Glutathione S-transferase Subunits Research Articles

Hypoxia exerts oxidative stress and changes in expression of antioxidant enzyme genes in gills of Mytilus galloprovincialis (Lamarck, 1819)

ABSTRACT Bivalve molluscs are widespread in coastal environments, where they are subjected to a wide range of environmental fluctuations, which can represent a significant impact to their antioxidant status. Hypoxia is a global problem affecting the world’s shallow waters, which is forecast to increase in coastal areas due to anthropogenic pressure and global warming. In this work, the effects of 24 h exposure to hypoxia (0.2 mg O2 l−1) on antioxidant parameters of Mytilus galloprovincialis were investigated. Functional parameters of gill samples (the number of dead cells and intracellular reactive oxygen species concentration) were measured by flow cytometry, and the expression level of antioxidant enzyme genes (CAT, GST, SOD) as well as NADH dehydrogenase subunit 2 were analysed using transcriptome analysis. Gills responded to oxygen depletion by an increased total number of intracellular reactive oxygen species, which was accompanied with high levels of mortality of cells (10 times higher compared with control). Significant increases of Mn-superoxide dismutase, glutathione S-transferase and NADH dehydrogenase subunit 2 gene expression levels were observed. Hypoxia did not cause changes in the expression of CAT. The results indicate an overall shift in antioxidant parameters in gill tissue that may represent a certain level of oxidative stress in mussels and decreased capacity of the antioxidant system to maintain redox homeostasis after exposure to hypoxia.

Novel benzo[b]xanthene derivatives: Bismuth(III) triflate-catalyzed one-pot synthesis, characterization, and acetylcholinesterase, glutathione S-transferase, and butyrylcholinesterase inhibitory properties.

In this study, 3,4-dihydro-12-aryl-1H-benzo[b]xanthene-1,6,11-(2H,12H)trione compounds were obtained through one-pot condensation of various substituted aromatic aldehydes, 2-hydroxy-1,4-naphthoquinone, and dimedone in the presence of Bi(OTf)3 as a green and reusable catalyst. The structural characterization of these novel substituted benzo[b]xanthenes was performed by spectroscopic methods, and their inhibitory actions against butyrylcholinesterase (BChE), acetylcholinesterase (AChE), and glutathione S-transferase (GST) were investigated. GST is an enzyme responsible for removing toxic molecules during Phase II reactions in the detoxification mechanism. The AChE and BChE enzymes, which are called cholinesterases, are among the enzymes that occur especially during dementia such as brain damage or Alzheimer's disease. Inhibition effects of the benzo[b]xanthene derivatives on AChE, BChE, and GST were found at the millimolar level. The best inhibitor for GST is compound 4a (31.18 ± 6.13 mM), for AChE, it is compound 4d (28.16 ± 3.46 mM), and for BChE, it is compound 4f (36.24 ± 3.19 mM). Compound 4a inhibited the dimerization of GST subunits, and compounds 4d and 4f directly inhibited the catalytic activity by interacting with the catalytic active site or a related site of the AChE and BChE enzymes, respectively.

Partial purification and characterization of glutathione S-transferase from the somatic tissue of Gastrothylax crumenifer (Trematoda: Digenea).

Aim:Aim of the present study was to carry out the partial purification and biochemical characterization of glutathione S-transferase (GST) from the somatic tissue of ruminal amphistome parasite, Gastrothylax crumenifer (Gc) infecting Indian water buffalo (Bubalus bubalis).Materials and Methods:The crude somatic homogenate of Gc was subjected to progressive ammonium sulfate precipitation followed by size exclusion chromatography in a Sephacryl S 100-HR column. The partially purified GST was assayed spectrophotometrically, and the corresponding enzyme activity was also recorded in polyacrylamide gel. GST isolated from the amphistome parasite was also exposed to variable changes in temperature and the pH gradient of the assay mixture.Results:The precipitated amphistome GST molecules showed maximum activity in the sixth elution fraction. The GST subunit appeared as a single band in the reducing polyacrylamide gel electrophoresis with an apparent molecular weight of 26 kDa. The GST proteins were found to be fairly stable up to 37°C, beyond this the activity got heavily impaired. Further, the GST obtained showed a pH optima of 7.5.Conclusion:Present findings showed that GST from Gc could be conveniently purified using gel filtration chromatography. The purified enzyme showed maximum stability and activity at 4°C.

