Drug residue formation from ronidazole, a 5-nitroimidazole. III. Studies on the mechanism of protein alkylation in vitro

Ronidazole (1-methyl-5-nitroimidazole-2-methanol carbamate) is reductively metabolized by liver microsomal and purified NADPH-cytochrome P-450 reductase preparations to reactive metabolites that covalently bind to tissue proteins. Kinetic experiments and studies employing immobilized cysteine or blocked cysteine thiols have shown that the principal targets of protein alkylation ara cysteine thiols. Furthermore, ronidazole specifically radiolabelled with 14C in the 4,5-ring, N-methyl or 2-methylene positions give rise to equivalent apparent covalent binding suggesting that the imidazole nucleus is retained in the bound residue. In contrast, the carbonyl-14C-labeled ronidazole gives approx. 6--15-fold less apparent covalent binding indicating that the carbamoyl group is lost during the reaction leading to the covalently bound metabolite. The conversion of ronidazole to reactive metabolite(s) is quantitative and reflects the amazing efficiency by which this compound is activated by microsomal enzymes. However, only about 5% of this metabolite can be accounted for as protein-bound products under the conditions employed in these studies. Consequently, approx. 95% of the reactive ronidazole metabolite(s) can react with other constituents in the reaction media such as other thiols or water. Based on these results, a mechanism is proposed for the metabolic activation of ronidazole.     

Drug residue formation from ronidazole, a 5-nitroimidazole. II. Involvement of microsomal NADPH-cytochrome P-450 reductase in protein alkylation in vitro

Purified liver microsomal NADPH-cytochrome P-450 reductase is able to catalyze the activation of [14C]ronidazole to metabolite(s) which bind covalently to protein. Like the reaction catalyzed by microsomes, protein alkylation catalyzed by the reductase is (1) sensitive to oxygen, (2) requires reducing equivalents, (3) is inhibited by sulfhydryl-containing compounds and (4) is stimulated several fold by either flavin mononucleotide (FMN) or methytlviologen. A cytochrome P-450 dependent pathway of ronidazole activation can be demonstrated as judged by the inhibition of the reaction by carbon monoxide, metyrapone and 2,4-dichloro-6-phenylphenoxyethylamine but the involvement of specific microsomal cytochrome P-450 isozymes has not been definitively established. Milk xanthine oxidase is also capable of catalyzing ronidazole activation. Polyacrylamide sodium dodecyl sulfate (SDS)-gel electrophoresis reveals that the reactive intermediate(s) of ronidazole does not alkylate proteins selectively.     

A convenient assay for mephenytoin 4-hydroxylase activity of human liver microsomal cytochrome P-450

A simple and rapid method for the determination of (S)-mephenytoin 4-hydroxylase activity by human liver microsomal cytochrome P-450 has been developed. [Methyl-14C] mephenytoin was synthesized by alkylation of S-nirvanol with 14CH3I and used as a substrate. After incubation of [methyl-14C]mephenytoin with human liver microsomes or a reconstituted monooxygenase system containing partially purified human liver cytochrome P-450, the 4-hydroxylated metabolite of mephenytoin was separated by thin-layer chromatography and quantified. The formation of the metabolite depended on the incubation time, substrate concentration, and cytochrome P-450 concentration and was found to be optimal at pH 7.4. The Km and Vmax rates obtained with a human liver microsomal preparation were 0.1 mM and 0.23 nmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450, respectively. The hydroxylation activity showed absolute requirements for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH in a reconstituted monooxygenase system. Activities varied from 5.6 to 156 pmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450 in 11 human liver microsomal preparations. The basic system utilized for the analysis of mephenytoin 4-hydroxylation can also be applied to the estimation of other enzyme activities in which phenol formation occurs.     

Drug residue formation from ronidazole, a 5-nitroimidazole. VIII. Identification of the 2-methylene position as a site of protein alkylation.

