Glutathione S-epoxide Transferase Research Articles

Molecular mechanisms associated with oxidative damage in the mouse testis induced by LaCl3.

China is the world's largest rare earth producer and exporter, previous studies have shown that rare earth elements can cause oxidative damage in animal testis. However, the molecular mechanisms underlying these observations have yet to be elucidated. In this paper, male mice were fed with different doses (10, 20, and 40 mg/kg BW) of LaCl3 for 90 consecutive days, regulatory role of nuclear factor erythroid-2 related factor 2 (Nrf-2)/antioxidant response element (ARE) pathway in testicular oxidative stress induced by LaCl3 were investigated. Analysis showed that LaCl3 exposure could lead to severe testicular pathological changes and apoptosis in spermatogenic cells, it up-regulated the peroxidation of lipids, proteins and DNA, and induced the excessive levels of reactive oxygen species (ROS) production in mouse testis, reduced the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathione S epoxide transferase (GST) as well as the glutathione (GSH) content. Furthermore, exposure to LaCl3 also downregulated the expression of Nrf2 and its target gene products, including heme oxygenase 1 (HO-1), glutamate-cysteine ligase catalytic subunit (GCLC), NAD(P)H dehydrogenase [quinine] 1(NQO1), protein kinase C (PKC), and phosphatidylinositol 3-kinase (PI3K), but upregulated the expression of Kelch-like ECH-related protein 1 (Keap1) in damaged mouse testes. Collectively, our data imply that the oxidative damage induced by LaCl3 in testis was related to inhibition of the Nrf-2/AREs pathway activation.

Treatment with geraniol ameliorates methionine-choline-deficient diet-induced non-alcoholic steatohepatitis in rats.

Non-alcoholic steatohepatitis (NASH) is one of the most common causes of chronic liver disease and is considered to be a causative factor of cryptogenic cirrhosis and hepatocellular carcinoma. The aim of this work was to investigate whether treatment with geraniol (a monoterpene) attenuated NASH induced by methionine-choline-deficient (MCD) diet in rats. Rats were fed with MCD diet to induce NASH and treated with geraniol (200 mg/kg/day) for 10 weeks. Treatment with geraniol reduced histological scores, fibrosis, and apoptosis in livers, lowered activities of alanine aminotransferase and aspartate aminotransferase in serum, and attenuated hepatic fat accumulation in rats fed with MCD diet. Treatment with geraniol preserved hepatic mitochondrial function, evidenced by reduced mitochondrial reactive oxygen species formation, enhanced adenosine triphosphate formation and membrane integrity, restored mitochondrial electron transport chain enzyme activity, and increased mitochondrial DNA content in rats fed with MCD diet. Treatment with geraniol reduced uncoupling protein 2 protein expression, and enhanced protein expression of prohibitin, mRNA expression of peroxisome proliferator-activated receptor α, and activity of mitochondrial carnitine palmitoyl transferase-I in livers of rats fed with MCD diet. Treatment with geraniol abated oxidative stress, evidenced by reduced malondialdehyde and 3-nitrotyrosine formation, enhanced activity of glutathione S-epoxide transferase, and down-regulated expression of inducible nitric oxide synthase and cytochrome P450 2E1 in livers of rats fed with MCD diet. Treatment with geraniol reduced myeloperoxidase activity and protein expression of tumor necrosis factor alpha and IL-6 in livers of rats fed with MCD diet. Treatment with geraniol attenuated MCD-induced NASH in rats.

Oxidative Stress Is Involved in the Renal Dysfunction Induced by Sinoaortic Denervation in Rats.

