Cytosolic GSH S-transferase Research Articles

Effect of diet on aflatoxin B1-DNA binding and aflatoxin B1-induced glutathione S-transferase placental form positive hepatic foci in the rat.

Effects of diets on hepatic aflatoxin B1 (AFB1)- DNA binding and AFB1-induced glutathione S- transferase placental (GST-P) form positive hepatic foci have been examined in young male Fischer rats. Animals were fed either AIN-76A or Purina Chow (PC) diet for 1 wk before AFB1- DNA binding studies in vivo and in vitro. Animals were injected i.p. with AFB1 (1 mug/kg body wt) and 3 days later were given either AIN-76A or PC diet with or without 0.1% phenobarbital (PB) in their drinking water. All animals were sacrificed 10 wks after AFB1 dosing for analysis of AFB1-induced GST-P positive hepatic foci by immunochemistry. Two h after i.p. injection of AFB1, hepatic AFB1-DNA binding in AIN-76A fed rats was twice as much as those in PC fed animals without affecting GSH levels. There was no significant effect of diet on either cytochrome P-450 content, GSH levels or microsomal cytochrome P-450 mediated AFB1-DNA binding to exogenous DNA. There was a 40% increase in cytosolic GSH S-transferase activity with 1-chloro-2,4-dinitrobenzene as a substrate in PC fed animals compared to those given AIN- 76A diet. The number and area of AFB1-induced GST-P positive hepatic foci were twice and fivefold as much in AIN-76A fed compared to those in PC fed rats. The number of AFB1-induced GST-P positive foci was increased 5-10 fold in the presence of PB in both groups. In summary, the present data indicate that feeding of PC diet compared to AIN-76A diet inhibits the initiation phase whereas AIN-76A stimulates the promotion phase of AFB1 hepatocarcinogenesis in rats by inhibiting AFB1-DNA binding and increasing AFB1-induced hepatic foci respectively.

Identification, characterization, and properties of a class alpha microsomal glutathione S-transferase.

Cytosolic glutathione (GSH) S-transferases (GST) are dimeric enzymes that exhibit both GSH-dependent conjugation and selenium-independent GSH-peroxidase (NonSe-GPX) activities [1]. of the various GST supergene families, class Alpha GST isozymes have attracted a lot of attention for their role in the conjugation and reduction of cellular lipid peroxidation products, including 4-hydroxy nonenal and lipid hydroperoxides. Morgenstern and his colleagues reported a non-homologous GST, designated as MGST, in rat and human liver microsomes that exhibited both these activities and differed from the cytosolic GSTs in primary structure [2].

Differential expression of glutathione S-transferase isoenzymes in murine small intestine and colon

Glutathione (GSH) S-transferase (GST) isoenzymes of the small intestine and colon of female A/J mice have been purified and characterized to determine their interrelationships with other murine GSTs. Cytosolic GST activity in the small intestine was at least due to six isoenzymes with isoelectric points (p I) of 9.5, 9.3, 9.1, 8.5, 6.2 and 5.5. Small intestine isoenzymes with p I values of 9.5, 9.3, 8.5, and 6.2 were identical to the mGSTA1-1 (Alpha class), mGSTP1-1 (Pi class), mGSTM1-1 (Mu class) and mGSTA4-4 (Alpha class), respectively, of other A/J mouse tissues on the basis of their reverse-phase HPLC elution profile, immunological cross-reactivity and/or N-terminal region amino acid sequence. Even though GST9.1 of the small intestine cross-reacted with the antibodies raised against Pi class GST, reverse-phase HPLC and N-terminal amino acid sequence analyses suggested that this isoenzyme may be structurally different from mGSTP1-1 as well as mGSTP2-2. Likewise, despite immunological similarity with the Mu class GSTs, small intestine GST5.5 appeared to be different from other Mu class murine GSTs characterized previously. Cytosolic GST activity in the colon was mainly due to four isoenzymes with p I values of 9.8, 9.4, 6.6 and 5.8. While the identity of colon GST6.6 could not be established due to its low abundance, GST9.8, GST9.4 and GST5.8 were identical to mGSTP1-1, mGSTM1-1 and mGSTA4-4, respectively, of other A/J mouse tissues including the small intestine. Isoenzymes corresponding to small intestine GST9.1 and GST5.5 could not be detected in the colon. The results of the present study indicate that the small intestine of female A/J mice is better equipped for protection against toxic effects of electrophiles than colon.

