Liver Glutathione S-transferase Research Articles

Acute cadmium intoxication induces alpha-class glutathione S-transferase protein synthesis and enzyme activity in rat liver

Acute cadmium intoxication affects glutathione S-transferase (GST) in rat liver. It has been found that 24 h after i.p. cadmium administration to rats, at a dose of 2.5 mg CdCl 2 kg −1 body weight, the activity of this enzyme in liver cytosol increased by 40%. A less stimulatory effect persisted till 48 h and thereafter the enzyme activity normalized. Since, GST isoenzymes belong to different classes in mammalian tissues, we used quantitative immunoassays to verify which family of GST isoenzymes is influenced by this intoxication. Only alpha-class glutathione S-transferase (α-GST) proteins were detected in rat liver cytosol and their level increased by about 25%, 24 h after cadmium treatment. No pi-GST isoforms were found in liver cytosol from either normal or cadmium-treated rats. Co-administration of actinomycin D with cadmium normalized both the protein level and the activity of α-GST, suggesting that some effect occurs on enzyme transcription of these isoenzymes by this metal. On the other hand, it seems unlikely that the stimulatory effect is due to the high level of peroxides caused by lipid peroxidation, since Vitamin E administration strongly reduced the TBARS level, but did not cause any GST activity decrease.

Purification and properties of the glutathione S‐transferases from the anoxia‐tolerant turtle, Trachemys scripta elegans

Glutathione S-transferases (GSTs) play critical roles in detoxification, response to oxidative stress, regeneration of S-thiolated proteins, and catalysis of reactions in nondetoxification metabolic pathways. Liver GSTs were purified from the anoxia-tolerant turtle, Trachemys scripta elegans. Purification separated a homodimeric (subunit relative molecular mass =34 kDa) and a heterodimeric (subunit relative molecular mass = 32.6 and 36.8 kDa) form of GST. The enzymes were purified 23-69-fold and 156-174-fold for homodimeric and heterodimeric GSTs, respectively. Kinetic data gathered using a variety of substrates and inhibitors suggested that both homodimeric and heterodimeric GSTs were of the alpha class although they showed significant differences in substrate affinities and responses to inhibitors. For example, homodimeric GST showed activity with known alpha class substrates, cumene hydroperoxide and p-nitrobenzylchloride, whereas heterodimeric GST showed no activity with cumene hydroperoxide. The specific activity of liver GSTs with chlorodinitrobenzene (CDNB) as the substrate was reduced by 2.6- and 8.7-fold for homodimeric and heterodimeric GSTs isolated from liver of anoxic turtles as compared with aerobic controls, suggesting an anoxia-responsive stable modification of the protein that may alter its function during natural anaerobiosis.

Effects of indoleacetic acid and kinetin on lipid peroxidation and antioxidant defense in various tissues of rats

This study aims to investigate the effects of indoleacetic acid (IAA) and kinetin (Kn), which are plant growth regulators (PGRs), on antioxidant defense systems [reduced glutathione (GSH), glutathione- S-transferase (GST), catalase (CAT)], and lipid peroxidation level (malondialdehyde, MDA) various tissues of rats. Rats (Sprague–Dawley albino) were exposed to 100 ppm IAA and Kn. One hundred parts per million of PGRs was administered orally to rats ad libitum for 21 days continuously. The PGRs treatments caused different effects on the content of MDA and antioxidant defense system in comparison to those of control rats. According to the results, the subchronic treatments of IAA caused significant decrease in the GSH concentration and CAT activity in erythrocyte. Kn decreased GSH concentration in erythrocyte too. While the MDA concentration in brain was increased significantly by IAA and Kn, Kn decreased significantly brain CAT and GST activity. The liver GST activity was decreased by IAA and Kn. But, liver CAT activity was increased by IAA. On the other hand, while IAA treatment caused a significant decrease kidney GST activity, Kn caused a significant decrease both kidney GST and CAT activity. Also, while heart CAT activity was decreased by IAA, heart GST activity was decreased by both IAA and Kn. Moreover, MDA concentration in heart was increased by Kn treatment. It was concluded that IAA might effect MDA and antioxidant defense on the animals at subchronic treatment.

