Microsomal and cytosolic epoxide hydrolase and glutathione S-transferase activities in the gill, liver, and kidney of the rainbow trout, Salmo gairdneri. Baseline levels and optimization of assay conditions

Microsomal and cytosolic epoxide hydrolase (mEH and cEH respectively) and glutathione S-transferase (GST) activities were measured in the liver, kidney, and gills of rainbow trout. Assays were optimized for time, pH, and temperature, using trans-stilbene oxide (TSO) and cis-stilbene oxide (CSO) as substrates for cEH and mEH, respectively. Optimal pH values for mEH, cEH, and GST were similar to mammalian values (i.e. 8.5, 7.5, and 9). Temperature optima differed between tissues and cell fractions. Specific activity of cEH-TSO was 3-14 times greater than mEH-CSO for all three tissues, and 8-60 times greater on a tissue weight basis. Liver and, to a lesser extent, kidney mEH were active against benzo[a]pyrene 4,5-oxide, whereas gill mEH was not active against this substrate. Liver cytosolic GST was active against CSO and 1-chloro-2,4-dinitrobenzene (CDNB) but not TSO, whereas gill and kidney cytosolic GST were active only against CDNB. Liver and kidney microsomal GST were active against CDNB, but no activity was found in gill microsomes. The results are discussed in relation to possible endogenous substrates and uninduced xenobiotic metabolizing capacities of different trout tissues.     

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.     

The toxicology and metabolism of chlorothalonil in fish. III. Metabolism,enzymatics and detoxication in Salmo spp. and Galaxias spp.

Hepatic glutathione S-transferase (GST) activity towards chlorothalonil, tetrachloroisophthalonitrile (TCIN), was established for the five fish species: Salmo gairdneri, S. trutta, Galaxias maculatus, G. truttaceus and G. auratus. Molecular weights and hepatic activity toward 1-chloro-2,4-dinitrobenzene (CDNB) of GST were determined in all species. Low level exposure to TCIN was found to induce higher GST activity in G. maculatus, G. truttaceus and S. gairdneri. Other properties of GST enzymes investigated in these fish included TCIN binding, reaction order and the effect of acephate on activity. The effect of TCIN exposure on liver thiol and glutathione (GSH) levels in S. gairdneri and G. truttaceus was investigated. Significant decreases occurred in liver thiol levels of fish when exposed to varying concentrations of TCIN for 96 h without feeding. Liver GSH levels increased in S. gairdneri exposed to 10 μg/l over 96 h with feeding. A marked increase in GST activity was observed during 96 h exposure to 10 μg/l. No significant changes in the activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were observed during exposure to 10 μg/l TCIN in vivo. A transient decrease in activity was observed during exposure to 30 μg/l TCIN. The previously reported in vitro inhibition of GAPDH by TCIN was studied in the context of liver cytosolic levels of GAPDH and GSH. Hepatic GSH and GST activity levels toward ¹⁴C-TCIN and CDNB were compared in S. gairdneri, G. maculatus and G. auratus. The order of asymptotic LC₅₀ values for the three species agreed with that for total hepatic GST activity toward ¹⁴C-TCIN consistent with a detoxication role for GSH-TCIN conjugation. A proposal for a screening process for Australian freshwater fish based on detoxication enzyme activity is discussed.     

Mouse strain differences in glutathione S-transferase activity and aflatoxin B1 biotransformation

