline hydroxylase were similar in the pectin-fed and cereal diet-fed rats. Hepatic HMG-CoA reductase activity, hepatic microsomal cytochrome b 5 content, and intestinal benzo[ a]pyrene hydroxylase activity were comparably lower in rats fed the purified diet with either dietary fiber when compared to those fed the cereal diet. It is concluded that dietary pectin and cellulose exert distinctly different influences on the hepatic microsomal mixed-function oxidase system for drug metabolism, but not on liver cholesterol synthesis or intestinal benzo[ a]pyrene hydroxylation, suggesting that different physiological mechanisms control these enzyme systems.

Carbon tetrachloride-induced changes in adrenal microsomal mixed-function oxidases and lipid peroxidation

Studies were carried out to compare the effects of carbon tetrachloride (CCl 4) in vivo and in vitro on adrenal and hepatic microsomal metabolism in guinea pigs. CCl 4 administration in vivo decreased adrenal and hepatic microsomal cytochrome P-450 concentrations and lowered benzphetamine (BZ) demethylase and benzo[ a]pyrene (BP) hydroxylase activities in both tissues. NADPH-cytochrome c reductase activity was decreased in hepatic but not in adrenal microsomes. Addition of CCl 4 to adrenal or hepatic microsomes in vitro produced a type I difference spectrum suggesting binding of CCl 4 to cytochrome(s) P-450; its magnitude was far greater in adrenal than in liver. Incubation of adrenal or hepatic microsomes in vitro with CCl 4 alone had little or no effect on mixed-function oxidase activity or on lipid peroxidation. However, when microsomes were incubated with CCl 4 + NADPH, the rates of BZ and BP metabolism were decreased, cytochrome P-450 concentrations were decreased, and lipid peroxidation was increased. The effects of CCl 4 + NADPH on enzyme activities were greater in adrenal than in hepatic microsomes. Addition of 1.0 m M EDTA or 0.1 m M MnCl 2 to the incubation medium blocked the effects of CCl 4 + NADPH on lipid peroxidation in adrenal and liver but had no effect on the decreases in mixed-function oxidase activities. The results indicate the following: (1) the adrenal cortex in the guinea pig is an active site of CCl 4 metabolism; (2) CCl 4 metabolism results in a loss of microsomal enzyme activities in the adrenal as well as liver; and (3) lipid peroxidation is not obligatory for the CCl 4-mediated destruction of microsomal enzymes.

Interaction of organic dyes with hepatic microsomal drug-metabolizing monoöxygenases in vitro.

Organic dyes such as malachite green, methylene blue, fuchsin, safranine T, neutral red, phenazine methosulphate, riboflavin, dichlorophenolindophenol, phenolphthalein, and fluorescein, inhibit hepatic microsomal mixed-function oxidases and, partly, enhance, partly, inhibit hepatic microsomal NADPH-dependent cytochrome c and neotetrazolium reductases, in contrast to other inhibitors of drug metabolism which do not affect cytochrome c reductase but only interact with cytochrome P-450.

Effect of halogenated benzenes on the toxicity and metabolism of malathion, malaoxon, parathion, and paraoxon in mice

The halogenated benzenes, inducers of xenobiotic metabolism, were studied for their effects on the metabolism of malathion, malaoxon, and paraoxon and on the toxicity and lethality of these organophosphorus insecticides and parathion. One millimole per kilogram of 1,4-dichlorobenzene, 1,2,4-trichlorobenzene, 1,4-dibromobenzene, 1,2,4-tribromobenzene, or hexabromobenzene or 0.1 mmol/kg hexachlorobenzene was administered po to male mice daily for 7 days. In general, the trihalogenated benzenes increased the LD50 of all four insecticides two- to sixfold. These increases were larger than those observed with the di- or hexahalogenated isomers. The bromine-substituted benzenes were usually more active than the chlorinated ones with the exception being hexabromobenzene. There was a good correlation between their effects on lethality and increases in in vitro carboxylesterase activity with either malathion or malaoxon as the substrate. The trihalogenated benzenes decreased the inhibitory effect of malathion on cholinesterase activity in the brain and to a lesser degree in the red blood cells, but not in liver or plasma. There was also a good correlation between protection against parathion and paraoxon lethality, protection against inhibition of cholinesterase in the brain and liver by paraoxon, and increases in the dearylation of paraoxon by microsomal mixed-function oxidases but not by hepatic or plasma esterases. It appears that the halogenated benzenes are able to protect against organophosphorus insecticide toxicity. With malathion increases in carboxylesterase activity may be important, and with paraoxon increases in microsomal mixed-function oxidase dearylation may play a role. Other changes such as in dealkylation and tissue binding cannot be excluded from contributing to the protection seen.