The hypothesis that oxidative stress contributes to renal dysfunction in sinoaortically denervated (SAD) rats was investigated. Rats were sinoaortically denervated and received treatment with tempol (0.5 mmol/L in drinking water) for 8 weeks. Although the tempol treatment of the SAD rats had no significant effect on blood pressure or blood pressure viability, it significantly ameliorated the renal dysfunction as indicated by increases in renal blood flow (RBF) and the glomerular filtration rate (GFR) and reductions in plasma creatinine, blood urea nitrogen (BUN), the urine albumin excretion rate (UAE), and the glomerular sclerosis score (GSS). The SAD rats treated with tempol exhibited decreased plasma and renal malondialdehyde (MDA) levels and reduced renal formation of reactive oxygen species (ROS), superoxide (O2-), peroxynitrite (OONO-) and 3-nitrotyrosine. Treatment with tempol suppressed the nuclear concentration of nuclear factor-kappaB (NF-κB) and reduced the renal levels of macrophage chemoattractant protein 1 (MCP-1) and interleukin-6 (IL-6). The tempol-treated SAD rats exhibited decreased renal advanced glycation end product (AGE) levels and decreased receptor for advanced glycation end products (RAGE) protein expression. The tempol treatment of the SAD rats restored mitochondrial adenosine triphosphate (ATP) formation, DNA content, membrane integrity and the renal oxygen consumption rate. Additionally, the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S epoxide transferase (GST), and catalase were decreased, and the activities of xanthin oxidase (XO) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were enhanced in the kidneys of the SAD rats. In conclusion, our work firstly provided direct evidence that oxidative stress played an important role in the renal dysfunction of SAD rats.

Strategies for advancement of short-term mutagenicity tests: on the optimal ionic strength for the liver microsomal assay.

The aim of this work was to optimize the ionic strength (tau) in the liver microsomal assay (LMA) in performing short-term genotoxicity tests. tau optimization would increase the sensitivity (i.e. decrease false negatives) and at the same time increase the specificity (decrease false positives). Such optimization depends upon the relative activities and stabilities of the liver polysubstrate cytochrome P450- and FAD-containing monooxygenase-dependent metabolizing enzymes present in the incubation mixtures. With regard to phase-I pathway, the expression of various P450-like activities (IA1, IA2, IIB1, IIE1, IIIA P450 classes) and thiobenzamide s-oxidase (as FAD-MFO marker), were examined in terms of their exact incubation conditions for the LMA during a period of preincubation (1 h) over the tau range 0.06-1.40. As a comparison with the phase-II pathway, the behaviour of glutathione S-transferases (total and pi class), glutathione S-epoxide transferase, epoxide hydrolase and UDP-glucuronosyl transferase were studied. Lipid peroxidation (LP) was also determined. Experiments were performed on S9 fractions derived from sodium phenobarbital, beta-naphthoflavone, isosafrol, ethanol and pregnenolone 16-alpha carbonitrile super-induced mouse liver. The maximal value of the mean specific activity (Asp), up to a 46% increase, was found at tau = 0.864 for oxidative reactions considered. On the contrary, a slight modulation of Asp for post-oxidative reactions was seen. LP was not changed appreciably by varying tau. In vitro DNA binding of the well-known premutagenic agent [14C]dimethylnitrosamine ([14C]DMNA), mediated by mouse hepatic microsomal enzymes, showed a significant increase of specific activity at tau = 0.864 (2.25-fold) compared to the usual tau (0.06) used. Additional confirmation of these results stems from mutagenesis experiments using DMNA on the diploid D7 strain of Saccharomyces cerevisiae as a biological test system. Indeed, a significant enhancement of mitotic gene conversion (up to 1.8-fold), mitotic crossing-over (2.6-fold) and reverse point mutation (2.6-fold) frequencies was achieved at tau = 0.86 compared to tau = 0.06 (traditional). These data show that tau = 0.86 can provide more convenient conditions for in vitro bioactivation (as exemplified by an increased Asp phase-I/Asp phase-II ratio), as well as DNA binding and genotoxic response.

Wide spectrum detection of precarcinogens in short-term bioassays by simultaneous superinduction of multiple forms of cytochrome P450 isoenzymes

