Epoxide Hydratase Activity Research Articles

Role of physiological factors in predicting the risk of oncological diseases on the basis of the polymorphism of the xenobiotic metabolism enzyme system

Summarized recent data on the polymorphism of the xenobiotic metabolism and detoxification enzyme (XMDE) system in humans are presented. The current notions on the molecular mechanisms of metabolic processes and the role of genetic polymorphism are reviewed. The roles of transport proteins and nuclear receptors in the regulation of the activity of the XMDE system are shown. The possibilities of using the polymorphism of the XMDE system as the basis for predicting the risk of oncological diseases are considered. Experimental modeling of different levels of the epoxide synthetase and epoxide hydratase activities revealed their close relationship with the toxic, mutagenic, and carcinogenic actions of polycyclic aromatic hydrocarbons. The data indicating the necessity of considering the physiological factors that could influence xenobiotic metabolism and the development of pathological changes are given.

Modulation of microsomal activity by potential chemopreventive agents of plant origin

AbstractTen compounds with potential chemopreventive activity were studied to determine their effects on microsomal mixed‐function oxidase and epoxide hydratase activity. In these studies, the highest nontoxic concentration of each compound was incubated with 1 mg rat liver microsomal protein and either [H3]benzo[a]pyrene 4,5‐oxide or [H3]benzo[a]pyrene 7,8‐dihydrodiol. High performance liquid chromatographic analysis of the metabolites was used to determine the ratio of metabolites to unmetabolized parent compound and then compared with control samples. Three of the ten compounds affected epoxide hydratase activity. Capsaicin inhibited; whereas, catechin and esculetin enhanced EH activity. Seven of the compounds tested inhibited microsomal mixed‐function oxidase activity. In order of potency, these seven compounds were tannic acid, d‐limonene, capsaicin, ellagic acid, propyl gallate, esculetin and catechin. Since the mutagenic and/or carcinogenic action of many chemicals is dependent upon the metabolic activity of microsomal mixed‐function oxidase and epoxide hydrase, the modulation of this activity by these chemicals provides further evidence in support of their potential chemopreventive properties.

Effects of monoterpenoids on in vivo DMBA-DNA adduct formation and on phase I hepatic metabolizing enzymes

We have previously demonstrated the anticarcinogenic effects of monocyclic monoterpenes such as limonene when given during the initiation phase of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary cancer in Wistar-Furth (WF) rats. Here we investigated the possible mechanisms for this chemoprevention activity including limonene's effects on DMBA-DNA adduct formation and hepatic metabolism of DMBA. Twenty-four hours after carcinogen administration, there were approximately 50% of the total DMBA-DNA adducts found in control animals formed in the liver, spleen, kidney and lung of limonene-fed animals. While circulating levels of DMBA and/or its metabolites were not different in control and limonene-fed rats, there was a 2.3-fold increase in DMBA and/or DMBA-derived metabolites in the urine of the limonene-fed animals. Studies of the effects of limonene and sobrerol, a hydroxylated monocyclic monoterpenoid with increased chemoprevention activity, on phase I metabolizing enzymes revealed that these terpenoids modulated cytochrome P450 (CYP) and epoxide hydratase (EH) activity. The 5% limonene diet increased total CYP to the same extent as phenobarbital (PB) treatment when compared to control, while 1% sobrerol (isoeffective in chemoprevention to 5% limonene) did not. However, both 5% limonene and 1% sobrerol diets greatly increased the levels of microsomal EH protein and associated hydrating activities towards benzo[a]pyrene 4,5-oxide when compared to control and PB treatment. These changes also modified the rate and regioselectivity of in vitro microsomal DMBA metabolism when compared to PB treatment or control. Identification of the specific isoforms of CYP induced by these terpenoids was performed using antibodies to CYP isozymes in Western blot analysis and inhibition studies of microsomal DMBA metabolism. Five per cent limonene was more effective than 1% sobrerol at increasing the levels of members of the CYP2B and 2C families but was equally effective at increasing EH. Furthermore, both terpenoid diets caused increased formation of the proximate carcinogen, DMBA 3,4-dihydrodiol. While these terpene-induced changes in hepatic CYP and EH do not explain the anticarcinogenic mechanism of these chemopreventive agents, or the ability of limonene systemically to reduce DMBA-DNA binding, they do reveal novel and selective induction mechanisms of hepatic enzymes.

