Effect of perfluorodecalin on activities of hepatic phase II xenobiotic biotransformation enzymes.

The activity of the hepatic phase II enzymes of xenobiotic biotransformation after intravenous administration of perfluorodecalin emulsion to rats was measured. Perfluorodecalin was found to increase the microsomal glutathione S-transferase and UDP-glucuronosyltransferase activities 1.4- and 2.3-fold, respectively. The activity of sulphotransferase was decreased 2-fold. These results show that perfluorodecalin is an inducer of both the enzymes of cytochrome P-450-dependent monooxygenase system [Mishin V. et al (1989) Chem.-Biol. Interactions, 72, 143-155.] and those catalyzing conjugation reactions: microsomal glutathione S-transferase and UDP-glucuronosyltransferase.     

Comparison of the impact of continuous and intermittent exposure to vinyl chloride, including phenobarbital effect

Rats were subjected to 4 h continuous and intermittent exposure to vinyl chloride (VC) at the time-weighted average concentration of 50,000 mg/m3. Prior to exposure, half of the animals obtained water, whereas the other half 0.1% sodium phenobarbital (PB) solution for seven consecutive days. The studies were focussed on: body weight, liver weight, activity of enzymes in the blood serum, activity of glutathione S-transferase in the liver cytoplasmatic and microsomal fraction, content of free non-protein sulfhydryl groups (NPSH) in the liver and urinary excretion of thiodiglycolic acid (TDGA). VC exposure, both continuous and intermittent, resulted in a decrease of body weight, NPSH depletion in the liver and TDGA urinary excretion. PB effects were manifested by the persistent decrease in rats' body weight, increase in the liver weight, increase in the cytoplasmatic activity of glutathione S-transferase in the liver and increase in TDGA urinary excretion. With none of the tested parameters, except TDGA, statistically significant differences between the continuous and intermittent VC exposure at the same time-weighted average concentration of 50,000 mg/m3, were found. TDGA urinary excretion was higher in rats poisoned in continuous exposure, as compared to the intermittent one.     

Stereochemistry of the microsomal glutathione S-transferase catalyzed addition of glutathione to chlorotrifluoroethene

The stereochemistry of S-(2-chloro-1,1,2-trifluoroethyl)glutathione formation was studied in rat liver cytosol, microsomes, N-ethylmaleimide-treated microsomes, 9000g supernatant fractions, purified rat liver microsomal glutathione S-transferase, and isolated rat hepatocytes. The absolute configuration of the chiral center generated by the addition of glutathione to chlorotrifluoroethene was determined by degradation of S-(2-chloro-1,1,2-trifluoroethyl)glutathione to chlorofluoroacetic acid, followed by derivatization to form the diastereomeric amides N-(S)-alpha-methylbenzyl-(S)-chlorofluoacetamide and N-(S)-alpha-methylbenzyl-(R)-chlorofluoroacetamide, which were separated by gas chromatography. Native and N-ethylmaleimide-treated rat liver microsomes, purified rat liver microsomal glutathione S-transferase, rat liver 9000g supernatant, and isolated rat hepatocytes catalyzed the formation of 75-81% (2S)-S-(2-chloro-1,1,2-trifluoroethyl)glutathione; rat liver cytosol catalyzed the formation of equal amounts of (2R)- and (2S)-S-(2-chloro-1,1,2-trifluoroethyl)glutathione. In rat hepatocytes, microsomal glutathione S-transferase catalyzed the formation of 83% of the total S-(2-chloro-1,1,2-trifluoroethyl)glutathione formed. These observations show that the microsomal glutathione S-transferase catalyzes the first step in the intracellular, glutathione-dependent bioactivation of the nephrotoxin chlorotrifluoroethene.     