Chemical and biochemical characteristics of O-demethylation of chlorotrianisene in the rat

The effects of NADPH concentration and of two inhibitors of the microsomal mixed function oxidase system [2-diethylaminoethyl-2,2-diphenyl valerate hydrochloride (SKF 525-A) and metyrapone] on rat liver microsomal O-demethylation of the triphenylethylene estrogen chlorotrianisene (CTA) were studied. Comparative data were obtained using untreated and phenobarbital-pretreated rats of both sexes. In the presence of microsomes from males, O-demethylation was induced slightly by phenobarbital (PB), and it was inhibited substantially by SKF 525-A, particularly with uninduced microsomes. Metyrapone had little inhibitory effect. In the presence of microsomes from females, O-demethylation was neither induced by PB nor inhibited significantly by SKF 525-A or metyrapone. Incubation of CTA with male rat microsomes afforded, after purification, a mixture of monophenolic metabolites which consisted primarily of a 1:1 mixture of E- and Z-desmethylchlorotrianisene (DMCTA).

Cytochrome P-450 and NADPH-Cytochrome c (P-450) Reductase in Reconstituted Phospholipid Vesicles as Demonstrated by Negative Staining Techniques

Artificial membrane vesicles or liposomes have become an accepted model for the study of enzyme-membrane interactions under controlled conditions.The development of reconstituted vesicle models for the microsomal mixed function oxidase enzymes has provided a system in which to study not only the biochemistry of these enzymes in a near physiologic milieu, but also structural aspects of the protein-phospholipid and protein-protein interactions. Ultrastructural studies on liposomes reconstituted with NADPH- cytochrome c (P-450) reductase (an amphipathic protein, 78,000 daltons, thought to be partially embedded in the microsomal membrane by its hydrophobic tail), and cytochrome P-450 (a hydrophobic protein, about 50,000 daltons, thought to be embedded within the lipid bilayer) either singly or together, should reveal information on whether these proteins are randomly arranged in the membrane or arranged in an ordered manner, an issue currently debated among cytochrome P-450 researchers.

Depression of hepatic mixed-function oxidase activity by B. pertussis in splenectomized and athymic nude mice

Administration of Bordetella pertussis (B. pertussis), Corynebacterium parvum (C. parvum), and several other immunoactive substances is known to cause a marked decrease in the activity of the hepatic microsomal mixed-function oxidase system. The effect of C parvum has been reported to involve the reticuloendothelial system. In the present study, the effect of B. pertussis administration to decrease hepatic microsomal drug metabolism was studied in unoperated, sham-operated, and splenectomized mice as well as in athymic nude (nu/nu) mice and their phenotypically heterozygous (+/nu) littermates. Administration of B. pertussis to the splenectomized, sham-operated, and unoperated mice resulted in a decrease in the activity of the microsomal enzyme system that was approximately the same for each of the three groups of animals. Administration of B. pertussis to nu/nu mice and the +/nu mice also decreased the microsomal enzyme activity measured 24 hr after injection. However, at 7 days after B. pertussis administration, the hepatic drug-metabolizing activity of the nu/nu mice was not significantly different from control values, whereas the activity of the +/nu mice was still significantly depressed. The failure of splenectomy to prevent the decrease in microsomal mixed-function oxidase activity caused by B. pertussis indicated that the effect of this agent differs from that of C. parvum, whose effect was prevented by splenectomy. Indeed, the results obtained with the athymic nude mouse suggests that the depression of hepatic mixed-function oxidase activity by B. pertussis may involve T-cell dependent responses.