Previous studies have suggested that mice are resistant to the carcinogenic effects of aflatoxin B1 (AFB1) and that this resistance is largely the result of expression of an isoenzyme of glutathione S-transferase (GST) with high activity toward AFB1-8,9-epoxide. Significant interstrain differences in cytosolic GST activities toward a variety of substrates have been reported in mice. If such differences exist for the conjugation of AFB1-8,9-epoxide, then there may be significant mouse strain differences in susceptibility to AFB1-induced hepatocarcinogenicity. The hepatic microsomal and cytosolic biotransformation of AFB1 was studied in 8 different strains of mice fed a purified diet. GST-mediated conjugation of AFB1-8,9-epoxide with glutathione and GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA) and cumene hydroperoxide (CHP) were determined with cytosolic fractions from 8-10 pooled livers. Specific activities of cytochrome-P-450-mediated oxidation of AFB1 to aflatoxin Q1 (AFQ1), aflatoxin M1 (AFM1), and aflatoxin P1 (AFP1), as well as the reactive intermediate AFB1-8,9-epoxide, were determined with hepatic microsomal fractions from each mouse strain. No striking differences in specific activity between mouse strains were observed for any of the P-450- or GST-mediated enzymatic pathways measured, although some statistically significant differences were found. GST specific activities toward AFB1-8,9-epoxide, CDNB, DCNB, ECA and CHP ranged from 1.5-2.1, 2,830-5,370, 81-144, 38-69 and 32-73 nmol/mg protein/min, respectively. The rate of formation of AFB1-8,9-epoxide ranged from 208 to 465 pmol/mg protein/min. The specific activities of AFQ1,AFM1, and AFP1 formation by microsomes ranged from 36-70, 161-326, and 252-426 pmol/mg protein/min, respectively. Mice fed a standard rodent chow diet showed evidence of microsomal and cytosolic enzyme induction when compared to mice fed a purified diet. The lack of substantial differences in enzyme specific activities between mouse strains suggests that interstrain variations in the hepatocarcinogenic effects of AFB1 in mice should not be large.     

Conjugation reactions in hepatocytes isolated from streptozotocin-induced diabetic rats

The activities of three drug conjugation reactions, glutathione, glucuronic acid and sulphate conjugation and the synthesis of glutathione, have been measured in hepatocytes isolated from streptozotocin-induced male diabetic rats. The intracellular content of reduced glutathione (GSH) was decreased in diabetic rat hepatocytes compared with controls. Following depletion of the intracellular GSH stores with diethylmaleate, the resynthesis of GSH in the presence of 0.5 mM L-methionine, occurred faster in diabetic rat hepatocytes than in those from control rats indicating that the cystathione pathway may be more efficient in the diabetic animals. In contrast, there was no significant difference in the resynthesis of GSH between control and diabetic rat hepatocytes in the presence of L-cysteine. The GSH conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and 3,4-dichloronitrobenzene (DCNB) was deficient in diabetic rat hepatocytes, although only the effect on the former reaction was statistically significant (P less than 0.05). The Vmax for CDNB conjugation was significantly lower (P less than 0.05) in cytosolic fractions prepared from diabetic rat liver than in control rat liver fractions. This was accompanied by an increase in the affinity of the enzyme for CDNB. In contrast, the Vmax and Km for the conjugation of DCNB in cytosolic fractions were unaffected by the induced-diabetes. Glucuronic acid conjugation of both 1-naphthol and phenolphthalein was markedly deficient in diabetic rat hepatocytes. The intracellular concentrations of the cofactor for glucuronidation, UDP-glucuronic acid, were decreased in diabetic rat liver and this was thought to contribute to the defect in glucuronidation. The sulphation of 1-naphthol was not significantly altered by the induced diabetes. Deficiencies in glutathione and glucuronic acid conjugation in streptozotocin-induced diabetic rats may result in an increased susceptibility to xenobiotic induced cytotoxicity.     

Mutagen activation of 1,2-dibromo-3-chloropropane by cytosolic glutathione S-transferases and microsomal enzymes

It is not clear whether glutathione (GSH) conjugation to 1,2-dibromo-3-chloropropane (DBCP) results in genotoxic activation. Therefore S9, cytosolic, and microsomal fractions from uninduced rat liver were evaluated for their relative ability to activate DBCP in a modified Ames system. The S9 enzymes, either alone or in combination with exogenous GSH, did not enhance the mutagenicity of DBCP; identical results were obtained with cytosolic enzymes. Significant mutagenic activation of DBCP was produced by either S9 or microsomal fractions in the presence of NADPH. Activation was proportional to cytochrome P-450 concentrations, and was diminished by exogenous GSH. The protection against genotoxicity exerted by GSH did not require cytosolic glutathione S-transferases (GST). Thus, mutagenic activation of DBCP as obtained with S9 fractions is primarily due to biotransformation by microsomal rather than by cytosolic enzymes. Kinetic studies of cytosol-catalyzed conjugation of GSH to DBCP revealed tissue-specific differences in apparent Km and Vmax. Renal and testicular GSTs were associated with 28-46% smaller Vmax values when compared to hepatic GSTs (31.2 +/- 1.9 nmol/min X mg protein). However, renal and testicular GSTs had relatively higher affinities for DBCP. Thus, extrahepatic tissues possess significant capacity to conjugate GSH to DBCP. DBCP-GSH conjugates may undergo enzymatic modification by extrahepatic peptidase and beta-lyase to yield other sulfur-containing moieties that perhaps mediate DBCP's extrahepatic toxicity.     

