Enhanced expression of rat hepatic microsomal epoxide hydrolase by methylthiazole in conjunction with liver injury

Microsomal epoxide hydrolase (mEH) is inducible by a number of xenobiotics. Induction of mEH by certain chemopreventive agents may implicate the protective effect. In contrast, many of carcinogenic agents also induce the enzyme. The hepatotoxicity and mEH expression by methylthiazoles, which are incorporated as functional groups in a number of therapeutic agents, were assessed in the rat liver to study the structural basis for the enzyme induction and the correlative enzyme expression with hepatotoxicity. Among the methylthiazoles examined, 4-methylthiazole (MT) at the daily dose of 1.17 mmol/kg body weight caused hepatic necrosis and degeneration after 1-3 consecutive daily treatment(s), whereas 4, 5-dimethylthiazole (DT) and 2,4,5-trimethylthiazole (TT) elicited no toxicity. Treatment of rats with MT at the daily dose of 1.17 mmol/kg increased the mEH mRNA by 17- and 7-fold at day 1 and day 3, respectively, relative to control. Whereas DT caused 5- and 2-fold increases in mEH mRNA at day 1 and day 3, respectively, TT minimally affected mEH expression. The mRNA increase was consistent with the protein induction. Hence, the methylthiazole causing hepatotoxicity was more active in inducing the enzyme. Whereas treatment with MT at the dose of 0.35 mmol/kg caused no hepatotoxicity, MT caused hepatic necrosis in starving rats. Northern blot analysis showed that the mEH mRNA level was increased to a greater extent by MT in starving rats than in control animals. Conversely, treatment of starving rats with either cysteine or methionine prior to MT prevented the hepatic necrosis. Elevation of the mEH mRNA by MT in starving animals was also inhibited by either cysteine or methionine pretreatment. These results demonstrated that the methylthiazole which caused hepatotoxicity also up-regulated mEH expression, whereas other methylthiazoles showing no toxicity minimally increased the gene expression. The observation that the extent of mEH expression by MT was highly associated with that of liver injury raised the notion that mEH expression by xenobiotics may not necessarily represent the beneficial and protective effects.     

Molecular basis for hepatic detoxifying enzyme induction by 2-(allylthio)pyrazine in rats in comparison with oltipraz: effects on prooxidant production and DNA degradation.

The expression of hepatic microsomal epoxide hydrolase (mEH) and glutathione S-transferases (GSTs) by 2-(allylthio)pyrazine (2-AP), an experimental chemopreventive agent, was investigated in rats. Northern blot analysis revealed that 2-AP caused increases in mEH, rGSTA2/3/5, and rGSTM1/2 mRNA levels. mEH and rGSTA2 proteins were also induced. Molecular basis of the enzyme induction by 2-AP was studied in comparison with oltipraz (Olt). Rats exposed to buthionine sulfoximine, a GSH-depleting agent, before treatment with either 2-AP or Olt exhibited greater increases in the mRNA levels than the individual treatment. Conversely, increases of the mRNAs were prevented by cysteine treatment, indicating that metabolic intermediates or reactive oxygens produced from the agents could be reduced by cysteine. Gel shift analysis revealed that nuclear factor-kappaB, which is associated with the altered cellular redox state, was not activated by the agents. Effects of these agents on the breakage of phix-174 DNA were compared in vitro. 2-AP effectively reduced the conversion of supercoiled DNA to the open circular form induced by benzenetriol and prevented benzenetriol- and iron-catalyzed degradation of DNA, whereas Olt failed to prevent strand breakage of DNA. These results provided evidence that: 1) 2-AP was effective in elevating the hepatic mEH and GST gene expression in rats, which might be mediated with the production of reactive oxygen species; 2) nuclear factor-kappaB activation was not involved in the induction of the detoxifying enzymes by either 2-AP or Olt in spite of their production of reactive oxygens in vivo; and 3) the antioxidant effect of 2-AP in vitro differed from that of Olt.     

Effects of lead on glutathione S-transferase expression in rat kidney: a dose-response study.

