Low Levels of GSTA1 Expression Are Required for Caco-2 Cell Proliferation

The colonic epithelium continuously regenerates with transitions through various cellular phases including proliferation, differentiation and cell death via apoptosis. Human colonic adenocarcinoma (Caco-2) cells in culture undergo spontaneous differentiation into mature enterocytes in association with progressive increases in expression of glutathione S-transferase alpha-1 (GSTA1). We hypothesize that GSTA1 plays a functional role in controlling proliferation, differentiation and apoptosis in Caco-2 cells. We demonstrate increased GSTA1 levels associated with decreased proliferation and increased expression of differentiation markers alkaline phosphatase, villin, dipeptidyl peptidase-4 and E-cadherin in postconfluent Caco-2 cells. Results of MTS assays, BrdU incorporation and flow cytometry indicate that forced expression of GSTA1 significantly reduces cellular proliferation and siRNA-mediated down-regulation of GSTA1 significantly increases cells in S-phase and associated cell proliferation. Sodium butyrate (NaB) at a concentration of 1 mM reduces Caco-2 cell proliferation, increases differentiation and increases GSTA1 activity 4-fold by 72 hours. In contrast, 10 mM NaB causes significant toxicity in preconfluent cells via apoptosis through caspase-3 activation with reduced GSTA1 activity. However, GSTA1 down-regulation by siRNA does not alter NaB-induced differentiation or apoptosis in Caco-2 cells. While 10 mM NaB causes GSTA1-JNK complex dissociation, phosphorylation of JNK is not altered. These findings suggest that GSTA1 levels may play a role in modulating enterocyte proliferation but do not influence differentiation or apoptosis.     

Human GSTA1-1 reduces c-Jun N-terminal kinase signalling and apoptosis in Caco-2 cells

The effect of GSTA1-1 (glutathione S-transferase Alpha 1-1) on JNK (c-Jun N-terminal kinase) activation was investigated in Caco-2 cells in which GSTA1 expression increases with degree of confluency, and in MEF3T3 cells with Tet-Off-inducible GSTA1 expression. Comparison of GSTA1 expression in pre-confluent, confluent and 8-day post-confluent Caco-2 cells revealed progressively increasing mRNA and protein levels at later stages of confluency. Exposure of pre-confluent cells to stress conditions including IL-1beta (interleukin-1beta), H2O2 or UV irradiation resulted in marked increases in JNK activity as indicated by c-Jun phosphorylation. However, JNK activation was significantly reduced in post-confluent cells exposed to the same stresses. Western-blot analysis of GSTA1-1 protein bound to JNK protein pulled down from cellular extracts showed approx. 4-fold higher GSTA1-1-JNK complex formation in post-confluent cells compared with pre-confluent cells. However, stress conditions did not alter the amount of GSTA1-1 bound to JNK. The role of GSTA1-1 in JNK suppression was more specifically revealed in Tet-Off-inducible MEF3T3-GSTA1-1 cells in which GSTA1 overexpression significantly reduced phosphorylation of c-Jun following exposure to IL-1beta, H2O2 and UV irradiation. Finally, the incidence of tumour necrosis factor alpha/butyrate-induced apoptosis was significantly higher in pre-confluent Caco-2 cells expressing low levels of GSTA1 compared with post-confluent cells. These results indicate that GSTA1 suppresses activation of JNK signalling by a pro-inflammatory cytokine and oxidative stress and suggests a protective role for GSTA1-1 in JNK-associated apoptosis.     

Glucocorticoid receptor (GR)-associated SMRT binding to C/EBPbeta TAD and Nrf2 Neh4/5: role of SMRT recruited to GR in GSTA2 gene repression