Effects of buthionine sulfoximine and diethyl maleate on glutathione turnover in the channel catfish.

Despite the growing use of fish in toxicological studies, little is known regarding glutathione (GSH) metabolism and turnover in these aquatic species. Therefore, we examined GSH metabolism in the liver and gills of channel catfish (Ictalurus punctatus), a commonly employed aquatic toxicological model. Treatment of channel catfish with L-buthionine-S,R-sulfoximine (BSO, 400 or 1000 mg/kg, i.p.), an inhibitor of GSH biosynthesis, did not deplete hepatic GSH in channel catfish. In addition, hepatic GSH concentrations did not fluctuate in catfish starved for 3 days, indicating relatively slow turnover of hepatic GSH. However, hepatic GSH concentrations were reduced significantly (P less than 0.05) after 7 days of starvation. Administration of the thiol alkylating agent diethyl maleate (DEM, 0.6 mL/kg, i.p.) resulted in depletion of 85% of hepatic GSH at 6 hr post-DEM, with complete GSH recovery observed at 24 hr post-DEM. Co-administration of BSO and DEM (1000 mg/kg, 0.6 mL/kg, respectively) substantially depleted gill GSH and eliminated detectable liver GSH. Following BSO/DEM, GSH recovery in hepatic mitochondria occurred more rapidly than did liver cytosolic GSH. gamma-Glutamylcysteine synthetase (GCS) activities were comparable in the 10,000 g supernatants of catfish liver and gills (204 +/- 21 and 268 +/- 20 nmol/min/mg protein, respectively) whereas gamma-glutamyltranspeptidase (GGT) activity was not detected in the 600 g post-nuclear fraction of either liver or gills. In conclusion, i.p. administration of DEM was an effective means for achieving short-term hepatic GSH depletion in channel catfish, whereas co-administration of BSO and DEM elicited prolonged and extensive hepatic GSH depletion in this species. Like rodents, channel catfish maintained physiologically distinct hepatic mitochondrial and cytosolic GSH pools, and also regulated hepatic GSH levels by in situ hepatic GSH biosynthesis. However, unlike rodents, there was no evidence for a labile hepatic cytosolic GSH pool in channel catfish. These similarities and differences need to be considered when designing toxicological studies involving the GSH pathway in channel catfish and possibly other fish species.     

In vitro interaction of organic mercury compounds with soluble glutathione S-transferases from rat liver

The in vitro interaction of organic mercury compounds with rat liver glutathione S-transferases (GST) was studied, using reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates. The inhibition of the GST activity was dose dependent, but not linear. The different GST isoenzymes were inhibited to different degrees. Kinetic studies never revealed competitive inhibition, with CDNB or with GSH as the variable substrate. Titration of remaining GSH in appropriate incubation mixtures with organomercurials revealed no GST catalyzed conjugation of these compounds with GSH. These experiments showed a spontaneous conjugation of the mercury compounds with GSH, explaining the parabolic inhibition observed in the kinetic studies with GSH as the variable substrate. Both organic and inorganic mercury are spontaneously conjugated with GSH, but interact with GST by direct binding to these proteins. This binding could have a protective function against mercury. No qualitative differences between organic and inorganic mercury were detected.     