Ronidazole protein-bound adducts were generated by the in vitro anaerobic incubation of [2-methylene-14C]ronidazole with microsomes from the livers of male rats. Acid hydrolysis of the protein adducts yielded an imidazole ring fragment bearing the radiolabel and an amino acid residue derived from the proteins. This fragment has been identified as carboxymethylcysteine by co-chromatography of the amino acid and its dansyl derivative with known standards under a variety of conditions. The carboxymethylcysteine was estimated to represent at least 15% of the radioactivity derived from the protein-bound adducts and provides unequivocal evidence that nucleophilic attack by protein cysteine thiols occurred at the 2-methylene position of ronidazole.     

Covalent binding of niridazole (Ambilhar) to tissue proteins of the rat

In vivo and in vitro experiments have shown that [14C] niridazole ( NDZ ) can covalently bind to the proteins of rat liver, kidney and testes, but not to the DNA in these tissues. The covalent binding was dose dependent, and the greatest amount of binding was found in the microsomal fraction. The binding of [14C] NDZ to microsomal protein was linear with time and with protein concentration. Reduced nicotinamide adenine dinucleotide phosphate was necessary for the binding, while cobaltous chloride pretreatment inhibited it, demonstrating that a cytochrome P-450 dependent mixed function oxidase mediated the binding. Pretreatment of rats with other compounds, such as phenobarbital, 3-methyl-cholanthrene and chloracetamide which alter the rate of metabolism of [14C] NDZ similarly affected the extent of hepatic binding of the radiolabelled metabolite. The possible relationships between these results and the cytotoxic effects of NDZ have been discussed.     

Comparison of aflatoxin B1 and aflatoxin G1 binding to cellular macromolecules in vitro, in vivo and after peracid oxidation; characterisation of the major nucleic acid adducts.

A comparison between [14C]aflatoxin B1 (AFB1) and [14C]aflatoxin G1 (AFG1) binding to rat liver and kidney cellular macromolecules has shown AFG1-DNA and-ribosomal RNA binding to be lower in both organs. For both mycotoxins more was bound to nucleic acids than to protein. Two hours after intraperitoneal injection (60 microgram/100 g) of [14C] AFB1, 40 ng, 151 ng/mg. Loss of radioactivity bound to liver DNA for both [14C]AFB1 and protein respectively and for [14C]AFG1 the respective figures were 10, 7 and 1 ng/mg. Loss of liver bound radioactivity to DNA for both [14C]AFG1 and [14C]AFG1 appeared to be biphasic indicating that an enzymic DNA repair process may be operating. In vitro binding studies also showed less AFG1 was bound to exogenous DNA after microsomal activation than AFB1. This difference was not a result of differences in the chemical reactivity of the "ultimate" electrophilic species, the respective expoxides, since chemical activation studies using 3-chloroperbenzoic acid showed similar amounts of AFG1 and AFB1 to be converted to the epoxides and to bind to DNA. Studies on the distribution coefficients of the two mycotoxins showed AFB1 to be more lipophilic than AFG1 and this may be an important factor in determining the weaker carcinogenicity of the latter compound. Characterisation of the major AFG1-DNA adduct formed in vitro, in vivo and after peracid oxidation showed it to have the structure trans-9,10-dihydro-9-(7-guanyl)-10-hydroxy-aflatoxin G1. This adduct is similar to that obtained from AFB1 by activation in vivo, in vitro and after peracid oxidation.     

Hepatic synthesis of carnitine from protein-bound trimethyl-lysine. Lysosomal digestion of methyl-lysine-labelled asialo-fetuin.

