Transient decrease of liver cytosolic glutathione S-transferase activities in rats given 1,2-dibromoethane or CCl4

In vivo treatment of fasted male rats with 1,2-dibromoethane (DBE) (0.4 mmol/kg) or carbon tetrachloride (CCl4) (4 mmol/kg) was found to rapidly alter the activities of liver cytosolic and microsomal glutathione S-transferases. Microsomal activities towards chloro-2,4-dinitrobenzene (CDNB) were increased 2 h after either treatment. Cytosolic activities towards CDNB and 3,4-dichloronitrobenzene (DCNB), but not 1,2-epoxy-3-(p-nitrophenoxy)-propane (ENPP), were selectively and transiently decreased after either treatment. Time course studies in DBE animals indicated that the decrease in cytosolic activity was not evident until 2 h although liver glutathione (GSH) concentrations were diminished within 15 min. In contrast, in CCl4 animals the decrease in cytosolic activity was evident within 15 min and was not accompanied by diminished GSH concentrations. By 4 h, cytosolic activities had rebounded to control levels in both DBE and CCl4-treated animals. Kinetic studies of the enzyme in liver cytosol from animals 2 h after treatment with DBE or CCl4 indicated that both treatments decreased the apparent Vmax while neither treatment altered the apparent Km. This pattern of change allows exclusion of a simple competitive mechanism of enzyme inhibition, but cannot distinguish between reversible non-competitive inhibition and irreversible inhibition. It is possible that the observed decreases in the activities of the abundant cytosal enzyme are due to 'sacrificial' covalent linkages between the enzyme and reactive metabolites of DBE or CCl4.     

Change of liver metabolism of 1,2-dibromoethane during simultaneous treatment with carbon tetrachloride

The combination of carbon tetrachloride (CCl₄) and 1,2-dibromoethane (DBE) in isolated rat hepatocytes led to a significant potentiation of both lipid peroxidation and of plasma membrane damage observed after a single treatment with CCl₄. Such a synergistic effect appeared to be related to the CCl₄-induced shift of DBE metabolism from the cytosolic conjugation with glutathione towards the microsomal transformation into toxic intermediates. In fact, CCl₄ significantly inactivated hepatocyte total GSH-transferase, i.e. the DBE detoxification pathway. Furthermore, while the microsomal metabolism of CCl₄ was not affected by the simultaneous presence of DBE, the amount of DBE reactive metabolities covalently bound to hepatocyte protein was significantly enhanced in the presence of CCl₄.     

Methyl linoleate ozonide as a substrate for rat glutathione S-transferases: reaction pathway and isoenzyme selectivity

The 9,10-mono-ozonide of methyl linoleate was shown to be a substrate for rat hepatic cytosolic, rat lung cytosolic and rat hepatic microsomal glutathione S-transferases (GST). The activities of lung cytosol and liver microsomes with methyl linoleate ozonide (MLO) were found to be high relative to the activity demonstrated by liver cytosol, as compared with their respective activities towards 1-chloro-2,4-dinitrobenzene (CDNB). Only a slight catalytic activity towards the ozonide was noticed for rat lung microsomes. Isoenzyme 2-2 exhibited the highest specific activity (208 nmol/min/mg) when isoenzymes 1-1, 1-2, 2-2, 3-3, 3-4, 4-4 and 7-7 were compared. This isoenzyme accounts for approx. 25% of cytosolic GST protein in rat lung, while in rat liver it represents approx. 9%. This may partly explain the high activity towards the ozonide noticed for rat lung cytosol. No stable conjugates were formed as products of the reaction of MLO with glutathione; although two glutathione-conjugates were noticed on TLC, they were only formed as intermediate compounds. Coupling of an aldehyde dehydrogenase assay or a glutathione reductase assay to the GST-catalyzed conjugation, demonstrated that oxidized glutathione and aldehydes are formed as the major products in the reaction. To further confirm the formation of aldehydes, the products of the GST-catalyzed reaction were incubated with 2,4-dinitrophenylhydrazine, which resulted in hydrazone formation. In conclusion, the activity of the GST towards the ozonide of methyl linoleate is similar to their peroxidase activity with lipid hydroperoxides as substrates.     