Glutathione S-transferases (GST, EC 2.5.1.18) are a family of phase II detoxification enzymes involved in the conjugation of glutathione to a highly diverse group of compounds. The purpose of this study was to evaluate the dose-response effects of lead acetate administration on the expression of rat kidney GST. Sprague-Dawley rats were injected with doses of lead acetate ranging from 0.11 to 114 mg/kg (0.3 to 300 mumol/kg) for three consecutive days and sacrificed 24 h later. Kidney GST activity, GST isoform HPLC profiles, blood lead analysis, and electron microscopy were performed. A dose of 1.1 mg/kg lead acetate resulted in a blood lead level of 26 micrograms/dl and produced a significant increase in GST activity which continued to increase with dose up to 38 mg/kg. Morphological changes were detected at 3.8 mg/kg and increasing severity of cellular damage paralleled dose, blood lead levels, and changes in body weight. Individual GST isoforms exhibited different thresholds and maxima; rGSTP1 and rGSTM1 had thresholds of 1.1 and 3.8 mg/kg, respectively, very similar rates of increase with dose, and a maximum yield that was 450% above control at a dose of 38 mg/kg for both enzymes. rGSTA1 and rGSTA3 showed similar thresholds (1.1 mg/kg) and maximal fold increase (275%) but varied in the relative response to each dose. These results indicate that renal GST increases occur at lead levels which are environmentally significant, that these changes precede cellular damage, and suggest that GST may serve as a tissue biomarker of lead exposure.     

Covalent binding of acrylonitrile to specific rat liver glutathione S-transferases in vivo.

Acrylonitrile (AN) is an industrial vinyl monomer that is acutely toxic. When administered to rats, AN covalently binds to tissue proteins in a dose-dependent but nonlinear manner [Benz, F. W., Nerland, D. E., Li, J., and Corbett, D. (1997) Fundam. Appl. Toxicol. 36, 149-156]. The nonlinearity in covalent binding stems from the fact that AN rapidly depletes liver glutathione after which the covalent binding to tissue proteins increases disproportionately. The identity of the tissue proteins to which AN covalently binds is unknown. The experiments described here were conducted to begin to answer this question. Male Sprague-Dawley rats were injected subcutaneously with 115 mg/kg (2.2 mmol/kg) [2,3-(14)C]AN. Two hours later, the livers were removed, homogenized, and fractionated into subcellular components, and the radioactively labeled proteins were separated on SDS-PAGE. One set of labeled proteins was found to be glutathione S-transferase (GST). Specific labeling of the mu over the alpha class was observed. Separation of the GST subunits by HPLC followed by scintillation counting showed that AN was selective for subunit rGSTM1. Mass spectral analysis of tryptic digests of the GST subunits indicated that the site of labeling was cysteine 86. The reason for the high reactivity of cysteine 86 in rGSTM1 was hypothesized to be due to its potential interaction with histidine 84, which is unique in this subunit.     

Effects of altered calcium homeostasis on the expression of glutathione S-transferase isozymes in primary cultured rat hepatocytes.

The effects of altered Ca2+ homeostasis on glutathione S-transferase (GST) isozyme expression in cultured primary rat hepatocytes were examined. Isolated hepatocytes were cultured on Vitrogen substratum in serum-free modified Chee's essential medium and treated with Ca2+ ionophore A23187 at 120 hr post-plating. GST activity increased slightly, albeit significantly, in a concentration-dependent manner in A23187-treated hepatocytes relative to untreated controls. Western blot analysis using GST class alpha and mu specific antibodies showed an approximately 1.6- and 1.5-fold increase in the class alpha, Ya and Yc subunits, respectively, whereas no significant increase (approximately 1.2-fold) in class mu GST expression was observed following A23187 treatment. Northern blot analysis revealed an approximately 5-fold increase in GST class alpha and an approximately 7-fold increase in class mu GST mRNA levels in ionophore-treated hepatocytes compared to untreated cells. Results of the Western and Northern blot analyses of the ionophore-treated hepatocytes were compared with those obtained for tert-butyl hydroperoxide-treated cells. Immunoblot analysis showed a significant increase in the expression of GST class alpha, Ya and Yc subunits, approximately 1.8- and 1.7-fold, respectively, for tert-butyl hydroperoxide-treated hepatocytes as compared to controls, with little or no increase in class mu GSTs. Northern blot analysis showed approximately 3- and 2-fold increases, respectively, in class alpha and mu GST mRNA levels, following the tert-butyl hydroperoxide treatment. The results of the present investigation show that alterations in Ca2+ homeostasis produced by either Ca2+ ionophore A23187 or tert-butyl hydroperoxide treatment of hepatocytes enhanced the expression of GST isozymes in primary cultured rat hepatocytes.     

