Identification and quantitation of glutathione in hepatic protein mixed disulfides and its relationship to glutathione disulfide

The amount of glutathione present in hepatic protein mixed disulfides was determined to be 20–30 nmole/g liver. This was established using two specific enzymatic methods: (a) the coupled assay with DTNB and glutathione (GSSG) reductase and (b) a newly developed test using GSH transferase and 1-chloro-2,4-dinitrobenzene for the estimation of GSH released from proteins after borohydride treatment; further, these results were confirmed by HPLC analysis. Thus, authentic glutathione makes up only 2–6% of the value for total protein mixed disulfides. The latter were determined with the generally employed o-phthalaldehyde assay, which is not necessarily specific for GSH. The amount of glutathione mixed disulfides depends linearly on the content of glutathione disulfide in the liver cell in the range studied. By increasing the GSSG levels from 20 to about 60 nmole/g liver with paraquat, nitrofurantoin or t-butyl hydroperoxide, glutathione protein mixed disulfides are increased by a similar amount.     

Conjugation of acrylamide with glutathione catalysed by glutathione-s-transferases of rat liver and brain

Acrylamide, an α,β unsaturated electrophile and a cumulative neurotoxin, was found to react with glutathione giving rise to an S-conjugate of acrylamide. Glutathione S-transferase of rat liver and brain cytosols (active on both acrylamide and 1-chloro 2,4 dinitrobenzene) emerged as a single major peak on elution from Sephadex G-75. The enzymic conjugation of acrylamide with glutathione increased with protein and was dependent on incubation time and pH of medium. Acrylamide inhibited glutathione-S-transferase activity towards 1-chloro 2,4-dinitro-benzene of both liver and brain cytosol, in a concentration and time dependent manner. Enzyme catalyzed conjugation of acrylamide with glutathione was induced significantly by phenobarbital and t-SO (tans-stilbene oxide). The enzymic conjugation of acrylamide increased two fold from neonatal to adult and then showed a decreasing pattern at subsequent ages.     

Effect of old age on paracetamol-induced lipid peroxidation in rat liver

Post-mitochondrial supernatants isolated from the livers of mature rats (3 to 6 months old) 2 h or more after the administration of a single large oral dose of paracetamol (800 mg/kg) showed rapid rates of lipid peroxidation when incubated in vitro. In similar experiments with old rats (27-30 months old) the time between administration of paracetamol and the onset of lipid peroxidation was much longer (6 h or more). In both age groups, lipid peroxidation was dependent on the depletion of glutathione from the liver.     

Detoxication of industrial pollutants by the glutathione glutathione-S-transferase system in the liver of Anabas testudineus (Bloch)

The interrelationship of reduced glutathione (GSH) and glutathione-S-transferase in the liver of a freshwater climbing perch Anabas testudineus (Bloch) exposed to common industrial pollutants has been studied. In both short- and long-term treatments there was a concomitant decrease in reduced glutathione profile and an increase in glutathione-S-transferase activity. It may be surmised that the majority of xenobiotics of industrial origin are detoxicated by the glutathione glutathione-S-transferase pathways enabling the fish to survive exposure to the additive and/or synergistic toxicity of mixtures of poisons.     

The influence of phenobarbital, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin on glutathione s-transferase activity of rat liver cytosol