Mouse liver glutathione S-transferase isoenzyme activity toward aflatoxin B1-8,9-epoxide and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide

As part of the studies of the biochemical basis for species differences in biotransformation of the carcinogen aflatoxin B1 (AFB1) and its modulation by phenolic antioxidants, we have investigated the role of mouse liver glutathione S-transferase (GST) isoenzymes in the conjugation of AFB1-8,9-epoxide. Isoenzymes of GST were purified to electrophoretic homogeneity from Swiss-Webster mouse liver cytosol by affinity chromatography and chromatofocusing. The isoenzyme fractions were characterized in terms of activity toward surrogate substrates and immunologic cross-reactivity with antisera to rat GSTs. The major isoenzymes were identified as SW 4-4, SW 3-3, and SW 1-1. The specific activity of SW 4-4 toward AFB1-8,9-epoxide was at least 50- and 150-fold greater than that of SW 3-3 and SW 1-1, respectively. Relatively high activity toward another epoxide carcinogen, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, was observed with both SW 4-4 and SW 3-3. SW 1-1 had the highest activity toward 1-chloro-2,4-dinitrobenzene (CDNB) whereas SW 4-4 had relatively low CDNB activity. Following pretreatment with 0.75% butylated hydroxyanisole in the diet, the fraction of total GST contributed by SW 1-1 appeared to increase dramatically, whereas in control mice SW 3-3 constituted the predominant isoenzyme. The high GST activity of mouse liver cytosol toward AFB1-8,9-epoxide is apparently due to an isoenzyme that contributes little to the overall cytosolic CDNB activity.     

In vitro interaction of dithiocarb with rat liver glutathione S-transferases

The in vitro interaction of dithiocarb (DTC) with rat liver glutathione S-transferase was studied, using reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates. The inhibition of the GST activity by DTC was dose dependent, but not linear. The different GST isoenzymes were inhibited to different degrees. Kinetic studies revealed uncompetitive inhibition towards GSH for GST AA, and an intermediate kinetic pattern between uncompetitive and noncompetitive inhibition for the other GST isoenzymes. With respect to CDNB, mixed type inhibition was found for most GST isoenzymes, and nearly uncompetitive inhibition for GST AA and M. Titration of remaining GSH in appropriate incubation mixtures with DTC revealed no GST catalyzed conjugation of DTC with GSH. It is concluded that DTC interact with GST by direct binding to these proteins. This binding could have a protective function against DTC.     

Conjugation of model substrates or microsomally-activated aflatoxin B1 with reduced glutathione, catalysed by cytosolic glutathione-S-transferases in livers of rats, mice and guinea pigs

Glutathione-S-transferase (GST) activity has been examined in liver cytosol fractions from guinea pigs, mice, control fed rats or rats with pre-neoplastic nodular liver lesions. The levels of activity in unfractionated cytosols have been assayed using the model substrates 1-chloro-2,4-dinitrobenzene (CDNB), 3,4-dichloronitrobenzene (DCNB) and monobromobimane (mBrB) with reduced glutathione (GSH). The order of activities in the various liver fractions using CDNB as substrate were: mouse greater than pre-neoplastic nodular rat greater than guinea pig greater than control rat and paralleled the capacities of the cytosols to catalyse the formation of aflatoxin B1-GSH from microsomally-activated aflatoxin B1 (AFB1) and GSH. Quantitative differences between the activities of the cytosols using the three model substrates were observed. In the mouse fractionation of GST activity by isoelectric focusing (I.E.F.) on preparative granular gels showed that the most basic component (isoelectric point pH 9.4) with the highest conjugating activity with respect to microsomally-activated AFB1 did not correspond with the peak of most activity for conjugating CDNB. In the pre-neoplastic nodular rat liver the CDNB conjugating activities of all fractions separated on granular I.E.F. gels, were higher than the corresponding fractions isolated from control rat liver, with particular enhancement of the peak containing the 3:3 isoenzyme. In contrast to control rat liver the 7:7 isoenzyme was detected in pre-neoplastic nodular liver preparations. These isoenzymes (3:3 and 7:7) did not contribute significantly to the enhanced level of AFB1-GSH formation catalysed by cytosol fractions prepared from pre-neoplastic nodular rat liver. The microsomally-activated AFB1-conjugating activity of unfractionated rat liver cytosols was increased to a relatively greater extent than CDNB conjugating activity during the induction of pre-neoplastic nodular liver lesions, and the elevated level of the activated AFB1-conjugating activity was found to be associated with the most basic fraction (isoelectric point pH 9.0). Analytical isoelectric focusing gels using mBrB as substrate demonstrated the presence of a basic GST isoenzyme in the pre-neoplastic nodular rat liver, not detected in preparations from the livers of control rats. The low level of activated AFB1-conjugating activity present in unfractionated guinea-pig cytosol was found to correspond with the fraction containing the peak of CDNB conjugating activity on preparative isoelectric focusing (isoelectric point pH 7.5). The lack of correlation between the conjugation of model substrates and the conjugation of xenobiotics could be of import     

Acetaminophen-glutathione conjugate formation in a coupled cytochrome P-450-glutathione S-transferase assay system mediated by subcellular preparations from adult and weanling rat tissues.