Differential activation and inactivation of benzo[ a]pyrene in various hepatoma cell lines

Treatment of pregnant rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a dose-dependent induction of a mixed-function oxidase system in fetal and maternal extra-hepatic tissues. At doses of 6 μmg/kg, aryl hydrocarbon hydroxylase (AHH) activity was increased 24-, 22- and 4-fold in fetal lung, kidney and skin, respectively, while maternal lung, kidney and adrenal AHH activity was increased 4-, 2- and 2-fold respectively. High-pressure liquid chromatographic (H.P.L.C.) analysis of benzo(a)pyrene (BP) metabolism after TCDD induction indicated that fetal lung, kidney and skin produced significant quantities of benzo(a)pyrene-7,8-dihydrodiol (BP-7,8-diol), benzo(a)pyrene-4,5-dihydrodiol (BP-4,5-diol) and 9- and 3-phenols of BP. The fetal liver produced benzo(a)pyrene-9,10-dihydrodiol (BP-9,10-diol), BP-4,5-diol, BP-7,8-diol and 9- and 3-phenols of BP. Maternal lung also produced BP-9,10-diol, while maternal adrenal gland yielded primarily the 9-phenol of BP. Epoxide hydratase activity was increased 2- to 3-fold in maternal lung, fetal lung and skin after TCDD pretreatment, but was not affected significantly in liver, kidney or placenta. Treatment of pregnant rats with TCDD increased the covalent binding of BP to DNA in preparations containing maternal liver, lung and placenta as well as fetal liver, lung and skin. Pretreatment with TCDD resulted in increased epoxide hydratase and AHH activities in extra-hepatic tissues but only AHH was increased in hepatic tissues, indicating that the inducing capabilities of TCDD differ from, but share some similarities with, both phenobarbital (PB) and 3-methylcholanthrene (MC). Thus, TCDD appears to provide an exceptionally potent and broad-spectrum transplacental induction of carcinogen-transforming enzymes in extra-hepatic tissues.

Effects of picloram on xenobiotic biotransformation in rat liver

1. The effect of picloram on model xenobiotic substrate biotransformation in vivo was studied in female and male rat liver. 2. Treatment with picloram had little effect on epoxide hydratase and glutathione S-transferase activity, but caused a dose-dependent increase in ethoxyresorufin-O-deethylase activity and a concomitant decrease in aldrin epoxidase activity in male rats. 3. Treatment of male rats with equivalent doses of 2-acetylaminofluorene, 2-amino-anthracene and picloram induced ethoxyresorufin-O-deethylase activity to the same degree. 4. Treatment of female rats with picloram resulted in dose-dependent increases in ethoxyresorufin and ethoxycoumarin-O-deethylation without decreasing aldrin epoxidase activity. 5. Picloram binds to liver microsomal preparations from rats pretreated with phenobarbitone and/or 3-methylcholanthrene, giving a type I spectrum. 6. The results indicate that picloram is a 3-methylcholanthrene-type inducer, and the implications are discussed.

Effects of Panax ginseng extract on the benzo(a)pyrene metabolizing enzyme system.

Ethanol extract of Panax ginseng C. A. Meyer, which has been used for centuries as a tonic in Asian countries, exhibited a selective induction of epoxide hydratase and cytosolic glutathione transferase activity without the concurrent induction of aryl hydrocarbon hydroxylase activity. Thus, Panax ginseng appears to have the potential to alter the metabolic patterns of benzo(a)pyrene and its reactive metabolites.

Induction of mammary cytochromes P-450: an essential first step in the metabolism of 7,12-dimethylbenz[a]anthracene by rat mammary epithelial cells.