Specific activities and apparent Michaelis-Menten kinetic parameters were determined for glutathione (GSH) S-transferase activity (E.C. 2.5.1.18) in rat liver cytosol, towards styrene oxide (STOX), 1,2-butylene oxide (BOX) and 1-chloro-2,4-dinitrobenzene (CDNB) as electrophilic substrates, before and after pretreatment with the drug-metabolizing enzyme inducers phenobarbital (PB), 3-methylcholanthrene (MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The measured GSH S-transferase activities appear to obey Michaelis-Menten kinetics. In non-induced animals the apparent K_m values of the transferase activities were equal for STOX vs GSH, but they differed by a factor of 2 for CDNB vs GSH and by a factor of 14 for BOX vs GSH. The apparent V_max values in each combination of GSH and electrophilic substrate were equal, but differed by one order of magnitude for the mutual substrate combinations. Pretreatment of the rats with MC resulted in enhancement of all measured activities expressed in terms of cytosol protein, while TCDD only enhanced the activities expressed as per gram body wt. PB enhanced both activities when STOX was employed as substrate, but when CDNB was used as the substrate, only the activity per gram body wt increased. All pretreatments increased the V_max values using CDNB as the substrate, while PB and MC had an enhancing effect using STOX; the V_max using BOX was enhanced after TCDD administration only. The K_m values using BOX as the substrate was lowered after MC pretreatment; TCDD pretreatment decreased the K_m using STOX, while it increased the K_m using CDNB. It is concluded that the GSH S-transferase system is inducible, but in contrast to the induction of the mixed function oxidase system, qualitative differences between the inducing effects of PB and MC were not observed. Use of TCDD as inducing agent, however. resulted in a different induction pattern, which may indicate that during induction with this agent different types of GSH S-transferases are involved.     

Interaction of chlorophenoxyalkyl acid herbicides with rat-liver glutathione S-transferases

The in vitro interaction of four chlorophenoxyalkyl (CPA) acid herbicides with rat-liver glutathione S-transferase (GST) was studied using reduced glutathione and 1-chloro-2,4-dinitrobenzene as substrates. Inhibition of GST activity by the CPA acids in crude extracts was dose dependent. Ring substitution and side-chain length were shown to be of importance in determining the extent of GST inhibition. While GST AA, an isoenzyme of GST, was stimulated by two CPA acids, each of the other GST isoenzymes (A, B, C, E and M) was inhibited, to different degrees. Kinetic studies revealed a mixed type inhibition of the isoenzymes. Conjugates of CPA acids with glutathione were not formed. These results indicate that CPA acids interact with GST by binding directly to these proteins, possibly at a different locus from that of the substrate. The binding of CPA acids to GST may, therefore, have a protective function against these herbicides.     

Effects of polysaccharide peptides from COV-1 strain of Coriolus versicolor on glutathione and glutathione-related enzymes in the mouse.

The effects of polysaccharide peptide (PSP), an immunomodulator isolated from Coriolus versicolor COV-1, on glutathione (GSH) and GSH-related enzymes was investigated in C57 mouse. Administration of PSP (1-4 micromole/kg, i.p.) produced a transient, dose-dependent depletion (10-37%) of hepatic GSH, with no effect on serum glutamic-pyruvic transaminase (SGPT) activity. Blood GSH was depleted (6-25%) at 3 h, followed by a rebound increase above the control GSH level (20%) at 18 h. The GSSG/GSH ratio, a measure of oxidative stress, was increased 3 h after PSP treatment but returned to normal levels at 24 h. Sub-chronic treatment of PSP (1-4 micromole/kg/day, i.p.) for seven days did not produce any significant changes in hepatic GSH levels and the GSSG/GSH ratio when measured 24 h after the final dose of PSP. PSP had little effect on glutathione transferase (GST), glutathione reductase (GSSG reductase) and glutathione peroxidase (GPX) activities in the liver. However, a dose-dependent increase in blood GPX activity (30-48%) was observed at 3h, which coincided with the increase in the GSSG/GSH ratio. The increase in blood GPX activity may be a responsive measure to deal with the transient oxidative stress induced by PSP treatment. The results showed that PSP only caused a transient perturbation on hepatic glutathione without affecting the GSH-related enzymes such as GST, GSSG reductase and GPX. The observed changes in blood GSH simply reflected the intra-organ translocation of glutathione, as the glutathione-related enzymes were not significantly affected by PSP treatment.     

Glutathione and glutathione S-transferase activities of mammalian cells in culture

Continuous cell cultures derived from various tissues of rat, mouse, hamster and man were assayed for their glutathione (GSH) content and glutathione S-transferase activities. GSH S-transferase activities were monitored toward the substrates 1-chloro-2,4-dinitro-benzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (PO). All cell lines tested contained appreciable amounts of GSH ranging from 10 to 65 mol/mg cellular protein. Likewise, all cell lines expressed GSH S-transferase activities. However, the various cell lines differed considerably in their relative transferase activities exhibiting some degree of species-specificity.     