The use of Aroclor 1254 to induce S9 liver fractions is a standard method for conducting short-term genotoxicity assays. An alternative induction procedure, using beta-naphthoflavone (beta-NF), as a safe (non-carcinogenic) substitute for polychlorinated biphenyls, combined with sodium phenobarbital (PB), was found to be equally effective. The aim of this work is to realize a novel schedule of induction for the preparation of metabolizing systems containing a wider spectrum of induced cytochrome P450s. Five inducers of different 'classes' such as PB (class IIB P450s), beta-NF (IA), isosafrol (IA2), ethanol (IIE1) and pregnenolone 16 alpha-carbonitrile (IIIA) were injected daily both separately (to achieve maximal monooxygenase induction) in male and female mice. Induction was monitored using specific P450-linked activities. In the optimal schedule for complete induction, the various monooxygenases were greater (2- to 4-fold) than those achieved by the classical schedule. More than a 14-fold increase of total P450 and 3.3-fold increase of NADPH-cytochrome (P450) c-reductase activity, over those uninduced, account for the above increase. For example, there was a marked increase in the deethylation of ethoxyresorufin (37-fold) compared to the uninduced mice that was considerably higher than classical induction (8-fold over uninduced). On the contrary, phase II reactions i.e. epoxide hydrolase, glutathione S-transferase, glutathione S-epoxide transferase and UDP-glucuronosyl transferase, examined to compare the phase I/phase II ratios in the traditional and proposed procedures, were increased to a lesser extent (2-fold over uninduced). No significant sex differences were seen. Five precarcinogens specifically metabolized by each of the induced P450s elicited a higher mutagenicity response in the presence of superinduced fractions with respect to the classical one, when tested on Salmonella typhimurium (cyclophosphamide, benzo[alpha]pyrene, 2-naphthylamine and dimethylnitrosamine) or Saccharomyces cerevisiae D7 strain (diethylstilbestrol). These novel metabolizing biosystems, with an enhanced spectrum of induced P450s and oxidative/post-oxidative reaction rates, are recommended for detecting unknown xenobiotics in genotoxicity studies.

Preliminary data on PAH metabolism in the marine mussel Mytilus galloprovincialis from Arcachon Bay, France

Oxidation of polycyclic aromatic hydrocarbons (PAH) and epoxide-conjugation capacities were examined from mussels, Mytilus galloprovincialis, collected from a “clean” site at the entrance to Arcachon Bay (south-west France) in 1984, using subcellular preparations from whole bodies and digestive glands. The existence of low-cytochrome P-450-dependent oxygenase activities [cytochrome P-450 content, NADPH cytochrome c reductase and benzopyrene monooxygenase (BaPMO) activities] was confirmed, as well as the presence of a comparatively important capacity for epoxide conjugation [epoxide hydrolase (EH), glutathione S epoxide-transferase (GST) activities and total cytosolic-glutathione content]. The optimal incubation temperatures for the individual in vitro enzyme activities in the mussel (25°C for BaPMO, 31°C for EH and GST) were lower than that required for optimal mammalian enzyme activities. Except for cytochrome P-450 content, there was no significant difference in enzyme activities recorded in whole-body and digestive gland fractions.

Stability of drug metabolizing enzymes during the incubation conditions of the liver microsomal assay with non-induced and induced mouse liver S-9 fractions.

The purpose of this work was to study the relative activities and stabilities of phase-I and phase-II drug metabolizing enzymes in incubation mixtures used in vitro genotoxicity testing in order to optimize the conditions of the assay, increase sensitivity and eliminate false negative results. Cytochrome P-450, NADPH-cytochrome P-450 (cytochrome c) reductase activity and various phase-I and phase-II enzyme activities of the drug-metabolizing system were determined in incubation mixtures used in liver microsomal assays. The behaviour of aminopyrine N-demethylase and p-nitroanisole O-demethylase activities as phase-I markers have been reported previously. Other activities measured were glutathione S-transferase, glutathione S-epoxide transferase and epoxide hydrase, and lipid peroxidation (LP) was determined. The experiments were carried out on liver S9 fractions derived from non-induced mice or mice induced with sodium phenobarbital (PB), and/or beta-naphthoflavone (beta-NF). The phase-II enzymes were much more stable (70-90% residual activity) than phase-I enzyme activities (35-60%) in all conditions tested. The residual cytochrome P-450 was approximately 70% stable and the remaining activity of NADPH-cytochrome c-reductase about 80%, indicating that this latter enzyme does not limit the rate of the monoxygenase system in these conditions. Phase-II enzymes were induced to a smaller extent (about 2 times) than in phase-I enzymes (5-6 times) by beta-NF + PB. NADPH-cytochrome c-reductase behaved as phase-II enzymes in this respect as well as for stability. LP was appreciably higher in non-induced than in induced animals. Treatment with the beta-NF + PB mixture, however, showed that induced enzymes were more stable than those obtained by simple induction with either beta-NF or PB alone. These results lead to the conclusion that prolonged incubation times in mutagenicity assays are unnecessary when considering the relative stabilities of the various phase-I and phase-II enzyme activities in the drug-metabolizing system.