Regeneration of serotonin from 5-methoxytryptamine by polymorphic human CYP2D6

Polymorphic cytochrome P450 2D6 (CYP2D6) is expressed in several types of central neurons but its function in human brain is currently unknown. Using recombinant enzymes and CYP2D6-transgenic mice, we established that 5-methoxytryptamine (5-MT), a metabolite and precursor of melatonin, is a specific and high-turnover endogenous substrate of CYP2D6. This potent serotonergic neuromodulator in numerous physiological systems binds tightly to recombinant CYP2D6 enzyme with an equilibrium dissociation constant (K(s)) of 23.4 micromol/l, and is O-demethylated to serotonin (5-hydroxytryptamine, 5-HT) with a high turnover of 51.7 min(-1) and low Michaelis-Menten constant of 19.5 micromol/l. The production of 5-HT from 5-MT catalyzed by CYP2D6 was inhibited by selective serotonin reuptake inhibitors, and their inhibition potency (K(i), micromol/l) decreased in the order of fluoxetine (0.411) > norfluoxetine (1.38) > fluvoxamine (10.1) > citalopram (10.9). Liver microsomes prepared from CYP2D6-transgenic mice showed about 16-fold higher 5-MT O-demethylase activity than that from wild-type mice. After the intravenous co-administration of 5-MT (10 mg/kg) and pargyline (20 mg/kg), serum 5-HT level was about 3-fold higher in CYP2D6-transgenic mice than wild-type mice. When dosed with alpha,alpha,beta,beta-d -5-MT, alpha,alpha,beta,beta-d4-5-HT was detected in transgenic mouse serum, and its content was much higher than wild-type mouse. alpha,alpha,beta,beta-d4-5-HT was not produced in CYP2D6-transgenic mice pretreated with quinidine. The regeneration of 5-HT from 5-MT provides the missing link in the serotonin-melatonin cycle. Up to 10% of the population lacks this enzyme. It is proposed that this common inborn error in 5-MT O-demethylation to serotonin influences a range of neurophysiologic and pathophysiologic events.     

Comparative expression of two alpha class glutathione S-transferases in human adult and prenatal liver tissues

The ability of the fetus to detoxify transplacental drugs and chemicals can be a critical determinant of teratogenesis and developmental toxicity. Developmentally regulated expression of alpha class glutathione S-transferases (GSTs) is of particular interest, since these isozymes have high activity toward peroxidative byproducts of oxidative injury that are linked to teratogenesis. The present study was initiated to examine the expression and catalytic activities of alpha class GST isozymes in human prenatal liver. Northern analysis demonstrated the presence of hGSTA1 and/or A2 (hGSTA1/2) and hGSTA4 steady-state mRNAs in second trimester prenatal livers. Western blotting of prenatal liver proteins provided corroborating evidence via detection of an hGSTA1/2-reactive protein in both cytosol and mitochondria and of hGSTA4-4-reactive protein in mitochondria alone. Catalytic studies demonstrated that prenatal liver cytosolic GSTs were active toward 1-chloro-2,4-dinitrobenzene (a general GST reference substrate), delta5-androstene-3,17-dione (relatively specific for hGSTA1-1), and 4-hydroxynonenal, a highly mutagenic alpha,beta-unsaturated aldehyde produced during oxidative damage and a substrate for hGSTA4-4. Total GSH-peroxidase and GST-dependent peroxidase activities were 9- and 18-fold higher, respectively, in adult liver than in prenatal liver. Multiple tissue array analyses demonstrated considerable tissue-specific and developmental variation in GST mRNA expression. In summary, our results demonstrate the presence of two important alpha class GSTs in second trimester human prenatal tissues, and indicate that mitochondrial targeting of GST may represent an important pathway for removal of cytotoxic products in prenatal liver. Furthermore, the relatively inefficient prenatal reduction of hydroperoxides may underlie an increased susceptibility to maternally transferred pro-oxidant drugs and chemicals.     

Chemical modification at subunit 1 of rat kidney Alpha class glutathione transferase with 2,3,5,6-tetrachloro-1,4-benzoquinone: close structural connectivity between glutathione conjugation activity and non-substrate ligand binding