Molecular Identification of Cytosolic Prostaglandin E2 Synthase That Is Functionally Coupled with Cyclooxygenase-1 in Immediate Prostaglandin E2Biosynthesis

Here we report the molecular identification of cytosolic glutathione (GSH)-dependent prostaglandin (PG) E(2) synthase (cPGES), a terminal enzyme of the cyclooxygenase (COX)-1-mediated PGE(2) biosynthetic pathway. GSH-dependent PGES activity in the cytosol of rat brains, but not of other tissues, increased 3-fold after lipopolysaccharide (LPS) challenge. Peptide microsequencing of purified enzyme revealed that it was identical to p23, which is reportedly the weakly bound component of the steroid hormone receptor/hsp90 complex. Recombinant p23 expressed in Escherichia coli and 293 cells exhibited all the features of PGES activity detected in rat brain cytosol. A tyrosine residue near the N terminus (Tyr(9)), which is known to be critical for the activity of cytosolic GSH S-transferases, was essential for PGES activity. The expression of cPGES/p23 was constitutive and was unaltered by proinflammatory stimuli in various cells and tissues, except that it was increased significantly in rat brain after LPS treatment. cPGES/p23 was functionally linked with COX-1 in marked preference to COX-2 to produce PGE(2) from exogenous and endogenous arachidonic acid, the latter being supplied by cytosolic phospholipase A(2) in the immediate response. Thus, functional coupling between COX-1 and cPGES/p23 may contribute to production of the PGE(2) that plays a role in maintenance of tissue homeostasis.

Kinetic studies of aflatoxin B1-glutathione conjugate formation in liver and kidneys of adult and weanling rats

Aflatoxin B1(AFB1)-glutathione(GSH) conjugation is the major pathway for the detoxification of aflatoxin metabolites. This reaction is catalysed by GSH S-transferase (GST) and play a major role in modulation of AFB1 adduct formation to nuclear DNA. Changes recorded in hepatic GST activity during development of rats can alter the balance between AFB1-GSH conjugation and AFB1-DNA adduct formation. Measurment of cytosolic GST using 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate showed that the enzyme activity is initially lower in weanling tissues as compared to that of adults. But nevertheless hepatic and renal cytosolic GST activity is increased significantly in growing rats pretreated with AFB1. Kinetic studies of AFB1-GSH conjugate formation in kidneys and livers of the two-age groups of rats treated with a single i.p. dose of AFB1 (400 μg/kg b.w.) revealed that at the end of 24 h of AFB1 administration the rate of the conjugate formation in kidneys of immature rats was approximately twice of that measured in adults. Age-related differences in the GST activity as well as AFB1-GSH conjugation was more pronounced in kidneys. The conjugate formation in kidneys of growing rats during 6–24 h following AFB1 administration shows that urinary excretion of aflatoxin metabolites is relatively rapid in growing rats. The major portion of the AFB1-GSH is formed in liver but contribution of the renal tissue to the formation of detoxification metabolites can not be ruled out. These data demonstrate that aflatoxin metabolites are eliminated more efficiently from kidneys of a growing rat. AFB1-induced GST induction in renal tissues of growing animals during 24 h of the carcinogen administration could be considered as an important mechanism for GSH conjugate formation and aflatoxin detoxification. Therefore GST induction in response to hepatotoxic drugs can confer resistance to young animals being exposed for the first time to such drugs. It is also worthmentioning that the GST activity measured before AFB1 administration does not reflect the rate of AFB1 detoxification via GSH conjugation.

Effect of the spine venom from the crown-of-thorns starfish, Acanthaster planci, on drug-metabolizing enzyme in rat liver.