Drug-induced Liver Disease

The various forms and manifestations of drug-induced liver disease depend on the operation and interaction of several potential factors. First, there is metabolism by the hepatic microsomal mixed-function oxidase (MFO) enzyme system, the most important component of which is cytochrome P-450. The microsomal membranes and the MFO enzymes are capable of induction (increase in structure and function). Second, for drug-induced liver damage to ensue, the drug must have certain structural properties so that upon metabolic conversion, activated (toxic) metabolites are produced. Third, in certain instances the normal tissue glutathione level is critical for normal excretion of a water-soluble metabolite; otherwise, with depletion of tissue macromolecules (mostly proteins) occurs, resulting in irreversible tissue necrosis. Lastly, although still a point of some controversy, in some instances genetically determined, metabolic, predisposing factors that favor the toxicity pathway may be present in a given patient, such as a patient who is a rapid acetylator. There may be other genetically determined abnormalities in drug metabolism that produce either abnormal metabolites or immunochemically reactive substances from a given agent, but these have not yet been identified.

Alterations in hepatic mixed-function oxidase activity of rainbow trout after acute treatment with polybrominated biphenyl isomers and FireMaster BP-6.

The effects of FireMaster BP-6 and several pure isomers on hepatic microsomal mixed-function oxidase (MFO) activity in rainbow trout have been investigated. After one parenteral injection of these agents at 150 mg/kg and sacrifice 5 d later, there was no elevation of hepatic cytochrome P-450. The noncoplanar isomers 2,4,5,2',4',5'-hexa and 2,3,4,5,2',4',5'-heptabromobiphenyl did not increase any MFO activity generally associated with an increase in cytochrome P-450 levels, including the dealkylations of ethoxycoumarin and benzphetamine. The coplanar isomer 3,4,5,3',4',5'-hexabromobiphenyl produced an increase in O-deethylation of both ethoxycoumarin and ethoxyresorufin, suggesting enhancement of cytochrome P-448 associated activity, but produced no increase in benzphetamine N-dealkylation. The "mixed inducer" 2,4,5,3',4',5'-hexabromobiphenyl, produced a small but insignificant elevation of cytochrome P-448 associated MFO activities and no increase in cytochrome P-450 associated activities. At both 150 and 500 mg/kg, the commercial mixture of polybrominated biphenyls FireMaster BP-6 produced marked elevations of both ethoxycoumarin and ethoxy-resorufin dealkylations but had no effect on demethylation of benzphetamine. When microsomes from rainbow trout treated with FireMaster BP-6 were examined electrophoretically, A coomassie Blue-staining band at 57,000 daltons was intensified, as seen after treatment of fish with the cytochrome P-448 inducer beta-naphthoflavone. It is concluded that coplanar polybrominated isomers produce an induction of hepatic cytochrome P-448 associated activity, while noncoplanar isomers are ineffective as inducers of cytochrome P-450 related MFO activities in rainbow trout.

Effect of rifampicin on the mouse hepatic mixed-function oxidase system

The properties of the mouse hepatic microsomal mixed-function oxidase system were compared following induction by rifampicin, β-naphthoflavone (BNP), phenobarbitone (PB) and pregnenolone 16α-carbonitrile (PCN). Close similarities were observed between rifampicin and PCN with regard to the profile of mixed-function oxidase activities induced. Testosterone 6β-hydroxylation was especially affected by both compounds which increased its activity disproportionately relative to PB and BNF. The responses of other mixed-function oxidase activities to in vitro additions of α-naphthoflavone, metyrapone, SKF 525A and sodium fluoride, and the spectral characteristics of the reduced cytochrome P-450-CO complex were also comparable after pretreatment with rifampicin and PCN, but different from those observed after PB or BNF. At the doses used, rifampicin was a less potent inducer of the mixed-function oxidase system than PCN and differences in potency could underlie the ability of PCN but not rifampicin to increase relative liver weight and microsomal protein content. However, it seems less likely that differences in potency could explain the dissimilarities in the reduced ethylisocyanide difference spectrum induced by rifampicin and PCN or the observation that, of the two microsomal polypeptide bands of 52–57000 mol.wt induced by rifampicin and PCN, only one appeared common to both inducers.

Chemical suppression of steroidogenesis.