Previous studies from this laboratory indicated that glutathione (GSH) conjugate formation with acetaminophen (APAP) is remarkably induced in liver of weanling rats in response to a single overdose of the drug administered intraperitoneally (ip). Increased APAP–GSH conjugation has been attributed to inducible glutathione S-transferases (GSTs) in dividing hepatocytes. In order to verify this finding, an in vitro reconstitution assay containing liver microsomes (source of cytochrome P-450) and cytosolic fractions (source of GST) from livers and kidneys of adult and weanling rats has been established. In vitro incubation of the reaction mixture was followed by solvent extraction, enzymatic digestion and HPLC analysis of the conjugate. Under controlled conditions, in vitro, the rate of APAP-GSH conjugation reflected the GST activity of cytosolic sample added to incubation system. The activity of cytosolic GST in catalyzing this reaction was measured using cytosols prepared from various tissue sources, particularly from animals pretreated with dietary butylated hydroxylanisole (BHA). The extent of APAP–GSH conjugate formation mediated by cytosols varied in this order: BHA-treated adult liver>BHA-treated weanling liver>control adult liver>control weanling liver>BHA-adult kidney>control adult kidney>BHA weanling kidney>control weanling kidney. In contrast to findings obtained from in vivo experiments, the rate of GST-dependent APAP conjugate formation with GSH in vitro is not induced in the presence of exogenous drug.     

Nitroglycerin metabolism in subcellular fractions of rabbit liver. Dose dependency of glyceryl dinitrate formation and possible involvement of multiple isozymes of glutathione S-transferases

The hepatic transformation of glyceryl trinitrate (GTN), commonly known as nitroglycerin, was studied in subcellular fractions prepared from rabbit livers. Both the cytosolic and microsomal fractions show activity toward GTN metabolism. Moreover, the formation of glyceryl dinitrates (GDNs) seems to be governed by different enzymatic processes in the two fractions. 1,2-GDN was preferentially formed in cytosolic fractions, whereas in microsomal fractions, 1,3-GDN was the predominant product. In cytosolic fractions, increasing starting concentrations of GTN led to a decrease in both the GTN degradation rate and the GDN ratio (1,2-GDN/1,3-GDN), which was mainly accounted for by saturation of the 1,2-GDN formation pathway. Various glutathione S-transferase (GST) inhibitors affected the rate of GDN formation differentially. In cytosolic fractions, 1-chloro-2,4-dinitrobenzene and iodomethane caused no change in the GDN ratio, while sulfobromophthalein, ethacrynic acid, and p-nitrobenzyl chloride decreased the GDN ratio, suggesting that different GST isozymes are inhibited by these agents. In microsomal fractions, no dose-dependent GTN metabolism and related change in the GDN ratios could be observed. With the exception of ethacrynic acid, addition of GST inhibitors did not decrease GDN metabolite production, and even in this case, no change in the GDN ratio was observed. The results suggest that different GTN metabolic pathways are present in the liver, most likely involving different GST isozymes.     

Subcellular distribution of glutathione S-transferase in Chinese fetal liver

Subcellular fractions were isolated from Chinese fetal liver at 4-8 months of age for the determination of glutathione S-transferase (GST). Using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, GST activity was found to be 66 +/- 34 nmol/(min.mg protein), mainly in the cytosol. The GST activities were detected principally in microsomes and their values were 66 +/- 31 and 144 +/- 83 nmol/(min.mg protein), respectively, when assayed with p-nitrobenzyl chloride (PNB) and ethacrynic acid (EA) as substrates. There were no age and sex-related differences in GST activities for any of the substrates studied during fetal development. The Km values of GST for CDNB, PNB and EA were 1112, 1039 and 205 mumol/L, respectively. The conjugation of GST may play an important role in fetal hepatic metabolism of toxic electrophiles.     