The effect of spine venom from the crown-of-thorns starfish (Acanthaster planci) on drug-metabolizing enzymes in rat liver was studied. The spine venom was prepared by saturation of spine homogenate with ammonium sulfate and the protein fraction precipitating 50% saturation was used as venom B. Venom A was the protein precipitated between 50 and 100% saturation. When venom B (100-200 mg/kg) was given to rats, liver microsomal GSH S-transferase and cytochrome P450 activities decreased while cytosolic GSH S-transferase activity was not changed. The decrease in these microsomal enzyme activities was seen from 12 hr to 24 hr after giving 100 mg/kg of venom B. Rats given venom A died, suggesting an involvement of the lethal factor in venom A. The data showed that the spine venom B from A. planci depressed microsomal GSH S-transferase and cytochrome P450 activities in rat liver and that this venom was distinct from the lethal factor of the spine venom.

Glutathione metabolism in hepatomous liver of rats treated with diethylnitrosamine

Glutathione metabolism was studied in rat liver during diethylnitrosamine (DEN) carcinogenesis. Some studies were also made in foetal rat liver. Endogenous GSH and non-protein thiols concentrations are increased in DEN-treated rats when compared to non-treated rats but no differences were found in cysteine, total thiols and protein thiols concentration. In foetal liver GSH concentration is only 35% of that in DEN-treated rat liver. The activities of several enzymes involved in glutathione metabolism are changed in DEN-treated rats. γ-Glutamyl transferase activity and cysteine formation from GSH by liver homogenates is increased sevenfold. γ-Glutamylcysteine synthetase activity, initial rate of [ 35S]cysteine incorporation in γ-glutamylcysteine and initial rate of GSH formation from [ 35S]cysteine are increased two-fold. Cytosolic GSH S-transferase activity is increased twofold in DEN-treated rats and so GSH S-conjugates concentration is probably also increased. In foetal rat liver γ-glutamyl transferase activity is about the same but γ-glutamylcysteine synthetase activity is only 10% of that in DEN-treated rat liver. The increased GSH concentration in DEN-treated rat liver is probably due to the simultaneous increase in the activities of γ-glutamyl transferase and γ-glutamylcysteine synthetase. Blood plasma total glutathione is increased 1.4 times in DEN-treated rats, but no differences are found in GSH hepatic arteriovenous gradient. This associated with the increased γ-glutamyl transferase activity suggests that sinusoidal GSH efflux is increased in DEN-treated rats.

Glutathione and glutathione-related enzyme activities of male and female rat hepatocytes under various culture conditions.

The effect of culture medium on glutathione (GSH) dependent detoxification defence system of primary cultured hepatocyte from either male or female rats was studied. Intracellular reduced (GSH) and oxidized glutathione (GSSG), and six GSH-related enzyme activities, including GSH peroxidase (GSH Px), GSH reductase (GSH Rd), cytosolic GSH S-transferase (cGST), microsomal GSH S-transferase (mGST), gamma-glutamyl transpeptidase (GTP), and gamma-glutamylcysteine synthetase (GCS), were investigated during a 6-day culture. Media free of fetal bovine serum (FBS) and with 2.5 or 10% FBS were used. Whatever the medium, there was an initial decrease of intracellular GSH and GSSG, a threefold increase of GSH at day 3 and fourfold increase of GSSG at day 4, later decreasing to their original level at day 6. The activities of all six GSH-related enzymes of male and female hepatocytes remained relatively stable during the first 72h, then gradually decreased to 50-80% of initial activities. With the exception of cGST, time-course profiles of other enzyme activities were not significantly different among various media. In both sexes, higher cGST activity was maintained for cells cultured in the presence of FBS. Results of immunoblotting analysis of cytosolic GST isozymes indicate that the placental form of GST (Yp) was markedly increased after plating and the extent of increase of Yp was higher in the presence of FBS. Despite the culture medium, the level of GST isoform Ya was maintained steadily for 6 days, however, Yb was maintained during the first 3 days and then decreased. In terms of the gender difference, GSH Px and GTP activities of hepatocytes from females were significantly greater than of males over the entire culture period. Results indicate that FBS seems not to be absolutely essential in maintaining GSH level and most of the GSH-related enzyme activities in rat hepatocytes. Furthermore, GSH levels and GSH-related enzyme activities of hepatocytes from female rats were similar to those from male rats.