2, 3, 5, 6-Tetrachloro-1, 4-benzoquinone (TCBQ) is a metabolite of pentachlorophenol known to react with cysteines of glutathione transferases (GSTs). TCBQ treatment of rat kidney rGSTA1-2 and rGSTA1-1 abolishes 70-80% conjugation of glutathione (GSH) to 1-chloro-2, 4-dinitrobenzene and results in strongly correlated quenching of intrinsic fluorescence of Trp-20 (R>0.96). rGSTA2-2 is only inhibited by 25%. Approximately 70% (rGSTA1-1) and 60% (rGSTA1-2) conjugation activity is abolished at TCBQ: GST stoichiometries near 1:1. The inactivation follows a Kitz/Wilson model with K(D) of 4.77+/-2.5microM for TCBQ and k(3) for inactivation of 0.036+/-0.01min(-1). A single tryptic peptide labelled with TCBQ was isolated from kidney rGSTA1-2 containing Cys-17 which we identify as the site of modification. Treatment with more than stoichiometric amounts of TCBQ modified other residues but resulted in only modest further inhibition of catalysis. We interpret these findings in terms of localised steric effects on the relatively rigid alpha-helix 1 adjacent to the catalytic site of subunit 1 possibly affecting the Alpha class-specific alpha-helix 9 which acts as a "lid" on the hydrophobic part of the active site. Homology modelling of rGSTA1-1 modified at Cys-17 of one subunit revealed only modest structural perturbations in the second subunit and tends to exclude global structural effects.     

Repeated administration of oxycodone modifies the gene expression of several drug metabolising enzymes in the hepatic tissue of male Sprague‐Dawley rats,including glutathione S‐transferase A‐5 (rGSTA5) and CYP3A2

Objectives Clinical use and illicit abuse of the potent opioid agonist oxycodone has dramatically increased over the past decade. Yet oxycodone remains one of the least studied opioids, particularly its interactions on the genomic level. The aim of this study was to examine potential alterations in gene expression of drug metabolising enzymes in the liver tissue of male Sprague‐Dawley rats chronically treated with oxycodone. Methods Rats were administered saline or oxycodone 15 mg/kg i.p. twice a day for 8 days. Changes in RNA levels were detected using microarray analysis validated by quantitative real‐time PCR; consequent changes in protein expression and functionality were further assessed by Western blotting and activity assays. Key findings The expression of several drug metabolising enzymes was modulated by oxycodone treatment: cytochrome P450 (CYP) 2B2, CYP2C13, CYP17A1, epoxide hydrolase 2, carboxylesterase 2, flavin‐containing monooxygenase 1, glutathione S‐transferase alpha 5 (rGSTA5) and CYP3A2. In particular, the mRNA level of rGSTA5 (formerly GSTYc₂) was up‐regulated by approximately 6.5 fold and CYP3A2 was down‐regulated by approximately 7.0 fold. Immunoblotting assays demonstrated a corresponding significant elevation of rGSTA5 protein and repression of CYP3A2 protein. The apparent cytosolic GST activity towards 1‐chloro‐2,4‐dinitrobenzene conjugation and reduction of cumene hydroperoxide were significantly higher in liver from oxycodone‐treated rats than that of saline‐treated rats. In addition, the microsomal activity of CYP3A2, measured via 6β‐hydroxylation of testosterone, was significantly decreased in oxycodone‐treated rats. Conclusions Repeated oxycodone administration is associated with a significant up‐regulation of rGSTA5 and concomitant down‐regulation of CYP3A2 mRNA, protein expression and functionality. These results support further in‐vivo studies into the clinical impact of our findings.     

Effect of thiabendazole (TBZ) on glutathione (GSH) and GSH related enzymes in mice liver

The present study examines the effects of thiabendazole (TBZ), its metabolites, 5-hydroxythiabendazole (5-OH TBZ) and 2-acetylbenzimidazole (ABI), and structural related compounds, thiazoles and thioamides on glutathione (GSH) concentration and GSH-related enzymes in the livers of ICR 11 week-old female mice. GSH concentration in liver and kidney of mice given orally TBZ 0.65 mol/kg (TBZ group) increased significantly compared with control mice from 24 h to 48 h after administration of TBZ. Even in mice to which TBZ at 0.175 mol/kg was administered in combination with L-buthionine sulfoximine (BSO) 4 mmol/kg (i.p.) (BSO-TBZ group), kidney GSH showed significant increase compared with BSO-control mice 48 h after the administration of TBZ. gamma-Glutamylcysteine synthetase (gamma-GCS) activity in the livers of the TBZ group markedly increased at 48 h and that of BSO-TBZ group increased from 24 h to 48 h. gamma-GCS in mice liver is thus enhanced by TBZ regardless of BSO administration. Hepatic glutathione peroxidase activity of the TBZ group did not change in response to cumene hydroperoxide assubstrate. That of BSO-treated mice decreased by TBZ-coadministration and significant differences was noted between BSO-control and BSO-TBZ group from 1 h to 48 h later. Hepatic glutathione S-transferase (GST) activity toward 1,2-dichloro-4-nitrobenzene (DCNB) was significantly elevated 24 h after administrations of TBZ in TBZ and BSO-TBZ groups. GST activity toward 1,2-epoxy-3-(p-nitrophenoxy) propane of TBZ group increased from 0.5 h to 24 h. Hepatic GST activity toward DCNB and 1-chloro-2,4-dinitrobenzene did not change by administration of 0.65 mol/kg 5-OH TBZ or ABI but increased by administrations of 0.33 mol/kg of thiazole, 4-methylthiazole, 4,5-dimethylthiazole or 2,4-dimethylthiazole. Increase in GSH concentration and GST activity in mice liver by TBZ administration may be considered to provide protection from TBZ or its active metabolites.     

