Cytosolic Glutathione S-transferase Activity Research Articles

Comparisons between human and rodent hepatic glutathione S-Transferase activities reveal sex and species differences

Glutathione S-transferases (GSTs) are conjugating enzymes involved in drug metabolism, antioxidant defence, and cell signalling. Herein, we investigated hepatic GST conjugation in several mouse and rat strains, including both sexes, with a direct comparison to humans. Using general and isoform-selective substrates, all mouse strains had significantly greater activities than humans for total cytosolic GST, GST-M, GST-T, and microsomal GST activities. Some strains had significantly greater GST-P activities compared to humans. Sex differences between males and females were evident in all strains for total cytosolic GST, GST-M, and GST-P, and sex differences in GST-T and microsomal GST activities within strains were noted. All rats had significantly greater activities than humans for GST-M and GST-T; only some strains were significantly greater than humans for GST-P, total cytosolic GST, and microsomal GST. Sex differences within strains showed significantly greater GST-M and GST-T activities in males compared to females. Select strains showed sex differences for total cytosolic and microsomal GST activities; there were no sex differences in GST-P activities. Significant differences in glutathione conjugation between humans and rodents exist, including sex differences. This highlights the need for careful animal selection in pre-clinical studies where GSTs are the primary metabolic pathway.

Titanium (IV) oxide anatase nanoparticles as vectors for diclofenac: assessing the antioxidative responses to single and combined exposures in the aquatic macrophyte Egeria densa

Titanium dioxide, frequently used in commonplace products, is now regularly detected in aquatic environments. Understanding its toxic effects on native biota is essential. However, combined toxicity with commonly occurring pollutants, such as the pharmaceutical diclofenac, may provide more insight into environmental situations. Therefore, the present study aimed to evaluate the effects of titanium dioxide and diclofenac, individually and combined, on the macrophyte Egeria densa. Diclofenac uptake and removal by the macrophyte were assessed. Diclofenac and titanium dioxide were mixed prior to exposure to allow binding, which was assessed. Toxicity of the individual compounds and the combination was evaluated by assaying enzymes as bioindicators of biotransformation and the antioxidative system. Cytosolic glutathione S-transferase and glutathione reductase activities were increased by diclofenac, titanium dioxide, and the combination. Both enzymes’ activities were more significantly elevated by diclofenac and the combination than nanoparticles alone. Microsomal glutathione S-transferase was unaffected by diclofenac exposure but inhibited with titanium dioxide and the mixture. Diclofenac elicited the most significant response. Based on the data, the cytosolic enzymes effectively prevented damage.

Inter-individual Variability in Activity of the Major Drug Metabolizing Enzymes in Liver Homogenates of 20 Individuals

Inter-individual variability in hepatic drug metabolizing enzyme (DME) activity is a major contributor to heterogeneity in drug clearance and safety. Accurate data on expression levels and activities of DMEs is an important prerequisite for in vitro-in vivo extrapolation and in silico based predictions. Characterization and assessment of inter-correlations of the major DMEs cytochrome P450s (CYPs) and UDP-glucuronosyltransferases (UGTs) have been extensively documented, but simultaneous quantification including other major DMEs has been lacking. Assessment of inter-donor variability and inter-correlations of CYPs, UGTs, sulfotransferases (SULTs), glutathione S-transferases (GSTs), NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH: quinone oxidoreductase 2 (NQO2) in a set of 20 individual liver homogenates. The main drug metabolizing isoforms of CYP and UGT have been reaction phenotype in individual liver microsomes and NQO1, NQO2, GSTT1 and GSTT2 in corresponding cytosol. In addition, we assessed overall SULT activity in liver cytosol using acetaminophen and 7-hydroxycoumarin as non-selective substrates and cytosolic GST activity using the non-selective substrate 1-chloro-2,4-dinitrobenzene (CDNB). Expression of GST isoforms was also assessed. While hepatic NQO1 activity was highly variable, NQO2 activity was more conserved. In addition, we found that of the hepatic GST isoforms, the variation in GSTM3 levels, which is poorly studied, was highest. The majority of significant correlations were found amongst CYP and UGT enzyme activities. The dataset presented provides the absolute quantification of the largest number of hepatic DME activities so far and constitute an essential resource for in silico toxicokinetic and metabolic modelling studies.