Rat mammary epithelial cells (RMEC) in culture have been shown to activate polycyclic aromatic hydrocarbon (PAH) carcinogens. This study investigates the role of mammary cytochrome P-450 monooxygenases in these metabolic processes. Monooxygenation of 7,12-dimethylbenz[a]anthracene (DMBA) by RMEC in culture exhibited a 6-h lag period before reaching a constant rate. The mechanism for this time-dependent expression of DMBA monooxygenase activity was investigated in lysed cells, where both conjugation and in situ induction of P-450 are prevented. Although metabolism of DMBA by untreated RMEC lysates was undetectable (less than 1 pmol/mg cell protein/h), prior exposure of cultured cells to benz[a]anthracene (BA) induced DMBA metabolism, (approximately 100 pmol/mg cell protein/h). BA pretreatment also eliminated the lag period for metabolism of DMBA by cultured RMEC but did not prevent additional induction of DMBA monoxygenase activity by the substrate. The distribution of monooxygenated DMBA metabolites formed by BA-induced cell lysates was clearly different from that obtained with purified P-450c, the predominant PAH-inducible isozyme in rat liver. For example, the carcinogen precursor DMBA 3,4-dihydrodiol, which is not formed by P-450c, was a clearly detectable product in RMEC. The low epoxide hydratase activity of BA-induced lysate (approximately 400-fold lower compared to that in the liver) limited formation of all DMBA dihydrodiols. The formation of DMBA 3,4-dihydrodiol increased by 5-fold following addition of exogenous purified epoxide hydratase. The DMBA monooxygenase activity of BA-induced RMEC lysates was completely inhibited by alpha-naphthoflavone but was only partially inhibited (50%) by a polyclonal antibody raised against cytochrome P-450c. Anti P-450c completely inhibited formation of some of the metabolites, partially inhibited formation of others and notably stimulated formation of DMBA 3,4-dihydrodiol by 60%. A polyclonal antibody that recognized both rat hepatic P-450a and a group of P-450 isozymes related to P-450h, and which totally inhibited DMBA 3,4-dihydrodiol formation by rat liver microsomes, did not inhibit formation of any DMBA metabolite in RMEC, including DMBA 3,4-dihydrodiol. Western blot analyses of RMEC homogenates demonstrated that BA pretreatment induces P-450c, but not P-450a or any of the P-450h-related isozymes. We conclude that metabolism of DMBA by RMEC depends on induction of P-450c and at least one additional form of cytochrome P-450 which is immunochemically distinct from rat hepatic P-450a and P-450h related isozymes, but is sensitive to alpha-naphthoflavone.

The effect of dietary lipids and antioxidants on the activity of epoxide hydratase in the rat liver and intestine

The effect of varying the fatty acid composition of the lipid components of the diet on the activity of epoxide hydratase in the rat liver and intestinal mucosa has been studied. Feeding a 10% cod liver oil diet (containing 18% C 20:5 and 11% C 22:6) resulted in a 3-fold increase in epoxide hydratase activity in the liver and a 1.6-fold increase in the intestine compared to rats fed a fat-free diet. The activity of epoxide hydratase in rats fed a cod liver oil diet was significantly greater than that for the group fed a lard diet (containing mainly saturated and mono-unsaturated fatty acids) containing the same quantity of vitamin E. Thus, the enhancing effect of the cod liver oil diet was due to the polyunsaturated fatty acids in this oil. Dietary corn oil (58% C 18:2) also stimulated epoxide hydratase activity in the liver but not in the intestine. Vitamin E levels of up to 500 mg/kg diet were ineffective at inducing epoxide hydratase activity in both the liver and intestine. Significant changes in the fatty acid composition of hepatic and intestinal microsomes took place when rats were fed diets of different fatty acid composition. These changes were such that the proportions of polyunsaturated fatty acids in the microsomal fractions reflected the amounts of these fatty acids in the dietary fat. Hepatic epoxide hydratase activity was found to be positively correlated to the proportion of polyunsaturated fatty acids in the microsomal fractions of the liver.