Differences between rats and rabbits in hepatic cytosolic glutathione S-transferase expression in response to nitrogen heterocycle and other inducers.

Glutathione S-transferase (GST) expression was examined in hepatic cytosol from rats and rabbits treated with 4-picoline, pyrrole, pyridine, pyrazine, imidazole, or piperidine using enzymatic activity, SDS-PAGE, and immunoblot analyses and the results were compared to those obtained with phenobarbital and 3-methylcholanthrene. SDS-PAGE and immunoblot analyses of hepatic cytosol prepared from rats treated with pyrazine revealed the induction of class alpha (Ya and Yc) and mu (Yb) bands with a corresponding 2.4-fold increase in metabolic activity using 1-chloro-2,4-dinitrobenzene as substrate. A new class alpha band migrating in the region of the Yc band was observed in the SDS-PAGE and detected in the immunoblot of cytosol from pyrrole-treated rats, whereas treatment with 4-picoline, imidazole, or piperidine failed to alter the expression of the major classes of GST isozymes in this species. SDS-PAGE and immunoblot analyses of rabbit hepatic cytosol revealed a unique species-dependent difference in the expression of GSTs. While phenobarbital and 3-methylcholanthrene induce class alpha and mu GST expression in rat hepatic cytosol, one of the most interesting observations was that neither of these agents stimulated GST expression in the rabbit. Immunoblot analysis of cytosol isolated from 4-picoline-treated rabbits using GST class alpha-specific IgG showed the appearance of a novel class alpha 28-kDa GST band and the concomitant disappearance of a class alpha 29-kDa GST band. In addition, SDS-PAGE and immunoblot analyses showed that treatment of rabbits with pyrrole, pyrazine, imidazole, or piperidine resulted in the disappearance of this class alpha 29-kDa GST band with no detectable expression of the class alpha 28-kDa GST band; the level of the class alpha 29-kDa band was unaffected by pyridine treatment. In contrast, immunoblot analyses of hepatic cytosol revealed that a 25.5-kDa class mu GST band disappeared following treatment with pyridine, but was unaffected by treatment with other nitrogen heterocycles. The Vmax of glutathione conjugation to the substrate 1-chloro-2,4-dinitrobenzene decreased by 52, 36, 59, 41, 37, and 32% in hepatic cytosol isolated from 4-picoline-, pyrrole-, pyridine-, pyrazine-, imidazole-, and piperidine-treated rabbits, respectively. The results suggest that nitrogen heterocycles differ in their ability to modulate glutathione S-transferase isozyme expression in rat and rabbit hepatic tissue and that rabbit hepatic GSTs are refractory to induction by agents such as pyrazine, phenobarbital, or 3-methylcholanthrene and hence these xenobiotics do not appear to be bifunctional inducers in this species.     

Modulating effects of thyroid state on the induction of biotransformation enzymes by 2,3,7,8-tetrachlorodibenzo-p-dioxin