Reducing Effect of Strobilanthes crispus Leaf Extract in Hepatocarcinogenesis Rats

The effect of Strobilanthes crispus extract and glycyrrhizin in on rats induced with carcinoma is reported. The results showed significant, increase of liver microsome glutathione s-transferase (GST) in rats after 12 weeks. Treatment with glycyrrhizin caused decrease in liver GST activity compared to control. Treatment with Strobilanthes crispus extract caused overall decrease in liver GST activity almost close to control groups. The microscopic observation of the lesion score during hepatocarcinogenesis revealed that cells of cance r group without treatment were severely necrotic at week 12. However, cells of cancer group with S. crispus treatment appeared normal at the same period.

Purple grape juice inhibits 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumorigenesis and in vivo DMBA-DNA adduct formation

There has been considerable interest in identifying specific foods and phytochemicals that may have breast cancer preventive properties. Concord grapes are rich in polyphenolic chemicals and anthocyanin pigments that may have biological properties which could suppress cancer such as having antioxidant, antiproliferative, and proapoptotic actions. To determine the potential breast cancer protective action of purple grape juice, we examined the effect of grape juice consumption on the initiation stage of 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumorigenesis and on the in vivo formation of rat mammary DNA adducts in female Sprague–Dawley rats. Consumption of grape juice significantly inhibited mammary tumor mass at termination and the growth of tumors for the first 5 weeks of detectable tumor development. Consumption of grape juice phenolics by rats also significantly inhibited in vivo mammary DMBA-DNA adduct formation by 34 and 56% for animals fed phenolics at 346 and 692 mg/dL, respectively, compared to controls. Mammary 8-oxo-deoxyguanosine (8-oxo-dG) levels decreased by 25 and 37%, respectively, but the differences were not statistically significant. Liver DMBA-DNA adducts decreased by 10–30%, while 8-oxo-dG adducts remained unchanged, following grape juice intake. Liver glutathione S-transferase activity was significantly increased following grape juice consumption, but only at the highest level of intake. In addition, liver activities of catalase increased and xanthine oxidase decreased significantly, but only at the highest grape juice dose. Thus, these studies indicate that specific constituents or combinations of phytochemicals in purple grape juice can block the initiation stage of DMBA-induced rat mammary tumorigenesis. This tumor inhibitory effect was associated with a suppression of mammary DMBA-DNA adduct formation, which in part may be explained by increased liver activity of the phase II metabolizing enzyme, glutathione S-transferase. Mammary and liver 8-oxo-dG levels were not significantly altered by grape juice consumption. Thus, grape juice constituents appear to have benefit in decreasing susceptibility of the rat mammary gland to the tumor-initiating action of DMBA.

Existence of no-observed effect levels for 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline on hepatic preneoplastic lesion development in BN rats

There is increasing evidence that dose–response curve of genotoxic carcinogen is nonlinear and a practical threshold dose exists. However, little is known about differences in the dose–response relationship of genotoxic carcinogen among different strain rats. Herein, we showed that low doses of genotoxic carcinogen 2-amino-3,8-dimethylimidazo[4,5- f] quinoxaline (MeIQx) had no effects on induction of liver glutathione S-transferase placental form (GST-P)-positive foci in both BN and F344 rats, and therefore demonstrated the existence of no-observed effect level for hepatocarcinogenicity of this genotoxic carcinogen irrespective of strains. These findings further support our notion that a practical threshold dose for MeIQx hepatocarcinogenicity exists in rats.