Enzymes involved in glutathione metabolism in rat liver and blood after carbon tetrachloride intoxication

Some glutathione-metabolizing enzymes in rat liver cytosol, blood plasma and urine of rats administered carbon tetrachloride (CCl₄) were investigated.After CCl₄ at different doses (0.3, 1.2, 2.5 ml/kg at 24, 12 and 3 or 6 h, respectively) liver glutathione reductase (GR), glyoxalase 1 (GLY-I) and glutathione S-transferases (GST) significantly decreased, while thioltransferase and glutathione peroxidase (GPX) did not change their activity.At the times under investigation the glutathione level significantly increased in the liver and the above-mentioned enzymes in plasma, thus evidencing a liver injury.The GR, GPX and GST assayed in urine within 72 h after CCl₄ administration showed a maximum of activity at 35–48 h.     

Effect of nitroheterocyclic drugs on lipid peroxidation and glutathione content in rat liver extracts

Incubation of rat liver cell-free extracts with an NADPH-generating system and with nifurtimox or benznidazole (two nitroheterocyclic drugs used in the treatment of Chagas' disease) produced oxidation of reduced glutathione (GSH) and increased lipid peroxidation, as shown by the generation of thiobarbituric-acid-reacting intermediates. Nifurtimox and benznidazole inhibited GSSG-reductase, but not GSH-peroxidase, the former inhibition contributing to GSH depletion. In every case, nifurtimox was more effective than benznidazole. Addition of GSH or free-radical scavengers (catalase, superoxide dismutase, mannitol, sodium benzoate or L-histidine) prevented the effect of nifurtimox on lipid peroxidation reactions. These results support the assumption [M. Dubin, S. N. J. Moreno, E. E. Martino, R. Docampo and A. O. M. Dubin, Biochem. Pharmac.32, 483 (1983)] that, in the rat liver, GSH exerts a protective action against oxygen radicals generated by the nitroheterocyclic drugs.     

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.     

Effects of dietary R-goitrin on hepatic and intestinal glutathione S-transferase, microsomal epoxide hydratase and ethoxycoumarin O-deethylase activities in the rat

The influence of dietary R-goitrin on components of the xenobiotic-metabolizing system was examined in the liver and small intestine of male Sprague-Dawley rats. Given at a level of 200 ppm in the diet for 14 days, the R-goitrin caused a statistically significant (P less than 0.05) 21% increase in liver weight relative to body weight. A less pronounced, but statistically significant, 11% increase in relative liver weight resulted from the administration of R-goitrin at 40 ppm in the diet. Hepatic glutathione S-transferase (GST) activity was significantly increased 1.5- and 2-fold over the basal level at concentrations of 40 and 200 ppm R-goitrin, respectively. Hepatic microsomal epoxide hydratase (EH) activity was also significantly increased. Hepatic EH activity was 1.6- and 3.3-fold greater in the 40- and 200-ppm R-goitrin groups, respectively, than in the control group given the basal diet. R-Goitrin at 200 ppm in the diet produced significant 1.2- and 1.4-fold increases of GST and microsomal EH activities, respectively, in the mucosa of the small intestine. The administration of R-goitrin at 40 or 200 ppm in the diet had no significant effect on either hepatic or intestinal ethoxycoumarin O-deethylase activity.     

The role of fluoroacetate-specific dehalogenase and glutathione transferase in the metabolism of fluoroacetamide and 2,4-dinitrofluorobenzene

2,4-Dinitrofluorobenzene (DNFB) reacts with glutathione to form a stable product similar to that formed with the model glutathione-S-transferase (GST) substrate, 1-chloro-2,4-dinitrobenzene (CDNB). DNFB is approx. 40 times als reactive as CDNB in this chemical reaction. The enzymatic defluorination of DNFB also proceeds at a more rapid rate than that of CDNB in the GST assay. Fluoroacetamide (FAM), like fluoroacetate (FAC), undergoes no discernable chemical defluorination. Its enzymatic defluorination is approx. 10% of that observed for FAC and only 0.2% of the rate for DNFB. An antibody raised to the fluoroacetate specific dehalogenase (FSD) precipitated both FAC and FAM defluorinating activity but had no effect on either CDNB or DNFB activity. The data are consistent with the hypothesis that DNFB is metabolized by the GST while FAM is metabolized by the FSD.     

