Glutathione S-transferases in nitrogen mustard-resistant and -sensitive cell lines

Tumor cell resistance to alkylating agents was studied by examining Walker 256 rat mammary carcinoma cells differentially sensitive to nitrogen mustards. A resistant subpopulation (WR) was selected by exposure to chlorambucil. WR cells showed approximately a 15-fold resistance to the cytotoxic effects of nitrogen mustards and elevated glutathione S-transferase (GST) activity when compared to the sensitive parent cell line (WS). To extend these findings, the GSTs from WR and WS were purified by affinity chromatography on S-hexylglutathione coupled to epoxy-activated agarose. Substrate specificity experiments using purified GSTs demonstrated different profiles of enzyme activity for WR and WS and suggested differential isoenzyme expression in these two cell lines. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis revealed that the major GST present in both WR and WS was a 26,000-Da subunit that was immunologically distinct from the rat liver GSTs. This GST subunit cross-reacted with antibodies against anionic human placental GST. In addition, three GST forms common to rat liver (29,500, 28,500 and 27,500 molecular weight) were also identified. Overexpression of the 29,500-Da protein was observed in WR cells. These data suggest that differential expression of GST subunits may contribute to the nitrogen mustard-resistant phenotype.     

Increased antitumour activity of chlorambucil following pretreatment with inducers of drug-metabolizing enzymes

Alkylating agent-sensitive and resistant lines of tumour cells growing either in the rat or in suspension culture have been pretreated with inducers of microsomal drug metabolizing enzymes prior to chlorambucil. This enhanced the cytotoxicity of chlorambucil in the sensitive tumour cells but was without effect on the resistant cells. In the intact animal the drug combination of phenobarbitone and chlorambucil did not appear to be more toxic to normal bone marrow cells than chlorambucil alone.     

Kinetics of propylene oxide metabolism in microsomes and cytosol of different organs from mouse, rat, and humans

Kinetics of the metabolic inactivation of 1,2-epoxypropane (propylene oxide; PO) catalyzed by glutathione S-transferase (GST) and by epoxide hydrolase (EH) were investigated at 37 degrees C in cytosol and microsomes of liver and lung of B6C3F1 mice, F344 rats, and humans and of respiratory and olfactory nasal mucosa of F344 rats. In all of these tissues, GST and EH activities were detected. GST activity for PO was found in cytosolic fractions exclusively. EH activity for PO could be determined only in microsomes, with the exception of human livers where some cytosolic activity also occurred, representing 1-3% of the corresponding GST activity. For GST, the ratio of the maximum metabolic rate (V(max)) to the apparent Michaelis constant (K(m)) could be quantified for all tissues. In liver and lung, these ratios ranged from 12 (human liver) to 106 microl/min/mg protein (mouse lung). Corresponding values for EH ranged from 4.4 (mouse liver) to 46 (human lung). The lowest V(max) value for EH was found in mouse lung (7.1 nmol/min/mg protein); the highest was found in human liver (80 nmol/min/mg protein). K(m) values for EH-mediated PO hydrolysis in liver and lung ranged from 0.83 (human lung) to 3.7 mmol/L (mouse liver). With respect to liver and lung, the highest V(max)/K(m) ratios were obtained for GST in mouse and for EH in human tissues. GST activities were higher in lung than in liver of mouse and human and were alike in both rat tissues. Species-specific EH activities in lung were similar to those in liver. In rat nasal mucosa, GST and EH activities were much higher than in rat liver.     

Glutathione-S-transferase activity toward aflatoxin epoxide in livers of mastomys and other rodents.

