Polymorphism- and species-dependent inactivation of glutathione transferase zeta by dichloroacetate

Glutathione transferase zeta catalyzes the glutathione-dependent oxidation or conjugation of a range of alpha-haloacids. Repeated administration of dichloroacetate to human subjects increases its plasma elimination half-life, and the activity of glutathione transferase zeta is decreased in rats given dichloroacetate. The objective of the studies presented here was to investigate the kinetics and mechanism of the dichloroacetate-induced decrease in glutathione transferase zeta activity. The rate constants (k(inact)) for the dichloroacetate-dependent inactivation of glutathione transferase zeta in liver cytosol are in the following order: rat > mouse > human; the half-maximal inhibitory concentration (K(inact)) of DCA did not differ among the species that were studied. In contrast to dichloroacetate, chlorofluoroacetate produced much less inactivation of mouse liver glutathione transferase zeta activity. Moreover, the addition of N-acetyl-L-cysteine or potassium cyanide did not fully block the dichloroacetate-induced inactivation of glutathione transferase zeta. The k(inact) values for the dichloroacetate-induced inactivation of four polymorphic variants of recombinant human glutathione transferase zeta (hGSTZ1-1) were in the following order: variant 1a-1a < 1b-1b approximately 1c-1c approximately 1d-1d. The dichloroacetate-induced inactivation of hGSTZ1-1 was irreversible. The binding of radioactivity from [1-(14)C]dichloroacetate and from [(35)S]glutathione to recombinant hGSTZ1c-1c was demonstrated, indicating covalent modification of the protein. These results show that dichloroacetate is a mechanism-based inactivator of glutathione transferase zeta and is biotransformed to electrophilic metabolites that covalently modify and, thereby, inactivate the enzyme.     

Inhibition of human hepatic glutathione S-transferase isozymes by ethacrynic acid and its metabolites.

The comparative inhibition of ethacrynic acid (EA) and its known metabolites against glutathione S-transferase (GST) was investigated using human livers procured from kidney donors. EA and all three metabolites of EA had an inhibitory effect against conjugation between 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH). The GSH adduct of EA (EA-GSH) was the most potent inhibitor of GSTs; EA-GSH was approximately one order of magnitude more potent than the parent EA, while L-cysteine conjugate of EA (EA-cysteine) and N-acetyl-L-cysteine conjugate of EA (EA-mercapturate) were approximately two orders of magnitude less potent than the parent EA. Further metabolism of EA-GSH conjugate is suggested to be a detoxification process in terms of GST activities.     

Effect of long-term ethanol exposure on the acinar distribution of hepatic glutathione S-transferase.

The ability of ethanol to affect the regional distribution of individual glutathione S-transferase (GST) isoenzymes in rat liver was investigated by analyzing the expression levels in cell lysates obtained from the periportal or perivenous liver region after in situ digitonin perfusion. In control rats, a significant perivenous dominance of GST proteins and activities measured by the substrates 1-chloro-2,4-dinitrobenzene (broad spectrum), 1, 2-dichloro-4-nitrobenzene (M1/M2-specific), and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (A1/A2-specific) was found. In pair-fed rats exposed to ethanol (36% of total calories) for 2 weeks, all GST activities measured were significantly increased in both acinar zones. However, the relative increase was greater in the perivenous region. The induction of the A1/A2-specific activity was the most pronounced. HPLC analysis revealed for both regions that this increase was largely confined to the A2 subunit, with only minor effects observed on the A1 subunit. At the mRNA level, the constitutive perivenous dominance of both GST A1 and GST A2 expression became more pronounced after ethanol administration. The results demonstrate that long-term ethanol exposure induces individual GST isoenzymes differently and might have a profound effect on xenobiotic-induced regional liver damage.     

Effect of garlic on the hepatic glutathione S-transferase and glutathione peroxidase activity in rat

It was attempted to observe the effect of garlic on the hepatic glutathione s-transferase and glutathione peroxidase activity in this study. Glutathione s-transferase (EC 2.5.1.18) are thought to play a physiological role in initiating the detoxication of potential alkylating agents, inclnding pharmacologically active compounds. Glutathione peroxidase (EC 1.11.1.9) might play an important role in the protection of cellular structures against oxidative challenge. The activities of glutathione s-transferase and glutathione peroxidase in rat liver were increased by the treatment of garlic juice. Allicin fraction, heat-treated allicin fraction and garlic butanol fraction markedly inhibited glutathione s-transferase activityin vitro, whereas glutathione peroxidase activity was significantly increased in heat-treated allicin fraction and garlic butanol fraction.     