Acetaminophen-Derived Activation of Liver Microsomal Glutathione S-Transferase of Rats

Effect of acetaminophen on glutathione (GSH) S-transferase and related drug metabolizing ;nzymes was studied in vivo. Rats were given acetaminophen (250 mg/kg, i.p.) 24 hr after the treatment with 3-methylcholanthrene (25 mg/kg, i.p.) and killed by decapitation at indicated times. Liver microsomal GSH S-transferase activity was increased to 331%, 193% and 158% of the control level at 3, 6 and 12 hr, respectively, after the administration of acetaminophen, while GSH content in the liver was markedly decreased at 3 and 6 hr. The increase in the transferase activity was not recovered by the treatment with dithiothreitol. Microsomal GSH peroxidase activity was significantly enhanced at 3 hr. Cytosolic GSH S-transferase and aniline hydroxylase in microsomes were gradually decreased with the increase in the time after administration of acetaminophen. Vmax values of both GSH S-transferase and GSH peroxidase activities in microsomes were increased at 3 hr. Two Km values were obtained for the peroxidase in the control, while only one was observed after the acetaminophen treatment. These results indicate that acetaminophen is converted via cytochrome P-450 to the reactive intermediate N-acetyl-p-benzoquinone imine, which binds to microsomal GSH S-transferase, resulting in the activation of the enzyme.

A Subfamily 2 Homo-Dimeric Glutathione S-Transferase mYrs-mYrs of Class Theta in Mouse Liver Cytosol

A homo-dimeric subfamily 2 glutathione (GSH) S-transferase (GST) mYrs-mYrs of the class theta was isolated from mouse liver cytosol and purified to homogeneity. The first 28 N-terminal amino acid sequence of the GST was completely identical to that of rat subfamily 2 GST Yrs-Yrs of the class theta. GST mYrs-mYrs cross-reacted with anti-rat GST Yrs-anti-sera but not with anti-sera raised against rat GSTs Ya-Ya (alpha), Yb1-Yb1 (mu), and Yp-Yp (pi) and represented more than 95% of the mouse liver cytosolic GST activity to scavenge the reactive sulfate ester 5-sulfoxymethylchrysene of the potent carcinogen 5-hydroxymethylchrysene. The mouse class theta GST had little activity toward 1-chloro-2,4-dinitrobenzene and was unretainable on GSH and an S-hexyl-GSH affinity columns. GST mYrs-mYrs had a much higher GSH peroxidase activity toward fatty acid hydroperoxides than did the other classes of mouse GSTs.

A new class of rat glutathione S-transferase Yrs-Yrs inactivating reactive sulfate esters as metabolites of carcinogenic arylmethanols.

A glutathione (GSH) S-transferase (GST), catalyzing the inactivation of reactive sulfate esters as metabolites of carcinogenic arylmethanols, was isolated from the male Sprague-Dawley rat liver cytosol and purified to homogeneity in 12% yield with a purification factor of 901-fold. The purified GST was a homo-dimeric enzyme protein with subunit Mr 26,000 and pI 7.9 and designated as Yrs-Yrs because of its enzyme activity toward "reactive sulfate esters." GST Yrs-Yrs could neither be retained on the S-hexylglutathione gel column nor showed any activity toward 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and 1,2-epoxy-3-(4'-nitrophenoxy)propane. 1-Chloro-2,4-dinitro-benzene was a very poor substrate for this GST. 1-Menaphthyl sulfate was the best substrate for GST Yrs-Yrs among the examined mutagenic arylmethyl sulfates. The enzyme had higher activities toward ethacrynic acid and cumene hydroperoxide. N-terminal amino acid sequence of subunit Yrs, analyzed up to the 25th amino acid, had no homology with any of the known class alpha, mu, and pi enzymes of the Sprague-Dawley rat. Anti-Yrs-IgG raised against GST Yrs-Yrs showed no cross-reactivity with any of subunits Ya, Yc, Yb1, Yb2, and Yp. Anti-IgGs raised against Ya, Yc, Yb1, Yb2, and Yp also showed no cross-reactivity with GST Yrs-Yrs. The purified enzyme proved to differ evidently from the 12 known cytosolic GSTs in various tissues of the rat in all respects. Immunoblot analysis of various tissue cytosols of the male rat indicated that apparent concentrations of the GST Yrs-Yrs protein were in order of liver greater than testis greater than adrenal greater than kidney greater than lung greater than brain greater than skeletal muscle congruent to heart congruent to small intestine congruent to spleen congruent to skin congruent to 0.