Biochemical characterization of metabolism-based atrazine resistance in Amaranthus tuberculatus and identification of an expressed GST associated with resistance.

SummaryRapid detoxification of atrazine in naturally tolerant crops such as maize (Zea mays) and grain sorghum (Sorghum bicolor) results from glutathione S‐transferase (GST) activity. In previous research, two atrazine‐resistant waterhemp (Amaranthus tuberculatus) populations from Illinois, U.S.A. (designated ACR and MCR), displayed rapid formation of atrazine‐glutathione (GSH) conjugates, implicating elevated rates of metabolism as the resistance mechanism. Our main objective was to utilize protein purification combined with qualitative proteomics to investigate the hypothesis that enhanced atrazine detoxification, catalysed by distinct GSTs, confers resistance in ACR and MCR. Additionally, candidate AtuGST expression was analysed in an F2 population segregating for atrazine resistance. ACR and MCR showed higher specific activities towards atrazine in partially purified ammonium sulphate and GSH affinity‐purified fractions compared to an atrazine‐sensitive population (WCS). One‐dimensional electrophoresis of these fractions displayed an approximate 26‐kDa band, typical of GST subunits. Several phi‐ and tau‐class GSTs were identified by LC‐MS/MS from each population, based on peptide similarity with GSTs from Arabidopsis. Elevated constitutive expression of one phi‐class GST, named AtuGSTF2, correlated strongly with atrazine resistance in ACR and MCR and segregating F2 population. These results indicate that AtuGSTF2 may be linked to a metabolic mechanism that confers atrazine resistance in ACR and MCR.

Schoolchildren's antioxidation genotypes are susceptible factors for reduced lung function and airway inflammation caused by air pollution

BackgroundWe recently reported the relationship between exposure to ambient air pollutants and changes in lung function and nasal inflammation among schoolchildren. A study was conducted to investigate whether antioxidation genotypes influence these associations. MethodsA follow-up study of 97 schoolchildren was conducted in New Taipei City, Taiwan. A structured respiratory health questionnaire was administered in September 2007, followed by monthly spirometry and measurement of nasal inflammation from October 2007 to November 2009. During the study period, complete daily monitoring data for air pollutants were obtained from the Environmental Protection Administration monitoring station and Aerosol Supersite. The genotypes of glutathione S-transferase (GST) subunits M1, T1, P1 and superoxide dismutases subunit 2 (SOD2) were characterized. Mixed-effects models were used, adjusting for known confounders. ResultGSTM1 null children had significant PM2.5-related increment in leukocyte (8.52%; 95% confidence interval (CI): 3.13–13.92%) and neutrophil (9.68%; 95% CI: 4.51–14.85%) in nasal lavage. Ozone levels were significantly and inversely associated with forced expiratory flow at 25% of forced vital capacity (FEF25%) (−0.43L/s; 95% CI: −0.58,−0.28L/s) in SOD2 Ala16 variant children. ConclusionIn this longitudinal study of schoolchildren. Our data provide evidence that antioxidation genotype modifies the airway inflammation caused by PM2.5. Antioxidation genotype also acts as an effect modifier, but not strong, in ozone-related small airway function response.

Biochemical responses in seabream (Sparus aurata) caged in-field or exposed to benzo(a)pyrene and paraquat. Characterization of glutathione S-transferases

Gilthead seabream (Sparus aurata) specimens were caged in-field at the Téboulba harbour or exposed to benzo(a)pyrene [B(a)P] or to paraquat [PQ] plus B(a)P, and several biochemical biomarker responses were investigated. Antioxidant enzymes, such as glutathione peroxidase, catalase and glutathione reductase, significantly increased in the in-field and B(a)P+PQ exposures, but were only moderately affected by B(a)P alone. Glucose-6-phosphate and 6-phosphogluconate dehydrogenases significantly diminished after in-field exposure. Different responses with biotransformation enzymes were observed: the P4501A-associated EROD activity was highly induced in response to B(a)P and B(a)P+PQ exposures, while total activity of the glutathione S-transferase (GST) was similar to control. However, after purification of the GST proteins by affinity chromatograpy and analysis by two-dimensional electrophoresis, nineteen highly reproducible isoforms were resolved. In addition, some of reproducible isoforms showed different and specific expression patterns in response to contaminants.Thus, proteomic analysis of the purified GST subunits is a reliable tool for ecotoxicological research, useful in polluted marine ecosystem as an effective biomarker of contamination.