Biochemical basis for the age-related sensitivity of broilers to aflatoxin B1

In this study, we investigated the mechanism underlying age-related susceptibility in broilers to aflatoxin B1 (AFB1). The results showed that AFB1 induced significant changes in serum alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT) activity & liver superoxide dismutase (SOD), malonaldehyde (MDA), glutathione peroxidase (GSH-Px) and glutathione S-transferase (GST) activity at day 7, 21 and 42 relative to control group. However, AFB1-induced changes in serum biochemical parameters and liver antioxidant activities become less severe with increasing age of broilers. Particularly, liver cytosolic GST activity increases with the age of broilers, crucial for the detoxification of AFB1. The mRNA expression level of Cytochrome P450 (CYP) enzymes was significantly higher at day 7, and decreases at day 21 and 42. While, the mRNA expression level of liver GSTA3, GSTA4 and EPHX1 increases with age of broilers. Maximum AFB1 residues level was detected at day 42 relative to day 7 and 21. While, AFM1 residues level increases (p < 0.05) from day 7 to 21, but decreases (p > 0.05) at day 42. Most importantly, our data confirmed the efficient AFB1-bioactivation by CYP enzymes and deficient detoxification of GST enzymes at younger age (∼7-day old) compared to older age. In summary, the age-related changes particularly in phase-I and phase-II enzymes mainly responsible for AFB1 bioactivation and detoxification may be partially accountable for the increased susceptibility of younger broilers (∼7-day old) compared to older broilers.

Responses of the antioxidative and biotransformation enzymes in the aquatic fungus Mucor hiemalis exposed to cyanotoxins

To investigate antioxidative and biotransformation enzyme responses in Mucor hiemalis towards cyanotoxins considering its use in mycoremediation applications. Catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx) in M.hiemalis maintained their activities at all tested microcystin-LR (MC-LR) exposure concentrations. Cytosolic glutathione S-transferase (GST) activity decreased with exposure to 100µg MC-LR l-1 while microsomal GST remained constant. Cylindrospermopsin (CYN) at 100µgl-1 led to an increase in CAT activity and inhibition of GR, as well as to a concentration-dependent GPx inhibition. Microsomal GST was inhibited at all concentrations tested. β-N-methylamino-L-alanine (BMAA) inhibited GR activity in a concentration-dependent manner, however, CAT, GPx, and GST remained unaffected. M. hiemalis showed enhanced oxidative stress tolerance and intact biotransformation enzyme activity towards MC-LR and BMAA in comparison to CYN, confirming its applicability in bioreactor technology in terms of viability and survival in their presence.

The effects of fenvalerate on hepatic and cerebral xenobiotic metabolizing enzymes in selenium and/or iodine deficient rats.

Particularly in developing countries, selenium and/or iodine deficiencies are encountered and use of pesticides in agriculture are not well-controlled. Fenvalerate is a pyrethroid insectide used in agriculture and has applications against a wide range of pests. This study was designed to evaluate the effects of fenvalerate on hepatic and cerebral xenobiotic metabolizing enzyme activities in the presence of iodine and/or selenium deficiency on a rat model. Iodine and/or selenium deficiency was induced by feeding three-week-old Wistar rats with a diet containing <0.005 mg selenium kg-1, and/or administering 1% sodium perchlorate in drinking water for 7 weeks. Test groups received fenvalerate (100 mg kg-1 BW IP) for the last 7 days. Hepatic and cerebral microsomal aniline hydroxylase (CYP2E1) and cytosolic glutathione S-transferase (GST) activities were determined. Besides, hepatic NADPH-cytochrome P450 reductase (P450R), ethoxyresorufin O-deethylase (EROD, CYP1A1/1A2) and penthoxyresorufin O-depenthylase (PROD, CYP2B1/2B2), activities were also measured. Fenvalerate had a general inductive effect on the hepatic and cerebral xenobiotic metabolizing enzyme activities. Moreover, enzyme activities were also altered by iodine and/or selenium deficiency, but the effects seemed to be enzyme- and tissue-specific. The inductive effect of fenvalerate, particularly in high dose exposures, may change the metabolism of several xenobiotics, including drugs, as well as endogenous substrates. The effects may vary depending on the selenium and/or iodine status of individual.