Glutathione S‐transferase tissue profiling by reporter peptide monitoring

Glutathione S-transferases (GSTs) form a widespread enzyme superfamily mainly involved in phase II detoxification. Differential expression of the various GST isoforms, differing in catalytic and structural properties, correlates with physiological and pathological states. Fast and simple determination of the GST profile is expected to be an important diagnostic tool in disease analysis. Here we propose a combined approach of high resolution separation techniques and electrospray mass spectrometric analyses for characterizing the spectrum of GSTs in male mouse liver. In this approach, the sensitivity and speed required for tissue GST profiling studies is achieved by tracking the reconstructed ion current of selected reporter peptides following chromatographic separation. This simple procedure, in which an affinity protein bait is followed by a chemical fragmentation and mass spectrometric analysis, could be sufficiently sensitive to detect the qualitative differences between physiological and pathological states.

Antioxidant and Protective Effects of Amrit Nectar Tablets on Adriamycin- and Cisplatin-Induced Toxicities

Maharishi herbal food supplements have been shown to inhibit the growth of mammary tumors and reduce free radical-mediated injuries. The purpose of this investigation is to evaluate the effects of aqueous and alcoholic extracts of Amrit Nectar tablets on rat liver microsomal lipid peroxidation and compare to other antioxidants. The protective effects of dietary Amrit Nectar tablets (MA-7; containing 38 herbs) on cisplatin-induced changes in glutathione (GSH) and glutathione-S-transferase (GST) activity in rat liver and kidney, and Adriamycin (Pharmacia S.p.A, Milan, Italy)-induced mortalities in mice were also investigated. Both aqueous and alcoholic extracts of MA-7 were more potent than other antioxidants tested under our experimental conditions. Adriamycin (15 mg/kg, intraperitoneally) caused 60% mortality during the period of 4 weeks in CDF1 mice. Dietary MA-7 (0.7%) treatment decreased the mortality to 20%. Dietary MA-7 (0.7%) supplementation with cisplatin treatment reversed the effects of cisplatin on liver and kidney GSH and GST activity. These results indicate that MA-7 is a powerful antioxidant. MA-7 supplementation with Adriamycin and cisplatin treatment may protect against their toxicities.

The drinking of a Salvia officinalis infusion improves liver antioxidant status in mice and rats

In this study, we evaluate the biosafety and bioactivity (antioxidant potential) of a traditional water infusion (tea) of common sage ( Salvia officinalis L.) in vivo in mice and rats by quantification of plasma transaminase activities and liver glutathione- S-transferase (GST) and glutathione reductase (GR) enzyme activities. The replacement of water by sage tea for 14 days in the diet of rodents did not affect the body weight and food consumption and did not induce liver toxicity. On the other hand, a significant increase of liver GST activity was observed in rats (24%) and mice (10%) of sage drinking groups. The antioxidant potential of sage tea drinking was also studied in vitro in a model using rat hepatocytes in primary culture. The replacement of drinking water with sage tea in the rats used as hepatocyte donors resulted in an improvement of the antioxidant status of rat hepatocytes in primary culture, namely a significant increase in GSH content and GST activity after 4 h of culture. When these hepatocyte cultures were exposed to 0.75 or 1 mM of tert-butyl hydroperoxide ( t-BHP) for 1 h, some protection against lipid peroxidation and GSH depletion was conferred by sage tea drinking. However, the cell death induced by t-BHP as shown by lactate dehydrogenase (LDH) leakage was not different from that observed in cultures from control animals. This study indicates that the compounds present in this sage preparation contain interesting bioactivities, which improve the liver antioxidant potential.

Inhibitory potency of some methoxyphenyl derivatives compounds on class mu GST activity in vitro

Methoxy phenyl derivative’s compounds i.e. curcumin, ferulic acid, and indomethacin have been reported to have inhibitory effects on rat’s liver glutathione S-transferases (GST) activity. Vaniline and ferulic acid have been known as degradation products of curcumin, and still have methoxy phenyl groups on the structures. Until the recent years, there is no information concerning the influences of methoxy phenyl derivative’s compounds i.e. vaniline, dimethylcurcumin and ethyl-p-methoxycinnamate on GST-activity. The aim of this research is to study the correlation of the inhibitory activity of class mu GST isolated from uninduced and phenobarbital-induced rat liver cytosol by curcumin, dimethylcurcumin, vaniline, indomethacin, ethyl pmethoxycinnamate, and ferulic acid. GST activity was measured spectrophotometrically on the conjugation of DCNB and GSH. The results showed that the inhibitory activity of mu class GST in uninduced or phenobarbital-induced rat liver GST were decreasing as follow : curcumin, dimethylcurcumin, vaniline, indomethacin, ethyl p-methoxycinnamate, and ferulic acid. Key words: methoxyphenyl derivative’s compounds, glutathione S-transferases activity.