Inhibition of rat and human glutathione S-transferase isoenzymes by ethacrynic acid and its glutathione conjugate

Ethacrynic acid, a potent inhibitor of glutathione S-transferases (GST), has been shown to enhance the cytotoxicity of chlorambucil in drug resistant cell lines, but a definite mechanism has not been established. Both covalent binding to GST and reversible inhibition of GST have been reported. In the present study no irreversible inhibition was observed: for all rat GST tested, inactivation was complete within 15 sec at 0 degree, and dialysis of GST after incubation with ethacrynic acid gave complete recovery of enzyme activity for all isoenzymes tested. Moreover, the inhibition was competitive towards 1-chloro-2,4-dinitrobenzene and non-competitive towards glutathione for rat isoenzyme 1-1. Strong inhibition of both human and rat GST of the alpha-, mu- and pi-classes was obtained with ethacrynic acid, while conjugation of ethacrynic acid with glutathione did not abolish its inhibiting properties. For the alpha-, mu- and pi-class I50 values (microM) were 4.6-6.0, 0.3-1.9 and 3.3-4.8, respectively for ethacrynic acid, and 0.8-2.8, less than 0.1-1.2 and 11.0, respectively for its glutathione conjugate. Of all isoenzymes tested the human isoenzyme mu is most sensitive to the action of both ethacrynic acid and its glutathione conjugate.     

Inhibition of purified rat liver glutathione S-transferase isozymes by diuretic drugs

Seven soluble rat liver glutathione S-transferase isozymes were isolated and the inhibition of these isozymes by selected diuretics was investigated using 1-chloro-2,4-dinitrobenzene as substrate. All isozymes were inhibited to some extent under the experimental conditions used, but there was significant isozyme dependent selectivity of inhibition. The greatest inhibitory effect (over 80%) was found when the phenoxyacetic acid diuretics and indacrynic acid were incubated with glutathione S-transferase 3-3, 3-4 and 4-4. The sulphamoylbenzoic acid diuretics, furosemide and bumetanide, were found to have a lesser effect on the isozymes studies. As glutathione S-transferase are thought to play an important protective role in the various tissues of animals and man, by catalysing the glutathione conjugation of electrophilic drugs and drug metabolites, their inhibition may be toxicologically important.     

Inhibition of various glutathione S-transferase isoenzymes by RRR-alpha-tocopherol.

The activity of human cytosolic glutathione S-transferases (GSTs) can positively or negatively be changed by various compounds. It is for instance known that RRR-alpha-tocopherol inhibits GST P1-1 [Haaften van R.I.M. et al. (2001) Alpha-tocopherol inhibits human glutathione S-transferase pi. BBRC 280, 631-633]. The effect of RRR-alpha-tocopherol on the other isoenzymes of GST in purified forms of the isoenzymes and in human liver cytosol (GST M and GST A) and lysate of human erythrocytes (GST P) is studied. It is found that all isoenzymes (purified enzymes and enzymes present in homogenates) are inhibited, in a concentration-dependent way, by RRR-alpha-tocopherol. GST P is in both cases inhibited with the highest potency compared to the other isoenzymes. It also appeared that the purified GST P1-1 isoenzyme is non-competitively inhibited by RRR-alpha-tocopherol. The IC(50) values of RRR-alpha-tocopherol for the purified isoenzymes of GST are much lower compared to the IC(50) values for human lysate and human liver cytosol. This is probably due to binding of RRR-alpha-tocopherol to proteins, e.g. albumin and hemoglobin, with higher affinity than to GST; so more RRR-alpha-tocopherol is needed to inhibit the enzyme. However, the inhibition of GSTs by RRR-alpha-tocopherol can still be of physiological relevance, because due to dermal application of cosmetic products very high concentrations vitamin E can be reached in the skin, where GST P1-1 is present. RRR-alpha-tocopherol might also be a good lead compound for the development of a new class of inhibitors of GST that can be used as adjuvant in cancer therapy.     