The expression of the glutathione S-transferase gene (GST), whose induction accounts for cancer chemoprevention, is regulated by activation of CCAAT/enhancer binding protein beta (C/EBPbeta) and NF-E2-related factor 2 (Nrf2). The present study investigated the repressing effects of activating glucocorticoid receptor (GR) on C/EBPbeta- and Nrf2-mediated GSTA2 gene induction and the mechanism. Dexamethasone that activates GR inhibited constitutive and oltipraz- or tert-butylhydroquinone (t-BHQ)-inducible GSTA2 expression in H4IIE cells. Also, dexamethasone repressed GSTA2 promoter-luciferase gene activity. Dexamethasone-GR activation did not inhibit nuclear translocation of C/EBPbeta or Nrf2 nor their DNA binding activities induced by oltipraz or t-BHQ. Deletion of the glucocorticoid response element (GRE) in the GSTA2 promoter abolished dexamethasone inhibition of the gene induction. Immunoprecipitation-immunoblotting, chromatin immunoprecipitation, and GST pull-down assays revealed that silencing mediator for retinoid and thyroid hormone receptors (SMRT), a corepressor recruited to steroid-GR complex for histone deacetylation, bound to TAD domain of C/EBPbeta and Neh4/5 domain of Nrf2. The GSTA2 promoter-luciferase activities were decreased by SMRT but not by truncated SMRTs. The small interference RNA (siRNA) against SMRT abolished SMRT repression of the gene induction by C/EBPbeta or Nrf2. The plasmid transfection and siRNA experiments directly evidenced the functional role of SMRT in GSTA2 repression. In conclusion, dexamethasone antagonizes C/EBPbeta- and Nrf2-mediated GSTA2 gene induction via ligand-GR binding to the GRE, and steroid-mediated GSTA2 repression involves inactivation of C/EBPbeta and Nrf2 by SMRT recruited to steroid-GR complex.     

Expression of rat liver glutathione-S-transferase GSTA5 in cell lines provides increased resistance to alkylating agents and toxic aldehydes

The glutathione-S-transferases (GST) are a major contributor to the eukaryotic cell's defences against chemical and oxidative stress. However, the role of individual GST isoenzymes in conferring resistance to xenobiotics has not been fully determined. We have examined the effect of the rat GSTA5 isoenzyme in the detoxication of alkylating agents and aldehydes by constructing a cell line in which it is stably expressed. The hamster fibroblast cell line V79 was transfected with a construct expressing GSTA5 from the CMV promoter. A stable clone (V79-GSTA5) was isolated after selecting for the neomycin phosphotransferase gene present on the introduced DNA. The cell line showed significantly increased levels of resistance towards the alkylating agents chorambucil and melphalan. Levels of resistance were 4-6-fold greater in V79-GSTA5 cells than in control cells. Increased levels of resistance were also observed towards the lipid peroxidation product acrolein (IC(50)=80 microM compared with 17 microM in control cells). The V79-GSTA5 cells also showed a 4-fold increase in resistance to trans, trans muconaldehyde (IC(50)=4 micro compared with l microM for control cells). GSTA5 did not protect against 4-hydroxynonenal, but it did provide greater levels of protection to hydrogen peroxide, with an IC(50) of 380 microM in V79-GSTA5 compared with 180 microM in control cells. In contrast, V79-GSTA5 cells were more sensitive to methyl glyoxal, suggesting that a methyl glyoxal-glutathione conjugate is more toxic that the parental compound. These data contribute towards the evaluation of the role of GSTA5 in the detoxication of these compounds.     

Differential effects of iron overload on GST isoform expression in mouse liver and kidney and correlation between GSTA4 induction and overproduction of free radicles.

We have investigated the effect of iron overload on the expression of mouse GSTA1, A4, M1, and P1 in liver, the main iron storage site during iron overload, and in kidney. In iron-overloaded animals, mRNA and protein levels of GSTA1, A4, and M1 were increased in liver. In kidney, GSTA4 protein level was also increased while, unexpectedly, GSTA1 and M1 expression was strongly decreased. We showed, by immunohistochemistry, that GSTA4 was more abundant in hepatocytes of periportal areas and in convoluted proximal tubular cells in normal liver and kidney, respectively. In iron-overloaded mice, GSTA4 staining was more intense in cells that preferentially accumulated iron, and conjugation of 4-hydroxynonenal, a specific substrate of GSTA4, was enhanced in both organs. Moreover an acute exposure of primary cultures of mouse hepatocytes to iron-citrate strongly induced oxidative stress and cellular injury and resulted in an increase in GSTA4 expression, while cotreatment with iron-citrate and either desferrioxamine or vitamin E prevented both toxicity and GSTA4 induction. These data demonstrate that GSTA1 and M1 are differentially regulated in liver and kidney while GSTA4 is induced in both organs during iron overload. Moreover, they support the view that iron-induction of GSTA4 is related to an overproduction of free radicals.     