Formation of acetaldehyde adducts of glutathione S-transferase A3 in the liver of rats administered alcohol chronically

Hepatic tissue damage induced by chronic exposure to alcohol is mediated through acetaldehyde and associated with reactive oxygen species, which impair cellular defense mechanisms. Because glutathione S-transferases (GSTs) play an important role in the detoxification of xenobiotics and reactive oxygen species, the current study was undertaken to test the effect of alcohol administration on structural and functional characteristics of rat (r) liver Alpha class rGSTs. Western blot analysis revealed an appreciable change in the expression of rGSTA3 subunit levels, whereas no change was observed in activity after chronic alcohol treatment. Reverse-phase high performance liquid chromatographic analysis of rat liver GSTs that were affinity purified with glutathione showed a 1.07-fold increase in rGSTA3 subunit levels in rats treated with alcohol chronically. In addition, liquid chromatographic–electrospray ionization mass spectrometric analysis of GSTs that were affinity purified with glutathione showed the formation of acetaldehyde adducts to the rGSTA3 subunit. Given the abundant expression of rGSTA3 subunit and acetaldehyde adduct formation, results of the current study support the suggestion that modification of rGSTA3 subunit, and thus its impaired function, in alcohol-exposed rats may contribute to the progression of alcohol-induced liver damage.

Effect of different oxygen tensions on weight gain, feed conversion, and antioxidant status in piapara, Leporinus elongatus (Valenciennes, 1847)

Juvenile piapara ( Leporinus elongatus) were progressively exposed to several levels of dissolved oxygen for 28 days, severe hypoxia, moderate hypoxia and normoxia, while weight gain, feed conversion, and some antioxidants and biomarkers of oxidative stress were measured in liver and blood. Fish exposed to severe hypoxia showed lower weight gain and feed conversion (1.6) when compared with moderate hypoxia (2.9) and normoxia (1.8). Lipoperoxidation in liver and blood was higher under moderate hypoxia and normoxia compared with severe hypoxia. Activities of catalase, glutathione reductase, and glutathione peroxidase in both liver and blood showed essentially no differences, while superoxide dismutase activities were higher in liver and blood in piapara exposed to severe hypoxia. Nevertheless, oxidized and total glutathione contents in blood and liver in fish exposed to hypoxia were higher than in fish exposed to normoxia. Liver glutathione S-transferase activity decreased with the increase in oxygen availability, while the level of microsomal cytochrome P450 1A increased with O 2 availability. Below a critical O 2 content, piapara showed decreased feed consumption, weight gain, and also cellular damage, but the antioxidant status was the same, or in some cases, higher than in normoxia. Despite the energetic limitations in oxidative metabolism under severe hypoxia, piapara appears to maintain essential antioxidant compensatory levels in such circumstances, probably to avoid the oxidative challenge that accompanies the increase in reactive oxygen species generation related to shifts in environmental oxygen availability. The results indicate that the balance between O 2 utilization and the antioxidant status in obligatory water-breathing organisms has a great importance for farming purposes.

Responses of the antioxidant defenses of the Goldfish Carassius auratus, exposed to 2,4-dichlorophenol

Goldfish Carassius auratus were exposed to 0.1 mg/l of 2,4-dichlorophenol (2,4-DCP), widely used as transportation power in China, for 2, 5, 10, 20 and 40 days, while one control group was designated for each exposure group. Antioxidant defenses consisting of contents of reduced glutathione (GSH) and glutathione disulfide (GSSG) and activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), selenium-dependent glutathione peroxidase (Se-GPx) and glutathione S-transferase (GST) in liver of freshwater fish were determined and the GSH–GSSG ratio and content of tGSH (total glutathione) were calculated. In the present study, the role of hepatic antioxidant defenses was evaluated and the possible poisoning mechanism of fish can be explained as an oxidative stress mechanism. In addition, hepatic SOD and GSH, especially tGSH, were sensitive to 2,4-DCP contamination and thus, can possibly be used in early assessment of 2,4-DCP-dominant polluted aquatic ecosystems.