Alteration of enzyme activities and kinetic properties of GST and NQO1 with naturally occurring phenolic compounds

Abstract Objective: Glutathione S-transferase (GST) and NAD(P)H:quinine oxidoreductase 1 (NQO1) are the enzymes important in cytoprotection and bioactivation of chemicals. This study has addressed effects of polyphenolic compounds; ellagic acid, quercetin, naringenin, resveratrol, rutin and hesperidin on rabbit liver GST and NQO1 enzyme activities. Methods: Cytosolic fractions were obtained from homogenized liver tissues of rabbit by differential centrifugation. After calculating IC50 values of individual enzymes for phenolic compounds, both Lineweaver-Burk and Dixon plots were drawn to determine the effect of phenolics on enzyme activity. Michaelis-Menten constant (Km), maximum velocity (Vmax), and inhibition constant (Ki) were calculated from Lineweaver-Burk and Dixon plots, respectively. Results: Resveratrol was found to be the most potent inhibitor for rabbit hepatic cytosolic GST activity with 75.9±2.06 μM IC50 value while naringenin was the least potent one with IC50 value of 260±1.92 μM. Hesperidin and quercetin were found to be the most and least potent inhibitors for NQO1 enzyme activity with IC50 values of 2.7±0.85 μM and 13.8±0.91 μM, respectively. Resveratrol and naringenin inhibited GST activity noncompetitively and mixed type with Ki of 6.2 μM and 245 μM, respectively; while both hesperidin with 0.64 μM Ki value and quercetin with 3.5 μM Ki value inhibited NQO1 activity in a competitive manner. Conclusion: These results indicate that phenolic compounds may modulate Phase II enzyme, GST and NQO1. Moreover, they can influence the metabolic activation of xenobiotic and toxic compounds metabolized by this enzyme.

Chemopreventive effects of korean red ginseng extract on rat hepatocarcinogenesis.

The objective of this study was to determine a chemopreventive activity of Korean red ginseng extract (KRG) in diethylnitrosamine (DEN) induced hepatocarcinogenesis in rats. After acclimatization for a week, Sprague-Dawley rats were randomized into five groups (n = 15) and fed either KRG (0.5, 1 or 2%) or control diets for 10 weeks. After two weeks of starting of experimental diets, the rats were initiated hepatocarcinogenesis by injection of DEN and were then subjected to two-thirds partial hepatectomy at five-week for developing the medium-term bioassay system. Both 0.5 and 1% KRG diets suppressed the area (55 and 60%; p= 0.0251 and 0.0144) and number (39 and 59%; p= 0.0433 and 0.0012) of glutathione S-transferase placental form (GST-P) positive foci when compared to the DEN-control group. The production of thiobarbituric acid reactive substances (TBARS) was significantly reduced in 0.5 and 1% KRG-treated rats. The supplementation of 1% KRG diet significantly elevated the levels of total glutathione (tGSH) and glutathione-related enzymes including cytosolic glutathione S-transferase (GST) and glutathione peroxidase (GPx) activities. It was also observed in cDNA microarray that the gene expressions (Cyp2c6, Cyp2e1, Cyp3a9, and Mgst1) involved in the xenobiotics metabolism via cytochrome P450 signaling pathway were down-regulated in the 1% KRG diet-treated group when compared to the DEN-control. The chemopreventive effects of KRG could be affected by 1) the decrease of lipid peroxidation, 2) the increase of tGSH content and GSH-dependent enzyme activities, and 3) the decrease of the gene expression profile involved in cytochrome P450 signaling pathway. These results suggest that KRG may prove to be a therapeutic agent against hepatocarcinogenesis.