Epoxide hydratase assay in human platelets using hepoxilin A 3 as a lipid substrate

1- 14C-labelled hepoxilin A 3 (8-hydroxy-11,12-epoxyeicosa-5,9,14-trienoic acid) was generated from 1- 14C-labelled arachidonic acid during incubation with a rat lung preparation lacking epoxide hydratase activity. The HPLC purified hepoxilin A 3 gave only two isomeric 8,11,12-triols (termed trioxilins A 3) upon incubation with a rat lung preparation containing epoxide hydratase activity. Based on this simple reaction an assay was developed using only 2000 cpm/tube of substrate and aliquots of a homogenate of platelet membranes from man. Products were assayed by thin-layer radiochromatography. Males were noted to have higher epoxide hydratase activity for this substrate than females.

Effects of commonly consumed vegetables on hepatic xenobiotic-metabolizing enzymes in the mouse

To investigate the effects of consuming a variety of common vegetables on the activities of xenobiotic-metabolizing enzymes in the mouse liver, male C57BL/6 mice were fed purified diets supplemented isocalorically with 20% freeze-dried powdered kidney bean, soya bean, alfalfa, cauliflower, mustard greens, carrot, kale, Brussels sprouts, beet, egg plant or onion or with 40% sweet potato. Hepatic ethoxycoumarin O-deethylase (ECD) activity was increased significantly ( P ⩽ 0.05) over the controls in the mice fed diets containing kidney bean (1.5-fold), alfalfa (1.6-fold), cauliflower (2.2-fold), mustard greens (1.2-fold), carrots (1.2-fold) and kale (1.3-fold). No significant increases in ECD activity were seen in the other groups. Aryl hydrocarbon hydroxylase (AHH) activity was assayed in the cauliflower and Brussels sprouts groups and was found to be unaffected by these diets. Glutathione S-transferase (GST) and epoxide hydratase (EH) activities were increased ( P ⩽ 0.05) by soya bean (1.2- and 1.6-fold respectively). Brussels sprouts (2.0- and 1.6-fold), cauliflower (1.2- and 1.6-fold), alfalfa (1.3- and 2.0-fold) and onion (1.8- and 2.3-fold). No significant increases in GST or EH activities were seen in the other groups. Of the twelve vegetables investigated, only three (sweet potato, beet and egg plant) had no statistically significant effects on any of the xenobiotic-metabolizing parameters tested. However, the beet diet caused an apparent inhibition of ECD activity (74% of control) and the sweet potato diet caused an apparent increase (1.3-fold) in GST activity, although statistical significance could not be established at P ⩽ 0.05.

Effects of dietary R-goitrin on hepatic and intestinal glutathione S-transferase, microsomal epoxide hydratase and ethoxycoumarin O-deethylase activities in the rat

The influence of dietary R-goitrin on components of the xenobiotic-metabolizing system was examined in the liver and small intestine of male Sprague-Dawley rats. Given at a level of 200 ppm in the diet for 14 days, the R-goitrin caused a statistically significant ( P < 0.05) 21% increase in liver weight relative to body weight. A less pronounced, but statistically significant, 11% increase in relative liver weight resulted from the administration of R-goitrin at 40 ppm in the diet. Hepatic glutathione S-transferase (GST) activity was significantly increased 1.5- and 2-fold over the basal level at concentrations of 40 and 200 ppm R-goitrin, respectively. Hepatic microsomal epoxide hydratase (EH) activity was also significantly increased. Hepatic EH activity was 1.6- and 3.3-fold greater in the 40- and 200-ppm R-goitrin groups, respectively, than in the control group given the basal diet. R-Goitrin at 200 ppm in the diet produced significant 1.2- and 1.4-fold increases of GST and microsomal EH activities, respectively, in the mucosa of the small intestine. The administration of R-goitrin at 40 or 200 ppm in the diet had no significant effect on either hepatic or intestinal ethoxycoumarin O-deethylase activity.