Inhibition of soluble glutathione S-transferase by diuretic drugs

Glutathione transferase are believed to play an important protective role in the various tissues of animals and man by catalysing the glutathione conjugation of electrophilic drugs and electrophilic drug metabolites. Many of these compounds have the potential to react with vital cellular macromolecules in the absence of this enzyme system. We have investigated the interaction of a number of high ceiling diuretics with the glutathione transferase contained in the cytosolic fraction of the rat liver. Of bumetanide, ethacrynic acid, furosemide, indacrynic acid and tienilic acid, only ethacrynic acid was conjugated with glutathione. Further experiments revealed that ethacrynic, indacrynic and tienilic acids are all potent inhibitors of glutathione S-aryltransferase. Glutathione S-alkyltransferase and glutathione S-epoxide transferase were also inhibited by the diuretics, but to a lesser extent than glutathione S-aryltransferase. The diuretics giving the greatest inhibition of these reactions were chemically related to ethacrynic acid. The concept where inhibition of glutathione- S-transferase by a drug may enhance its own toxicity is considered. This mechanism has also the potential of enhancing the toxicity of other concurrently administered drugs which normally require glutathione S-transferase for detoxication.

Evolution of activation-detoxification enzyme patterns during precarcinogenesis in synergism: 3-methylcholanthrene and dimethylnitrosamine.

The effects of 3-methylcholanthrene (3MC) and dimethylnitrosamine (DMN), administered singly or in combination at chronic carcinogenic doses, on the evolution of activity patterns of enzymes involved in the metabolism of DMN and polycyclic hydrocarbons were studied in the liver and lung of Sprague-Dawley rats and Swiss mice. In the pulmonary tissues of rats, during the early stage of administration (7 weeks), there was substantial increase of epoxide hydrase and glutathione S-epoxide transferase activity; from then on these detoxifying enzyme activities rapidly decreased toward 25 weeks, and this decreasing trend coincided with a substantial surge of aryl hydrocarbon hydroxylase (AHH) activity at 15 weeks; 65%, 95%, and 50% increase with 3MC, DMN, and DMN+3MC, respectively. The activity of DMN-demethylase was slightly increased following treatment with DMN or DMN+3MC for 15 weeks. On the other hand, decrease of DMN-demethylase activity in the liver of rats throughout most of the experimental period was apparent and, after an initial small increase at 7 weeks, there was a trend of decreasing AHH activity from 7 toward 25 weeks with DMN+3MC; the 7-week increase in AHH was moreover compensated by the large increases in the activity of the three detoxifying enzymes, epoxide hydrase, glutathione S-epoxide transferase, and UDP-glucuronyl transferase at the same period. The data suggest that the previously observed pulmonary syncarcinogenesis between 3MC and DMN in rats (Hoch-Ligeti et al. 1968) is probably due to increase of reactive metabolites of both 3MC and DMN. In the mouse liver, a combination of initial increase of epoxide hydrase and UDP-glucuronyl transferase activity at 5 weeks, together with decrease of AHH activity at 10 weeks, suggests a decrease of activated hydrocarbon metabolites available for binding to macromolecules. While AHH activity showed a strongly decreasing trend in the mouse lung, increases of 21%, 30%, and 113% in DMN-demethylase activity were observed after the mice were given 3MC, DMN, or DMN+3MC, respectively, for 10 weeks. The pulmonary syncarcinogenic effect of 3MC and DMN in mice (Cardesa et al. 1973; Argus et al. 1982) appears to be due primarily to increase of reactive metabolite(s) of DMN. The data are consistent with previous observations (Arcos et al. 1978) that activated metabolites originating from liver are unlikely to be contributors in DMN+3MC pulmonary syncarcinogenesis.

Tissue differences in the ontogeny of inducibility of drug-metabolizing enzymes by 3-methylcholanthrene in the rabbit.