Reduced glutathione inhibits the alkylation by N-nitrosodimethylamine of liver DNA in vivo and microsomal fraction in vitro

The transfer of radioactivity from N-nitroso-[14C]dimethylamine to trichloroacetic acid precipitable macromolecules in the microsomal fraction of rat liver was investigated. This transfer was found to depend on N-nitrosodimethylamine being metabolized. Cytosolic fraction and cytosol enriched with reduced glutathione inhibited the binding of radioactivity to acid insoluble proteins. Depletion of glutathione in rat liver with diethylmaleate prior to i.v. administration of 10 mg N-nitroso-[14C]dimethylamine/kg led to an increase in O6-methylguanine and N-7-methylguanine in DNA. If rats were fed disulfiram for 6 days (2 g/kg feed), glutathione and glutathione S-transferase were enhanced, and the degree of methylation of guanine by N-nitrosodimethylamine was greatly reduced, as was the metabolism of N-nitrosodimethylamine in the intact animal. Fasting rats for 24 h did not change the N-nitrosodimethylamine-demethylase activity in vitro but greatly enhanced the methylation of guanine in vivo, while the glutathione content and glutathione S-transferase activity were not changed compared to fed animals.     

Urinary ascorbic acid—HPLC determination and application as a noninvasive biomarker of hepatic response

A high-performance liquid chromatograph (HPLC) procedure has been developed for the determination of rat urinary ascorbic acid, a major metabolite cf the hepatic glucuronic acid pathway. The presence of EDTA and HC1 effectively inhibited degradation of ascorbic acid during the collection of urine specimens. The reliability of the procedure was demonstrated by its high recovery (90%), specificity (characteristic absorption maximum and discrimination from iso-ascorbic acid), and reproducibility (2–3% coefficient of variation). The usefulness of this assay as an indicator of hepatic response was demonstrated in preliminary experiments where increases in urinary ascorbic acid excretion were detected in male rats treated with PCB 126 (3,3′,4,4′,5-pentachloro-biphenyl) or PCB 105 (2,3,3′,4,4′-pentachlorobi-phenyl). The HPLC measurement also showed that the two PCB congeners differed markedly in their potency in stimulating urinary ascorbic acid excretion. For example, 10 μ/kg bw/day of PCB 126 was sufficient to cause a fourfold increase in urinary ascorbic excretion while 5000 μ/kg bw/day of PCB 105 was required for a sevenfold increase. In response to the administration of PCB 105 or PCB 126, urinary ascorbic acid appeared to increase to the same extent as increases in hepatic ethoxyresorufin O-deethylase (EROD) and UDP-glucuronosyltransferase (UGT) activities, and to a much higher extent than changes in liver weight and hematological and serum clinical chemical parameters. The sensitivity and specificity, the ease in obtaining timed specimens, and the noninvasive nature make this assay a useful biomarker of hepatic response in dose-finding and various acute and chronic studies.     

Effects of excess nicotinamide administration on the urinary excretion of nicotinamide N-oxide and nicotinuric acid by rats

We investigated a useful chemical index for an excessive nicotinamide intake and how this excessive nicotinamide intake affects the tryptophan-nicotinamide metabolism in rats. Weaning rats were fed on a tryptophan-limited and nicotinic acid-free diet containing no, 0.003%, 0.1%, 0.2%, or 0.3% nicotinamide for 21 days. Urine samples were collected on the last day and analyzed the intermediates and metabolites on the tryptophan-nicotinamide pathway. Nicotinamide N-oxide, nicotinic acid and nicotinuric acid, metabolites of nicotinamide, were detected when nicotinamide at more than 0.1% had been taken. An intake of nicotinamide of more than 0.1% increased the urinary excretion of quinolinic acid, an intermediate on the pathway. Nicotinamide N-oxide and nicotinuric acid increased with increasing dietary concentration of nicotinamide. These results show that the measurements of nicotinamide N-oxide and nicotinuric acid in urine would be useful indices for an excessive nicotinamide intake.     

Dietary administration of 2(3)-t-butyl-4-hydroxyanisole elevates mouse liver microsome-mediated DNA binding and mutagenicity of aflatoxin B1

Administration of the phenolic antioxidant 2(3)-t-butyl-4-hydroxyanisole (BHA) to mice resulted in a 2-3-fold increase in the liver microsome catalyzed irreversible binding of aflatoxin B1 (AFB1) to calf thymus DNA and up to a 5-fold increase in the ability to induce mutations in Salmonella typhimurium TA98. Maximum induction of AFB1 binding to DNA occurred after 2 days of BHA administration whereas cytosolic glutathione S-transferase was maximally induced (6-fold) only after 10 days of BHA feeding. The induction of a new cytochrome P-450 species was indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and an enhanced sensitivity to inhibition by metyrapone and alpha-naphthoflavone. Addition of control cytosol (containing glutathione S-transferase) + glutathione to control microsomes decreased AFB1 binding to DNA by 26%. However, replacement of control cytosol by BHA cytosol which contained 6 times more glutathione S-transferase only marginally enhanced the inhibition to 38%. These data suggest that BHA may exert its effect in the liver primarily through an alteration of the cytochrome P-450 dependent activation process although an increase in the conjugation of reactive metabolite may play a contributory role.     