Metabolic activation of the nephrotoxic haloalkene 1, 1, 2-trichloro-3, 3, 3-trifluoro-1-propene by glutathione conjugation

1, 1, 2-Trichloro-3, 3, 3-trifluoro-1-propene (TCTFP) is structurally closely related to the stable and non-toxic tetrachloroethylene. However, in TCTFP, the trifluoromethyl group enhances chemical reactivity with nucleophiles. This fact suggested that TCTFP may be metabolized intensively by glutathione (GSH) conjugation and therefore, like hexachlorobutadiene, would be expected to be nephrotoxic. We have investigated the nephrotoxicity and metabolism of TCTFP. Administration of 20 and 40 mg/kg to male rats resulted in a large, dose-dependent increase in urinary excretion of γ-glutamyl transpeptidase (GGT) indicative of proximal tubular damage. No increase in plasma transaminase concentrations indicative of liver damage was found. In rats, N- acetyl-S-(1,2- dichloro-3, 3, 3- trifluoro-1- propenyl)- l - cysteine was a major urinary metabolite of TCTFP. TCTFP was transformed by microsomal and cytosolic GSH S-transferases from rat liver to S-(1,2-dichloro-3, 3, 3-trifluoro-1-propenyl)glutathione (DCTFPG) (identified by NMR and mass spectrometry). DCTFPG was toxic to rat renal cortex cells. Inhibition of GGT and cysteine conjugate β-lyase blocked DCTFPG cytotoxicity. These results suggest the following TCTFP bioactivation: conjugation with GSH in the liver, catabolism of the GSH S-conjugate to the cysteine S-conjugate and cleavage of the cysteine S-conjugate by β-lyase with formation of reactive intermediates in the kidney.

Effect of β-naphthoflavone on the metabolism of aflatoxin B 1 in hamsters

The effect of β-naphthoflavone (BNF) pretreatment of hamsters on the hepatic metabolism of aflatoxin B 1 (AFB 1) has been examined in studies in vitro and in vivo. Pretreatment with BNF not only increased microsomal cytochrome P-450 by 50–80% but also increased microsome-mediated AFB 1 epoxidation as measured by AFB 1-DNA binding 2.6 fold without significantly affecting other hydroxylations. Neither cytosolic GSH S-transferases' activities nor AFB 1-GSH (AFB 1-SG) conjugation were affected. In vivo, hepatic AFB 1-DNA binding was also increased about 3–4-fold. These results in contrast to those observed in the rat indicate that induced species of cytochrome P-450 are primarily responsible for higher epoxidation of AFB 1 in the hamster.

Mechanism of Inhibition of Aflatoxin B1- Dna Binding in the Liver by Butylated Hydroxyanisole Pretreatment of Rats

Previous studies have demonstrated that pretreatment of rats with butylated hydroxyanisole (BHA) inhibited aflatoxin B1 (AFB1)-DNA binding and AFB1 hepatocarcinogenesis. Our detailed studies on the effect of BHA pretreatment of rats on both AFB1-DNA binding and AFB1-glutathione (AFB1-SG) conjugation with hepatosubcellular fractions, isolated hepatocytes and in intact animals are reviewed. For these studies, young male rats were fed a semisynthetic diet with or without 0.75% BHA for 2 wks. Even though there were no significant differences either in microsomal cytochrome P-450 content or microsome-mediated AFB1 binding to exogenous DNA with cytochrome P-450 from control or treated rats, there were large differences in GSH S-transferase activity with treated cytosols showing 100% higher activity than the controls. Kinetics of cytosolic inhibition of microsome-mediated AFB1-DNA binding and formation of AFB1-SG conjugate indicated that the inhibition of AFB1-DNA binding was greater with cytosols from treated compared to the controls with the concomitant formation of AFB1-SG conjugate. Reconstitution studies with intact nuclei, microsomes and cytosol indicated more AFB1-DNA binding with the control than with BHA-treated animals. In isolated hepatocyte system, at 2 μM level of AFB1, AFB1-DNA binding in treated hepatocytes was about 15% of controls whereas thiol conjugation was 5 fold higher in the treated than in control hepatocytes. Addition of 1 mM styrene oxide caused about 100% and 9 fold increase in AFB1-DNA binding in control and treated hepatocytes respectively with corresponding decreases in thiol conjugation. In intact rats, BHA treatment reduced hepatic AFB1-DNA binding to 15% of controls with concomitant increase in biliary excretion of AFB1-SG conjugate. These results indicate that the induced cytosolic GSH S-transferases after BHA treatment of rats play a significant role in inhibiting hepatic AFB1-DNA binding and AFB1 hepatocarcinogenesis by inactivation of the reactive AFB1-epoxide.