1. The effects of treatment of rabbits of different ages with 3-methylcholanthrene on cytochrome P-450 content, and benzo[a]pyrene hydroxylase, epoxide hydrolase, glutathione S-epoxide transferase, 1-naphthol glucuronyl transferase and morphine glucuronyl transferase activities of liver, kidney and lung have been investigated. 2. The cytochrome P-450 content of liver and kidney was inducible at all ages, whereas that of lung was inducible up to only seven days old. Benzo[a]pyrene hydroxylase activity was inducible more than two-fold in kidney at all ages, but in liver and lung up to only seven days old. 3. Epoxide hydrolase activity was inducible, only in liver and lung of younger animals, to an extent of less than 50%. 4. Glutathione S-epoxide transferase activity was inducible, in the liver of young animals and in the kidney of adult animals, to extents of less than 50%. 5. Both 1-naphthol and morphine glucuronly transferase activities were significantly induced in the liver of animals up to three days old, the former being induced to the greater extent. These enzymes were not studied in kidney or lung. 6. It is concluded that 3-methylcholanthrene treatment of rabbits has tissue-specific and age-specific effects on various drug-metabolizing enzyme.

Effect of selected induction of microsomal and nuclear aryl hydrocarbon monooxygenase and epoxide hydrolase as well as cytoplasmic glutathione S-epoxide transferase on the covalent binding of the carcinogen benzo(a)pyrene to rat liver DNA in vivo.

Groups of four adult male rats [ZUR:SIV-Z] were pretreated with corn oil (control; 2ml/kg/day i.p. for 3 days), trans-stilbene-oxide (SO; 200 mg/kg/day i.p. for 2 days), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10 μg/kg i.p. once, 4 days before killing), phenobarbital (PB; 1 g/liter in the drinking water for 8 days), and dieldrin (20 mg/kg/day i.p. for 3 or 9 days). They received an injection of [G-3H]benzo(a)pyrene (BaP, 31 μg/kg, 7.4·109 dpm/kg;i.v.) 16 h before killing. In the liver of each rat, five enzymatic activities and the covalent binding of BaP to DNA have been determined. The microsomal aryl hydrocarbon monooxygenase activity (AHM) ranged from 75% of control (SO) to 356% (TCDD), the nuclear AHM from 63% (SO) to 333% (TCDD). Microsomal epoxide hydrolase activity (EH) was induced up to 238% (PB), nuclear EH ranged from 86% (TCDD) to 218% (PB). A different extent of induction was observed in the two compartments. Highest induction of glutathione S-epoxide transferase activity (GST) was found with PB (202%). The DNA binding of BaP was modulated within 79% (dieldrin, 9 days) and 238% of control (TCDD). An enzyme digest of control DNA was analysed by Sephadex LH-20 chromatography. Multiple linear regression analysis with all data expressed as % of control yielded the following equation: DNA Binding= 1.49· Microsomal AHM-1.07 · Nuclear AHM+0.33 · Microsomal EH-0.52 · Nuclear EH+0.11 · Cytoplasmic GST+58.2. From this analysis it is concluded that (1) AHM located in the endoplasmic reticulum is most important in the formation of DNA-binding metabolites, (2) EH in the same compartment is not determinative in this respect nor has it a protective effect, (3) both membrane-bound enzyme activities located in the nucleus may inactivate potential ultimate carcinogens, and (4) cytoplasmic GST probably cannot reduce DNA binding due to its subcellular localization.

The biological fate of vinylidene chloride in rats

The main eliminative route for [ 14C]vinylidene chloride ([ 14C]DCE) after intragastric, i.v. or i.p. administration to rats is pulmonary; both unchanged DCE and DCE-related CO 2 are excreted by that route and other DCE metabolites via the kidneys. Part of the urinary 14C is of biliary origin. After intragastric dosing, the plot of the pulmonary output of unchanged DCE against the logarithm of reciprocal doses in biphasic. Pulmonary elimination of DCE and CO 2 and urinary excretion of DCE metabolites after an intragastric dose occupy 3 days. In comparison, 80% of a small i.v. dose is excreted unchanged within 1 h of injection; more than 60% within 5 min. Biotransformation of DCE affords thiodiglycollic acid, and an N-acetyl- S-cysteinyl-acetyl derivative as major urinary metabolites together with substantial amounts of chloroacetic acid, dithioglycollic acid and thioglycollic acid. It is probable that chloroacetic acid, which is a DCE metabolite per se, lies on a main metabolic pathway for DCE, since it affords several metabolites in common with DCE. Furthermore, electrolysis of one molecular proportion of the [ 14C]thiodiglycollate metabolite from [1- 14C]DCE or [1- 14C]chloroacetic acid gives 1 equivalent of 14CO 2, and this evidence is consistent with the transformation of DCE into chloroacetic acid by a mechanism involving the migration of one Cl atom and the loss of the other one. CO 2 (and hence urea) may be produced through the action of epoxide hydratase on 1,1-dichloroethylene oxide or by a minor oxidative pathway for chloroacetic acid. The N-acetyl- S-cysteinyl-acetyl derivative is probably formed via the reaction of 1,1-dichloroethylene oxide and glutathione S-epoxide transferase.