The effect of selenium on the hepatic drug metabolism and inducibility in rat

1. Rats were fed either a normal or selenium-deficient diet for 4 weeks. The subgroup on selenium deficient diet had selenium supplementation as 3 ppm Se in the drinking water. Benzo(a)pyrene was given intraperitoneally as an inducer. 2. Se deficiency decreased glutathione peroxidase and cytochrome c-reductase activities while other activities were unchanged as compared to normal diet. 3. Selenium deficiency was a prerequisite for the induction of glutathione peroxidase, S-reductase and S-transferase enzymes. 4. Benzo(a)pyrene increased hepatic microsomal cytochrome P-450 content in rats on normal and selenium supplemented diet but not in the selenium deficient group. 5. The 7-ethoxyresorufin and 7-ethoxycoumarin deethylase, aryl hydrocarbon hydroxylase and cytochrome c-reductase activities were increased by benzo(a)pyrene in all the dietary groups. 6. The UDP-glucuronosyltransferase activity was also increased by benzo(a)pyrene in all the experimental groups and this was true with p-nitrophenol and phenolphthalein as aglycons.     

Enzymatic Mechanisms Involved in Phenanthrene Degradation by the White Rot Fungus Pleurotus ostreatus

The enzymatic mechanisms involved in the degradation of phenanthrene by the white rot fungus Pleurotus ostreatus were examined. Phase I metabolism (cytochrome P-450 monooxygenase and epoxide hydrolase) and phase II conjugation (glutathione S-transferase, aryl sulfotransferase, UDP-glucuronosyltransferase, and UDP-glucosyltransferase) enzyme activities were determined for mycelial extracts of P. ostreatus. Cytochrome P-450 was detected in both cytosolic and microsomal fractions at 0.16 and 0.38 nmol min(sup-1) mg of protein(sup1), respectively. Both fractions oxidized [9,10-(sup14)C]phenanthrene to phenanthrene trans-9,10-dihydrodiol. The cytochrome P-450 inhibitors 1-aminobenzotriazole (0.1 mM), SKF-525A (proadifen, 0.1 mM), and carbon monoxide inhibited the cytosolic and microsomal P-450s differently. Cytosolic and microsomal epoxide hydrolase activities, with phenanthrene 9,10-oxide as the substrate, were similar, with specific activities of 0.50 and 0.41 nmol min(sup-1) mg of protein(sup-1), respectively. The epoxide hydrolase inhibitor cyclohexene oxide (5 mM) significantly inhibited the formation of phenanthrene trans-9,10-dihydrodiol in both fractions. The phase II enzyme 1-chloro-2,4-dinitrobenzene glutathione S-transferase was detected in the cytosolic fraction (4.16 nmol min(sup-1) mg of protein(sup-1)), whereas aryl adenosine-3(prm1)-phosphate-5(prm1)-phosphosulfate sulfotransferase (aryl PAPS sulfotransferase) UDP-glucuronosyltransferase, and UDP-glucosyltransferase had microsomal activities of 2.14, 4.25, and 4.21 nmol min(sup-1) mg of protein(sup-1), respectively, with low activity in the cytosolic fraction. However, when P. ostreatus culture broth incubated with phenanthrene was screened for phase II metabolites, no sulfate, glutathione, glucoside, or glucuronide conjugates of phenanthrene metabolites were detected. These experiments indicate the involvement of cytochrome P-450 monooxygenase and epoxide hydrolase in the initial phase I oxidation of phenanthrene to form phenanthrene trans-9,10-dihydrodiol. Laccase and manganese-independent peroxidase were not involved in the initial oxidation of phenanthrene. Although P. ostreatus had phase II xenobiotic metabolizing enzymes, conjugation reactions were not important for the elimination of hydroxylated phenanthrene.     