Functional Characterization of Alpha-Class Glutathione S-Transferases from the Turkey (Meleagris Gallopavo)

Six Alpha-class glutathione S-transferase (GST) subunits were cloned from domestic turkey livers, which are one of the most susceptible animals known to the carcinogenic mycotoxin aflatoxin B₁. In most animals, GST dysfunction is a risk factor for susceptibility toward AFB₁, and we have shown that turkeys lack GSTs with affinity toward the carcinogenic intermediate exo-aflatoxin B(1)-8-9-epoxide (AFBO). Conversely, mice are resistant to AFB₁ carcinogenesis, due to high constitutive expression of mGSTA3 that has high affinity toward AFBO. When expressed in Escherichia coli, all six tGSTA subunits possessed conjugating activities toward substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA), and cumene hydroperoxide (CHP) with tGSTA1.2 appearing most active. Interestingly, tGSTA1.1, which lacks one of the four Alpha-class signature motifs, possessed enzymatic activities toward all substrates. All had comparable activities toward AFBO conjugation, an activity absent in turkey liver cytosols. E. coli-expressed mGSTA3 conjugated AFBO with more than 3-fold greater activity than that of tGSTAs and had higher activity toward GST prototype substrates. Mouse hepatic cytosols had approximately 900-fold higher catalytic activity toward AFBO compared with those from turkey. There was no apparent amino acid profile in tGSTAs that might correspond to specificity toward AFBO, although tGSTA1.2, which had slightly higher AFBO-trapping ability, shared Tyr¹⁰⁸ with mGSTA3, a residue postulated to be critical for AFBO trapping activity in mammalian systems. The observation that recombinant tGSTAs detoxify AFBO, whereas their hepatic forms do not, implies that the hepatic forms of these enzymes are silenced by one or more regulatory mechanisms.

Biochemical studies on glutathione S-transferase from the bovine filarial worm Setaria digitata

Setaria digitata is a filarial worm of the cattle used as a model system for antifilarial drug screening, due to its similarity to the human filarial parasites Wuchereria bancrofti and Brugia malayi. Since filarial glutathione S-transferase (GST) is a good biochemical target for antifilarial drug development, a study has been undertaken for the biochemical characterization of GST from S. digitata. Cytosolic fraction was separated from the crude S.digitata worm homogenate by ultracentrifugation at 100,000 g and subjected to ammonium sulfate precipitation followed by affinity chromatography using GSH-agarose column. The kinetic parameters K (m) and V (max) values with respect to GSH were 0.45 mM and 0.105 μmol min(-1) mL(-1) respectively. With respect to 1-chloro-2,4-dinitrobenzene, the K (m) and V (max) values were 1.21 and 0.117 μmol min(-1) mL(-1) respectively. The effect of temperature and pH on GST enzyme activity was studied. The protein retained its enzyme activity between 0°C and 40°C, beyond which it showed a decreasing tendency, and at 80°C, the activity was lost completely. The enzyme activity was varying with change in pH, and the maximum GST activity was observed at pH 7.5. Gel filtration chromatographic studies indicated that the protein has a native molecular mass of about 54 kDa. The single band of GST subunit appeared in sodium dodecyl sulfate polyacrylamide gel electrophoresis was found to have molecular mass of ∼27 kDa. This shows that cytosolic S. digitata GST protein is homodimeric in nature.

Dietary Diacetylene Falcarindiol Induces Phase 2 Drug-Metabolizing Enzymes and Blocks Carbon Tetrachloride-Induced Hepatotoxicity in Mice through Suppression of Lipid Peroxidation