The chemistry and biogenesis of the S-containing metabolites of vinyl chloride in rats

In order to determine whether vinyl chloride yields chloroethylene oxide in vivo, the biogenesis of the various urinary S-containing metabolites in rats has been investigated. N-Acetyl- S-(2-hydroxyethyl)cysteine is a major vinyl chloride metabolite in rats, but according to the method of protective esterification that is used, so either N-acetyl- S-(2-chloroethyl)cysteine or N-acetyl- S-(2-hydroxyethyl)cysteine may be isolated from the body fluids. N-Acetyl- S-vinylcysteine is a second related metabolite. These S-containing vinyl chloride metabolites are not mutagenic in S. typhimurium. Neutral methanol methylates N-acetyl- S-(2-hydroxyethyl)cysteine. N-Acetyl- S-(2-methoxyethyl)cysteine plus N-acetyl- S-vinylcysteine degrade to give the volatile S-(2-methoxyethyl)(prop-1 or 2-enyl)sulphide. Administration of several vinyl chloride metabolites and closely related compounds to rats shows that chloroacetaldehyde and S-(carboxymethyl)cysteine, but not chloroacetic acid, lie on a pathway or pathways connecting vinyl chloride with thiodiglycollic acid. The fact (a) that chloroacetaldehyde affords both thiodiglycollic acid and N-acetyl- S-(2-hydroxyethyl)cysteine in the animal and (b) that S-(carboxymethyl)cysteine has been identified amongst the hydrolytic products from an hepatic extract prepared from vinyl chloride-treated animals is consistent with the formation of chloroacetaldehyde, and with the reaction of chloroethylene oxide or chloroacetaldehyde with glutathione in the presence of a glutathione S-epoxide transferase to give the identified S-containing metabolites.

Mouse liver and lung glutathione S-epoxide transferase: Effects of phenobarbital and 3-methylcholanthrene administration

The effects of 3-methylcholanthrene (3MC) and phenobarbital (PB) administration on the levels of glutathione-S-epoxide transferase activity in supernatant preparations of liver and lung were studied in a number of different strains of mice, C57Bl/6, C3H, C3H f −, Balb/c −, A + and DBA/2 +. Three epoxide substrates, 3MC-11,12-oxide, styrene oxide (SO) and 3,3,3-trichloro-1,2-epoxypropane (TCPO), were employed in this investigation. PB administration (75 mg/kg body weight for 3 days) resulted in 13–57% increases in enzyme activity in the liver supernatant but was ineffective in inducing activity in lung. 3MC administration (40 mg/kg body weight for 2 days) on the other hand was without any effect on glutathione-S-epoxide transferase activity in both liver and lung.

Radiochemical assay of glutathione S-epoxide transferase and its enhancement by phenobarbital in rat liver in vivo

A rapid and sensitive assay of glutathione S-epoxide transferase was developed using tritium-labeled styrene oxide as the epoxide substrate. The method is based on the separation of unreacted styrene oxide from the glutathione conjugate formed after the incubation by the extraction with light petroleum. The radioactivity of the conjugate remaining in the aqueous phase can be determined subsequently by liquid scintillation. Using the present radiochemical method we found, after the intraperitoneal administration of phenobarbital (80 mg/kg) in vivo, a significant enhancement (about 56%) in the conjugation of styrene oxide with glutathione catalyzed by rat liver soluble fraction. No enhancement in the enzyme activity could be detected, on the other hand, after administration with styrene (1 g/kg) or 20-methylcholanthrene (20 mg/kg).

The metabolism of carcinogenic polycyclic hydrocarbons by tissues of the respiratory tract.