Activation of rat liver microsomal glutathione S-transferase by reduced oxygen species

The effect of enzymatically generated reduced oxygen metabolites on the activity of hepatic microsomal glutathione S-transferase activity was studied to explore possible physiological regulatory mechanisms of the enzyme. Noradrenaline and the microsomal cytochrome P-450-dependent monooxygenase system were used to generate reduced oxygen species. When noradrenaline (greater than 0.1 mM) was incubated with rat liver microsomes in phosphate buffer (pH 7.4), an increase in microsomal glutathione S-transferase activity was observed, and this activation was potentiated in the presence of a NADPH-generating system; the glutathione S-transferase activity was increased to 180% of the control with 1 mM noradrenaline and to 400% with both noradrenaline and NADPH. Superoxide dismutase and catalase inhibited partially the noradrenaline-dependent activation of the enzyme. In the presence of dithiothreitol and glutathione, the activation of the glutathione S-transferase by noradrenaline, with or without NADPH, was not observed. In addition, the activation of glutathione S-transferase activity by noradrenaline and glutathione disulfide was not additive when both compounds were incubated together. These results indicate that the microsomal glutathione S-transferase is activated by reduced oxygen species, such as superoxide anion and hydrogen peroxide. Thus, metabolic processes that generate high concentrations of reduced oxygen species may activate the microsomal glutathione S-transferase, presumably by the oxidation of the sulfhydryl group of the enzyme, and this increased catalytic activity may help protect cells from oxidant-induced damage.     

Inhibition of glutathione S-transferase by bile acids.

The effects of bile acids on the detoxification of compounds by glutathione conjugation have been investigated. Bile acids were found to inhibit the total soluble-fraction glutathione S-transferase activity from rat liver, as assayed with four different acceptor substrates. Dihydroxy bile acids were more inhibitory than trihydroxy bile acids, and conjugated bile acids were generally less inhibitory than the parent bile acid. At physiological concentrations of bile acid, the glutathione S-transferase activity in the soluble fraction was inhibited by nearly 50%. This indicates that the size of the hepatic pool of bile acids can influence the ability of the liver to detoxify electrophilic compounds. The A, B and C isoenzymes of glutathione S-transferase were isolated separately. Each was found to be inhibited by bile acids. Kinetic analysis of the inhibition revealed that the bile acids were not competitive inhibitors of either glutathione or acceptor substrate binding. The microsomal glutathione S-transferase from guinea-pig liver was also shown to be inhibited by bile acids. This inhibition, however, showed characteristics of a non-specific detergent-type inhibition.     

Effect of large doses of ascorbic acid on the hepatic and extra-hepatic drug-metabolizing enzymes in guinea pig

Water solubility and non-toxic properties of ascorbic acid are taken as criteria for beneficial effects of large doses of the vitamin. In the present study, male guinea pigs, dosed daily with 15, 30 or 50 mg/100g body weight for 10 weeks, demonstrated no differences in effect on liver and lung weights, body growth and microsomal protein contents of liver and lung when compared with controls. When guinea pigs were fed excessive ascorbic acid, there was a small non-significant increase (p less than 0.05) in hepatic and pulmonary cytochrome P-450, and significant increase (p less than 0.05) in hepatic cytochrome b5 which was accompanied with a significant increase in arylhydrocarbon hydroxylase activity in the two organs. Activity of NADPH-dependent cytochrome c-reductase was decreased in liver and remained unaffected in lung and colon. Drug detoxifying enzymes responded in different ways to increased intake of ascorbic acid. Activity of UDP-glucuronyltransferase remained unchanged on feeding excessive ascorbic acid, whereas glutathione S-transferase was decreased significantly in liver and was unaltered in lung and colon. Reduced glutathione was decreased only in the lung. The observed changes in drug activating and detoxifying enzymes appear to be important from drug pharmacokinetics and carcinogenesis point of view.     