Falcarindiol is a diacetylenic natural product containing unique carbon-carbon triple bonds. Mice were orally administrated falcarindiol (100 mg/kg), and drug-metabolizing and antioxidant enzymes were monitored in several tissues of mice. Treatment with falcarindiol was found to increase glutathione S-transferase (GST) and NAD(P)H: quinone oxidoreductase 1 activities in liver, small intestine, kidney, and lung. No changes were observed in cytochrome P450 (CYP) 1A known to activate procarcinogens. Western blot analysis revealed that various GST subunits including GSTA4, which plays an important role in the detoxification of alkenals produced from lipid peroxides, were induced in liver, small intestine, and kidney of falcarindiol-treated mice. Additionally, we investigated the protective effects of falcarindiol against hepatotoxicity induced by carbon tetrachloride (CCl(4)) and the mechanism of its hepatoprotective effect. Pretreatment with falcarindiol prior to the administration of CCl(4) significantly suppressed both an increase in serum alanine transaminase/aspartate transaminase (ALT/AST) activity and an increase in hepatic thiobarbituric acid reactive substance levels without affecting CCl(4)-mediated degradation of CYP2E1. Formation of hexanoyl-lysine and 4-hydroxy-2(E)-nonenal-histidine adducts, lipid peroxidation biomarkers, in homogenates from the liver of CCl(4)-treated mice was decreased in the group of mice pretreated with falcarindiol. These results suggest that the protective effects of falcarindiol against CCl(4) toxicity might, in part, be explained by anti-lipid peroxidation activity associated with the induction of the GSTs including GSTA4.

Biochemical warfare on the reef: the role of glutathione transferases in consumer tolerance of dietary prostaglandins.

BackgroundDespite the profound variation among marine consumers in tolerance for allelochemically-rich foods, few studies have examined the biochemical adaptations underlying diet choice. Here we examine the role of glutathione S-transferases (GSTs) in the detoxification of dietary allelochemicals in the digestive gland of the predatory gastropod Cyphoma gibbosum, a generalist consumer of gorgonian corals. Controlled laboratory feeding experiments were used to investigate the influence of gorgonian diet on Cyphoma GST activity and isoform expression. Gorgonian extracts and semi-purified fractions were also screened to identify inhibitors and possible substrates of Cyphoma GSTs. In addition, we investigated the inhibitory properties of prostaglandins (PGs) structurally similar to antipredatory PGs found in high concentrations in the Caribbean gorgonian Plexaura homomalla.Principal Findings Cyphoma GST subunit composition was invariant and activity was constitutively high regardless of gorgonian diet. Bioassay-guided fractionation of gorgonian extracts revealed that moderately hydrophobic fractions from all eight gorgonian species examined contained putative GST substrates/inhibitors. LC-MS and NMR spectral analysis of the most inhibitory fraction from P. homomalla subsequently identified prostaglandin A2 (PGA2) as the dominant component. A similar screening of commercially available prostaglandins in series A, E, and F revealed that those prostaglandins most abundant in gorgonian tissues (e.g., PGA2) were also the most potent inhibitors. In vivo estimates of PGA2 concentration in digestive gland tissues calculated from snail grazing rates revealed that Cyphoma GSTs would be saturated with respect to PGA2 and operating at or near physiological capacity.SignificanceThe high, constitutive activity of Cyphoma GSTs is likely necessitated by the ubiquitous presence of GST substrates and/or inhibitors in this consumer's gorgonian diet. This generalist's GSTs may operate as ‘all-purpose’ detoxification enzymes, capable of conjugating or sequestering a broad range of lipophilic gorgonian compounds, thereby allowing this predator to exploit a range of chemically-defended prey, resulting in a competitive dietary advantage for this species.

Modulation of oxidative phosphorylation machinery signifies a prime mode of anti-ageing mechanism of calorie restriction in male rat liver mitochondria

Mitochondria being the major source and target of reactive oxygen species (ROS) play a crucial role during ageing. We analyzed ageing and calorie restriction (CR)-induced changes in abundance of rat liver mitochondrial proteins to understand key aspects behind the age-retarding mechanism of CR. The combination of blue-native (BN) gel system with fluorescence Difference Gel Electrophoresis (DIGE) facilitated an efficient analysis of soluble and membrane proteins, existing as monomers or multi-protein assemblies. Changes in abundance of specific key subunits of respiratory chain complexes I, IV and V, critical for activity and/or assembly of the complexes were identified. CR lowered complex I assembly and complex IV activity, which is discussed as a molecular mechanism to minimize ROS production at mitochondria. Notably, the antioxidant system was found to be least affected. The GSH:GSSG couple could be depicted as a rapid mean to handle the fluctuations in ROS levels led by reversible metabolic shifts. We evaluated the relative significance of ROS generation against quenching. We also observed parallel and unidirectional changes as effect of ageing and CR, in subunits of ATP synthase, cytochrome P450 and glutathione S-transferase. This is the first report on such 'putatively hormetic' ageing-analogous effects of CR, besides the age-retarding ones.