In this study we investigated to what extent the induction of detoxification enzymes by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is modulated by concomitant TCDD-induced changes in thyroid state. Euthyroid (Eu) male Sprague-Dawley rats, surgically thyroidectomized (Tx) rats and Tx rats receiving substitution doses of 3,3',5-triiodothyronine (Tx+T3) or thyroxine (Tx+T4) by osmotic minipumps were treated with a single ip injection of 10 μg TCDD/kg/bwt or with vehicle (corn oil). Three days after TCDD administration, rats were sacrificed and blood and livers were collected for analysis. Total hepatic cytochrome P450 (CYP) content was increased by ≈50% by TCDD in all groups but was not affected by thyroid state. In Eu rats, TCDD increased CYP1A1/1A2 activity 90-fold, CYP1A1 protein content 52-fold and CYP1A1 mRNA levels ≈5.8-fold. Similar findings were obtained in Tx, Tx+T3 and Tx+T4 rats except that TCDD-induced CYP1A1 activity was significantly decreased in Tx rats. NADPH cytochrome P450 reductase activity was not affected by TCDD but was decreased in Tx rats, which may explain the diminished TCDD-induced CYP1A1 activity in Tx rats. Hepatic p-nitrophenol UDP-glucuronyltransferase (UGT) activity was induced ≈4-fold by TCDD in Eu rats. Similar basal and TCDD-induced activities were observed in Tx+T3 and Tx+T4 rats, but TCDD-induced activities were significantly lower in Tx rats. TCDD did not have a significant effect on overall glutathione-S-transferase (GST) activity or hepatic GST 2-2, 3-3 or 4-4 protein levels but produced a marked increase in GST 1-1 protein levels. Thyroid state did not affect basal or TCDD-induced GST activity or subunit pattern. Iodothyronine sulfotransferase (ST) activity was not affected by TCDD treatment and was slightly but not significantly lower in Tx rats than in Eu, Tx+T3 and Tx+T4 rats. These results suggest that the changes in thyroid hormone levels associated with TCDD treatment have little modulating effects on the induction of hepatic detoxification enzymes in Sprague-Dawley rats exposed to this compound.     

Glutathione S-transferases in wild-type and doxorubicin-resistant MCF-7 human breast cancer cell lines

1. Overexpression of glutathione S-transferases (GST) in breast cancer cells is hypothesized to be a component of the multifactorial doxorubicin-resistant phenotype. 2. We have characterized the expression of GST enzymes at the catalytic activity, protein and mRNA levels in wild-type MCF-7 (MCF-7/WT) human breast cancer cells and a line selected for resistance to doxorubicin (MCF-7/ADR), with the goal of modulating GST activity to overcome resistance. 3. The MCF-7/ADR cells were 30-65-fold more resistant to doxorubicin than the MCF-7/WT cells. 4. Total cytosolic GST catalytic activity was elevated 23-fold in the MCF-7/ADR cells as compared with the MCF-7/WT cells, and the MCF-7/ADR cells also showed 3-fold increases in catalytic activity toward GST mu and alpha class-selective substrates. Neither cell line showed detectable catalytic activity with a GST mu class-selective substrate. 5. MCF-7/ADR cells showed pronounced overexpression of GST mu protein and GST P1 mRNA in comparison with the wild-type cell line. Neither cell line displayed detectable GST alpha or mu at the protein level. 6. A glutathione analogue that functions as a selective GST alpha inhibitor was more potent at inhibiting total cytosolic GST catalytic activity in the MCF-7/ADR cell line than GST alpha and mu class-selective inhibitory glutathione analogues and the non-selective GST inhibitor ethacrynic acid. 7. The multidrug resistance-associated protein, which can function as a glutathione-conjugate transporter, appeared weakly overexpressed in the MCF-7/ADR cells in comparison with the MCF-7/WT cells.     

Metabolism and urinary excretion of chlormethiazole in humans.

1. Chlormethiazole and five of its metabolites excreted in urine in man have been investigated by g.l.c.-mass spectrometry. 2. Four metabolites have been identified by comparison with authentic compounds as 5-acetyl-4-methylthiazole, 5-(1-hydroxyethyl)-4-methylthiazole, 5-(2-hydroxyethyl)-4-methylthiazole and 4-methyl-5-thiazoleacetic acid; 4-methyl-5-thiazoleacetaldehyde is proposed for the other metabolite. 3. The amounts of chlormethiazole and its identified metabolites excreted in urine have been quantitatively determined after a single oral dose in three healthy adults. Approximately 16% of the dose was excreted as chlormethiazole, 5-acetyl-4-methylthiazole, 5-(1-hydroxyethyx)-4-methylthiazole and 4-methyl-5-thiazoleacetic acid.     

Purification and characterization of hepatic glutathione transferases from an insectivorous marsupial,the brown antechinus (Antechinus stuartii)