The biosynthesis of carnitine in the rat was studied by following the metabolism of two radioactive derivatives of asialo-fetuin. The first contained 14C-labelled methyl groups covalently bound to the 6-N-amino fraction of its lysine residues as 6-N-monomethyl- and dimethyl-lysine. By treating this protein with iodomethane, a second derivative was produced in which the radioactivity was preferentially incorporated as 6-N-[Me-14C]-trimethyl-lysine. These desialylated glycoproteins, like other asialo-proteins, were immediately cleared from the blood by rat liver. Within hepatocyte lysosomes, the 14C-labelled proteins were rapidly hydrolysed, producing free amino acids containing the various 6-N-[Me-14C]methylated lysine residues. The radioactive amino acids crossed the lysosomal membrane and were further metabolized in the cytosol. Carnitine was the major radioactive metabolite detected in extracts of the rat carcass and liver after intravenous injection of 6-N-[Me-14C]trimethyl-lysine-labelled asialo-fetuin. Within 3h, at least 34.6% of the trimethyl-lysine in the administered protein was converted into carnitine. Similarly, an isolated perfused rat liver converted 30% of the added peptide-bound trimethyl-lysine into carnitine within 90 min. On the other hand, in numerous attempts we failed to detect radioactive carnitine in both rat liver and carcass between 20 min and 22 h after injection of 6-N-[Me-14C]-monomethyl- and -dimethyl-lysine-labelled asialo-fetuin. These data provide evidence for a pathway of carnitine biosynthesis that involves trimethyl-lysine as a peptide-bound precursor as proposed by R.A. Cox & C.L. Hoppel [(1973) Biochem. J. 136, 1083-1090] and V. Tanphaichitr & H.P. Broquist [(1973) J. Biol. Chem. 248, 2176-2181]. The findings also show that rat liver can synthesize carnitine without the aid of other tissues, but cannot convert free partially methylated lysines into trimethyl-lysine.     

Drug protein conjugates--III. Inhibition of the irreversible binding of ethinylestradiol to rat liver microsomal protein by mixed-function oxidase inhibitors, ascorbic acid and thiols

The metabolism of [6,7-3H]ethinylestradiol [( 3H]EE2) by rat liver microsomes was studied in vitro. After incubation of [3H]EE2 with rat liver microsomes for 20 min, 90% of the substrate was metabolised and 18% of the 3H-labelled material irreversibly bound to microsomal protein. Ascorbic acid (1 mM) decreased irreversible binding of 3H and produced an accumulation of 2-hydroxyethinylestradiol (2OH-EE2), while mixed-function oxidase inhibitors (0.5 mM) decreased binding of 3H to protein by inhibiting EE2 2-hydroxylation. Addition of thiols gave water-soluble metabolites which were characterised as 1(4)-thioether derivatives of 2OH-EE2 by co-chromatography with synthetic products. The results are consistent with the hypothesis that the chemically reactive metabolite of EE2 formed in vitro is either a quinone or o-semiquinone derived from 2OH-EE2 [1].     

Drug residue formation from ronidazole,a 5-nitroimidazole. VII. Comparison of protein-bound products formed in vitro and in vivo

In vivo experiments were conducted with ronidazole radiolabelled in the 2-¹⁴CH₂-, 4,5-¹⁴C-, N¹⁴CH₃- and 4-³H-positions. The hepatic protein-bound residues, assessed by the radioactivity of exhaustively washed protein samples, were independent of the radiolabel position and occurred with 4-³H loss (>80%) in excellent agreement to previous results obtained in vitro with anaerobic incubations of liver microsomes (Miwa et al., Chem. Biol. Interact., 41 (1982) 297).HPLC analysis of acid hydrolyzed in vivo protein-bound residues, obtained from [2-¹⁴CH₂] ronidazole, produced a radiochromatographic profile which was virtually identical to that obtained from a similarly treated in vitro sample. Moreover, almost quantitative (76–96%) liberation of radiolabelled methylamine was obtained from hydrolysates of in vivo and in vitro residue samples formed from [N¹⁴CH₃] ronidazole. With 4,5-ring labeled ronidazole the distribution of total radioactivity of the protein hydrolysate on cation exchange resin and the fraction of the residue recovered as oxalic acid were nearly identical for the in vivo and in vitro products.We interpret these data to indicate that ronidazole alkylates proteins with retention of most of the carbon framework of the molecule, in vivo. It is also concluded that the in vitro model, previously used to examine the mechanism of protein alkylation, accurately reflects the salient process initially occuring in the intact animal during the formation of protein-bound residues of this drug.     