Reversible inhibition of rat hepatic glutathione S-transferase 1-2 by bilirubin

The inhibition of rat hepatic glutathione (GSH) S-transferase 1-2 by bilirubin exhibited pseudo first-order kinetics with k(obs) values of 0.0214 +/- 0.0005 and 0.040 +/- 0.008 sec-1 at 4 and 8 microM bilirubin, when followed to 72 and 84% completion respectively. These correspond to calculated second-order rate constants of 5.3 +/- 0.1 x 10(3) and 5.0 +/- 1.0 x 10(3)/M.sec. The extent of inhibition of the transferase increased with bilirubin concentration, with half-maximal inhibition at 4 microM bilirubin. Inhibition was reversed by 10-fold dilution of bilirubin or by increasing the pH from 6.0 to 7.4. Premixing 0.2 to 0.5 microM albumin, hemoglobin or aldolase with bilirubin prevented inhibition of GSH S-transferase 1-2. Protection by these proteins occurred at a selected high concentration (0.2 to 0.4 microM) at which they reduced free bilirubin to concentrations (less than 0.5 microM) that did not inhibit isoenzyme 1-2 significantly. No protection was afforded by a selected low protein concentration (0.001 to 0.01 microM) which did not strikingly reduce bilirubin levels in solution. We conclude that bilirubin inhibition of GSH S-transferase 1-2 appears to be a second-order process; the reaction is clearly first-order with respect to GSH S-transferase and appears also to be first-order with respect to bilirubin. It is proposed that (a) inhibition of GSH S-transferase 1-2 results from slow, reversible bilirubin binding, and (b) added proteins appear to prevent GSH S-transferase inhibition by binding high molar ratios of bilirubin.     

Hepatic mixed function oxygenase activity and glutathione S-transferase activity in mice following ethanol consumption and withdrawal

The effect of an 8 day liquid diet containing 7% v/v ethanol and the effect of ethanol withdrawal on several drug metabolizing enzyme activities, cytochrome P-450 content and glutathione S-transferase activity (GST) has been studied in male C57/BL mice. After treatment, hepatic microsomal activities toward benzphetamine (BNZ), biphenyl (BPH) and dimethylnitrosamine (DMN) and cytosolic GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) were determined. Ethanol treatment caused a differential time dependent increase in the metabolism of the 4 xenobiotics. Increased BPH-4-hydroxylase activity correlated most closely with that of the increased concentration of hepatic P-450. That is, both values were increased (5.8-fold) over controls after 8 days of ethanol treatment. Ethanol withdrawal (24 h) resulted in a 46% reduction in the P-450 content and a 26% reduction in BPH-4-hydroxylase activity compared to the elevated values at day 8. By 48 h, the values were no different from controls. DNA-N-demethylase, BNZ-N-demethylase and GST activities all increased after 4 days of ethanol treatment and remained the same at 8 days. However, ethanol withdrawal resulted in differential time dependent changes in the activities towards BNZ, DMN, and CDNB. While DMN-N-demethylase activity returned to control activity within 24 h, BNZ-N-demethylase activity did not change for the first 24 h of withdrawal, but returned to control activity by 48 h. GST activity had not decreased by 48 h of withdrawal. These data suggest that ethanol induces several cytochrome P-450 isozymes that have a time difference in induction by ethanol and reduction following ethanol withdrawal. Furthermore, ethanol induction of GSTs occurs quickly (4 days) and remains elevated at least 48 h after ethanol withdrawal.     

Effects of lead exposure on the glutathione metabolism of rat lenses

Abstract

Effects of lead (Pb) were studied on rat lenses after oral exposure to lead acetate (0.1 %) in drinking water given ad libitum for up to 3 months. Activities of the key enzymes involved in the glutathione metabolic pathway were found to be severely impaired along with an increase of blood/lens Pb level. Activity of superoxide dismutase, which is responsible for scavenging the free radicals, was also significantly decreased. Depletion of reduced glutathione content with a concomitant elevation of peroxide levels showed an oxidative stress in the ocular lens as a result of Pb exposure. The results of the present study demonstrate that Pb significantly impairs the defense mechanism of the lens, which comprises both the glutathione and superoxide dismutase systems.     

Reduction in cutaneous and hepatic glutathione contents,glutathione S-transferase and aryl hydrocarbon hydroxylase activities following topical application of acrylamide to mouse

A single topical application of acrylamide to mouse resulted in time-dependent depletion in cutaneous and hepatic glutathione contents, glutathione S-transferase and aryl hydrocarbon hydroxylase activities.     

Papaverine, an opium alkaloid influences hepatic and pulmonary glutathione S-transferase activity and glutathione content in rats.

The present study evaluates the effect of oral administration of papaverine at differential dosing regimens (100 mg/kg bw and 200 mg/kg bw) on the hepatic and pulmonary glutathione S-transferase (GST) activity and glutathione content (GSH) in male Wistar rats. Papaverine treatment caused a pronounced increase in GST activity and GSH content at the higher dosing level in the rat liver and lung. We conclude that papaverine, can possibly act as a chemopreventive agent against chemical carcinogenesis.     