Activation of microsomal glutathione transferase activity by reactive intermediates formed during the metabolism of phenol

The activity of microsomal glutathione transferase was increased 1.7-fold in rat liver microsomes which carried out NADPH dependent metabolism of phenol. Known phenol metabolites were therefore tested for their ability to activate the microsomal glutathione transferase. The phenol metabolites benzoquinone and 1,2,4-benzenetriol both activated the glutathione transferase in microsomes 2-fold independently of added NADPH. However, NADPH was required to activate the enzyme in the presence of hydroquinone. Catechol did not activate the enzyme in microsomes. The purified enzyme was activated 6-fold and 8-fold by 5 mM benzenetriol and benzoquinone respectively. Phenol, catechol or hydroquinone had no effect on the purified enzyme. When microsomal proteins that had metabolized [14C]phenol were examined by SDS polyacrylamide gel electrophoresis and fluorography it was found that metabolites had bound covalently to a protein which comigrated with the microsomal glutathione transferase enzyme. We therefore suggest that reactive metabolites of phenol activate the enzyme by covalent modification. It is discussed whether the binding and activation has general implications in the regulation of microsomal glutathione transferase and, since some reactive metabolites might be substrates for the enzyme, their elimination through conjugation.     

Irreversible protein binding of norethisterone (norethindrone) epoxide.

14,15-3H-Norethisterone-4 beta, 5 beta-epoxide, a metabolite of norethisterone, was incubated with several proteins and nucleic acids. After 30 min incubation 0.19 nmol of the epoxide were irreversibly bound per mg albumin which contains free sulfhydryl groups; proteins without SH-groups, such as concanavalin A, gamma-globulin, DNA and RNA, did not irreversibly bind norethisterone epoxide. A superoxide (O2) generating enzyme system comprised of xanthine oxidase and hypoxanthine was capable of catalyzing the irreversible binding of the parent compound, norethisterone, to albumin, indicating that an oxidation product was formed which reacted with the protein. When norethisterone epoxide was incubated for 60 min with hepatic microsomes of rats in absence of NADPH, about 2.0 nmol of the epoxide were irreversibly incorporated per mg microsomal protein. This binding was increased to 5.2 nmol by addition of a NADPH regenerating system. Addition of glutathione and cytosol decreased only the NADPH-dependent protein binding; phenobarbital pretreatment of rats induced this NADPH-dependent binding of norethisterone epoxide to microsomal protein by a factor of 2. In presence of NADPH, binding of the epoxide to microsomal protein depended on substrate concentration used. The results indicate that norethisterone epoxide is able to chemically react with proteins. In addition, hepatic microsomal enzymes convert the epoxide to another metabolite which also can react with proteins.     

Paradoxical enhancement of the toxicity of 1,2-dibromoethane by O6-alkylguanine-DNA alkyltransferase

The presence of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) paradoxically increases the mutagenicity and cytotoxicity of 1,2-dibromoethane (DBE) in Escherichia coli. This enhancement of genotoxicity did not occur when the inactive C145A mutant of human AGT (hAGT) was used. Also, hAGT did not enhance the genotoxicity of S-(2-haloethyl)glutathiones that mimic the reactive product of the reaction of DBE with glutathione, which is catalyzed by glutathione S-transferase. These experiments support a mechanism by which hAGT activates DBE. Studies in vitro showed a direct reaction between purified recombinant hAGT and DBE resulting in a loss of AGT repair activity and a formation of an hAGT-DBE conjugate at Cys(145). A 2-hydroxyethyl adduct was found by mass spectrometry to be present in the Gly(136)-Arg(147) peptide from tryptic digests of AGT reacted with DBE. Incubation of AGT with DBE and oligodeoxyribonucleotides led to the formation of covalent AGT-oligonucleotide complexes. These results indicate that DBE reacts at the active site of AGT to generate an S-(2-bromoethyl) intermediate, which forms a highly reactive half-mustard at Cys(145). In the presence of DNA, the DNA-binding function of AGT facilitates formation of DNA adducts. In the absence of DNA, the intermediate undergoes hydrolytic decomposition to form AGT-Cys(145)-SCH(2)CH(2)OH.     