Ribose. An oxidation product of glucose 6-phosphate in microsomal fraction

An oxidative metabolism of glucose 6-phosphate was studied in rat liver microsomal fraction. Although radioactive 14CO2 was formed from [1-14C]glucose 6-phosphate in the microsomal fraction (Hino, Y., and Minakami, S. (1982) J. Biochem. (Tokyo) 92, 547-557), the formation was negligible when [2-14C]glucose 6-phosphate was used as a starting substrate. These results indicated an inability of the microsomal fraction to rearrange [2-14C]glucose 6-phosphate to form [1-14C] glucose 6-phosphate, and it was expected that a certain compound derived from glucose 6-phosphate accumulated as an end-product of the reaction. We, therefore, have tried to identify the product by high performance liquid chromatography, and found that ribose accumulated as the end-product. The formation of ribose was inhibited in the same manner as that of 14CO2 by antibodies against rat liver microsomal hexose-6-phosphate dehydrogenase, and the ratios of ribose to 14CO2 formed in the reaction were 0.5-0.8 on a molar basis. The finding of ribose formation further suggested the involvement of ribose phosphate isomerase and phosphatase activities in the reaction.     

Covalent binding of 4-nitrobenzyl mercaptan S-sulfate to the sulfhydryl groups of hepatic cytosolic proteins and bovine serum albumin with mixed disulfide bond formation

4-Nitrobenzyl [35S]mercaptan S-sulfonic acid ([35S]NBM S-sulfate), a new type of reactive metabolite of the thiol [35S]NBM in rat liver cytosol fortified with 3'-phosphoadenosine 5'-phosphosulfate, bound rapidly and covalently at pH 7.4 and 37 degrees C to the sulfhydryl groups of rat liver cytosolic proteins with formation of disulfide bonds. From the radioactive proteins was isolated and identified the sole amino acid adduct, S-([35S]NBM)cysteine, after their acid hydrolysis under the anaerobic conditions. Bovine serum albumin (BSA), a model protein with a single SH group, also reacted readily with radioactive NBM S-sulfate to form a disulfide bond in stoichiometric manner. S-([35S]NBM)-cysteine was also isolated and identified as the sole amino acid adduct from the well-washed, radioactive BSA after the same anaerobic acid hydrolysis. A normal hepatic level of GSH not only retarded the BSA-NBM adduct formation completely, but also detached the radioactivity from BSA by the reduction of the disulfide bond with formation of [35S]NBM and its disulfide. Of twenty-one amino acids examined at pH 7.4 and 37 degrees C, only cysteine reacted with NBM S-sulfate and afforded S-(NBM)cysteine with concomitant formations of S-sulfocysteine, cystine, NBM, and its disulfide.     

The metabolism of benzyl isothiocyanate and its cysteine conjugate.

1. The corresponding cysteine conjugate was formed when the GSH (reduced glutathione) or cysteinylglycine conjugates of benzyl isothiocyanate were incubated with rat liver or kidney homogenates. When the cysteine conjugate of benzyl isothiocyanate was similarly incubated in the presence of acetyl-CoA, the corresponding N-acetylcysteine conjugate (mercapturic acid) was formed. 2. The non-enzymic reaction of GSH with benzyl isothiocyanate was rapid and was catalysed by rat liver cytosol. 3. The mercapturic acid was excreted in the urine of rats dosed with benzyl isothiocyanate or its GSH, cysteinyl-glycine or cysteine conjugate, and was isolated as the dicyclohexylamine salt. 4. An oral dose of the cysteine conjugate of [14C]benzyl isothiocyanate was rapidly absorbed and excreted by rats and dogs. After 3 days, rats had excreted a mean of 92.4 and 5.6% of the dose in the urine and faeces respectively, and dogs had excreted a mean of 86.3 and 13.2% respectively. 5. After an oral dose of the cystein conjugate of [C]benzyl isothiocyanate, the major 14C-labelled metabolite in rat urine was the corresponding mercapturic acid (62% of the dose), whereas in dog urine it was hippuric acid (40% of the dose). 5. Mercapturic acid biosynthesis may be an important route of metabolism of certain isothiocyanates in some mammalian species.     