Effect of diallyl disulfide on the hepatic glutathione s-transferase activity in rat: Diallyl disulfide effect on the glutathione S-transferase

Glutathione s-transferase is thought to play a key role in initiating the detoxication of potential alkylating agents, including pharmacologically active compounds. It is widely accepted that garlic contains allin which is converted to allicin by alliinase. Allicin is easily degraded to diallyl disulfide and other components. This report attempted to observe the effect of diallyl disulfide on some biological activities. It was observed that the activity of serum transaminase was not changed by the treatment of diallyl disulfide. The liver cytosolic glutathione s-transferase was significantly increased. whereas the microsomal glutathione s-transferase was not increased.     

Role of glutathione and glutathione S-transferase in chlorambucil resistance.

A chlorambucil (CLB)-resistant cell line, N50-4, was developed from the established mouse fibroblast cell line NIH 3T3, by multistep drug selection. The mutant cells exhibited greater than 10-fold resistance to CLB. Alterations in GSH and glutathione S-transferase (GST) were found in CLB-resistant variants. A 7-10-fold increase in cellular GSH content and a 3-fold increase in GST activity were detected in N50-4 cells, compared with parental cells, as determined by enzymatic assays. An increase in steady state levels of the GST-alpha isozyme mRNA was found in the CLB-resistant cells, as analyzed by Northern blotting. No GST gene amplification or rearrangement was shown by Southern blot analysis. To test the relative roles of GSH and GST in CLB resistance, a number of GSH- and GST-blocking agents were used. The CLB toxicity was significantly enhanced in N50-4 cells by administration of either the GSH-depleting agent buthionine sulfoximine or the GST inhibitors ethacrynic acid or indomethacin. The resistance to CLB cytotoxicity in N50-4 cells, however, was still significantly higher than that of parental cells. The resistance of N50-4 cells to CLB was almost completely abolished by combination pretreatment yielding both GSH depletion and GST inhibition. The results indicate that both increased cellular GSH content and increased GST activity play major roles in CLB resistance in N50-4 mutant cells.     

Inhibitory effect of anthocyanidins on hepatic glutathione S-transferase,UDP-glucuronosyltransferase and carbonyl reductase activities in rat and human

Abstract

1. Anthocyanins and their aglycone anthocyanidins represent the most abundant flavonoids in human diet and popular constituents of various dietary supplements. The aim of this study was to evaluate inhibitory effect of four anthocyanidins (delphinidin, cyanidin, malvidin and pelargonidin) on three families of important drug-metabolizing enzymes: carbonyl reductases (CBRs), glutathione S-transferases (GSTs) and UDP-glucuronosyltransferases (UGT).

2. Human or rat hepatic subcellular fractions were incubated with or without pure anthocyanidins (100?µM) and the activities of CBR, GST and UGT were assayed using menadione, 1-chloro-2,4-dinitrobenzene and p-nitrophenol as substrates, respectively. For the most potent inhibitors, half maximal inhibitory concentrations (IC₅₀) were determined and the inhibition kinetics study was performed.

3. Anthocyanidins inhibited weakly the activity of GST and moderately the activities of CBR and UGT. Cyanidin was the most potent inhibitor of human UGT with IC₅₀?=?69?µM (at 200?µM substrate concentration) and competitive type of action. Delphinidin acted as significant non-competitive inhibitor of human CBR with IC₅₀?=?16?µM (at substrate concentration 500?µM). The inhibitory potency of anthocyanidins differed in rat and human samples significantly.

4. Anthocyanidins are able to inhibit CBR and UGT in vitro. Possible interference of anthocyanidins (in high-dose dietary supplements) with simultaneously administered drugs, which are UGT or CBR substrates, should be checked.     

Inhibition of tyrosine hydroxylase activity by methyl bromide exposure.

Rats were exposed to methyl bromide gas (16-250 ppm) for 8 hr, and tyrosine hydroxylase (TH) activity in the striatum, hypothalamus, frontal cortex, midbrain, and medulla oblongata was measured in brain homogenates from exposed rats, and in vivo following administration of decarboxylase inhibitor. Exposure to methyl bromide dose-dependently inhibited both in vitro and in vivo TH activity. Of the five brain areas, TH activity in the hypothalamus was most sensitive to methyl bromide. The time course of enzyme inhibition after exposure was similar to those of decreases in catecholamine concentrations, locomotor activity change, and body temperature reported previously. These results suggest methyl bromide reduces catecholaminergic neuronal activity in the brain via inhibition of TH activity.