A large number of chemicals are known to interfere with steroidogenesis in the adrenal cortex and other tissues. Many xenobiotics inhibit steroid hormone production as a result of interactions with cytochrome P-450-containing hydroxylases in adrenal mitochondria or microsomes. For example, metyrapone, a compound used clinically in the evaluation of pituitary-adrenocortical function, binds to various cytochromes P-450 in the adrenal, preventing the interactions of steroid substrates with the enzymes and inhibiting steroidogenesis. The mineralocorticoid antagonist, spironolactone, and its major circulating metabolite, canrenone, also competitively interact with adrenal steroid hydroxylases. In addition, spironolactone is converted by adrenal microsomes to an unknown metabolite which promotes the destruction of cytochromes P-450, decreasing the activities of steroid hydroxylases. Carbon tetrachloride is similarly "activated" by adrenal microsomal mixed function oxidases resulting in a decline in steroidogenic enzyme activity. Carbon tetrachloride (in the presence of NADPH) initiates lipid peroxidation in adrenal microsomes but its toxic effects on steroid hydroxylases are fully demonstrable when lipid peroxidation is inhibited by EDTA. A number of heavy metals, including cadmium, also inhibit adrenal steroid hydroxylases. When incubated with adrenal microsomes, cadmium does not affect cytochrome P-450 levels but decreases basal and substrate stimulated NADPH-cytochrome P-450 reductase activity. Although inhibitory effects of many chemicals on steroidogenesis have been described, the toxicological significance as well as definitive mechanisms of action have in most cases yet to be determined.

Chemical Suppression of Steroidogenesis

A large number of chemicals are known to interfere with steroidogenesis in the adrenal cortex and other tissues. Many xenobiotics inhibit steroid hormone production as a result of interactions with cytochrome P-450-containing hydroxylases in adrenal mitochondria or microsomes. For example, metyrapone, a compound used clinically in the evaluation of pituitary-adrenocortical function, binds to various cytochromes P-450 in the adrenal, preventing the interactions of steroid substrates with the enzymes and inhibiting steroidogenesis. The mineralocorticoid antagonist, spironolactone, and its major circulating metabolite, canrenone, also competitively interact with adrenal steroid hydroxylases. In addition, spironolactone is converted by adrenal microsomes to an unknown metabolite which promotes the destruction of cytochromes P-450, decreasing the activities of steroid hydroxylases. Carbon tetrachloride is similarly "activated" by adrenal microsomal mixed function oxidases resulting in a decline in steroidogenic enzyme activity. Carbon tetrachloride (in the presence of NADPH) initiates lipid peroxidation in adrenal microsomes but its toxic effects on steroid hydroxylases are fully demonstrable when lipid peroxidation is inhibited by EDTA. A number of heavy metals, including cadmium, also inhibit adrenal steroid hydroxylases. When incubated with adrenal microsomes, cadmium does not affect cytochrome P-450 levels but decreases basal and substrate stimulated NADPH-cytochrome P-450 reductase activity. Although inhibitory effects of many chemicals on steroidogenesis have been described, the toxicological significance as well as definitive mechanisms of action have in most cases yet to be determined.

719 AN ALTERNATIVE INTERPRETATION OF THE ENZYME DEFICIENCY IN THE GUNN RAT

Bilirubin glucuronyl transferase activity (BGT) is negligible in jaundiced (jj) Gunn rats, with intermediate and higher levels in heterozygous (Jj) and outbred (JJ) rats, respectively. This could be explained either by a defective enzyme or structural defects in the endoplasmic reticulum in which the enzyme is compartmented. If the latter interpretation is correct, similarly segregating activities might be expected among other microsomal enzymes. The activity of aminopyrine demethylase (AD), a hepatic microsomal mixed-function oxidase, was assayed in male jj, Jj, JJ Gunn and Wistar (Wi) rats. BGT (μg bilirubin conjugated/30 min/mg protein N) and AD (nmoles CH2O produced/min/mg protein) are shown below (mean±SD). Results: jj AD jj or JJ rats (p < .05). A correlation (r=.81) exists between BGT and AD (p < .01) in JJ rats, but not in Jj rats. The data suggests a microsomal membrane defect in enzyme compartmentation may be the primary abnormality in the Gunn rat and possibly in Crigler-Najjar Syndrome.