Biochemical study on the hypoglycemic effects of onion and garlic in alloxan-induced diabetic rats

The present study was carried out to investigate the effects of onion ( Allium cepa Linn) and garlic ( Allium sativum Linn) juices on biochemical parameters, enzyme activities and lipid peroxidation in alloxan-induced diabetic rats. Alloxan was administered as a single dose (120 mg/kg BW) to induce diabetes. A dose of 1 ml of either onion or garlic juices/100 g body weight (equivalent to 0.4 g/100 g BW) was orally administered daily to alloxan-diabetic rats for four weeks. The levels of glucose, urea, creatinine and bilirubin were significantly ( p < 0.05) increased in plasma of alloxan-diabetic rats compared to the control group. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and alkaline and acid phosphatases (AlP, AcP) activities were significantly ( p < 0.05) increased in plasma and testes of alloxan-diabetic rats, while these activities were decreased in liver compared with the control group. Brain LDH was significantly ( p < 0.05) increased. The concentration of thiobarbituric acid reactive substances and the activity of glutathione S-transferase in plasma, liver, testes, brain, and kidney were increased in alloxan-diabetic rats. Treatment of the diabetic rats with repeated doses of either garlic or onion juices could restore the changes of the above parameters to their normal levels. The present results showed that garlic and onion juices exerted antioxidant and antihyperglycemic effects and consequently may alleviate liver and renal damage caused by alloxan-induced diabetes.

Sparus aurata L. liver EROD and GST activities, plasma cortisol, lactate, glucose and erythrocytic nuclear anomalies following short-term exposure either to 17β-estradiol (E 2) or E 2 combined with 4-nonylphenol

Immature Sparus aurata L. (gilthead seabream) were exposed to 17β-estradiol (E 2) 4000 ng/l and to the same E 2 concentration mixed with 50,000 ng/l 4-nonylphenol (E 2+NP) during 4, 8, 12 and 16 h. E 2 availability and E 2 plasma level variations were assessed. Liver biotransformation capacity was measured as ethoxyresorufin- O-deethylase (EROD) and glutathione- S-transferase (GST) activities. Plasma cortisol, lactate and glucose were also determined. Genotoxicity was assessed through erythrocytic nuclear anomalies (ENA) frequency. Liver EROD activity significantly decreased during the whole experiment for both treatments, with the exception of 16 h exposure to E 2. Liver GST activity was significantly increased after 8 and 12 h of exposure either to E 2 or E 2+NP. An endocrine disruption expressed as plasma cortisol decrease was observed after 16 h exposure under both tested conditions, concomitantly with a plasma lactate increase. No genotoxic responses, measured as ENA frequency, were detected. Analyzing the E 2 water concentration in aquaria without fish it was demonstrated an intense and fast E 2 loss, considerably reducing its availability to fish. In the presence of fish, E 2 water levels were drastically reduced after 4 h exposure, being this reduction more pronounced in E 2 aquarium when compared to E 2+NP aquarium. In addition, it was demonstrated a rapid E 2 uptake from the water since the highest E 2 plasma concentrations were observed after 4 h exposure, followed by a continuous decrease, which became more pronounced between 8 and 12 h of exposure. Furthermore, during the first 8 h exposure to E 2 and E 2+NP, seabream plasma E 2 concentrations were higher than the initial water exposure concentration. Comparing the E 2 plasma levels in both seabream-exposed groups, it was clear that its concentration is always higher in E 2+NP-treated fish. Despite the previous results, no significant differences were found in the measured responses between E 2 and E 2+NP.