Cystamine restores GSTA3 levels in Vanin-1 null mice

Free cysteamine levels in mouse tissues have been strictly correlated to the presence of membrane-bound pantetheinase activity encoded by Vanin-1. Vanin-1 is involved in many biological processes in mouse, from thymus homing to sexual development. Vanin-1 -/- mice are fertile and grow and develop normally; they better control inflammation and most of the knockout effects were rescued by cystamine treatment. Gene structure analysis showed the presence of an oxidative stimuli-responsive ARE-like sequence in the promoter. In this paper we investigate antioxidant-detoxifying enzymatic activities at the tissue level, comparing Vanin-1 -/- and wild-type mice. In Vanin-1 null animals we pointed out a decrease in the Se-independent glutathione peroxidase activity. The decrease in enzymatic activity appeared to be correlated to an impairment of GST isoenzyme levels. In particular a significant drop in GSTA3 together with a minor decrement in GSTM1 and an increase in GSTP1 levels was detected in Vanin-1 -/- livers. Cystamine administration to Vanin-1 -/- mice restored specifically GSTA3 levels and the corresponding enzymatic activity without influencing protein expression. A possible role of cystamine on protein stability/folding can be postulated.     

Phosphorylation enhances mitochondrial targeting of GSTA4-4 through increased affinity for binding to cytoplasmic Hsp70

Recently we showed that three different isoforms of cytosolic glutathione S-transferases (GST), including GSTA4-4, are also localized in the mitochondrial compartment. In this study, we have investigated the mechanism of mouse GSTA4-4 targeting to mitochondria, using a combination of in vitro mitochondrial import assay and in vivo targeting in COS cells transfected with cDNA. Our results show that the mitochondrial GSTA4-4 is more heavily phosphorylated compared with its cytosolic counterpart. Protein kinase activators (cAMP, forskolin, or phorbol-12-myristate-13-acetate) markedly increased GSTA4-4 targeting to mitochondria, whereas kinase inhibitors caused its retention in the cytosol. Immunoinhibition and immunodepletion studies showed that the Hsp70 chaperone is required for the efficient translation of GSTA4-4 as well as its translocation to mitochondria. Co-immunoprecipitation studies showed that kinase inhibitors attenuate the affinity of GSTA4-4 for cytoplasmic Hsp70 suggesting the importance of phosphorylation for binding to the chaperone. Mutational analysis show that the putative mitochondrial targeting signal resides within the C-terminal 20 amino acid residues of the protein and that the targeting signal requires activation by phosphorylation at the C-terminal-most protein kinase A (PKA) site at Ser-189 or protein kinase C (PKC) site at Thr-193. We demonstrate for the first time that PKA and PKC modulate the cytoplasmic and mitochondrial pools of GSTA4-4.     

CYP2E1-mediated oxidative stress regulates HO-1 and GST expression in maneb- and paraquat-treated rat polymorphonuclear leukocytes

Cytochrome P4502E1 (CYP2E1), glutathione-S-transferase A4-4 (GSTA4-4), and inducible nitric oxide synthase (iNOS) are implicated in maneb- and paraquat-induced toxicity leading to various pathological conditions. The study aimed to investigate the role of CYP2E1 in maneb- and paraquat-induced oxidative stress in rat polymorphonuclear leukocytes (PMNs) and its crosstalk with iNOS-mediated nitrosative stress and GSTA4-4-linked protective effect, if any and their consequent links with the nuclear factor erythoid 2-related factor 2 (Nrf2) activation and heme oxygenase-1 (HO-1) expression. Rats were treated with/without maneb and/or paraquat for 1, 2, and 3 weeks along with vehicle controls. Subsets of rats were also treated with diallyl sulfide (DAS) or aminoguanidine (AG) along with the respective controls. Maneb and paraquat augmented the reactive oxygen species (ROS), lipid peroxidation (LPO) and 4-hydroxy nonenal (4-HNE) contents, and superoxide dismutase (SOD) activity in the PMNs. However, maneb and paraquat attenuated the reduced glutathione (GSH) level and the expression/activity of total GST and GST-pi. Maneb and paraquat increased the expression/activity of CYP2E1, GSTA4-4, iNOS, Nrf2 and HO-1, and nitrite content. CYP2E1 inhibitor, DAS noticeably alleviated maneb- and paraquat-induced ROS, LPO, 4-HNE, SOD, Nrf2 and HO-1, GST, GSH, and GST-pi while iNOS, nitrite content and GSTA4-4 levels were unchanged. Conversely, AG, an iNOS inhibitor, attenuated maneb- and paraquat-directed changes in nitrite, LPO, iNOS but it did not alter ROS, GSH, SOD, GST, GST-pi, Nrf2, HO-1, CYP2E1, and GSTA4-4. The results demonstrate that CYP2E1 induces iNOS-independent free radical generation and subsequently modulates the Nrf2-dependent HO-1 and 4-HNE-mediated GST expression in maneb- and paraquat-treated PMNs.     