Pulegone mediated hepatotoxicity: evidence for covalent binding of R(+)-[14C]pulegone to microsomal proteins in vitro

Incubation of R(+)-[14C]pulegone with rat liver microsomes in the presence of NADPH resulted in covalent binding of radioactive material to macromolecules. Covalent binding was much higher in phenobarbital-treated microsomes as compared to 3-methylcholanthrene treated or control microsomes. The Km and Vmax of covalent binding was 0.4 mM and 1.7 nmol min-1 mg-1, respectively. Covalent binding was drastically inhibited (93%) in the presence of piperonyl butoxide. Antibodies to phenobarbital-induced cytochrome P-450 and NADPH-cytochrome P-450 reductase inhibited covalent binding to an extent of 72% and 47%, respectively. Cysteine and semicarbazide also inhibited NADPH dependent binding of radiolabel from R(+)-[14C]pulegone to microsomal proteins. The results suggest the involvement of liver microsomal cytochrome P-450 in the bioactivation of R(+)-pulegone to reactive metabolite(s) which might be responsible for covalent binding to macromolecules resulting in toxicity.     

Epoxy derivatives of aromatic polycyclic hydrocarbons. The synthesis of dibenz[a,c]anthracene 10,11-oxide and its metabolism by rat liver preparations

The synthesis of dibenz[a,c]anthracene 10,11-oxide is described. The oxide was unstable and was rapidly decomposed with cold mineral acid into a mixture of 10- and 11- hydroxydibenz[a,c]anthracene. The oxide was converted by rat liver microsomal preparations and homogenates into a product that is probably 10,11-dihydro-10,11-dihydroxydibenz[a,c]anthracene and which was identical with the metabolite formed when dibenz[a,c]anthracene was metabolized by rat liver homogenates. The oxide did not react either chemically or enzymically with GSH. 10,11-Dihydrodibenz[a,c]anthracene and 10,11-dihydrodibenz[a,c]anthracene 12,13-oxide were both metabolized by rat liver preparations into trans-10,11,12,13-tetrahydro-10,11-dihydroxydibenz[a,c] anthracene and the oxide was converted chemically into this dihydroxy compound, and it reacted chemically but not enzymically with GSH. In the alkylation of 4-(p-nitrobenzyl)pyridine, the ;K-region' epoxide, dibenz[a,h]anthracene 5,6-oxide, was more active than either dibenz[a,c]anthracene 10,11-oxide or 10,11-dihydrobenz[a,c]anthracene 12,13-oxide.     

The biosynthesis of tyramine glucuronide by liver microsomal fractions.

Labelled tyramine glucuronide was synthesized in vitro from UDP-[14C]glucuronic acid, [14C]tyramine or [3H]tyramine. The glucuronidation was carried out at pH9.2 in the presence of a monoamine oxidase inhibitor, trans-2-phenylcyclopropylamine. The Km values for tyramine were 69 and 125 micrometer and those for UDP-glucuronic acid were 260 and 290 micrometer respectively for guinea-pig and rat liver microsomal preparatons. The specific activities of microsomal glucuronyltransferase measured in fresh hepatic preparations of guinea pig, mouse and rat were respectively 601, 251 and 235 pmol of [14C]tyramine glucuronide/min per mg of protein. Increase in activity ranged from 2- to 6-fold in preparations which were frozen and thawed once and 5.4- to 10-fold when the freezing and thawing was repeated. Rabbit liver has very low activity, and monkey liver and intestine were completely devoid of this conjugating capacity.     