In order to study the liver glutathione-S-transferase (GST) activity toward aflatoxin B1 (AFB1) epoxide in mastomys in comparison with other rodents, we performed in vitro studies of the cytosolic GST activity toward AFB1-epoxide using mastomys, rat, mouse and hamster liver. Also AFB1 metabolism by liver microsomes including formation of AFB1-DNA adducts was studied. Cytosolic GST activity toward AFB1-epoxide was highest in mastomys liver, and higher in the hamster and mouse livers than in the rat liver, correlating well with the differences of the sensitivity of these species to the toxicity of AFB1. However, no relationship was noted between the sensitivity of a given species to the toxicity of AFB1 and the microsomal activity of binding of AFB1 to DNA or metabolizing AFB1 to AFM1, AFQ1 and AFP1. These results demonstrate the importance of the GST mediated AFB1-epoxide conjugation with glutathione in determining the differing sensitivities of these species to AFB1 toxicity. The extremely high activity of GST in mastomys indicates that this species would be a good model animal for studying GST toward AFB1-epoxide.     

The effect of tridiphane (2-(3,5-dichlorophenyl)-2-(2,2,2-trichloroethyl)oxirane) on hepatic epoxide-metabolizing enzymes: indications of peroxisome proliferation

Administration of tridiphane (Tandem, DOWCO 356, 2-(3,5-dichlorophenyl)-2-(2,2,2-trichloroethyl)oxirane) to male Swiss-Webster mice for 3 days at 100, 250, and 500 mg/kg (ip) resulted in increases in liver weight accompanied by an increase in mitotic index and increases in large particle and microsomal protein. Epoxide hydrolase (EH) activity towards cis-stilbene oxide (CSO, microsomal EH) was elevated in microsomes and cytosol, a decrease in microsomal cholesterol EH was found, and hydrolysis of trans-stilbene oxide (TSO, cytosolic EH) was elevated in the cytosol but not in the microsomes. Glutathione S-transferase (GST) activity was elevated in cytosol for CSO, TSO, and 1,2-dichloro-4-nitrobenzene (DCNB), with inconsistent responses found with 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (ENPP). Microsomal GST was not consistently effected by tridiphane. Clofibrate (500 mg/kg, 3 daily ip injections) treatment resulted in similar responses in liver size, microsomal protein, and the EHs. The increase in cytosolic EH activity previously has been noted only in animals treated with peroxisome proliferators. Examination of livers from mice treated with 250 mg/kg tridiphane revealed that an increase in hepatic peroxisomes was apparent after 3 days of treatment. This was accompanied by decreases in serum cholesterol and triglyceride levels and increases in liver carnitine acetyl transferase and cyanide-insensitive oxidation of palmitoyl-CoA. This study demonstrates that tridiphane does have in vivo effects on mammalian epoxide-metabolizing enzymes and extends the association of increased cytosolic epoxide hydrolase activity with peroxisome proliferation.     

In vitro metabolism of amiodarone by rabbit and rat liver and small intestine

The present studies were designed to investigate whether amiodarone (Am) is metabolized in the major organs and tissues of the rat and rabbit. Incubations using Am and tissue homogenates (600 g supernatant) of rabbit and rat lung, liver, kidney, and gut revealed formation of desethylamiodarone (DEA) by the liver and gut. Subsequent experiments using the post-mitochondrial, cytosolic, and microsomal fractions of these tissues indicated that metabolism of Am was greatest in the microsomal fractions. In both species, greater DEA formation was detected for microsomes of hepatic origin. The hepatic microsomal mediated production of DEA was altered by protein concentration in both the rabbit and rat preparations with protein concentrations of 5 mg providing the greatest DEA production. DEA formation by gut microsomes was greatest at 3 mg of protein for the rabbit but exhibited no significant change from 1 mg to 10 mg of protein for the rat. In vitro metabolism of Am by rabbit and rat hepatic microsomal preparations was significantly reduced by 1 mM piperonyl butoxide, SKF 525-A, n-octylamine, and carbon monoxide. Effects of these inhibitors on rabbit and rat gut microsomal incubations were inconclusive. HPLC analysis of incubation samples revealed a species difference in the metabolism of Am as demonstrated by the detection of three metabolites in addition to DEA. The unidentified metabolites (I, II, III) were detected in rabbit hepatic microsomal incubations. Metabolite II was also detected in incubations using rabbit duodenal tissue microsomes. No metabolites other than DEA were found in incubations using rat tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of microsomal and cytosolic glutathione S-transferase activities by S-nitrosylation