Purification of a new acidic glutathione S-transferase, GST-Yn1Yn1, with a high leukotriene-C4 synthase activity from rat brain

A new acidic form of glutathione S-transferase (GST, pI 6.2) was purified from rat brain by S-hexylglutathione affinity chromatography followed by chromatofocusing. This form occupied 20-25% of the total activity bound to the affinity column. It had a molecular mass (subunit 26 kDa) similar to that of a major GST form of rat testis (MT or 6-6) on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. However, it differed from the MT in isoelectric point, activity towards 1,2-dichloro-4-nitrobenzene and immunological properties. On two-dimensional gel electrophoresis the brain form gave a spot which was identical in molecular mass, isoelectric point and immunological properties to a less acidic one (Yn1) of two spots (Yn1 and Yn2) of the testis GST-MT. Therefore, the brain acidic form is a homodimer, and named GST-Yn1Yn1. The activity was inhibited by sulfasalazine, an inhibitor of leukotriene-C4 synthase. This form (GST-Yn1Yn1) showed the highest leukotriene-C4 synthase activity, 496 nmol/mg protein in 5 min, among nine cytosolic GST isoenzymes from the rat. The Km values for leukotriene A4 and glutathione were 26 microM and 3.5 mM respectively. A major GST form of rat brain, occupying about 40% of the total activity, was identical with GST-P (7-7) purified from rat liver bearing preneoplastic hyperplastic nodules and localized at astroglias. GST-P also showed the significant leukotriene-C4 synthase activity, 67.2 nmol/mg protein in 5 min, but the Km for leukotriene A4 was 100 microM, fourfold higher than that of GST-Yn1 Yn1. These results suggest that mainly GST-Yn1 Yn1 may be involved in leukotriene-C4 synthesis in rat brain.     

Irreversible binding of ethynyl-estradiol metabolites to protein and nucleic acids as catalyzed by rat liver microsomes and mushroom tyrosinase

It is known that most carcinogenic chemicals can be bound irreversibly to proteins or nucleic acids. A study is presented that explored the irreversible binding of 17alpha-ethinyl estradiol to proteins and nucleic acids by the catalytic action of rat liver microsomes and muschroom tyrosinase. The microsomal binding reaction was inhibited by glutathione and cysteine and its derivatives though it was not affected by lysine and amines. Binding reactivity in the tyrosinase system was inhibited by glutathione, cysteine and its derivates, lysine, and amines. Polylysine did not bind the metabolite of ethinyl estradiol, which suggests the NH2 groups do not bind to the intermediate in the microsomal reaction. However, polylysine did bind irreversibly with esthinyl estradiol metabolites in tyrosinase catalysis. The nonenzymatic reaction of 17beta-hydroxy-4,10(1)-estradiene-2,3-dione with cysteine, lysine, and lysine derivates was found to support the thesis that estrogen o-quinones are the intermediates involved in the protein binding of estrogens in tyrosinase catalysis. An irreversible binding of ethinyl estradiol to DNA and RNA occurred with tyrosinase but not with rat liver microsomes. It was concluded that the results of rat liver microsome catalysis make it unlikely that estrogens are chemical carcinogens.     

Estrogen 1,2-epoxides or estrogen quinones/semiquinones

Metabolic activation of estradiol leading to the formation of catechol estrogens is a prerequisite for its genotoxic activity. Both estrogen-o-quinones/semiquinones and estrogen 1,2-epoxides have been proposed to be responsible for this activity. Incubations of [3H]estradiol and [3H]1 alpha,2 alpha-epoxy-4-estrene-3-one-17 beta-ol (ketotautomer of estradiol 1,2-epoxide) with rat liver microsomal and cytosol preparations were carried out in the presence of SKF 525A, ascorbic acid, glutathione and cysteine. Ascorbic acid decreased binding to proteins and aqueous-soluble fraction with both [3H] estradiol and [3H]epoxyestrenolone in incubations with microsomes but no effect with cytosol fraction. Incubations of microsomes with thiols gave water-soluble metabolites which were characterized as 1(4)-thioether derivatives of 2-hydroxyestradiol and incubations of [3H]epoxyestrenolone with cytosol and thiols gave estradiol-2-thioether. Incubations with ascorbic acid and thiols resulted in decreased formation of water-soluble metabolites in microsomal incubations but not in cytosol incubations. These studies indicate that the major pathway for irreversible binding of estrogens to macromolecules involves estrogen-o-quinones/semiquinones and not estrogen 1, 2-epoxide.     