The course of CCl4 induced hepatotoxicity is altered in mGSTA4-4 null (-/-) mice

Glutathione S-transferases (GSTs) play a key role in cellular detoxification of environmental toxicants through their conjugation to glutathione (GSH). Recent studies have shown that the alpha-class GSTs also provide protection against oxidative stress and lipid peroxidation (LPO). GSTA4-4 is a member of a sub group of the alpha-class GSTs. It has been shown to metabolize 4-hydroxynonenal (4-HNE) with high catalytic efficiency through its conjugation to glutathione (GSH) and has been suggested to be a major component of cellular defense against toxic electrophiles such as 4-HNE generated during LPO. Since the hepatotoxicity of carbon tetrachloride (CCl(4)) has been suggested to be due to the generation of free radicals leading to membrane LPO, the present studies were designed to compare hepatotoxicity of CCl(4) in GSTA4-4 null (-/-) and wild type (+/+) mice. The results show that administration of a single dose of CCl(4) (1 ml/kg i.p.) resulted in time dependent hepatotoxicity in both -/- and +/+ mice; the extent of cellular damage by serum enzymes suggests that progression was more rapid in -/- mice, although injury was similar by 24 h. Histopathologic examination showed similar degrees of centrilobular necrosis by 24 h but much greater surrounding degenerative change, including cellular swelling, disarray, and vacuolization, in the liver of -/- mice. As expected -/- mice did not show any expression of mGSTA4-4; after CCl(4) a compensatory increase in the activities of total GST activity was noted at 24 h. Major alterations in other antioxidant enzymes was not observed. 4-HNE levels in the liver of -/- mice were about four-fold higher than in +/+ mice, suggesting a positive correlation between 4-HNE levels and the altered course of CCl(4) hepatotoxicity. These studies suggest that GSTA4-4 is an important component during the early stages (1-6 h) of cellular defense against oxidative stress and LPO although, it is not effective in protecting against the ultimate degree of overall cell injury.     

The inhibition of human glutathione S-transferases activity by plant polyphenolic compounds ellagic acid and curcumin.

Glutathione S-transferases (GSTs) are multifunctional detoxification proteins that protect the cell from electrophilic compounds. Overexpression of GSTs in cancer results in resistance to chemotherapeutic agents and inhibition of the over expressed GST has been suggested as an approach to combat GST-induced resistance. The inhibition of human recombinant GSTs by natural plant products was investigated in this study. Using 1-chloro-2,4 dinitrobenzene (CDNB) as a substrate, ellagic acid and curcumin were shown to inhibit GSTs A1-1, A2-2, M1-1, M2-2 and P1-1 with IC(50) values ranging from 0.04 to 5 microM whilst genistein, kaempferol and quercetin inhibited GSTs M1-1 and M2-2 only. The predominant mode of inhibition with respect to the G and H-sites were mixed inhibition and uncompetitive to a lesser extent. The K(i) (K(i)(')) values for ellagic acid and curcumin with respect to GSH and CDNB were in the range 0.04-6 microM showing the inhibitory potency of these polyphenolic compounds. Ellagic acid and curcumin also showed time- and concentration-dependent inactivation of GSTs M1-1, M2-2 and P1-1 with curcumin being a more potent inactivator than ellagic acid. These results facilitate the understanding of the interaction of human GSTs with plant polyphenolic compounds with regards to their role as chemomodulators in cases of GST-overexpression in malignancies.     