There is increasing evidence that S-nitrosylation is a mechanism for the regulation of protein function via the modification of critical sulfhydryl groups. The activity of rat liver microsomal glutathione S-transferase (GST) is increased after treatment with N-ethylmaleimide (NEM), a sulfhydryl alkylating reagent, and is also increased under conditions of oxidative stress. In the present study, preincubation of purified rat liver microsomal GST with S-nitrosoglutathione (GSNO) or the nitric oxide (NO) donor, 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO), resulted in a 2-fold increase in enzyme activity. This increase in activity was reversed by dithiothreitol. The initial treatment of microsomal GST with either GSNO or DEA/NO was associated with an 85% loss of free sulfhydryl groups. After removal of the nitrosylating agents over a 6-hr period, approximately 50% of the enzyme was still nitrosylated, as determined by redox chemiluminescence. Furthermore, preincubation of either purified enzyme or hepatic microsomes with GSNO or DEA/NO prevented further enzyme activation by NEM, suggesting that NEM and the NO donors interact with a common population of sulfhydryl groups in the enzyme. In contrast, both NEM and NO donors partially inhibited the activity of cytosolic GST isoforms. The inhibitory activity of NEM and NO donors was much more evident when the GST pi isoform was used instead of a mixture of GST isoforms. These data suggest that there may be differential regulation of microsomal and cytosolic GST activities under conditions of nitrosative stress.     

Acinar distribution of glutathione-dependent detoxifying enzymes. Low glutathione peroxidase activity in perivenous hepatocytes

The acinar distribution of glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G-6-PDH) was examined by analyzing periportal (p.p.) and perivenous (p.v.) rat hepatocytes selectively isolated by the digitonin-collagenase perfusion. The cytosolic GST activity was higher in p.v. cells, but the microsomal GST and cytosolic GR were found to be evenly distributed in the acinus. In contrast, the activity of both the Se-dependent GPx and the microsomal (Se-independent) GPx, as well as G-6-PDH, was much lower in the p.v. than in the p.p. cells. The heterogeneous distribution of GST, GPx and G-6-PDH was confirmed by analyzing liver perfusion effluents collected after ante- or retrograde digitonin infusion. The relatively low activities of GPx and G-6-PDH in the p.v. cells could partly explain the susceptibility of this region to chemical injury.     

Metabolism of CDRI-85/92, a new potent anti-ulcer agent, involving cis-trans conversion

CDRI 85/92, an anti-ulcer drug, is a new proton pump inhibitor, currently in an advanced stage of drug development. To know more about the drug it was our objective to delineate/identify the metabolic pathway as well as the enzymes responsible for the formation of metabolites. Metabolism of CDRI-85/92 (cis-5-styryl-2-oxazolidinone-4-carboxylic acid) was investigated in rat liver cellular fractions (S9, microsomes and cytosol) using reverse-phase HPLC and mass spectrometry techniques. Two major metabolites were produced by rat liver S9 fractions and reducing factor generating system from either untreated rats or phenobarbitone (PB)-pretreated rats. Incubation of CDRI-85/92 with postmitochondrial fraction (S9) for 24 h resulted in a cis to trans conversion (metabolite M2). Further cis-trans metabolizing capacity was measured separately in the cytosolic and microsomal fractions. Incubation with the cytosolic fraction resulted in an increased rate of cis-trans conversion, while the microsomal fraction showed no cis to trans conversion, thereby restricting the cis to trans conversion to Phase II enzymes, which are mainly located in the cytosol. Studies with PB-pretreated rat liver S9 fractions resulted in an increased rate of cis to trans conversion. Another metabolite was also present (M1) which was identified as an oxygenated metabolite by mass spectrometry. The major urinary metabolite from CDRI-85/92-treated Sprague-Dawley rats (20 mg/kg p.o.) was identified as M2. Studies using sulfobromophthalein and N-ethylmaleimide, as specific inhibitors of GST, showed a complete absence of metabolism, thus indicating the involvement of GST in the metabolism of CDRI-85/92. This study will be helpful in providing clues about factors influencing the bioavailability of CDRI-85/92 as well as drug-drug interactions.     