The binding of decomposition products of UDP-galactose to the microsomes and polyribosomes isolated from rat liver

UDP-D-[U-14C]galactose is decomposed to [U-14C]galactose-1-phosphate and [U-14C]galactose by rat liver microsomal and crude polyribosomal fractions, under conditions commonly used to assay of glycosyltransferase activities. UDP-D-[U-14C]galactose, at neutral pH, is also chemically degraded to the [U-14C]galactose-1,2-cyclic phosphate. The 1,2-cyclic phosphate derivative of galactose also exists in the commercial UDP-D-[U-14C]galactose. It is a very important finding that products of the UDP-D-[U-14C]galactose decomposition are tightly, although nonenzymatically, bound to tested subcellular fractions and may create a false impression of protein glycosylation. The application of controls containing all radioactive substances present in suitable samples is recommended in order to avoid incorrect interpretations of the results.     

Multi-step metabolic activation of benzene. Effect of superoxide dismutase on covalent binding to microsomal macromolecules,and identification of glutathione conjugates using high pressure liquid chromatography and field desorption mass spectrometry

Incubation of [¹⁴C]benzene or [¹⁴C]phenol with liver microsomes from untreated rats, in the presence of a NADPH-generating system, gave rise to irreversible binding of metabolites to microsomal macromolecules. For both substrates this binding was inhibited by more than 50% by addition of superoxide dismutase to the incubation mixtures. The decrease in binding was compensated for by accumulation of [¹⁴C]hydroquinone, indicating superoxide-mediated oxidation of hydroquinone as one step in the activation of benzene to metabolites binding to microsomal macromolecules. Since our previous work had shown that binding occurred mainly with protein rather than ribonucleic acid and was virtually completely prevented by glutathione, suggesting identity of metabolite(s) responsible for binding to protein and glutathione, a conjugate was chemically prepared from p-benzoquinone and reduced glutathione (GSH) and identified by field desorption mass spectrometry (FDMS) as 2-(S-glutathionyl) hydroquinone. Microsomal incubations, containing an NADPH-generating system, with benzene, phenol, hydroquinone or p-benzoquinone in the presence of [³H]glutathione or, alternatively, with [¹⁴C]benzene or [¹⁴C]phenol in the presence of unlabeled glutathione, were performed. All of these incubations gave rise to a peak of radioactivity eluting from the high pressure liquid chromatograph (HPLC) at a retention time identical to that of the chemically prepared 2-(S-glutathionyl) hydroquinone, whilst microsomal incubation of catechol in the presence of [³H]glutathione led to a conjugate with a very different retention time which was not observed after incubation of benzene or phenol. The microsomal metabolites of p-benzoquinone, hydroquinone and phenol thus eluting from the HPLC were further identified as the 2-(S-glutathionyl) hydroquinone by field desorption mass spectrometry. The glutathione adduct formed from benzene during microsomal activation eluted from HPLC with the same retention time and its mass spectrum also contained the molecular ion (MH⁺) (m/e 416) of this conjugate as an intense peak, but the fragmentation patterns did not allow definite assignments probably due to the considerably smaller amounts of ultimate reactive metabolites formed from this pre-precursor and thus relatively larger amounts of impurities.The results indicate that rat liver microsomes activate benzene via phenol and hydroquinone to p-benzosemiquinone and/or p-benzoquinone as quantitatively important reactive metabolites.     