Proteomic identification, cDNA cloning and enzymatic activity of glutathione S-transferases from the generalist marine gastropod, Cyphoma gibbosum

Glutathione S-transferases (GST) were characterized from the digestive gland of Cyphoma gibbosum (Mollusca; Gastropoda), to investigate the possible role of these detoxification enzymes in conferring resistance to allelochemicals present in its gorgonian coral diet. We identified the collection of expressed cytosolic Cyphoma GST classes using a proteomic approach involving affinity chromatography, HPLC and nano-spray liquid chromatography–tandem mass spectrometry (LC–MS/MS). Two major GST subunits were identified as putative mu-class GSTs; while one minor GST subunit was identified as a putative theta-class GST, apparently the first theta-class GST identified from a mollusc. Two Cyphoma GST cDNAs (CgGSTM1 and CgGSTM2) were isolated by RT-PCR using primers derived from peptide sequences. Phylogenetic analyses established both cDNAs as mu-class GSTs and revealed a mollusc-specific subclass of the GST-mu clade. These results provide new insights into metazoan GST diversity and the biochemical mechanisms used by marine organisms to cope with their chemically defended prey.

Effects of the Anticonvulsant, Valproate, on the Expression of Components of the Cytochrome-P-450-Mediated Monooxygenase System and Glutathione S-Transferases

It has been shown previously that the anticonvulsant agent, sodium valproate, induces certain cytochrome P-450 monooxygenase activities and decreases glutathione S-transferase activity. We have used Western blotting, RNase protection assays and Northern blot hybridization to determine the effects of valproate on the abundance of individual components of the cytochrome P-450 monooxygenase and of glutathione S-transferase subunits. Due to the short half-life of the drug in rats we have used an in vitro experimental system comprised of rat hepatocytes co-cultured with rat primitive biliary epithelial cells. Valproate was shown to be a potent inducer of two members of the cytochrome P-450 (CYP)2B subfamily, CYP2B1 and 2B2. The induction of the proteins was mediated at the level of the mRNAs, with the mRNA for CYP2B1 being more highly induced than that for CYP2B2. The drug also induced, but to a much lesser extent, two important components of the cytochrome-P-450-mediated monooxygenase system, NADPH-dependent cytochrome P-450 reductase and cytochrome b5, and their corresponding mRNAs. Thus, the effects of valproate on cytochromes P-450 and other components of the cytochrome-P450-mediated monooxygenase system mimic those of another, structurally diverse, antiepileptic drug, phenobarbital. However, in contrast to phenobarbital, which induces glutathione S-transferase subunits 1, 2, 3, 4 and 7, valproate selectively decreases the abundance of subunits 3 and/or 4. It has been shown previously that CYP2B1 is involved in the production of metabolites of valproate implicated in hepatotoxicity. The induction of this protein by valproate would thus contribute substantially to the hepatotoxic effects associated with the drug.

Cloning and characterization of a tau glutathione S-transferase subunit encoding gene in Gossypium hirsutum

A predicted tau Glutathione S-transferase (GST) subunit encoding gene, named GhGST, was isolated from Gossypium hirsutum with RACE method from SSH library based on Verticillium dahliae stress. The data revealed an open reading frame of 678 bp encoding a protein of 225 amino acids with a molecular weight of 25.821 kDa. Semi-quantitative RT-PCR analysis showed that the mRNA of GhGST was expressed in root, stem and leaf. And the content of GhGST expression increased under Verticillium dahliae stress in root. The expression of GhGST gene was verified by transformation in E. coli BL21 (DE3) strain with the recombinant expression vector pET-32A. GST activity assay showed the crude GhGST protein had obvious activity to 1-chloro-2,4-dinitrobenzene (CDNB) substrate.

Enhanced glutathione S-transferase expression in 2-hydroxyamino- 1-methyl-6-phenylimidazo[4,5-b]pyridine-resistant IEC-18 cells

In the present study we show that repeated exposure of the rat intestinal epithelial cell line IEC-18 to 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP), from a toxicological point of view the most relevant phase-1 metabolite of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP, the main heterocyclic aromatic amine present in processed meat), led to the selection of N-OH-PhIP-resistant IEC-18 cells. This phenomenon was accompanied by a fivefold increase in total glutathione S-transferase (GST) activity, measured with the broad-spectrum substrate 1-chloro-2,4-dinitrobenzene, in the N-OH-PhIP-resistant IEC-18 cells. Furthermore, a Western blotting analysis revealed that the expression of GST subunits A1, A3, A4, M1 and P1 was enhanced in the N-OH-PhIP-resistant IEC-18 cells.