Reductase-mediated metabolism of motexafin gadolinium (Xcytrin) in rat and human liver subcellular fractions and purified enzyme preparations

The biotransformation of motexafin gadolinium (MGd, Xcytrin) was investigated in subcellular rat and human liver fractions. Microsomal MGd metabolism was dependent on NADPH in both species. Cytosolic metabolism in rat and human livers was dependent on NADPH or NADH. Under anaerobic conditions, MGd metabolism increased in liver microsomes and purified enzyme preparations. Cytochrome P450 (CYP450) inhibitors ketoconazole, proadifen, and carbon monoxide increased NADPH-dependent MGd metabolism in microsomes. Treatment of rats with beta-naphthoflavone increased cytosolic metabolism of MGd twofold, but had no effect on microsomal metabolism. Conversely, in liver preparations from phenobarbital treated rats microsomal metabolism of MGd was enhanced twofold, but not in cytosolic preparations. Purified CYP450 reductase from phenobarbital-treated rabbit or untreated human livers metabolized MGd suggesting involvement of CYP450 reductase. Dicumarol, a potent DT-diaphorase inhibitor, inhibited MGd metabolism in both rat and human liver cytosol. These data suggest MGd metabolism in rat liver involves CYP450 reductase and/or DT-diaphorase. In human liver preparations only CYP450 reductase is directly involved in MGd metabolism. A metabolite identified in microsomes and cytosol is a metal-free, reduced form of MGd, indicating that both enzymes generate metabolite 1, which appears to be PCI-0108, a synthetic precursor to MGd.     

Kinetic modeling of beta-chloroprene metabolism: I. In vitro rates in liver and lung tissue fractions from mice, rats, hamsters, and humans.

Beta-chloroprene (2-chloro-1,3-butadiene, CD) is carcinogenic by inhalation exposure to B6C3F1 mice and Fischer F344 rats but not to Wistar rats or Syrian hamsters. The initial step in metabolism is oxidation, forming a stable epoxide (1-chloroethenyl)oxirane (1-CEO), a genotoxicant that might be involved in rodent tumorigenicity. This study investigated the species-dependent in vitro kinetics of CD oxidation and subsequent 1-CEO metabolism by microsomal epoxide hydrolase and cytosolic glutathione S-transferases in liver and lung, tissues that are prone to tumor induction. Estimates for Vmax and Km for cytochrome P450-dependent oxidation of CD in liver microsomes ranged from 0.068 to 0.29 micromol/h/mg protein and 0.53 to 1.33 microM, respectively. Oxidation (Vmax/Km) of CD in liver was slightly faster in the mouse and hamster than in rats or humans. In lung microsomes, Vmax/Km was much greater for mice compared with the other species. The Vmax and Km estimates for microsomal epoxide hydrolase activity toward 1-CEO ranged from 0.11 to 3.66 micromol/h/mg protein and 20.9 to 187.6 microM, respectively, across tissues and species. Hydrolysis (Vmax/Km) of 1-CEO in liver and lung microsomes was faster for the human and hamster than for rat or mouse. The Vmax/Km in liver was 3 to 11 times greater than in lung. 1-CEO formation from CD was measured in liver microsomes and was estimated to be 2-5% of the total CD oxidation. Glutathione S-transferase-mediated metabolism of 1-CEO in cytosolic tissue fractions was described as a pseudo-second order reaction; rates were 0.0016-0.0068/h/mg cytosolic protein in liver and 0.00056-0.0022 h/mg in lung. The observed differences in metabolism are relevant to understanding species differences in sensitivity to CD-induced liver and lung tumorigenicity.     