Interaction of 1,4-benzoquinone and 2,4-dichlorophenoxyacetic acid with microsomal glutathione transferase from rat liver

Glutathione transferase (GST) was purified from the microsomes of rat liver by glutathione affinity chromatography. The interaction of 2,4-dichlorophenoxyacetic acid (2,4-D) and 1,4-benzoquinone with microsomal GST was investigated and compared with cytosolic GST. The kinetic inhibition pattern of 1,4-benzoquinone towards microsomal GST was found to be different from that towards cytosolic GST. Microsomal GST purified by affinity chromatography was inhibited by 2,4-D in a non dose-dependent manner, while the crude microsomal GST was inhibited in a dose-dependent manner. This difference was shown to be induced by a reaction on the affinity column, and not by Triton X-100 (also shown to be a GST inhibitor), glutathione, or the elution buffer 0.2% Triton X-100 and 5 mM glutathione in 50 mM Tris-HCl, pH 9.6. The binding of microsomal GST to the affinity matrix caused a partial inactivation of the active site for 2,4-D interaction. The results show that the properties of soluble GST enzymes may not be extrapolated to the microsomal ones.     

Inactivation of cytochrome P-450 by 2-isopropyl-4-pentenamide and other xenobiotics leads to heme-derived protein adducts

When cytochrome P-450 in phenobarbital-induced rat liver microsomes was destroyed by 2-isopropyl-4-pentenamide (AIA) in vitro, 50% of the degraded heme was recovered as heme-derived products irreversibly bound to microsomal proteins. In contrast, less than 50% of the degraded heme was accounted for as N-alkylated porphyrins. Furthermore, 64% of the irreversibly bound products was bound specifically to a 54-kD form of cytochrome P-450. Several other compounds which have been reported to destroy cytochrome P-450 by forming N-alkylated porphyrins also produced heme-derived protein adducts. These findings indicate that the formation of heme-derived protein adducts may represent an important pathway for the irreversible degradation of cytochrome P-450 by many xenobiotics.     

Enzymatic reaction of chlorotrifluoroethene with glutathione: 19F NMR evidence for stereochemical control of the reaction

Chlorotrifluoroethene, a potent nephrotoxin, is a substrate for the glutathione S-transferases present in the cytosolic and microsomal fractions of rat liver. The glutathione conjugate formed by both subcellular fractions has been identified as S-(2-chloro-1,1,2-trifluoroethyl)glutathione by 1H and 19F NMR and by secondary ion mass spectrometry. The conjugate formed by the cytosolic fraction is an equimolar mixture of two diastereomers, whereas the conjugate formed by the microsomal fraction is predominantly one diastereomer, as judged by the 19F NMR spectra. No evidence for the formation of S-(trihalovinyl)glutathione derivatives by an addition/elimination reaction was found. High-performance liquid chromatography was employed to measure the rates of glutathione conjugate formation in vitro. The rates of S-(2-chloro-1,1,2-trifluoroethyl)glutathione formation were 75-107 nmol min-1 (mg of protein)-1 and 151-200 nmol min-1 (mg of protein)-1 catalyzed by the cytosolic and microsomal fractions, respectively (measured at pH 7.4, 37 degrees C, with 5 mM glutathione). These results suggest that glutathione conjugation occurs at high rates in vivo to produce the highly nephrotoxic S-(2-chloro-1,1,2-trifluoroethyl)glutathione.     

Evidence for binding of metabolically activated 1,2-dibromoethane to chromatin of forestomach and liver

The covalent binding of metabolically activated 1,2-dibromoethane (DBE), a potent carcinogen, to chromatin constituents of forestomach and liver was examined $\text{in vitro}$. Chromatin was prepared from forestomach and liver of B6C3F1 mice and characterized. In order to activate DBE, microsomes and cytosol were isolated from mouse forestomach and liver and incubated with [¹⁴C]-DBE in the presence of a NADPH regenerating system. Results demonstrate that DBE bound covalently to the same extent to protein of microsomes and chromatin isolated from forestomach and liver. On the contrary, DBE bound significantly more to chromatin DNA of forestomach or liver than it did to salmon sperm DNA. It appears from these results that the metabolically activated DBE is more reactive to homologous DNA than exogenous DNA. Fractionation of DBE-bound chromatin protein into histone and nonhistone proteins resulted in higher binding of DBE to non-histone than to histone proteins isolated from forestomach and liver.     