Evidence that 1-naphthol is not an obligate intermediate in the covalent binding and the pulmonary bronchiolar necrosis by naphthalene

Recent studies of a number of volatile aromatic hydrocarbons have suggested that the formation of covalently bound metabolites arises solely through the intermediate formation of phenols. This study further examines the involvement of 1-naphthol in the in vivo and in vitro formation of covalently bound metabolites and pulmonary bronchiolar necrosis by naphthalene. Marked differences were observed in the rate of 1-naphthol formation in lung and liver microsomal incubations without correspondingly large differences between the rates of formation of covalently bound metabolites from naphthalene and 1-naphthol. Glutathione decreased covalent binding in hepatic microsomal incubations containing 14[C]1-naphthol but did not result in the formation of any of the glutathione adducts isolated from identical incubations containing 14[C]naphthalene. Tissue levels of covalently bound radioactivity in mice treated with 14[C]1-naphthol or 14[C]naphthalene were similar; however, in contrast to studies with naphthalene, 1-naphthol administration did not deplete tissue glutathione nor result in detectable tissue injury. These studies indicate that 1-naphthol is not an obligate intermediate in the formation of covalently bound metabolites from naphthalene nor does it appear to be a more proximate lung toxic metabolite.     

Proteolytic conversion of proinsulin into insulin. Identification of a Ca2+-dependent acidic endopeptidase in isolated insulin-secretory granules.

The metabolism of cysteine and cysteinesulphinate was studied in freshly isolated rat hepatocytes. Over 80% of the 14CO2 formed from [1-14C]cysteinesulphinate could be accounted for by production of hypotaurine plus taurine in incubations of rat hepatocytes with either 1 mM- or 25 mM-cysteinesulphinate. In similar incubations with 1 mM- or 25 mM-cysteine, less than 10% of 14CO2 evolution from [1-14C]cysteine could be accounted for by production of hypotaurine plus taurine. In incubations with cysteine, but not with cysteinesulphinate, the production of urea and ammonia was substantially increased above that observed in incubations without substrate. Addition of unlabelled cysteinesulphinate did not affect 14CO2 production from [1-14C]cysteine. Addition of 2-oxoglutarate resulted in a marked increase in cysteinesulphinate catabolism via the transamination pathway, but addition of neither 2-oxoglutarate nor pyruvate to the incubation system had any effect on cysteine catabolism. Inhibition of cystathionase with propargylglycine decreased 14CO2 production from [1-14C]cysteine about 50% and markedly decreased production of ammonia plus urea N; cysteinesulphinate catabolism by cysteinesulphinate-independent pathways in the rat hepatocyte and, furthermore, that cleavage of cyst(e)ine by cystathionase may be an important physiological pathway for cysteine catabolism in rat liver.     

Reduced glutathione protection against rat liver microsomal injury by carbon tetrachloride. Dependence on O2.

Rat liver microsomal membranes contain a reduced-glutathione-dependent protein(s) that inhibits lipid peroxidation in the ascorbate/iron microsomal lipid peroxidation system. It appears to exert its protective effect by scavenging free radicals. The present work was carried out to assess the effect of this reduced-glutathione-dependent mechanism on carbon tetrachloride-induced microsomal injury and on carbon tetrachloride metabolism because they are known to involve free radicals. Rat liver microsomes were incubated at 37 degrees C with NADPH, EDTA and carbon tetrachloride. The addition of 1 mM-reduced glutathione (GSH) markedly inhibited lipid peroxidation and glucose 6-phosphatase inactivation and, to a lesser extent, inhibited cytochrome P-450 destruction. GSH also inhibited covalent binding of [14C]carbon tetrachloride-derived 14C to microsomal protein. These results indicate that a GSH-dependent mechanism functions to protect the microsomal membrane against free-radical injury in the carbon tetrachloride system as well as in the iron-based systems. Under anaerobic conditions, GSH had no effect on chloroform formation, carbon tetrachloride-induced destruction of cytochrome P-450 or covalent binding of [14C]carbon tetrachloride-derived 14C to microsomal protein. Thus, the GSH protective mechanism appears to be O2-dependent. This suggests that it may be specific for O2-based free radicals. This O2-dependent GSH protective mechanism may partly underlie the observed protection of hyperbaric O2 against carbon tetrachloride-induced lipid peroxidation and hepatotoxicity.