Piperine effects on the expression of P4502E1, P4502B and P4501A in rat

Treatment of rat with piperine (PIP) (1·4 mmol/kg, 3 days ip injections) resulted in an approximate two-fold increase in total liver microsomal P450 content relative to that in uninduced animals.

2. 4-Nitrophenol and aniline hyroxylase activities in the hepatic microsomes prepared from rat treated with PIP decreased by 30 and 28% respectively as compared with control. Immunoblot analyses also revealed decreased P4502E1 levels in hepatic microsomes from PIP-treated animals.

3. In contrast with P4502E1 suppression, hepatic 2B1 and 2B2 levels were significantly increased in PIP-induced animals, as evidenced by both metabolic activity and immunoblot analysis of the liver microsomal fractions. The rate of hexobarbital hydroxylase activity in microsomes from PIP-treated animals was markedly elevated and was inhibited by approximately 62% in the presence of monoclonal anti-P4502B IgG. Immunoblot analyses demonstrated that P4502B1 ana 2B2 levels in hepatic microsomes from PIP-treated animals were comparable with those from phenobarbital-treated animals.

4. 7-Ethoxycoumarin deethylase activity was elevated approximately two-fold in PIP-induced animals and was 17% of that derived from 3-methylcholanthrene-induced animals. 7-ethoxycoumarin deethylase activity in PIP-induced hepatic microsomes was inhibited 63% in the presence of monoclonal anti-P4501 A antibody. Immunoblot analysis confirmed the increase in P4501A levels by PIP, which was 15% of that in hepatic microsomes from 3-methylcholanthrene-induced animals.

5. PIP treatment failed to affect microsomal epoxide hydrolase (mEH) and glutathione S-transferases (GST) expression, as indicated by immunoblot analyses using polyclonal antibodies toward mEH and GST subunits Ya, Yb1, Yb2 and Yc.

6. These results demonstrate that PIP treatment suppressed P4502E1 expression and enhanced 2B and 1A expression, whereas this agent failed to affect hepatic mEH and GST expression.     

Glutathione S-transferase-mediated chlorothalonil metabolism in liver and gill subcellular fractions of channel catfish

Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile) is a broad spectrum fungicide that is a potent acute toxicant to fish. Therefore, the metabolism of chlorothalonil was investigated in liver and gill cytosolic and microsomal fractions from channel catfish (Ictalurus punctatus) using HPLC. All fractions catalyzed the metabolism of chlorothalonil to polar metabolites. Chlorothalonil metabolism by cytosolic fractions was reduced markedly when glutathione (GSH) was omitted from the reaction mixtures. The lack of microsomal metabolism in the presence of either NADPH or an NADPH-regenerating system indicated direct glutathione S-transferase (GST)-catalyzed conjugation with GSH without prior oxidation by cytochrome P450. Cytosolic and microsomal GSTs from both tissues were also active toward 1-chloro-2,4-dinitrobenzene (CDNB), a commonly employed reference substrate. In summary, channel catfish detoxified chlorothalonil in vitro by GST-catalyzed GSH conjugation in the liver and gill. The present report is the first to confirm microsomal GST activity toward CDNB in gill and toward chlorothalonil in liver, and also of gill cytosolic GST activity towards chlorothalonil, in an aquatic species.     