Trichinellosis in mice: effect of albendazole on the glutathione transferase in the intestines

The glutathione S-transferases (GSTs) are a family of multifunctional enzymes involved in cellular detoxification. The aim of this study was to evaluate the effect of albendazole--drug of choice for trichinellosis--on the total activity and kinetics of cytosolic GST in the mouse intestines during experimental trichinellosis. Our results showed a statistically significant decrease in the total GST activity both in the small and large intestines of the mice infected with the nematode Trichinella spiralis (Owen, 1835) and treated with albendazole, compared with the control mice that were infected but untreated with the drug. Furthermore, albendazole administration modified the kinetics of substrate saturation of GST in the intestines of the infected mice because the drug caused changes in Michaelis constant values of this enzyme. Based on our observations, we suggest that the quaternary structure of GST from the mouse intestines is impacted by this drug during trichinellosis.

Changes in the activity and kinetics of mouse intestinal glutathione transferase during experimental trichinellosis

The glutathione S-transferases (GSTs) are a group of multifunctional enzymes, which play a critical role in cellular detoxification. Our investigations deal with the contribution of GST in the biochemical defense against Trichinella spiralis infection. The aim of this study was to examine the effect of T. spiralis infection on the total activity and kinetic properties of cytosolic GST in the intestine during the intestinal phase of experimental trichinellosis in mice. Our results showed a statistically significant increase (relative to the uninfected control) in the total GST activity both in the small and large intestines of the infected mice. Moreover, we observed changes in the kinetics of substrate saturation of GST. Trichinellosis in the small intestine caused a 12-fold decrease in the low K (m) value and a sixfold increase in the high K (m) value. In the large intestine, infection with T. spiralis caused only a fivefold increase in the low K (m) value, whereas the high K (m) value remained unchanged. We suggest that GST from the mouse small intestine could be involved in the detoxification of parasite excretory-secretory products released to the host intestine during trichinellosis and that these products influence the quaternary structure of this enzyme.

Mechanisms underlying iron and copper ions toxicity in biological systems: Pro-oxidant activity and protein-binding effects

Iron and copper ions, in their unbound form, may lead to the generation of reactive oxygen species via Haber–Weiss and/or Fenton reactions. In addition, it has been shown that copper ions can irreversibly and non-specifically bind to thiol groups in proteins. This non-specific binding property has not been fully addressed for iron ions. Thus, the present study compares both the pro-oxidant and the non-specific binding properties of Fe 3+ and Cu 2+, using rat liver cytosol and microsomes as biological systems. Our data show that, in the absence of proteins, Cu 2+/ascorbate elicited more oxygen consumption than Fe 3+/ascorbate under identical conditions. Presence of cytosolic and microsomal protein, however, differentially altered oxygen consumption patterns. In addition, Cu 2+/ascorbate increased microsomal lipid peroxidation and decreased cytosolic and microsomal content of thiol groups more efficiently than Fe 3+/ascorbate. Finally, Fe 3+/ascorbate and Cu 2+/ascorbate inhibited in different ways cytosolic and microsomal glutathione S-transferase (GST) activities, which are differentially sensitive to oxidants. Moreover, in the absence of ascorbate, only Cu 2+ decreased the content of cytosolic and microsomal thiol groups and inhibited cytosolic and microsomal GST activities. Catechin partially prevented the damage to thiol groups elicited by Fe 3+/ascorbate and Cu 2+/ascorbate but not by Cu 2+ alone. N-Acetylcysteine completely prevented the damage elicited by Cu 2+/ascorbate, Fe 3+/ascorbate and Cu 2+ alone. N-Acetylcysteine also completely reversed the damage to thiol groups elicited by Fe 3+/ascorbate, partially reversed that of Cu 2+/ascorbate but failed to reverse the damage promoted by Cu 2+ alone. Our data are discussed in terms to the potential damage that the accumulation of iron and copper ions can promote in biological systems.