The gene for the microsomal glutathione S-transferase is on human chromosome 12

The microsomal glutathione S-transferase (GST) is a unique membrane-bound GST structurally distinct from the cytosolic GSTs. A cDNA encoding this 154 amino acid protein has recently been isolated and characterized. Using the cDNA as the hybridization probe, we now report the assignment of the human microsomal GST gene to chromosome 12 through the use of a panel of mouse-human somatic cell hybrid lines. This locus has recently been designated as GST 12. In addition, genomic Southern blotting data suggest that the human microsomal GST is encoded by a single- or very-low-copy gene. Therefore, the human GST gene superfamily resides on at least four separate chromosomes: 1 (GST 1), 6 (GST 2), 11 (GST 3), and 12 (GST 12).     

Isolation and purification of glutathione S-transferases from Brachionus plicatilis and B. calyciflorus (Rotifera)

1. The enzyme glutathione S-transferase (GST), a critical element in xenobiotic metabolism, was isolated from the marine rotifer Brachionus plicatilis and its freshwater congener B. calyciflorus. 2. In B. plicatilis, GST comprised 4.2% of cytosolic protein and was present as three separate isozymes with mol. wts 30,000, 31,400 and 33,700. Specific activity of crude homogenates was 56 nmol min-1 mg-1 protein, while that of affinity chromatography purified GST was 1850. 3. In B. calyciflorus, GST was present as two isozymes with mol. wts of 26,300 and 28,500, representing 1.0% of cytosolic protein. Crude GST specific activity was 1750 nmol min-1 mg-1 protein and purified was 72,400. 4. Rotifer GSTs are unusual because they are monomers whereas all other animals thus far investigated posses dimeric GSTs.     

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.     

Biochemical Characterization and Distribution of Glutathione S-Transferases in Leaping Mullet (Liza saliens)

In this study, feral leaping mullet (Liza saliens) liver cytosolic glutathione S-transferases (GSTs) were investigated and characterized using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA) as substrates. The average GST activities towards CDNB and EA were found to be 1365 +/- 41 and 140 +/- 20 nmol/min per mg protein, respectively. The effects of cytosolic protein amount and temperature ranging from 4 to 70 degrees C on enzyme activities were examined. While both activities towards CDNB and EA showed similar dependence on protein amount, temperature optima were found as 37 and 42 degrees C, respectively. In addition, the effects of pH on GST-CDNB and -EA activities were studied and different pH activity profiles were observed. For both substrates, GST activities were found to obey Michaelis-Menten kinetics with apparent V(max) and K(m) values of 1661 nmol/min per mg protein and 0.24 mM and 157 nmol/min per mg protein and 0.056 mM for CDNB and EA, respectively. Distribution of GST in Liza saliens tissues was investigated and compared with other fish species. Very high GST activities were measured in tissues from Liza saliens such as liver, kidney, testis, proximal intestine, and gills. Moreover, our results suggested that GST activities from Liza saliens would be a valuable biomarker for aquatic pollution.     

DNA adduction by polychlorinated biphenyls: adducts derived from hepatic microsomal activation and from synthetic metabolites

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants and complete carcinogens in rodents. Metabolism of lower chlorinated congeners with rat liver microsomes was investigated in earlier studies and DNA adduction was also reported. The current study was designed to compare DNA adducts formed after bioactivation of PCBs with rat, mouse and human hepatic microsomes, and to investigate the role of quinoid PCB metabolites in DNA adduct formation. Eight congeners ranging from mono- to hexachlorinated biphenyls were tested. Metabolites obtained through microsomal bioactivation as well as synthetic quinoid metabolites of 4-monochlorobiphenyl (4-CB) were incubated with calf-thymus DNA (CT-DNA), and the resulting adducts were analyzed by the 32P-post-labelling method. DNA adducts were formed with mono- di- and tri-chlorinated congeners, but not with higher chlorinated congeners. Similar adduct patterns were observed for 2-monochlorobiphenyl (2-CB) activated with hepatic microsomes from rat, mouse and human, while 4-CB, 3,4-dichlorobiphenyl (3,4-CB) and 3,4,5-trichlorobiphenyl (3,4,5-CB) showed similar patterns for two out of the three microsomal systems tested. 4,4' -trichlorobiphenyl (4,4' -CB) showed different adduct patterns in all microsomal systems. Higher adduct levels were obtained with the rodent microsomes compared with human microsomes and were related to higher cytochrome P450 activity. When adducts derived from microsomal activation of 4-CB were compared by co-chromatography with those derived from the incubation of DNA with synthetic 2-(4' -chlorophenyl)-1,4-benzoquinone (4-BQ), one adduct co-migrated in three different chromatography systems. This study demonstrates that rodents as well as human hepatic enzymes metabolize lower chlorinated biphenyl congeners to reactive intermediates that form DNA adducts in vitro and shows that the para-quinone metabolites of PCBs are, in part, involved in direct DNA adduction.     