The polymorphic human glutathione transferase T1-1, the most efficient glutathione transferase in the denitrosation and inactivation of the anticancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea

A member of the Theta class of human glutathione transferases (GST T1-1) was found to display the greatest catalytic activity towards the cytostatic drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) of the GSTs studied. In this investigation (the most extensive to date), enzymes from four classes of the soluble human GSTs were heterologously expressed, purified, and kinetically characterized. From the 12 enzymes examined, only GST M2-2, GST M3-3 and GST T1-1 had significant activities with BCNU. This establishes that the activity is not a characteristic of a particular class of GSTs. Although GST M3-3 was previously reported to have the greatest activity with BCNU, the current investigation demonstrates that GST M2-2 is equally active and that GST T1-1 has an approximately 20-fold higher specific activity than either of the Mu class enzymes. A more rigorous kinetic analysis of GST T1-1 gave the following parameters with BCNU: a k(cat) of 0.035 +/-0.003s(-1) and a K(M) of 1.0 +/- 0.1mM. The finding that GST T1-1 has the highest activity towards BCNU is significant since GST T1-1 is expressed in the brain, a common target for BCNU treatment. Furthermore, the existence of a GST T1-1 null allele in up to 60% in some populations, may influence both the sensitivity of tumors to chemotherapy and the severity of adverse side-effects in patients treated with this agent.     

Extension of the blood half-life of glyceryl trinitrate. Inhibition of glutathione organic nitrate ester reductase activity in the rat and guinea pig

Cephalothin, penicillin G and probenecid inhibited GSH organic nitrate ester reductase (ONER) and several other enzymatic activities of GSH-S-transferases (EC 2.5.1.18) from rat and guinea pig liver. Erythrityl tetranitrate, a substrate for ONER, inhibited the aryl and alkyl transferase activities of two guinea pig liver GSH-S-transferases. These findings support the concept that ONER is one of the several activities possessed by the GSH-S-transferases. In an examination of possible in vivo action, parenteral administration of these inhibitors 2–30 min prior to i.v. administration of [¹⁴C]glyceryl trinitrate resulted in a 50–100 per cent increase in the half-time of the metabolism phase of [¹⁴C]glyceryl trinitrate clearance from the blood and postponed the appearance of metabolites. This presumably occurs through the in vivo inhibition of GSH-ONER activity of the GSH-S-transferases and suggests a possible means of prolonging the pharmacologie action of nitrate esters.     

Collagen gel sandwich and immobilization cultures of rat hepatocytes: Problems encountered in expressing glutathione S-transferase activities

Collagen gel sandwich and immobilization cultures of rat hepatocytes are two recently developed organotypical culture models. Basic information with respect to the maintenance of xenobiotic biotransformation pathways and the expression of key enzyme activities, however, is lacking, making their use in pharmaco-toxicological studies rather speculative. The expression of the glutathione S-tranferase (GST; EC 2.5.1.18) activity, a key phase II enzyme, has been chosen to study the various problems that may arise in expressing the results of cytosolic enzyme activities when rat hepatocytes are cultured using both new culture models. Collagen gel matrix easily entraps culture medium proteins. These interfere with the cytosolic protein content, a parameter versus which cytosolic enzyme activities, including GSTs, are usually expressed. The following solutions are proposed: expression of the cytosolic enzyme activity results versus either (i) microsomal proteins, these are not contaminated by medium proteins, or versus (ii) cytosolic proteins after a complete collagenase digestion (0.05% collagenase type I of Sigma, 45 min, 37 °C) of the collagen matrix. Expression of enzyme activities versus cellular DNA appears to be unacceptable since unreliable results were obtained due to entrapped DNA in the collagen matrix. Once it was known how to express cytosolic enzyme activity, the maintenance of GST activities was investigated in both culture models using 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) as substrates for total and Mu class GSTs, respectively. Two culture media were compared, control medium (DMEM) with and without supplementation of -proline (final concentration 60 μg/ml). In both culture models, after an initial decrease, total GST activities increased significantly up to values higher than those observed for freshly isolated cells. The Mu class GST activities were maintained constant for 7 days and increased thereafter. -Proline supplementation of the culture medium prevented the initial decline in total and Mu class GST activities in both culture configurations but did not seem to be of crucial importance in the maintenance of GST activities in both culture models.