Assessment of pollution in sewage ponds using biomarker responses in wild African sharptooth catfish (Clarias gariepinus) in Tanzania

The interactive effects of mixed pollutants in sewage wastewater on biomarker responses were investigated using wild male African sharptooth catfish (Clarias gariepinus) in Morogoro, Tanzania. A total of 58 fish were used, of which 21 were from Mindu dam (reference site) and 22, 9 and 10 from Mafisa, Mazimbu and Mzumbe sewage ponds, respectively. Liver somatic index (LSI) and gonadosomatic index (GSI) were significantly greater (two- to threefold) and (five- to sixfold), respectively, in fish from all sewage ponds. Haemoglobin concentration and gill filament 7-ethoxyresurufin O-deethylase (EROD) activities were significantly higher (1.2-fold and twofold, respectively) in fish from Mzumbe sewage ponds than in fish from Mindu dam, whereas liver EROD activity was significantly higher in fish from Mzumbe and Mafisa sewage ponds (5-fold). A HPLC method for determination of enzymatically formed p-nitrophenyl-glucuronide (PNPG) was developed and applied to measure UDP-glucuronosyl transferase (UGT) activities that was significantly higher in fish from all sewage ponds (2-2.5-fold) than in fish from Mindu dam. Kinetic characteristics and assay dependence of UGT were studied with microsomal preparations. Metallothionein (MT) content was significantly lower (three- to fourfold) in fish from sewage ponds than in fish from Mindu dam, and corresponded with cumulative levels of cadmium, lead and mercury. Condition factor, vitellogenin (Vtg), acetylcholinesterase (AChE) activities in plasma, eyes and brain, haematocrit, plasma protein and cytosolic glutathione S-transferase (GST) activities were comparable in fish from sewage ponds and Mindu dam. Although specific pollutants other than the metals were not identified by chemical analysis, application of a suite of biomarkers in C. gariepinus demonstrated that all sewage ponds were contaminated by pollutants of public health concern.

Comparative Effects of Superoxide Anion and Hydrogen Peroxide on Microsomal and Cytosolic Glutathione S-Transferase Activities of Rat Liver

Glutathione S-transferases (GSTs) are isoenzymes occurring in the cytoplasm and as integral membrane proteins. In addition to their role in drug metabolism by conjugating electrophilic and lipophilic compounds with glutathione (GSH), these enzymes display multiple functions in cells, including antioxidant action. It has been generalized that reactive oxygen species (ROS) inhibit cytosolic GSTs and activate microsomal GSTs; some evidence shows, however, that different ROS-generating systems can inhibit microsomal GST activity. We therefore tested the effect of Fe3+/ascorbate, another ROS-generating system, on cytosolic and microsomal GST activities from rat liver cytosol and microsomes, respectively, and compared it to that of hydrogen peroxide (H2O2). We found that, while both agents displayed similar inhibitory effects on cytosolic GST activity, they promoted opposite effects on microsomal GST activity. Using specific antioxidant enzymes, we corroborated that the effect of Fe3+/ascorbate involves generation of O2(.-) without dismutation into H2O2. Since these ROS have physicochemical properties and redox potentials that are very distinct, their reactivity is different, and their oxidative action is likely to have different targets. We discuss how these properties are related with the oxidative potency of ROS, especially those of O2(.-) and H2O2.

Comparison of citrus coumarins on carcinogen-detoxifying enzymes in Nrf2 knockout mice