Positive correlation exists between glutathione S-transferase activity and aflatoxin formation in Aspergillus flavus.

The presence of glutathione (GSH) S-transferase activity, using 1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate, has been established in the cytosolic fraction of the toxigenic (aflatoxin producing) and nontoxigenic strains of Aspergillus flavus. Significant differences in the GSH S-transferase activity were observed between the toxigenic and non-toxigenic strains. A positive correlation has been demonstrated for the first time between aflatoxin formation and a biochemical parameter, namely GSH S-transferase activity. The evidence in support of A. flavus GSH S-transferase induction by endogenous aflatoxins is as follows: (i) the age-related production of aflatoxin follows the same pattern as the cytosolic GSH S-transferase activity profile; (ii) significantly higher enzyme activity was associated with mycelia of a toxigenic strain grown in medium supporting high aflatoxin production (sucrose-low-salts medium) while the enzyme activity was low in medium producing less aflatoxin (glucose-ammonium nitrate medium). The GSH S-transferase activity of the non-toxigenic strain was hardly affected by a change in the medium as it produces no aflatoxins; and (iii) the toxigenic strain demonstrated significantly higher apparent Vmax. with no change in Km as compared with the non-toxigenic strain. This indicates that the enzyme induction by endogenous aflatoxins is similar to the action of phenobarbitol and other inducing drugs (Kaplowitz et al., 1975).

Effect of subchronic cholestasis on microsomal mixed-function oxidases and the glutathione-conjugating enzyme system in rat liver

Bile duct ligation in male rats for two weeks led to a marked increase in both serum sorbitol dehydrogenase activity and serum bile acid concentration indicating cholestatic liver injury. Furthermore, a rise in the hepatic hydroxyproline level indicating collagen accumulation was observed. As a consequence of these alterations, the hepatic microsomal mixed-function oxidase system was impaired as evidenced by a decrease in cytochrome P-450 content and in the activities of NADPH-cytochrome c reductase and aminopyrine-demethylase. While the hepatic glutathione content remained unaffected, the cytosolic GSH S-transferase activity was clearly suppressed due to subchronic cholestasis.

Comparative study of hepatic microsomal and cytosolic enzyme activities in three murine strains

A comparative study on hepatic microsomal p-nitroanisole- O-demethylase, aminopyrine- N-demethylase, NADPH-cytochrome c reductase and cytosolic glutathione (GSH) S-transferase was performed in both sexes of C3Hf, C57BL/6J and DBA/2 mice. In control and phenobarbital (PB)-induced mice similarities in hepatic microsomal activities were found with slight variations among strains and sexes. The Ah locus nonresponsive DBA/2 mice showed uninduced microsomal activities after β-naphthoflavone (BNF), as expected; the other two strains were induced by BNF to a similar extent. Differences in cytosolic GSH S-transferase were detected among strains and sexes; C57BL/6J mice showed the highest basal and induced levels. The males of all the three strains examined were found to have higher GSH S-transferase activity than females.

Differences in inducibility of particulate and cytosolic rat liver glutathione S-transferase activities.

1. Rat liver mitochondrial microsomal glutathione (GSH) S-transferase activities with 1-chloro-2,4-dinitrobenzene as substrate were 11.0 and 11.6%, and with 1,2-dichloro-4-nitrobenzene as substrate were 27.7 and 15.4%, of the corresponding cytosolic activities respectively. 2. Marked difference in inducibility of cytosolic and particulate GSH S-transferase activities were seen after pretreatment of animals with enzyme-inducing agents. 3-Methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin enhanced cytosolic GSH S-transferase activity only. Phenobarbital induced the cytosolic and microsomal enzymes only.