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.     

Organ distribution and kinetics of Glutathione transferase from African river prawn, Macrobrachium vollenhovenii (Herklots)

The distribution of glutathione transferase (GST) in the major organs of African river prawn (Macrobrachium vollenhovenii) was studied. All the organs studied had GST activity. The specific activity of the extract from the hepatopancreas was highest while that from the muscle lowest. Purified GST from the hepatopancreas which could conjugate glutathione (GSH) with only 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBDCl) among some electrophilic substrates tested, had a K(m)(NBDCl) of 2.2+/-0.12 mmol l(-1) while the K(m)CDNB was 2.03+/-0.29 mmol l(-1). Chloride ion, a product of the enzymatic reaction readily inhibited the conjugation of CDNB with GSH with an I50 of 0.12 mmol l(-1), whereas chloride ion up to 0.6 mol l(-1) had no inhibitory effect on the conjugation of GSH with NBDCl. However, nitrite inhibited the two reactions but the K(i) for the conjugation of NBDCl was lower than the K(i) for the conjugation of CDNB. The enzyme had an optimum temperature of 40 degrees C and an activation energy of 35.1 kJ/mol. The overall results show that M. vollenhovenii GST (mvGST) uses different mechanisms for different electrophilic substrates. The high K(m) of mvGST for the electrophilic substrates may be a special physiological adaptation for effective xenobiotic detoxication.     

Inhibition of glutathione S-transferases by thonningianin A, isolated from the African medicinal herb, Thonningia sanguinea, in vitro.

There is evidence that increased expression of glutathione S-transferase (EC: 2.5.1.18, GST) is involved in resistance of tumor cells against chemotherapeutic agents. In this study we investigated the inhibitory effects of thonningianin A (Th A), a novel antioxidant isolated from the medicinal herb, Thonningia sanguinea on uncharacterized rat liver GST and human GST P1-1. Using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, rat liver cytosolic GST activity was inhibited by Th A in a concentration dependent manner with 50% inhibition concentration (IC50) of 1.1 microM. When Th A was compared with known potent GST inhibitors the order of inhibition was tannic acid>cibacron blue>hematin>Th A>ethacrynic acid with CDNB as substrate. Th A also exhibited non-competitive inhibition towards both CDNB and glutathione. Furthermore, using 1,2-dichloro-4-nitrobenzene, ethacrynic acid and 1,2-epoxy-3-(p-nitrophenoxy) propane as substrates Th A at 1.0 microM inhibited cytosolic GST by 2%, 12% and 36% respectively. Human GST P1-1 was also inhibited by Th A with an IC50 of 3.6 microM. While Th A showed competitive inhibition towards CDNB it exhibited non-competitive inhibition towards GSH of the human GST P1-1. These results suggest that Th A represents a new potent GST in vitro inhibitor.     

Acetaminophen–glutathione conjugate formation in a coupled cytochrome P-450-glutathione S-transferase assay system mediated by subcellular preparations from adult and weanling rat tissues

Previous studies from this laboratory indicated that glutathione (GSH) conjugate formation with acetaminophen (APAP) is remarkably induced in liver of weanling rats in response to a single overdose of the drug administered intraperitoneally (ip). Increased APAP–GSH conjugation has been attributed to inducible glutathione S-transferases (GSTs) in dividing hepatocytes. In order to verify this finding, an in vitro reconstitution assay containing liver microsomes (source of cytochrome P-450) and cytosolic fractions (source of GST) from livers and kidneys of adult and weanling rats has been established. In vitro incubation of the reaction mixture was followed by solvent extraction, enzymatic digestion and HPLC analysis of the conjugate. Under controlled conditions, in vitro, the rate of APAP-GSH conjugation reflected the GST activity of cytosolic sample added to incubation system. The activity of cytosolic GST in catalyzing this reaction was measured using cytosols prepared from various tissue sources, particularly from animals pretreated with dietary butylated hydroxylanisole (BHA). The extent of APAP–GSH conjugate formation mediated by cytosols varied in this order: BHA-treated adult liver>BHA-treated weanling liver>control adult liver>control weanling liver>BHA-adult kidney>control adult kidney>BHA weanling kidney>control weanling kidney. In contrast to findings obtained from in vivo experiments, the rate of GST-dependent APAP conjugate formation with GSH in vitro is not induced in the presence of exogenous drug.     