Replacement Surgery with Unnatural Amino Acids in the Lock-and-Key Joint of Glutathione Transferase Subunits

Proteins contain amino acid residues essential to structure and function. Ribosomal protein synthesis is typically limited to the 20 amino acids of the genetic code, but posttranslational chemical modifications can greatly expand the diversity of side chain functionalities. In this investigation, a natural aromatic residue in the lock-and-key joint at the subunit interface of the dimeric glutathione transferase P1-1 was replaced by an S-alkylcysteine residue to give a functional enzyme. Introduction of Cys in the key position inactivates the enzyme, but subsequent alkylation of this residue enhances the catalytic efficiency up to 27,000-fold. Combinatorial modification of Cys by a mixture of reagents facilitated identification of an n-butyl group as the most efficient activator. Alkylation also enhanced binding affinity for active-site ligands and stabilized the enzyme against chemical denaturation and thermal inactivation.

Several glutathione S-transferase isozymes that protect against oxidative injury are expressed in human liver mitochondria

The mitochondrial environment is rich in reactive oxygen species (ROS) that may ultimately peroxidize membrane proteins and generate unsaturated aldehydes such as 4-hydroxy-2-nonenal (4HNE). We had previously demonstrated the presence of hGSTA4-4, an efficient catalyst of 4HNE detoxification, in human liver mitochondria to the exclusion of the cytosol. In the present study, GSH-affinity chromatography was used in conjunction with biochemical and proteomic analysis to determine the presence of additional cytosolic glutathione S-transferases (GSTs) in human hepatic mitochondria. HPLC-subunit analysis of GSH affinity-purified liver mitochondrial proteins indicated the presence of several potential mitochondrial GST isoforms. Electrospray ionization-mass spectrometry analysis of eluted mitochondrial GST subunits yielded molecular masses similar to those of hGSTP1, hGSTA1 and hGSTA2. Octagonal matrix-assisted laser desorption/ionization time of flight mass spectrometry and proteomics analysis using MS-FIT confirmed the presence of these three GST subunits in mitochondria, and HPLC analysis indicated that the relative contents of the mitochondrial GST subunits were hGSTA1 > hGSTA2 > hGSTP1. The mitochondrial localization of the alpha and pi class GST subunits was consistent with immunoblotting analysis of purified mitochondrial GST. Enzymatic studies using GSH-purified mitochondrial GST fractions demonstrated the presence of significant GST activity using the nonspecific GST substrate 1-chloro-2,4-dinitrobenzene (CDNB), as well as 4HNE, δ 5-androstene-3,17-dione (ADI), and cumene hydroperoxide (CuOOH). Interestingly, the specific mitochondrial GST activities toward 4HNE, a highly toxic α,β-unsaturated aldehyde produced during the breakdown of membrane lipids, exceeded that observed in liver cytosol. These observations are suggestive of a role of GST in protecting against mitochondrial injury during the secondary phase of oxidative stress, or modulation of 4HNE-mediated mitochondrial signaling pathways. However, other properties of mitochondrial GST, such as conjugation of environmental chemicals and binding of lipophilic non-substrate xenobiotics and endogenous compounds, remain to be investigated.

Deficiency of Glutathione Transferase Zeta Causes Oxidative Stress and Activation of Antioxidant Response Pathways