Interindividual variation and organ-specific patterns of glutathione S-transferase alpha,mu, and pi expression in gastrointestinal tract mucosa of normal individuals

Glutathione S-transferase (GST) protein in gastrointestinal (GI) tracts of 16 organ donors, from whom all or substantial portions of the GI tract (stomach-colon) were available, was quantitated by HPLC and examined for interindividual variability/consistency of organ-specific patterns of expression. GSTP1, GSTA1, and GSTA2 were major components, and GSTM1 and GSTM3 were minor components. Consistent patterns of organ-specific expression were evident despite a high degree of interindividual variation of expression. GSTP1 was expressed throughout the GI tract and showed a decrease of expression from stomach to colon. GSTA1 and GSTA2 were expressed at high levels in duodenum and small intestine and expression decreased from proximal to distal small intestine. In contrast, GSTA1 and GSTA2 expression in colon and stomach of all subjects was low, particularly for colon where GSTA1 expression was 20- to 800-fold lower than that in corresponding small intestine. These consistent patterns of expression would suggest that compared to duodenum and small intestine, colon and to a lesser extent stomach always have low potential for GST-dependent detoxification of chemical carcinogens and are therefore at greater risk of genotoxic effects, particularly via substrates that are specific for GSTA1. This may be a factor in the greater susceptibility of stomach and colon to cancers compared to duodenum/small intestine.     

Molecular cloning and characterization of alpha-class glutathione S-transferase genes from the hepatopancreas of red sea bream, Pagrus major

Two distinct cDNAs corresponding to GSTA1 and GSTA2 genes encoding glutathione S-transferases (GSTs) from the hepatopancreas of red sea bream, Pagrus major were cloned and sequenced. A comparison of the nucleotide sequences of GSTA1 and GSTA2 revealed 98% identity and their derived amino acid sequences had 96% similarity. Both genes could be classified as alpha-class GSTs on the basis of their amino acid sequence identity with other species. Genomic DNA cloning showed that both GSTA1 and GSTA2 genes consisted of six exons and five introns. In a comparison of genomic DNAs, the structures of GSTA1 and GSTA2 differed. In addition, Southern-blot analysis indicated that at least two kinds of alpha-class GSTs existed in the P. major genome. In order to biochemically characterize the recombinant enzymes (pmGSTA1-1 and pmGSTA2-2), both clones were highly expressed in Escherichia coli. The purified pmGSTA1-1 and pmGSTA2-2 exhibited glutathione conjugating activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide, while neither pmGSTs show detectable activity toward 1,2-dichloro-4-nitrobenzene, ethacrynic acid, 4-hydroxynonenal, or p-nitrobenzyl chloride. Despite their high level of amino acid sequence identity, the pmGSTs had quite different enzyme-kinetic parameters.     

Maneb and paraquat-induced modulation of toxicant responsive genes in the rat liver: comparison with polymorphonuclear leukocytes

Experimental studies have shown that toxicant responsive genes, cytochrome P450s (CYPs) and glutathione S-transferases (GSTs) play a critical role in pesticide-induced toxicity. CYPs play pro-oxidant role and GSTs offer protection in maneb (MB) and paraquat (PQ)-induced brain and lung toxicities. The present study aimed to investigate the effect of repeated exposures of MB and/or PQ on lipid peroxidation (LPO), glutathione content (GSH) and toxicant responsive genes, i.e., CYP1A1, 1A2, 2E1, GSTA4-4, GSTA1-1 and GSTA3-3 in the liver and to correlate the same with polymorphonuclear leukocytes (PMNs). A significant augmentation in LPO and reduction in GSH content was observed in a time of exposure dependent manner in the liver and PMNs of MB and/or PQ treated animals. The expression and catalytic activity of CYP2E1 and GSTA4-4 were significantly increased following MB and/or PQ exposure both in the liver and PMNs. Although the expression of GSTA3-3 was increased, the expression of GSTA1-1 was unaltered after MB and/or PQ treatment in both the liver and PMNs. MB augmented the expression and catalytic activity of CYP1A1 in the liver, however, CYP1A2 was unaffected. PQ, on the other hand, significantly increased hepatic CYP1A2 expression and catalytic activity. MB and/or PQ did not produce any significant changes in CYP1A1 and CYP1A2 in PMNs. The results of the study thus demonstrate that MB and PQ differentially regulate hepatic CYP1A1 and CYP1A2 while LPO, GSH, CYP2E1, GSTA4-4 and GSTA3-3 are modulated in the similar fashions both in the liver and PMNs.     

High-affinity binding of fatty acyl-CoAs and peroxisome proliferator-CoA esters to glutathione S-transferases effect on enzymatic activity.