Responses of hepatic xenobiotic metabolizing enzymes of mouse, rat and guinea-pig to nickel.

The effects of nickel (Ni) on hepatic monooxygenase activities (aniline 4-hydroxylase, AH; ethylmorphine N-demethylase, EMND; aminopyrine N-demethylase, AMND), cytochrome P-450, cytochrome b5, microsomal haem and reduced glutathione (GSH) levels, and glutathione S-transferase (GST) activities toward several substrates (1, chloro-2-4-dinitrobenzene, CDNB; 1,2 dichloro-4-nitrobenzene, DCNB; ethacrynic acid, EAA) in mice, rats and guinea-pigs were studied. Ni (59.50 mg NiCl2.6H2O/kg, subcutaneously) was administered to the animals 16 hr prior to sacrifice. Ni significantly inhibited AH, EMND, AMND activities, and decreased cytochrome P-450, cytochrome b5 (except in the livers of rats), and microsomal haem levels in the livers of all the animal species examined. However, the depressions were more profound in livers of mice than in those of the other two species. The hepatic GSH level was significantly inhibited in mice whereas no alteration was observed in rats. In guinea-pigs, the hepatic GSH level was significantly increased by Ni. The hepatic GST activity toward the substrate CDNB was significantly depressed in mice, unaltered in rats and significantly increased in guinea-pigs by Ni. The hepatic GST activity toward DCNB was significantly inhibited in mice whereas no significant alteration was observed in rats. In guinea-pigs, Ni caused significant increase in hepatic GST activity for DCNB. However, hepatic GST activity toward EAA was significantly inhibited in mice whereas significantly increased in rats and guinea-pigs. These results seem to indicate that i) there exists quantitative, but not qualitative, differences among the hepatic monooxygenases of rodents in response to Ni, mice being more sensitive than rats and guinea-pigs, ii) the influence of Ni on hepatic GSH level varies depending on the animal species and iii) the hepatic GSTs of rodents are differentially regulated by Ni.     

Effect of protein-calorie malnutrition on cytochromes P450 and glutathione S-transferase.

Protein-calorie malnutrition (PCM) can develop both from inadequate food intake and as a consequence of diseases such as cancer and AIDS. Several studies have shown that PCM can alter drug clearance but little information is available on the effect of PCM on individual cytochrome P450 isoforms and phase II conjugation enzymes. The aim of the present study was to begin a systematic evaluation of the effect of PCM on the activity of individual drug metabolizing enzymes in a rat model of PCM. Control and PCM rats received isocaloric diets which contained either 21% or 5% (deficient) protein. After 3 weeks, the animals were sacrificed and microsomal and cytosolic fractions prepared. Ethoxyresorufin O-deethylation (EROD), chlorzoxazone 6-hydroxylation, dextromethorphan N- and O-demethylation and 1-chloro-2,4-dinitrobenzene (CDNB) conjugation were used as measures of CYP1A, CYP2E1, CYP3A2, CYP2D1 and glutathione S-transferase (GST) activity, respectively. Additionally, NADPH-cytochrome P450 reductase activity was measured in the liver microsomes. PCM significantly reduced the maximum velocity (Vmax) of all model reactions studied. However, differential effects were observed with respect to K(m) values of the reactions. The K(m) values for EROD and dextromethorphan N-demethylation were significantly increased in PCM animals, whereas the K(m) values for chlorzoxazone 6-hydroxylation and dextromethorphan O-demethylation were decreased. In contrast, the K(m) value for CDNB conjugation was unchanged. When NADPH-cytochrome P450 reductase activity was compared, a 29% reduction in reductase activity was noted in PCM animals as compared to controls. Thus, it appears that PCM decreases the overall activity of certain phase I and phase II metabolism enzymes in rat liver while exhibiting differential effects on K(m). Furthermore, this reduction in activity may be due in part to diminished activity of cytochrome P450 reductase.