1. Five unique glutathione transferase isoenzymes were purified from the hepatic cytosol of an insectivorous marsupial, the brown antechinus. The purified GSTs were characterized by structural and catalytic properties including apparent molecular weight andisoelectricpoint,specificity towards modelsubstrates,kineticparameters,sensitivityto inhibitors and cross-reactivity with antisera raised against human GSTs. 2. An alpha class GST, Antechinus GST 1-1, predominated in the hepatic cytosol, representing 71% of the total GST purified. The substrate specificity of Antechinus GST 1-1 was similar to that of other alpha class GSTs, particularly with respect to its high activity with cumene hydroperoxide. The mu class was represented by three GST isoenzymes, Antechinus GST 3-3, GST 3-4 and GST 4-4. These isoenzymes represented 8, 2 and 10% of the total GST purified respectively. A single GST, Antechinus GST 22, belonged to the pi class of GSTs and represented 12% of the total GST purified. The hepatic GST isoenzyme ratio (by class) observed in the brown antechinus was more similar to that observed in the human than in rat. 3. A previous study investigating a herbivorous marsupial, the brushtail possum (Trichosurus vulpecula) also identified a predominant hepatic GST belonging to the alpha class and displaying peroxidase activity. The evolutionary conservation of a similar predominant GST isoenzyme in these marsupials suggests that they play an important role in the detoxication metabolism of these unique mammals.     

Metabolism and Urinary Excretion of Chlormethiazole in Humans

1. Chlormethiazole and five of its metabolites excreted in urine in man have been investigated by g.l.c.-mass spectrometry.

2. Four metabolites have been identified by comparison with authentic compounds as 5-acetyl-4-methylthiazole, 5-(1-hydroxyethyl)-4-methylthiazole, 5-(2-hydroxyethyl)-4-methylthiazole and 4-methyl-5-thiazoleacetic acid; 4-methyl-5-thiazoleacetaldehyde is proposed for the other metabolite.

3. The amounts of chlormethiazole and its identified metabolites excreted in urine have been quantitatively determined after a single oral dose in three healthy adults. Approximately 16% of the dose was excreted as chlormethiazole, 5-acetyl-4-methylthiazole, 5-(1-hydroxyethyl)-4-methylthiazole and 4-methyl-5-thiazoleacetic acid.     

Complementary DNA cloning, protein expression, and characterization of alpha-class GSTs from Macaca fascicularis liver.

Large species differences exist in sensitivity to aflatoxin B(1) (AFB(1))-induced liver cancer. Mice are resistant to AFB(1)-induced liver cancer because they express an alpha-class GST (mGSTA3-3) that has high activity toward the reactive intermediate aflatoxin B(1)-8,9-epoxide (AFBO). Rats constitutively express only small amounts of a GST with high AFBO activity (rGSTA5-5) and thus are sensitive to AFB(1)-induced hepatocarcinogenesis, although induction of rGSTA5-5 can confer resistance in rats. In contrast to rodents, constitutively expressed human hepatic alpha-class GSTs have little or no AFBO detoxifying activity. Recently, we found that the nonhuman primate, Macaca fascicularis (Mf), has significant constitutive hepatic GST activity toward AFBO and most of this activity belongs to mu-class GSTs. To determine if any alpha-class GSTs in Mf liver have AFBO activity, a cDNA library from a male Mf liver was constructed and screened using the human alpha-class GstA1 cDNA as a probe. Three different cDNA clones with full-length open reading frames were identified from the Mf hepatic cDNA library. Analyses of the cDNA deduced protein sequences indicated that these three alpha-class cDNA clones were 97-98% homologous with each other, and shared 93, 95, and 95% identity with human GSTA1, and were named mfaGSTA1, mfaGSTA2, and mfaGSTA3, respectively. Bacterially expressed mfaGSTA1-1 recombinant protein had similar activities toward classic GST substrates such as DCNB, CHP, and ECA, but slightly lower CDNB conjugating activity relative to human GSTA1-1. However, similar to hGSTA1-1, mfaGSTA1-1 had no AFBO conjugating activity. In addition, similar to human GSTA1 gene, cDNA-derived amino acid sequence analyses demonstrated that all of these Mf alpha-class GSTs genes (mfaGSTA1, mfaGSTA2, and mfaGSTA3) had none of the six critical residues that were identified previously to confer high AFBO activity in mouse alpha-class GSTA3-3. Thus, in contrast to rodents but similar to humans, alpha-class GSTs from the nonhuman primate, Mf, have little conjugating activity toward AFBO.     