Properties of the microsomal and cytosolic glutathione transferases involved in hexachloro-1:3-butadiene conjugation

Hexachloro-1,3-butadiene (HCBD) is a substrate for the hepatic microsomal glutathione transferases and is metabolised at higher rates by these enzymes than their cytosolic counterparts. Conjugation reactions catalysed by the microsomal and cytosolic transferases have been studied and characterized using this substrate and 1-chloro-2,4-dinitrobenzene (CDNB). In rat liver microsomes the Km values for HCBD and CDNB were 0.91 and 0.012 mM and in cytosol 0.51 and 0.10 mM respectively. Vmax values for HCBD were 1.39 and 0.35 nmol conjugate formed/min/mg protein for microsomes and cytosol respectively. In microsomal systems HCBD was a potent competitive inhibitor of the metabolism of CDNB with a Ki value of approximately 10 microM. However, CDNB did not inhibit HCBD metabolism significantly. These data suggest that more than one microsomal enzyme is involved in HCBD metabolism. The microsomal membrane could be solubilized without significant inhibition of HCBD activity; however, some detergents did inhibit the conjugation reaction. Activity was also lost on treatment of microsomal membranes with trypsin indicating the enzyme is localized on the cytoplasmic surface of the endoplasmic reticulum. Pretreatment of the rats with Aroclor 1254, 3-methylcholanthrene or phenobarbital did not change the microsomal conjugation of HCBD or CDNB with glutathione. Of seven species investigated, a human liver sample showed the highest ratio of microsomal to cytosolic glutathione transferase activity for HCBD (in microsomes 40-fold higher specific activity than in cytosol). Glutathione conjugation appears to play a critical role in the toxicity and carcinogenicity of some halogenated hydrocarbons. These data substantiate the potentially important role for the microsomal glutathione transferase in catalysing these reactions.     

Metabolic inactivation of five glycidyl ethers in lung and liver of humans, rats and mice in vitro

1. Some glycidyl ethers (GE) have been shown to be direct mutagens in short-term in vitro tests and consequently GE are considered to be potentially mutagenic in vivo. However, GE may be metabolically inactivated in the body by two different enzymatic routes: conjugation of the epoxide moiety with the endogenous tripeptide glutathione (GSH) catalysed by glutathione S-transferase (GST) or hydrolysis of the epoxide moiety catalysed by epoxide hydrolase (EH). 2. The metabolic inactivation of five different GE, the diglycidyl ethers of bisphenol A (BADGE), 4,4'-dihydroxy-3,3',5,5'-tetramethylbiphenyl (Epikote YX4000) and 1,6-hexanediol (HDDGE) and the GE of 1-dodecanol (C12GE) and o-cresol (o-CGE), has been studied in subcellular fractions of human, C3H mouse and F344 rat liver and lung. 3. All GE were chemically very stable and resistant to aqueous hydrolysis, but were rapidly hydrolysed by EH in cytosolic and microsomal fractions of liver and lung. The aromatic GE were very good substrates for EH. In general, microsomal EH is more efficient than cytosolic EH in hydrolysis of GE, and human microsomes are more efficient than rodent microsomes. 4. The more water-soluble GE, o-CGE and HDDGE, were good substrates for GST whereas the more lipophilic GE, YX4000 and C12GE, were poor substrates for GST. In general, rodents are more efficient in GSH conjugation of GE than humans. 5. In general, the epoxide groups of YX4000 are the most and those of HDDGE the least efficiently inactivated of the five GE under study. For the other three GE no general trend was observed: the relative efficiency of inactivation varied with organ and species. 6. The large variation in metabolism observed with five representative GE indicate that GE have variable individual properties and should not be considered as a single, homogenous class of compounds.     

Biphasic kinetics of imipramine N-oxidation in rat brain microsomes

Imipramine (IMI) N-oxidase activity in brain microsomes from rats of both sexes was determined by high performance liquid chromatography, and compared with the results in rat liver microsomes. Brain and liver microsomal IMI N-oxidation was sensitive to thermal inactivation and had an optimal pH at around 9.0. IMI N-oxidase activity (15.54 pmol/min/mg protein) in brain microsomes was about one-hundredth that of liver microsomes (2.08 nmol/min/mg protein) at a substrate concentration of 5 mM. IMI N-oxidase activities in both brain and liver microsomes displayed biphasic kinetics that associated a low Km-low Vmax element with a high Km-high Vmax component. Furthermore, a significant sex difference was observed in Vmax values (male>female) in both phases, but Km values were similar between male and female rats, resulting in a significant sex difference (male>female) in intrinsic clearance values (Vmax/Km) of the low-Km and the high-Km phases.     