Naturally occurring coumarins possess anti-carcinogenic activities in part by inducing carcinogen-detoxifying enzymes glutathione S-transferase (GST) and/or NAD(P)H quinone oxidoreductase (NQO1). Our goal was to determine whether citrus coumarins induce hepatic GST and/or NQO1 via activation of Nrf2 and the antioxidant response element (ARE). First, HepG2 cells stably transfected with the ARE and a green fluorescent protein (GFP) reporter were treated with increasing concentrations of coumarins and compared to positive controls. tert-Butylhydroquinone (TBHQ) and oltipraz increased GFP fluorescence, as did coumarin, limettin, auraptene, imperatorin, and 7,8-benzoflavone, suggesting that they activate the ARE, whereas isopimpinellin did not increase GFP fluorescence. Next, the effects of orally administered coumarins and oltipraz on hepatic GST and NQO1 activities were compared in Nrf2 knockout mice or Nrf2 heterozygous mice exhibiting the wild-type phenotype. Oltipraz, auraptene, imperatorin, isopimpinellin, and auraptene all significantly increased liver cytosolic GST activities in Nrf2 heterozygous mice. This effect was abrogated in Nrf2(−/−) mice dosed with oltipraz, attenuated in mice Nrf2(−/−) mice treated with auraptene and imperatorin, and still significant in Nrf2(−/−) mice treated with isopimpinellin. Of these compounds, only isopimpinellin significantly increased liver cytosolic NQO1 activities, and this effect was not attenuated in Nrf2(−/−) mice. These results strongly suggest that imperatorin and auraptene induce murine liver cytosolic GST activities via the Nrf2/ARE mechanism. Although structurally similar, isopimpinellin did not appear to activate HepG2–ARE–GFP and the Nrf2 knockout mouse study suggests that isopimpinellin may induce GST and NQO1 via additional mechanisms.

Modulation of rat pulmonary carcinogen-metabolising enzyme systems by the isothiocyanates erucin and sulforaphane

The objective of this study was to evaluate the potential of the structurally related aliphatic isothiocyanates erucin and sulforaphane to modulate the pulmonary carcinogen-metabolising enzyme systems in rat lung, a target organ of their chemopreventive activity. Precision-cut rat lung slices were prepared and incubated for 24 h with a range of concentrations of either erucin or sulforaphane, up to 50 μM. Neither compound modulated the O-deethylation of ethoxyresorufin whereas they elevated markedly CYP1A1 and, to a lesser extent, CYP1B1 apoprotein levels. Neither compound influenced the O-depentylation of pentoxyresorufin or CYP2B apoprotein levels, but sulforaphane caused a modest increase in CYP3A2 apoprotein levels. Pulmonary quinone reductase activity, monitored using 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide as substrate, was markedly up-regulated by both compounds and was paralleled by a similar rise in protein levels. Both compounds increased cytosolic glutathione S-transferase activity, measured using 1-chloro-2,4-dinitrobenzene as the accepting substrate; a modest rise was seen in GSTα protein levels, determined immunologically, whereas GSTπ levels were un-affected by the same treatment. Finally, both erucin and sulforaphane increased total glutathione concentration in lung cytosol. It is concluded that these aliphatic isothiocyanates have the potential to antagonise the carcinogenicity of pulmonary carcinogens by stimulating the in situ detoxication of their DNA-binding genotoxic metabolites.

Induction of Hepatic Glutathione S-Transferase and UDP-Glucuronosyltransferase Activities by Ketamine in Rats

Ketamine has been shown to induce rat cytochrome P-450 in a way similar to phenobarbital. However, whether ketamine is able to induce glutathione S-transferase (GST) and UDP-glucuronosyltransferase (UGT), two major phase II drug-metabolizing enzymes, remains unclear. The present study aimed to investigate the effect of ketamine on GST and UGT activities in rats. In a dose-response study, male adult Wistar rats were treated with 10, 20, 40 or 80 mg/kg ketamine intraperitoneally twice daily for 4 days. Livers were removed 1 day after ketamine treatment and hepatic GST and UGT activities were determined. In a reversibility study, rats were treated with 80 mg/kg ketamine intraperitoneally twice daily for 4 days and killed 1, 2, 3 or 4 days after the last dose of ketamine. Livers were removed and hepatic GST and UGT activities were determined. The results of the dose-response study showed that treatment of rats with 10, 20, 40, or 80 mg/kg ketamine produced 19%, 20%, 18%, and 25% increases respectively in the catalytic activity of hepatic cytosolic GST, and 41%, 41%, 35%, and 38% increases respectively in the catalytic activity of microsomal UGT. The results of the reversibility study showed that the GST activities of the rats killed 1, 2, 3, or 4 days after ketamine treatment were 62%, 88%, 46% and 65% higher than the activity of the control group. The UGT activities of the rats killed 1, 2, 3, or 4 days after ketamine treatment were 56%, 53%, 54% and 72% higher than the activity of the control group. Ketamine is able to induce the activities of hepatic GST and UGT in rats. The induced GST and UGT activities persist for at least 4 days after cessation of ketamine. The results suggest the possibility of interactions of drugs related to phase II enzyme induction in chronic ketamine users.