A new and highly sensitive method for measuring 3-methoxytyramine using HPLC with electrochemical detection. Studies with drugs which alter dopamine metabolism in the brain

3-Methoxytyramine (3-MT) is a minor metabolite of dopamine which is suggested to reflect the turnover and utilization of dopamine. A novel, isocratic HPLC method has been developed which can be used to analyse 3-MT in homogenates of rat brain without the need for additional purification procedures. Furthermore, the coulometric electrochemical detection system is sensitive enough to measure 3 pg of 3-MT (equivalent to 0.6 ng/g tissue wet weight). 3-Methoxytyramine was measured in the striatum and n. accumbens after decapitation and rapid freezing, using 3-methoxy-4-hydroxybenzylamine as the internal standard. The effects of dopaminergic and other drugs on this metabolite were examined using this method. -Methyl-p-tyrosine (200 mg/kg i.v.) produced parallel linear decreases in dopamine and 3-MT in naive rats, but not those pretreated with tranylcypromine (5 mg/kg i.p.). Methamphetamine (0.3–10 mg/kg i.p.) and amphetamine (0.3–10 mg/kg i.p.) both dose-dependently increased 3-MT in naive and tranylcypromine-pretreated rats. In naive animals, 3-MT was not altered by intraperitoneal injection of the dopamine reuptake inhibitors, bupropion (10 mg/kg) and nomifensine (10 mg/kg) or by sibutramine HCl (3 mg/kg), amitriptyline (10 mg/kg), desipramine (10 mg/kg) and zimeldine (10 mg/kg). 3-Methoxy-tyramine was decreased by apomorphine (5 mg/kg i.p.) and also by large doses of the selective D₂ antagonist, BRL 34778 (5 mg/kg i.p.) or -DOPA (50 mg/kg i.p.). The selective D₁ antagonist, SCH 23390 (0.1 or 5 mg/kg i.p.) was without effect. In tranylcypromine-pretreated rats, 3-MT was dose-dependently reduced and increased by apomorphine (0.01–5 mg/kg i.p.) and BRL 34778 (0.1–5 mg/kg i.p.), respectively. The drug SCH 23390 (0.1–5 mg/kg i.p.) produced much smaller increases in 3-MT which were probably mediated through the striatonigral pathway. Overall, the data suggest that measurement of 3-MT, after inhibition of monoamine oxidase, is a useful index of the release and utilization of dopamine. However, after substantial and prolonged depletion of dopamine, levels of 3-MT in naive animals are a better index. Also, the formation of 3-MT in naive rats provides a sensitive method for distinguishing between dopamine releasing agents and reuptake inhibitors.     

Endogenous gamma-aminobutyric acid (GABA)(A) receptor active neurosteroids and the sedative/hypnotic action of gamma-hydroxybutyric acid (GHB): a study in GHB-S (sensitive) and GHB-R (resistant) rat lines

In the rat brain, gamma-hydroxybutyric-acid (GHB) increases the concentrations of 3alpha-hydroxy,5alpha-pregnan-20-one (allopregnanolone, 3alpha,5alpha-THP) and 3alpha,21-dihydroxy,5alpha-pregnan-20-one (allotetrahydrodeoxycorticosterone/3alpha,5alphaTHDOC), two neurosteroids acting as positive allosteric modulators of gamma-aminobutyric acid (GABA)(A) receptors. This study was aimed at assessing whether neurosteroids play a role in GHB-induced loss of righting reflex (LORR). Basal and GHB-stimulated brain concentrations of endogenous 3alpha,5alpha-THP and 3alpha,5alpha-THDOC were analyzed in two rat lines, GHB-sensitive (GHB-S) and GHB-resistant (GHB-R), selectively bred for opposite sensitivity to GHB-induced sedation/hypnosis. Basal neurosteroid concentrations were similar in brain cortex of the two rat lines. However, in male GHB-S rats, administration of GHB (1000 mg/kg, i.p., 30 min) increased brain cortical concentrations of 3alpha,5alpha-THP and 3alpha,5alpha-THDOC 7- and 2.5-fold, respectively, whilst male GHB-R animals displayed only a 4- and 2-fold increase, respectively. In GHB-S rats this increase lasted up to 90 min and declined 180 min following GHB administration, a time course that matches LORR onset and duration. In contrast, in GHB-R rats, which failed to show GHB-induced LORR, brain cortical 3alpha,5alpha-THP and 3alpha,5alpha-THDOC had returned to control values within 90 min. At onset of LORR, a similar increase in brain cortical levels of 3alpha,5alpha-THP and 3alpha,5alpha-THDOC (2-3-fold) was observed in GHB-S female rats and in the few female GHB-R rats that lost the righting reflex after GHB administration, but not in female GHB-R rats failing to show LORR. Sub-hypnotic doses (7.5 and 12.5 mg/kg, i.p.) of pregnanolone, administered 10 min before GHB, dose-dependently facilitated the expression of GHB-induced LORR in GHB-R male rats. These results suggest that the GHB-induced increases of brain 3alpha,5alpha-THP and 3alpha,5alpha-THDOC concentrations are implicated in the eliciting of the sedative/hypnotic action of GHB.