Oxidative metabolism of seleno-L-methionine to L-methionine selenoxide by flavin-containing monooxygenases

The roles of flavin-containing monooxygenases (FMOs) in the oxidation of seleno-l-methionine (SeMet) to l-methionine selenoxide (MetSeO) were investigated using cDNA-expressed human FMOs, purified rat liver FMOs, and rat liver microsomes. MetSeO and the N-2,4-dinitrophenyl-derivatives of SeMet and MetSeO were synthesized and characterized by 1H-NMR and ESI/MS. These reference compounds were then used to develop a sensitive HPLC assay to monitor SeMet oxidation to MetSeO. The formation of MetSeO in rat liver microsomes was time-, protein concentration-, SeMet concentration-, and NADPH-dependent. The microsomal activity exhibited a SeMet Km value (mean +/- S.D.; n = 4) of 0.91 +/- 0.29 mM and a Vmax value of 44 +/- 8.0 nmol MetSeO/mg protein/min. The inclusion of 1-benzylimidazole, superoxide dismutase, or deferoxamine caused no inhibition of the rat liver microsomal activity. Because these results suggested the involvement of FMOs in the oxidation of SeMet in rat liver microsomes, the formation of MetSeO was also examined using cDNA-expressed human and purified rat FMOs. The results showed that both rat and human FMO1 and FMO3 but not FMO5 can catalyze the reaction. The SeMet kinetic constants were obtained with purified rat liver FMO3 (Km = 0.11 mM, Vmax = 280 nmol/mg protein/min) and rat liver FMO1 (Km = 7.8 mM, Vmax = 1200 nmol/mg protein/min). Because SeMet has anti-cancer, chemopreventive, and toxic properties, the kinetic results suggest that FMO3 is likely to play a role in the biological activities of SeMet at low exposure conditions.     

Metabolic inactivation of five glycidyl ethers in lung and liver of humans,rats and mice in vitro

1. Some glycidyl ethers (GE) have been shown to be direct mutagens in short-term in vitro tests and consequently GE are considered to be potentially mutagenic in vivo. However, GE may be metabolically inactivated in the body by two different enzymatic routes: conjugation of the epoxide moiety with the endogenous tripeptide glutathione (GSH) catalysed by glutathione S-transferase (GST) or hydrolysis of the epoxide moiety catalysed by epoxide hydrolase (EH). 2. The metabolic inactivation of five different GE, the diglycidyl ethers of bis phenol A (BADGE), 4,4'-dihydroxy-3,3',5,5'-tetramethylbiphenyl (Epikote YX4000) and 1,6- hexanediol (HDDGE)and the GE of 1-dodecanol (C₁₂GE)and o-cresol (o-CGE), has been studied in subcellular fractions of human, C3H mouse and F344 rat liver and lung. 3. All GE were chemically very stable and resistant to aqueous hydrolysis, but were rapidly hydrolysed by EH in cytosolic and microsomal fractions of liver and lung. The aromatic GE were very good substrates for EH. In general, microsomal EH is more efficient than cytosolic EH in hydrolysis of GE, and human microsomes are more efficient than rodent microsomes. 4. The more water-soluble GE, o-CGE and HDDGE, were good substrates for GST whereas the more lipophilic GE, YX4000 and C₁₂GE, were poor substrates for GST. In general, rodents are more efficient in GSH conjugation of GE than humans. 5. In general, the epoxide groups of YX4000 are the most and those of HDDGE the least efficiently inactivated of the five GE under study. For the other three GE no general trend was observed: the relative efficiency of inactivation varied with organ and species. 6. The large variation in metabolism observed with five representative GE indicate that GE have variable individual properties and should not be considered as a single, homogenous class of compounds.