Comparison of the affinities of newly identified human bile acid binder and cationic glutathione S-transferase for bile acids

The bile acid binding properties of the newly identified bile acid binder (Mr = 36,000) (FEBS Lett. 1984. 177: 31-35) and the major cationic glutathione (GSH) S-transferase (Mr = 50,000) in human liver cytosol were compared. Binding affinities were measured by the competitive displacement by bile acids of 1-anilino-8-naphthalene sulfonate (ANS) bound to the proteins and, in some cases, by direct methods of flow dialysis and equilibrium dialysis. The binding affinities for various bile acids by the human bile acid binder were 2-5 orders of magnitude greater than those by human cationic GSH S-transferase. This suggests an important physiologic role for the former protein in intracellular transfer of bile acids in human liver.     

Regulation of rat liver microsomal glutathione S-transferase activity by thiol/disulfide exchange

The regulation of purified glutathione S-transferase from rat liver microsomes was studied by examining the effects of various sulfhydryl reagents on enzyme activity with 1-chloro-2,4-dinitrobenzene as the substrate. Diamide (4 mM), cystamine (5 mM), and N-ethylmaleimide (1 mM) increased the microsomal glutathione S-transferase activity by 3-, 2-, and 10-fold, respectively, in absence of glutathione; glutathione disulfide had no effect. In presence of glutathione, microsomal glutathione S-transferase activity was increased 10-fold by diamide (0.5 mM), but the activation of the transferase by N-ethylmaleimide or cystamine was only slightly affected by presence of glutathione. The activation of microsomal glutathione S-transferase by diamide or cystamine was reversed by the addition of dithiothreitol. Glutathione disulfide increased microsomal glutathione S-transferase activity only when membrane-bound enzyme was used. These results indicate that microsomal glutathione S-transferase activity may be regulated by reversible thiol/disulfide exchange and that mixed disulfide formation of the microsomal glutathione S-transferase with glutathione disulfide may be catalyzed enzymatically in vivo.     

Regulation of hepatic drug metabolism by elaidic and linoleic acids in rats.

Elaidic and linoleic acids were administered at doses of 40 and 200 mg/kg i.p. every second day for 4 weeks to rats fed a fat-free diet. The fatty acids had only a slight effect on the weight gain of the animals. The amount of microsomal protein was slightly decreased with the higher dose of linoleic acid. The higher dose level of both fatty acids decreased the microsomal phospholipid content. The relative amounts of microsomal phospholipid fatty acids were also altered due to fatty acid administration. The activity of microsomal NADPH cytochrome c reductase and microsomal cytochrome P-450 contents were decreased by the higher dose of linoleic acid. The hepatic aryl hydrocarbon hydroxylase and p-nitroanisole O-demethylase activities decreased in fatty acid-treated rats. The UDP-glucuronosyltransferase activity was also lowered after the fatty acid administration. The results suggest that fatty acid-induced changes in the activities of drug-metabolizing enzymes may be due to the microenvironmental changes of membrane-bound enzymes.     

Purification and characterization of cytosolic liver protein facilitating heme transport into apocytochrome b5 from mitochondria. Evidence for identifying the heme transfer protein as belonging to a group of glutathione S-transferases

The results of the present experiment are shown in terms of the transport of protoheme from mitochondria to apocytochrome b5 when fresh rat liver mitochondria, apocytochrome b5, and cytosol were incubated. The heme transfer protein was purified from rat liver cytosol up to approximately 133-140-fold with a 43% yield by the procedure discussed herein, including Sephadex G-75 and CM-cellulose column chromatography. The final preparation showed apparent homogeneity upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Its native form was found to be a dimeric protein with a Mr = 45,000 which consists of a subunit with a Mr = 23,000. In the transporting system, the heme transfer depended on the concentration of mitochondria (donor), apocytochrome b5 (acceptor), and purified transfer protein, respectively. Omission of one of these components led to an almost complete loss of the transfer activity. The transport of mitochondrial protoheme was a rapid reaction which showed approximate linearity until 1.5 min and after that it became saturated. When the functional capacity was tested by the NADH-cytochrome c reductase system, the reconstituted cytochrome b5 expressed its complete original catalytic properties, as well as its characteristic absorption spectra for the hemoprotein. Furthermore, the detailed physicochemical and immunological characterization of the transfer protein provided evidence that the protein is identical with soluble glutathione S-transferase, which conjugates glutathione with a variety of electrophilic compounds. At least one of the glutathione S-transferase isozymes observed was identified as GST-C2, which comprises the subunit of Yb'Yb' by the immunoprecipitation reaction using various anti-glutathione S-transferase isozyme antibodies.     