Epoxide hydratase activity in mouse-liver peroxisomes

Epoxide hydratase activity in mouse-liver peroxisomes

A comparison of the effect of selenium on the mutagenicity and metabolism of benzo[ a]pyrene in rat and hamster liver S9 activation systems

When selenium (Na 2SeO 3) was included in the incubation mix containing rat or hamster liver S9 preparations both the metabolism and mutagenicity of benzo[ a]pyrene (BaP) and several of its metabolites were altered. At non-toxic concentrations selenium inhibited the S9 dependent mutagenicity of BaP and number of its metabolites on Salmonella typhimurium strain TA100 as indicated by the number of histidine independent revertants observed. High performance liquid chromatographic analysis of S9 generated metabolites of BaP from rat and hamster liver indicated that selenium caused quantitative differences in the amounts of the metabolic products. In hamster liver S9 differences were reflected in decreased amounts of strongly mutagenic BaP-7,8-dihydrodiol and increased amounts of 4,5- and 9,10-dihydrodiols that were weakly mutagenic to TA100 in that system. In rat liver S9 selenium caused quantitatively similar decreases in BaP-7,8- and 9,10-dihydrodiol and 3-hydroxy-BaP. When used as substrate 3-hydroxy-BaP was the most mutagenic to TA100 in the rat activation system whereas BaP-7,8-dihydrodiol was most mutagenic in the hamster S9 system. Assays that measured the formation of water-soluble conjugates of BaP indicated that selenium did not significantly alter the formation of sulfate ester or glutathione conjugates although a 12–17% reduction of labeled metabolites bound to the glucuronide fraction was observed. Results described in this report suggest that selenium modified the metabolism and hence the mutagenicity of BaP to TA100 by affecting mixed-function oxidase and/or epoxide hydratase activity in both the rat and hamster liver S9 activation systems.

Arachidonic acid epoxides. Demonstration through [18O]oxygen studies of an intramolecular transfer of the terminal hydroxyl group of (12S)-hydroperoxyeicosa-5,8,10,14-tetraenoic acid to form hydroxyepoxides.

Purified 12-hydroperoxyeicosa - 5, 8, 10, 14 - tetrae noic acid (12-HPETE) containing deuterium atoms at 5, 6, 8, 9, 11, 12, 14, and 15 was prepared by incubating octadeuterated arachidonic acid with a platelet preparation in air or [18O]oxygen gas. A mixture of 12-HPETE containing 16O16O:18O18O (5:4) was subsequently prepared and incubated with hematin in phosphate buffer (pH 7.4); in another experiment the same mixture of 12-HPETE was incubated with a rat lung preparation (0-30% ammonium sulfate) which lacked epoxide hydratase activity. Gas chromatography-mass spectrometry negative ion chemical ionization analysis of the extracted products after conversion into pentafluorobenzyl-trimethylsilyl derivatives indicated that the products from both incubations, i.e. 12-hydroxyeicosa - 5,8,10,14 - tetraenoic acid (12 - HETE), 8 - hydroxy - 11,12-epoxyeicosa - 5,9,14 - trienoic acid (8H-11,12-EPETE), 10H - 11,12-epoxyeicosa - 5,8,14 - trienoic acid (10H-11,12-EPETE), and 8,11,12-trihydroxyeicosa-5,9,14-trienoic acid, all retained either two [16O]oxygen or two [18O]oxygen atoms of starting 12-HPETE and that no mixed species existed which contained one [16O]oxygen and one [18O]oxygen atom. These results demonstrate for the first time in the arachidonic acid series an intramolecular transfer of the terminal hydroxyl group of the hydroperoxide of 12-HPETE to the C-8 or C-10 alkyl positions to form the hydroxyepoxides 8H-11,12-EPETE and 10H-11,12-EPETE. This reaction carried out by both hematin in the absence of protein and the rat lung preparation is suggestive of a metal-hydroperoxide-olefin "cage" complex facilitating a concerted reaction in which the terminal hydroxyl group of the hydroperoxide is trapped by alkyl free radical centers.