Carcinogenic polycyclic hydrocarbons undoubtedly require metabolic activation, a process currently thought to involve epoxide formation. Previous studies have shown that the NADPH-dependent microsomal mono-oxygenase that catalyses the formation of epoxides is present in preparations of human and rat lung tissue (Grover et al., 1973, 1974). These tissues are also capable of the further metabolism of hydrocarbon epoxides since, with both species, the conversion of benz[a]anthracene 5,doxide into the corresponding dihydrodiol, catalysed by microsomal epoxide hydrase, and into the corresponding glutathione conjugate, catalysed by glutathione S-epoxide transferase, was detected (Grover et al., 1973; Grover, 1974). The polycyclic hydrocarbons that are present in tobacco smoke (Wynder H 3-hydroxybenzo[a]pyrene was also detected. The results obtained show that rat

Metabolism of benz[a]anthracene epoxides by rat-liver

Benz[a]anthracene is metabolized to 5,6-dihydro-5,6-dihydroxybenz[a]anthracene and 8,9-dihydro-8,9-dihydroxybenz[a]anthracene by the microsomal fraction of ratliver and NADPH and these metabolites are formed at similar rates. When the soluble liver fraction and GSH are also present, the rate of formation of the 8,9-dihydrodiol is not affected while that of the 5,6-isomer is considerably reduced. Since epoxides are the intermediates formed in the metabolism of aromatic hydrocarbons, the further metabolism of benz[a]anthracene 5,6- and 8,9-oxide into dihydrodiols and into GSH conjugates has been investigated. Each epoxide is converted into a dihydrodiol by microsomal “epoxide hydrase” at a similar rate, whereas conjugation with GSH. a reaction catalysed by “glutathione S-epoxide transferase” in the soluble liver fraction, proceeds considerably more rapidly with the 5,6- than with the 8,9-isomer. When a mixture of the two epoxides of benz[a]anthracene is incubated both with “epoxide hydrase” and with “GSH transferase”, the 5,6-isomer is converted largely into the GSH conjugate while the main route of metabolism of the 8,9-isomer is by hydration to the dihydrodiol.

K-region epoxides of polycyclic hydrocarbons: Formation and further metabolism by rat-lung preparations

Epoxides of 7-methylbenz[a]anthracene and of benzo[a]pyrene that have been identified as the K-region epoxides, 7-methylbenz[a]anthracene 5,6-oxide and benzo[a]pyrene 4,5-oxide, have been detected as microsomal metabolites using preparations from the lungs of rats that had been pretreated with the microsomal mixed function oxidase inducer, 3-methylcholanthrene. It was also possible, using lung microsomal preparations from uninduced animals, to demonstrate the formation of an epoxide identified as the K-region derivative, benz[a]anthracene 5,6-oxide, as a microsomal metabolite of benz[a]anthracene. The K-region epoxides of 7-methylbenz[a]anthracene and of benzo[a]pyrene could not always be detected as metabolites when lung microsomal preparations from uninduced rats were used. The activities of two other enzymes present in pulmonary tissue fractions that are involved in the further metabolism of polycyclic hydrocarbon epoxides have also been measured and the values compared with those obtained with rat-liver. When benz[a]anthracene 5,6-oxide was used as substrate, much lower levels of microsomal epoxide hydrase activity were found in lung than in liver, but soluble-supernatant fractions of rat-lung appeared to possess higher levels of glutathione S-epoxide transferase activity than were present in rat-liver. The significance of these results in relation to the metabolic activation of polycyclic hydrocarbons by epoxide formation and to the induction of tumours of the respiratory tract by members of this class of chemical carcinogens is discussed.

AN ENZYME CATALYSING THE CONJUGATION OF EPOXIDES WITH GLUTATHIONE

1. Liver supernatant preparations from rats and ferrets catalyse the conjugation of some epoxides with glutathione. The enzyme involved might be called ;glutathione S-epoxidetransferase', as it is different from glutathione S-aryltransferase, the enzyme catalysing the conjugation of 1,2-dichloro-4-nitrobenzene, 4-nitro-pyridine N-oxide and other cyclic compounds with glutathione and from the enzyme catalysing the conjugation of iodomethane and glutathione. 2. The enzyme does not catalyse the reaction with cysteine. It is not inactivated by dialysis but is unstable at pH 5.0. 3. The role of the enzyme in metabolism of foreign compounds is discussed.