Inhibition of glutathione S-transferase zeta and tyrosine metabolism by dichloroacetate: a potential unifying mechanism for its altered biotransformation and toxicity.

Dichloroacetate (DCA) inhibits its own metabolism and is converted to glyoxylate by glutathione S-transferase zeta (GSTz). GSTz is identical to maleylacetoacetate isomerase, an enzyme of tyrosine catabolism that converts maleylacetoacetate (MAA) to fumarylacetoacetate and maleylacetone (MA) to fumarylacetone. MAA and MA are alkylating agents. Rats treated with DCA for up to five days had markedly decreased hepatic GSTz activity and increased urinary excretion of MA. When dialyzed cytosol obtained from human liver was incubated with DCA, GSTz activity was unaffected. In contrast, DCA incubation inhibited enzyme activity in dialyzed hepatic cytosol from rats. Incubation of either rat or human hepatic cytosol with MA led to a dose dependent inhibition of GSTz. These data indicate that humans or rodents exposed to DCA may accumulate MA and/or MAA which inhibit(s) GSTz and, consequently, DCA biotransformation. Moreover, DCA-induced inhibition of tyrosine catabolism may account for the toxicity of this xenobiotic in humans and other species.     

Differential induction of rat hepatic cytochrome P-448 and glutathione S-transferase B messenger RNAS by 3-methylcholanthrene

Liver poly(A⁺)-RNA isolated from untreated and 3-methylcholanthrene treated rats has been translated in the rabbit reticulocyte cell-free system in order to determine the level of translationally active cytochrome P-448, glutathione S-transferase B and serum albumin mRNAs. Translatable cytochrome P-448 mRNA was not detected in untreated rats; however in animals treated with 3-methylcholanthrene cytochrome P-448 mRNA was elevated markedly. Functional rat liver glutathione S-transferase B mRNA was elevated 2-fold by 3-methylcholanthrene administration, whereas the serum albumin mRNA level was decreased by 50%. Our results indicate that 3-methylcholanthrene is not just a specific inducer of drug metabolizing enzymes but can alter the mRNA level encoding other polypeptides and thus affect cellular homeostasis.     

Activation of microsomal glutathione S-transferase activity by sulfhydryl reagents.

Rat liver microsomes exhibit glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as the second substrate. This activity can be stimulated 8-fold by treatment of the microsomes with N-ethylmaleimide and 4-fold with iodoacetamide. The corresponding glutathione S-transferase activity of the supernatant fraction is not affected by such treatment. These findings suggest that rat liver microsomes contain glutathione S-transferase distinct from those found in the cytoplasmic and that the microsomal transferase can be activated by modification of microsomal sulfhydryl group(s).     

Dietary influence of selenium on the incidence of N-nitrosodiethylamine-induced hepatoma with reference to drug and glutathione metabolizing enzymes

The dietary administration of selenium (sodium selenite; 4 p.p.m.) daily has been found to be highly effective in reducing the incidence of cancer induced by N-nitrosodiethylamine (DEN) in Wistar strain rats. Selenium treatment either before initiation, during initiation and selection/phenobarbital promotion phases of hepatocarcinogenesis has been found to be effective in elevating hepatic microsomal cytochrome b(5), NADPH-cytochrome C reductase and cytosolic aryl hydrocarbon hydroxylase activities to a statistically significant level measured either in the hyperplastic nodule or in the surrounding liver tissues compared to control animals. Moreover, selenium treatment throughout the study, decreases the cytosolic glutathione S-transferase and microsomal UDP-glucuronyl transferase activities by a significant degree when compared to control rats. Alterations in glutathione metabolizing enzyme activities (glutathione reductase, gamma-glutamyl transpeptidase, gamma-glutamylcysteine synthetase and glucose-6-phosphate dehydrogenase) were also observed in selenium-treated groups. Our results confirm the fact that selenium is particularly protective in limiting the action of DEN during the initiation phase of hepatocarcinogenesis.