Hemoglobin- and hemin-catalyzed transformation of 12 l-hydroperoxy-5,8,10,14-eicosatetraenoic acid

12 L-Hydroperoxy-5,8,10,14-eicosatetraenoic acid (12 L-HPETE) containing 2H at positions 5,6,8,9,11,12,14,15 was prepared during brief incubations of rat platelets with a mixture of [1- 14C]arachidonic acid and 5,6,8,9,11,12,14,15-[ 2H 8]arachidonic acid. The labelled 12L-HPETE was converted into a mixture of products, including 8- and 10-hydroxy-11,12-epoxyeicosatrienoic acids, during incubations in phosphate buffer containing bovine hemin. Hemoglobin could replace hemin in this reaction, which did not proceed in buffer alone. Mass spectral analysis showed these hydroxy epoxides to be identical to those previously identified from rat lung. These epoxides were hydrolyzed by a rat lung enzyme previously shown to contain epoxide hydratase activity. These results show that the hydroxy epoxides could be formed from 12 L-HPETE nonenzymatically.

Variations in induction of drug-metabolizing enzymes by trans-Stilbene oxide in rodent species

trans-Stilbene oxide (400 mg/kg) produced a 500% increase in the microsomal in the microsomal epoxide hydratase activity in rat and mouse with little change in the soluble enzyme activity. However, in guinea pig, the soluble epoxide hydratase activity increased by about 33% with only a small increase (47.6%) in the microsomal enzyme activity. The soluble glutathione S-transferase activities were also induced in both rat and mouse, with little change in that of the guinea pig. Increasing dosage of trans-stilbene oxide from 400 mg/kg to 1000 mg/kg had little effect on the above enzyme activities. That the guinea pig was not relatively refractory to all inducing agents was shown by the fact phenobarbital (100 mg/kg) and 3-methylcholanthrene (25 mg/kg) produced relatively similar increases in the activities of aniline hydroxylase and P-aminopyrine P-demethylase in rat, mouse and guinea pig. However, these inducers produced only a 15–20% stimulation in the soluble glutathione S-transferase and microsomal epoxide hydratase activities in guinea pig, when compared to a 50–80% increase in rat and mouse, suggesting a general resistance to induction by the phase II enzymes in guinea liver. In all animal models, the inducer markedly increased th emicrosomal total phospholipid content, although the sphingomyelin content itself was decreased. In both rat and mouse, the microsomal cholesterol content was significantly decreased while that in guinea pig was unaffected. Possible factors responsible for the observed species differences are discussed.

Inhibition by 17-α-ethinyl estradiol of benzo[ a]pyrene metabolism in isolated adult rat hepatocytes

The effects of 17-α-ethinyl estradiol [EE] were studied on benzo[ a]pyrene (BP) metabolism and covalent binding to DNA and on some enzymes involved in the activation/inactivation pathway. Suspensions of hepatocytes freshly isolated from adult rats either untreated or pretreated with phenobarbital or methylcholanthrene were used as an experimental model closer to the in vivo situation than subcellular fractions. The formation of water-soluble and of organic metabolites of benzo[ a]pyrene was inhibited by EE at 10 −4 M in hepatocytes from untreated rats and at 10 −3 M in those isolated from rats pretreated with phenobarbital. Dihydrodiols were the main class of metabolites affected by EE. The pretreatment of rats with 3-methylcholanthrene rendered hepatocytes insensitive to the effect of EE. The effect of EE on BP metabolism was well correlated to its inhibitory effect on BP metabolite binding to DNA in untreated and phenobarbital-treated rats. These data could not however be related to a quantitative decrease in cytochrome P-450 of hepatocytes incubated in the presence of EE. Epoxide hydratase, UDP-glucuronyl-transferase and glutathione S-transferase activities were lowerea by 36, 27 and 19% respectively in the presence of 10 −4 M EE. The impairment of BP metabolism by EE might be due to a functional alteration specific for cytochrome P-450, or to a non-destructive mechanism and/or to a competition of both chemical. for a number of enzymatic pathways common to their metabolism.