Juvenile sea bass biotransformation, genotoxic and endocrine responses to β-naphthoflavone, 4-nonylphenol and 17β-estradiol individual and combined exposures

Juvenile sea bass, Dicentrarchus labrax L., were exposed during 2, 4, 8, and 24 h to 0.9 μM β-naphthoflavone (BNF), 131 nM 17β-estradiol (E 2), 4.05 μM 4-nonylphenol (NP), as well as to BNF combined either to E 2 or NP (maintaining the previous concentrations). Liver cytochrome P450 content (P450), ethoxyresorufin- O-deethylase (EROD), and glutathione S-transferase (GST) activities were measured in order to evaluate biotransformation responses. Genotoxicity was assessed as erythrocytic nuclear abnormalities (ENA) frequency. The effects on endocrine function were evaluated as plasma cortisol and glucose. Cortisol was not affected by xeno/estrogens tested, either in single exposure or mixed with BNF. Nevertheless, the intermediary metabolism was affected since glucose concentration increased after 4 h exposure to E 2, and after all BNF + NP exposure lengths. Moreover, a synergism between BNF and NP was thoroughly demonstrated, whereas a sporadic antagonistic interaction was found at 4 h BNF + E 2 exposure. Liver EROD and GST activities were not significantly altered by single E 2 or NP exposure. However, both compounds were able to induce EROD activity in the presence of BNF. NP single exposure was able to significantly increase liver P450 content, while its mixture with BNF displayed an antagonistic interference. Considering the xeno/estrogens single exposures, only NP induced an ENA increase; however, both mixtures (BNF + E 2 and BNF + NP) displayed genotoxic effects. Fish responses to mixtures of xenobiotics are complex and the type of interaction (synergism/potentiation or antagonism) in a particular mixture can vary with the evaluated biological response.

Effect of acute phosgene inhalation on antioxidant enzymes, nitric oxide and nitric-oxide synthase in rats

To study the effect of acute phosgene inhalation on the antioxidant enzymes, nitric oxide (NO) and nitric oxide synthase (NOS) in rats. Phosgene was produced by decomposing bis (trichdomethyl) carbonate in the presence of N,N-dimethyl formamide. SD rats were randomly divided into two groups: control and phosgene exposure groups. In a special experimental device with equipment modulating the gas flow, phosgene exposed rats inhaled phosgene quantitatively for five minutes. Two hours later, all the rats were sacrificed and the ratio of wet weight to dried weight of lung (WW/DW) was calculated. Peripheral blood, serum and liver were collected to examine the activities of antioxidant enzymes including glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), NOS, and NO level. The total content of proteins were also determined. The WW/DW ratio of lung in phosgene exposure group was much higher than that in control group (P < 0.01). The activities of GST in serum and liver of phosgene exposure group increased significantly (P < 0.05). The activities of SOD, CAT, GSHPx and NOS in serum or blood and liver of phosgene exposure group were also increased significantly (P < 0.05). But the content of NO was significantly decreased (P < 0.01). Acute phosgene inhalation may cause a dramatically changes of several antioxidant enzyme activities, and acute injury of liver to some extent in rats. The latter is related to reactive oxygen species. But the elevation of antioxidant enzyme activities suggests that antioxidative treatment for acute phosgene poisoning should not be considered first.

The mouse liver content of carbonic anhydrase III and glutathione S-tranferases A3 and P1 depend on dietary supply of methionine and cysteine