Glutathione S-transferase (GST) zeta (GSTZ1-1) plays a significant role in the catabolism of phenylalanine and tyrosine, and a deficiency of GSTZ1-1 results in the accumulation of maleylacetoacetate and its derivatives maleylacetone (MA) and succinylacetone. Induction of GST subunits was detected in the liver of Gstz1(-/-) mice by Western blotting with specific antisera and high-performance liquid chromatography analysis of glutathione affinity column-purified proteins. The greatest induction was observed in members of the mu class. Induction of NAD(P)H:quinone oxidoreductase 1 and the catalytic and modifier subunits of glutamate-cysteine ligase was also observed. Many of the enzymes that are induced in Gstz1(-/-) mice are regulated by antioxidant response elements that respond to oxidative stress via the Keap1/Nrf2 pathway. It is significant that diminished glutathione concentrations were also observed in the liver of Gstz1(-/-) mice, which supports the conclusion that under normal dietary conditions, the accumulation of electrophilic intermediates such as maleylacetoacetate and MA results in a high level of oxidative stress. Elevated GST activities in the livers of Gstz1(-/-) mice suggest that GSTZ1-1 deficiency may alter the metabolism of some drugs and xenobiotics. Gstz1(-/-) mice given acetaminophen demonstrated increased hepatotoxicity compared with wild-type mice. This toxicity may be attributed to the increased GST activity or the decreased hepatic concentrations of glutathione, or both. Patients with acquired deficiency of GSTZ1-1 caused by therapeutic exposure to dichloroacetic acid for the clinical treatment of lactic acidosis may be at increased risk of drug- and chemical-induced toxicity.

TRANSCRIPTIONAL REGULATION OF THE NAD(P)H:QUINONE OXIDOREDUCTASE 1 AND GLUTATHIONES-TRANSFERASE YA GENES BY MERCURY, LEAD, AND COPPER

Recently, we demonstrated the ability of heavy metals, particularly Hg2+, Pb2+, and Cu2+, to differentially modulate in Hepa 1c1c7 cells the expression of the phase II xenobiotic metabolizing enzymes NAD(P)H:quinone oxidoreductase 1 (Nqo1) and glutathione S-transferase subunit Ya (Gst ya) genes, yet the mechanisms involved remain unknown. To investigate the molecular mechanisms involved in the regulation of Nqo1 and Gst ya genes by heavy metals, Hepa 1c1c7 cells were treated with Hg2+, Pb2+, or Cu2+ in the presence and absence of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a potent inducer of Nqo1, Gst ya, and Cyp1a1 genes. Analysis of the time-dependent effect of heavy metals revealed that Hg2+ and Pb2+ increased whereas Cu2+ inhibited the constitutive and inducible expression of Nqo1 and Gst ya mRNAs in a time-dependent manner. The RNA synthesis inhibitor actinomycin D significantly inhibited the Nqo1 and Gst ya mRNA induction in response to metals, indicating a requirement of de novo RNA synthesis. The protein synthesis inhibitor cycloheximide significantly inhibited metal-mediated induction of Nqo1 and Gst ya mRNAs, which coincided with a decrease in the nuclear factor erythroid 2-related factor 2 (Nrf2) protein expression, implying the requirement of Nrf2 protein synthesis for the induction of these genes. Furthermore, inhibition of Nrf2 protein degradation by carbobenzoxy-L-leucyl-L-leucyl-leucinal (MG-132), a 26S proteasome inhibitor, significantly reversed the cycloheximide-mediated inhibition of Nqo1 and Gst ya mRNAs, which coincided with an increase in the expression of Nrf2, confirming that a transcriptional mechanism is involved. Nqo1 and Gst ya mRNA and protein decay experiments revealed lack of post-transcriptional and post-translational mechanisms. This is the first demonstration that heavy metals regulate the expression of Nqo1 and Gst ya genes through a transcriptional mechanism.

Interaction of Glutathione S-Transferase with Hypericin: A Photophysical Study

The photophysics of hypericin have been studied in its complex with two different isoforms, A1-1 and P1-1, of the protein glutathione S-transferase (GST). One molecule of hypericin binds to each of the two GST subunits. Comparisons are made with our previous results for the hypericin/human serum albumin complex (Photochem. Photobiol. 1999, 69, 633-645). Hypericin binds with high affinity to the GSTs: 0.65 microM for the A1-1 isoform and 0.51 microM for the P1-1 isoform (Biochemistry 2004, 43, 12761-12769). The photophysics and activity of hypericin are strongly modulated by the binding protein. Intramolecular hydrogen-atom transfer is suppressed in both cases. Most importantly, while there is significant singlet oxygen generation from hypericin bound to GST A1-1, binding to GST P1-1 suppresses singlet oxygen generation to almost negligible levels. The data are rationalized in terms of a simple model in which the hypericin photophysics depends entirely upon the decay of the triplet state by two competing processes, quenching by oxygen to yield singlet oxygen and ionization, the latter of these two are proposed to be modulated by A1-1 and P1-1.