Species variations in the sensitivity of the endoplasmic reticulum to the herbicide i.i.i. trifluoro-n-(2-methyl-4-phenyl sulfonyl phenyl) methane sulfonamide

1. The effects of the herbicide I.I.I. trifluoro- N-(2-methyl-4-phenyl sulfonyl phenyl) methane sulfonamide on the drug metabolising enzyme activities in livers of rat, mouse and guinea pig have been compared. 2. In all three animal models, the herbicide increased the activities of both aniline hydroxylase and p-aminopyrine demethylase. The greatest inductive effect was seen in the rat, while the least effect was evident in the guinea pig. 3. In mouse and guinea pig, 1.1.1. trifluoro- N-(2-methyl-4-phenyl sulfonyl phenyl) methane sulfonamide had no effect on the soluble or microsomal epoxide hydratases or the glutathione S-transferases. In the rat. however, the herbicide significantly decreased the microsomal epoxide hydratase activity, as well as the soluble and microsomal glutathione S-transferase activities. 4. These results are discussed in relation to factors responsible for species differences in the response to foreign compounds.

Alterations in the enzyme activity and polypeptide composition of rat hepatic endoplasmic reticulum during acute exposure to 2-acetylaminofluorene

Studies have been made of the morphology, enzyme activity and protein composition of liver endoplasmic reticulum in rats exposed to acute doses of the carcinogen, 2-acetylaminofluorene (2-AAF). Electron microscopic examination revealed numerous ultrastructural changes in the hepatocyte; most consistent alterations were the disorganisation of endoplasmic reticulum system with apparent increase of smooth endoplasmic reticulum. Administration of 2-AAF to rats immediately depressed microsomal glucose-6-phosphatase activity and eventually induced epoxide hydratase activity 6–7-fold over control activity. The induction was time-dependent and maximal rates of induction were observed at dosages greater than 40 mg/kg body wt. The treatment also induced cytochrome b 5 content, NADH and NADPH cytochrome c reductase activities (1.0–1.5-fold). Only very small changes in the total content of cytochrome P-450 were noted. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of microsomal proteins from 2-AAF pretreated animals showed time-dependent induction of two polypeptides which differed slightly in migration, in the region of M r = 48 000; the faster-migrating induced polypeptide has been identified as epoxide hydratase. Two-dimensional PAGE analysis of microsomal proteins from 2-AAF exposed rats showed a reproducible deletion of a protein with molecular weight in the region of 67 000. The basis for the alterations in the protein composition of endoplasmic reticulum in response to 2-AAF treatment is discussed.

Metabolism of benzo[ a]pyrene by guinea pig adrenal and hepatic microsomes

Studies were carried out to compare the metabolism of benzo[ a]pyrene (BP) by adrenal and hepatic microsomes obtained from adult male guinea pigs. Adrenal microsomes produced fluorescent metabolites (primarily phenols) approximately three to four times more rapidly than hepatic microsomes, but the differences in the rates were considerably smaller when total BP metabolism was assessed using an isotopic assay. The apparent discrepancy between the two assays is attributable to differences in the profiles of BP metabolites produced by adrenal and liver. Separation of metabolites by high pressure liquid chromatography revealed that adrenal microsomes converted BP to primarily a phenolic metabolite with a retention time identical to that of 3-hydroxy-BP. Liver microsomes, by contrast, produced approximately equal amounts of compounds co-chromatographing with 3-hydroxy-BP and BP-4,5-dihydrodiol. Small amounts of other metabolites were also produced by adrenal and hepatic microsomes. Liver microsomes catalyzed the conversion of BP to metabolites that became covalently bound to exogenous DNA. The amount of binding was dependent upon the duration of incubation and concentration of microsomal protein. Adrenal microsomes, by contrast, did not promote BP binding to DNA. Inhibition of microsomal epoxide hydratase activity with trichloropropene oxide (TCPO) blocked the formation of dihydrodiol metabolites of BP by adrenal and liver microsomes. In the presence of TCPO, liver microsomes produced large amounts of a BP metabolite co-chromatographing with BP-4,5-oxide. TCPO also increased the rate of production of DNA-binding roetabolites by liver microsomes but had no effect on the formation of DNA-binding metabolites by adrenal microsomes. The results demonstrate major differences in the pathways of BP metabolism by guinea pig adrenal and hepatic microsomes. Although adrenal microsomes metabolize BP more rapidly than hepatic microsomes, far greater amounts of reactive metabolites are produced by the liver. Thus, adrenal metabolism of BP may be of little toxicological significance.