The contents of glutathione S-transferase (GST) subunits, carbonic anhydrase III (CAIII), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a 230 kDa protein are affected by protein deprivation in mouse liver. In order to know if particular amino acids control these contents, the effects of feeding for 5 days with diets containing different amino acids were examined. After an exploration using SDS-PAGE analysis, the action of selected diets was further examined by distinct techniques. The 230 kDa protein was identified as fatty acid synthase (FAS) by both mass spectrometry and amino acid sequence analyses. Dietary tests showed that: (1) a protein-free diet (PFD) increased the content of glutathione S-transferases P1 and M1, and glyceraldehyde-3-phosphate dehydrogenase, while the content of glutathione S-transferase A3, fatty acid synthase and carbonic anhydrase III decreased; (2) a protein-free diet having either methionine or cysteine preserved the normal contents of glutathione S-transferases P1, A3, M1 and carbonic anydrase III; (3) a protein-free diet having threonine preserved partially the normal contents of glutathione S-transferases P1, A3, M1 and carbonic anhydrase III; (4) a protein-free diet having methionine, threonine and cysteine prevented in part the loss of fatty acid synthase; and (5) the glyceraldehyde-3-phosphate dehydrogenase content was controlled by increased carbohydrate level and/or by lower amino acid content of diets, but not by any specific amino acid. These data indicate that methionine and cysteine exert a main role on the control of liver glutathione S-transferases A3 and P1, and carbonic anhydrase III. Thus, they emerge necessary to prevent unsafe alterations of liver metabolism caused by protein deprivation.

Rat liver glutathione S-transferase activity stimulation following acute cadmium or manganese intoxication.

The effect of cadmium or manganese administration on rat liver glutathione S-transferase (GST) has been investigated. The activity of this enzyme in liver cytosol, where almost all the cellular activity is present, had increased by more than 36% 24 h after a single i.p. injection of CdCl(2) (2.5 mg kg(-1) b.w.) or MnCl(2) (2.0 mg kg(-1) b.w.). After shorter and longer time intervals, a lower enzyme activity stimulation was observed in both cases. When liver cytosol was incubated for 10 min with 75 microM CdCl(2) or 40 microM MnCl(2), no effect was observed on enzyme activity. The increase in GST following cadmium or manganese administration was blocked by prior administration of actinomycin D, indicative of a possible transcription-dependent response. The liver soluble GST from both control and metal-treated rats was not at all affected by Vitamin E, in the range of 20-300 microM. By contrast, hematin was seen to be a competitive inhibitor of this liver enzyme from both types of rats by using CDNB as substrate and the K(i) value was equal to 0.22 microM. The possibility that under the conditions used class alpha GST isoenzymes are affected by cadmium or manganese is discussed.

Molecular cloning and characterization of a glutathione S-transferase from largemouth bass ( Micropterus salmoides) liver that is involved in the detoxification of 4-hydroxynonenal

We are currently investigating the role of detoxification pathways in protecting against the sublethal effects of chemicals in largemouth bass ( Micropterus salmoides). To this end, previous work in our laboratory indicated a remarkable ability of bass liver glutathione S-transferases (GSTs) to detoxify 4-hydroxynonenal (4HNE), a common mutagenic and cytotoxic α,β-unsaturated aldehyde produced during the peroxidation of lipids. In the current study, we observed that GST-mediated 4HNE conjugation in bass liver follows high efficiency single-enzyme Michaelis–Menten kinetics, suggesting that an individual GST isoform is involved in 4HNE detoxification. Using 5′ and 3′ rapid amplification of cDNA ends (RACE), a full-length GST cDNA of 957 base pairs (bp) in length, containing an open reading frame of 678 bp and encoding a polypeptide of 225 amino acids, has been cloned. Interestingly, a search of the BLAST protein database revealed the presence of homologous GST proteins in the plaice ( Pleuronectes platessa), European flounder ( Platichthys flesus) and fathead minnow ( Pimephales promelas), but not in other fish species. Furthermore, the bass GST protein exhibited little homology with the mammalian GSTA4 subclass of proteins which rapidly metabolize 4HNE. The recombinant 6x His-tagged expressed GST protein showed high catalytic activity towards 4HNE, while showing moderate or low activity toward other class specific GST substrates. HPLC-GST subunit analysis, followed by sequencing, demonstrated that the isolated bass liver GST subunit constitutes the major GST protein in bass liver, with a molecular mass of 26.4 kDa. In summary, the presence of a highly expressed GST isozyme in bass and several evolutionarily divergent fish species indicates the conservation of an important and distinct detoxification protein that protects against oxidative damage in certain aquatic organisms.