Characterization of hepatic glutathione S-transferases in coho salmon ( Oncorhynchus kisutch)

The glutathione S-transferases (GSTs) are a family of phase II detoxification enzymes which protect against chemical injury. In contrast to mammals, GST expression in fish has not been extensively characterized, especially in the context of detoxifying waterborne pollutants. In the Northwestern United States, coho salmon ( Oncorhynchus kisutch) are an important species of Pacific salmon with complex life histories that can include exposure to a variety of compounds including GST substrates. In the present study we characterized the expression of coho hepatic GST to better understand the ability of coho to detoxify chemicals of environmental relevance. Western blotting of coho hepatic GST revealed the presence of multiple GST-like proteins of approximately 24–26 kDa. Reverse phase HPLC subunit analysis of GSH affinity-purified hepatic GST demonstrated six major and at least two minor potential GST isoforms which were characterized by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI MS–MS) and Fourier transform-ion cyclotron resonance (FT-ICR) MS analyses. The major hepatic coho GST isoforms consisted of a pi and a rho-class GST, whereas GSTs representing the alpha and mu classes constituted minor isoforms. Catalytic studies demonstrated that coho cytosolic GSTs were active towards the prototypical GST substrate 1-chloro-2,4-dinitrobenzene, as well as towards ethacrynic acid and nitrobutyl chloride. However, there was no observable cytosolic GST activity towards the pesticides methyl parathion or atrazine, or products of oxidative stress, such as cumene hydroperoxide and 4-hydroxynonenal. Interestingly, coho hepatic cytosolic fractions had a limited ability to bind bilirubin, reflecting a potential role in the sequestering of metabolic by-products. In summary, coho salmon exhibit a complex hepatic GST isoform expression profile consisting of several GST classes, but may have a limited a capacity to conjugate substrates of toxicological significance such as pesticides and endogenous compounds associated with cellular oxidative stress.

Effects of metals and detergents on biotransformation and detoxification enzymes of leaping mullet ( Liza saliens)

In this study, feral leaping mullet ( Liza saliens) liver microsomal 7-ethoxyresorufin O-deethylase (EROD), and cytosolic glutathione S-transferases (GSTs) activities were investigated using 7-ethoxyresorufin, 1-chloro-2,4-dinitrobenzene (CDNB), and ethacrynic acid (EA) as substrates, respectively. The average EROD activity was found as 1139±175 pmol resorufin/min/mg protein. The average GST activities towards CDNB and EA were found as 1364±41 and 140±19 nmol/min/mg protein, respectively. We have, then, investigated the in vitro effects of some metals and detergents on CYP1A and GST activities in leaping mullet liver. Leaping mullet liver microsomal EROD activity was significantly inhibited by Hg (0.1 mM), Ni (0.1 mM), Cd (0.1 mM), Cu (0.1 mM), Zn (0.1 mM), Sb (0.1 mM), Fe 2+ (1 mM), Co (1 mM), Al (1 mM), and Fe 3+ (1 mM), with the percent inhibition of 80, 80, 77, 75, 70, 69, 56, 53, 46, and 44, respectively. Similarly, conjugation of CDNB catalyzed by GST was inhibited significantly to lesser extend by Hg (0.1 mM), Sb (0.1 mM), Cd (0.1 mM), Cu (0.1 mM), Zn (0.1 mM), Fe 3+ (1 mM), Co (1 mM), and Fe 2+ (1 mM), with the percent inhibition of 70, 69, 65, 61, 54, 51, 47, and 43, respectively. The degrees of inhibition observed on GST catalyzed EA conjugation by Hg (0.1 mM), Cd (0.1 mM), Sb (0.1 mM), Cu (0.1 mM), and Zn (0.1 mM) were 86, 78, 69, 51, and 42, respectively. In addition to metals, the effect of various detergents on leaping mullet liver EROD, GST-CDNB, and GST-EA activities were studied. It was found that ionic detergents strongly inhibited the EROD activity, whereas much less inhibitions were observed with GST catalyzed activities. Therefore, the CYP1A inhibition potencies of metals and detergents suggest that their contribution to the overall CYP1A induction in polycyclic aromatic hydrocarbons contaminated environmental samples has to be taken into account for better interpretation of environmental studies.