Human cytochrome-P450 enzymes metabolize N-(2-methoxyphenyl)hydroxylamine, a metabolite of the carcinogens o-anisidine and o-nitroanisole, thereby dictating its genotoxicity

N-(2-Methoxyphenyl)hydroxylamine is a component in the human metabolism of two industrial and environmental pollutants and bladder carcinogens, viz. 2-methoxyaniline (o-anisidine) and 2-methoxynitrobenzene (o-nitroanisole), and it is responsible for their genotoxicity. Besides its capability to form three deoxyguanosine adducts in DNA, N-(2-methoxyphenyl)-hydroxylamine is also further metabolized by hepatic microsomal enzymes. To investigate its metabolism by human hepatic microsomes and to identify the major microsomal enzymes involved in this process are the aims of this study. N-(2-Methoxyphenyl)hydroxylamine is metabolized by human hepatic microsomes predominantly to o-anisidine, one of the parent carcinogens from which N-(2-methoxyphenyl)hydroxylamine is formed, while o-aminophenol and two N-(2-methoxyphenyl)hydroxylamine metabolites, whose exact structures have not been identified as yet, are minor products. Selective inhibitors of microsomal CYPs, NADPH:CYP reductase and NADH:cytochrome-b(5) reductase were used to characterize human liver microsomal enzymes reducing N-(2-methoxyphenyl)hydroxylamine to o-anisidine. Based on these studies, we attribute the main activity for this metabolic step in human liver to CYP3A4, 2E1 and 2C (more than 90%). The enzymes CYP2D6 and 2A6 also partake in this N-(2-methoxyphenyl)hydroxylamine metabolism in human liver, but only to ～6%. Among the human recombinant CYP enzymes tested in this study, human CYP2E1, followed by CYP3A4, 1A2, 2B6 and 2D6, were the most efficient enzymes metabolizing N-(2-methoxyphenyl)hydroxylamine to o-anisidine. The results found in this study indicate that genotoxicity of N-(2-methoxyphenyl)hydroxylamine is dictated by its spontaneous decomposition to nitrenium/carbenium ions generating DNA adducts, and by its susceptibility to metabolism by CYP enzymes.     

Is fluoroacetate-specific defluorinase a glutathione S-transferase?

Fluoroacetate-specific defluorinase (FSD) is a critical enzyme in the detoxication of fluoroacetate. This study investigated whether FSD can be classed as a glutathione S-transferase (GST) isoenzyme with a high specificity for fluoroacetate detoxication metabolism. The majority of FSD and GST activity, using 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as GST substrates, in rat liver was cytosolic. GSTT1 specific substrate, EPNP caused a slight non-competitive inhibition of FSD activity. CDNB, a general substrate of GST isoenzyme, was a more potent non-competitive inhibitor of FSD activity. The fluoroacetate defluorination activity by GST isoenzymes was determined in this study. The results showed that the GSTZ1C had the highest fluoroacetate defluorination activity of the various GST isoenzymes studied, while GSTA2 had a limited activity toward fluoroacetate. The human GSTZ1C recombinant protein then was purified from a human GSTZ1C cDNA clone. Our experiments showed that GSTZ1C catalysed fluoroacetate defluorination. GSTZ1 shares many of the characteristics of FSD; however, it accounts only for 3% of the total cytosolic FSD activity. GSTZ1C based enzyme kinetic studies has low affinity for fluoroacetate. The evidence suggests that GSTZ1 may not be the major enzyme defluorinating fluoroacetate, but it does detoxify the fluoroacetate. To clarify the identity of enzymes responsible for fluoroacetate detoxication, further studies of the overall FSD activity are needed.