Inhibition of cytosolic glutathione S-transferase activity from rat liver by copper

H(2)O(2) inactivation of particular GST isoforms has been reported, with no information regarding the overall effect of other ROS on cytosolic GST activity. The present work describes the inactivation of total cytosolic GST activity from liver rats by the oxygen radical-generating system Cu(2+)/ascorbate. We have previously shown that this system may change some enzymatic activities of thiol proteins through two mechanisms: ROS-induced oxidation and non-specific Cu(2+) binding to protein thiol groups. In the present study, we show that nanomolar Cu(2+) in the absence of ascorbate did not modify total cytosolic GST activity; the same concentrations of Cu(2+) in the presence of ascorbate, however, inhibited this activity. Micromolar Cu(2+) in either the absence or presence of ascorbate inhibited cytosolic GST activity. Kinetic studies show that GSH but no 1-chloro-2,4-dinitrobenzene prevent the inhibition on cytosolic GST induced by micromolar Cu(2+) either in the absence or presence of ascorbate. On the other hand, NEM and mersalyl acid, both thiol-alkylating agents, inhibited GST activity with differential reactivity in a dose-dependent manner. Taken together, these results suggest that an inhibitory Cu(2+)-binding effect is likely to be negligible on the overall inhibition of cytosolic GST activity observed by the Cu(2+)/ascorbate system. We discuss how modification of GST-thiol groups is related to the inhibition of cytosolic GST activity.

The Role of Retinoid X Receptor α in Regulating Alcohol Metabolism

There is substantial overlap in retinol and alcohol metabolism. Mice that lack retinoic acid (RA) receptor retinoid X receptor alpha (RXRalpha) expression in the liver are more susceptible to alcoholic liver disease. To investigate the interaction between RXRalpha and alcoholic liver disease, ethanol metabolism was studied in hepatocyte RXRalpha-deficient [RXRalpha knockout (KO)] mice. Hepatocyte RXRalpha deficiency resulted in a significant increase in hepatic alcohol dehydrogenase (ADH) activity, ADH1 protein, but not Adh1 mRNA. Polysomal distribution analysis indicated that more polysome-associated Adh1 mRNA was present in the mutant mouse livers, suggesting increased ADH1 protein synthesis in RXRalpha KO mice compared with wild-type mice. However, ADH2 and ADH3 enzyme activities were not affected by RXRalpha deficiency. Although ethanol clearance was increased, acetaldehyde elimination was reduced when RXRalpha was not expressed in the liver. Both mitochondrial aldehyde dehydrogenase (ALDH) 2 and cytosolic ALDH activities were reduced in the mutant mice compared with the wild type. Western blot analysis revealed that the levels of ALDH1A1 and ALDH1A2 were decreased in the mutant mice. Semiquantitative reverse transcriptase-polymerase chain reaction indicated that liver Aldh1a1 mRNA level was also reduced due to the lack of RXRalpha expression. Thus, RXRalpha differentially affects ADH and ALDH activity, leading to an increase in alcohol clearance, but a reduction in acetaldehyde elimination. In addition, CYP2E1 as well as mitochondrial and cytosolic glutathione S-transferase activities were significantly lower in RXRalpha KO mice than in wild-type mice. Our results reveal the central role of RXRalpha in ethanol metabolism.