Acyl-CoAs are present at high concentrations within the cell, yet are strongly buffered by specific binding proteins in order to maintain a low intracellular unbound acyl-CoA concentration, compatible with their metabolic role, their importance in cell signaling, and as protection from their detergent properties. This intracellular regulation may be disrupted by nonmetabolizables acyl-CoA esters of xenobiotics, such as peroxisome proliferators, which are formed at relatively high concentration within the liver cell. The low molecular mass acyl-CoA binding protein (ACBP) and fatty acyl-CoA binding protein (FABP) have been proposed as the buffering system for fatty acyl-CoAs. Whether these proteins also bind xenobiotic-CoA is not known. Here we have identified new liver cytosolic fatty acyl-CoA and xenobiotic-CoA binding sites as glutathione S-transferase (GST), using fluorescent polarization and a acyl-etheno-CoA derivative of the peroxisome proliferator nafenopin as ligand. Rat liver GST and human liver recombinant GSTA1-1, GSTP1-1 and GSTM1-1 were used. Only class alpha rat liver GST and human GSTA1-1 bind xenobiotic-CoAs and fatty acyl-CoAs, with Kd values ranging from 200 nM to 5 microM. One mol of acyl-CoA is bound per mol of dimeric enzyme, and no metabolization or hydrolysis was observed. Binding results in strong inhibition of rat liver GST and human recombinant GSTA1-1 (IC50 at the nanomolar level for palmitoyl-CoA) but not GSTP1-1 and GSTM1-1. Acyl-CoAs do not interact with the GSTA1-1 substrate binding site, but probably with a different domain. Results suggest that under increased acyl-CoA concentration, as occurs after exposure to peroxisome proliferators, acyl-CoA binding to the abundant class alpha GSTs may result in strong inhibition of xenobiotic detoxification. Analysis of the binding properties of GSTs and other acyl-CoA binding proteins suggest that under increased acyl-CoA concentration GSTs would be responsible for xenobiotic-CoA binding whereas ACBP would preferentially bind fatty acyl-CoAs.     

Activation or detoxification of mutagenic and carcinogenic compounds in transgenic Drosophila expressing human glutathione S-transferase.

Sensitivity of transgenic Drosophila melanogaster with expression of a human gene encoding the glutathione S-transferase alpha subunit (GSTA1-1) to 1,2:5,6-dibenzanthracene (DBA) and 1,2-dichloroethane (DCE) was investigated in the somatic mutation and recombination test (SMART). We performed the same assay in control transgenic flies expressing the bacterial lacZ gene. Three types of transgenic Drosophila strains carrying GSTA1-1 were used: two transgenic strains homozygous for the second chromosome with a single-copy transgene insertion and one strain with two transgene insertions. Larvae carrying the lacZ gene were significantly more sensitive to genotoxic effects of DBA than those carrying three copies of the GSTA1-1 gene. The larvae with lacZ expression showed significantly lower sensitivity to DCE compared with those expressing GSTA1-1. Finally, a pretreatment with buthionine-sulphoximine (BSO) in experiment with DCE significantly decreased the frequency of mutation events in larvae with three GSTA1-1 copies in comparison with others.     

Role of multidrug resistance protein 1 (MRP1) and glutathione S-transferase A1-1 in alkylating agent resistance. Kinetics of glutathione conjugate formation and efflux govern differential cellular sensitivity to chlorambucil versus melphalan toxicity

We investigated the role of phase II (conjugation) and phase III (efflux) detoxification of the anticancer drugs melphalan (MLP) and chlorambucil (CHB). Although both drugs are substrates of Alpha-class glutathione S-transferases (GST) and the monoglutathionyl conjugates formed in these enzymatic reactions are transported by MRP1, we found that GSTA1-1 and MRP1 acted in synergy to confer resistance to CHB but not to MLP (Morrow, C. S., Smitherman, P. K., Diah, S. K., Schneider, E., and Townsend, A. J. (1998) J. Biol. Chem. 273, 20114-20120). To explain this selectivity of MRP1/GST-mediated resistance, we report results of side-by-side experiments comparing the kinetics of MLP- versus CHB-glutathione conjugate: formation, product inhibition of GSTA1-1 catalysis, and transport by MRP1. The monoglutathionyl conjugate of CHB, CHB-SG, is a very strong competitive inhibitor of GSTA1-1 (K(i) 0.14 microM) that is >30-fold more potent than that of the corresponding conjugate of MLP, MLP-SG (K(i) 4.7 microM). The efficiency of GSTA1-1-mediated monoglutathionyl conjugate formation is more than 4-fold higher for CHB than MLP. Lastly, both CHB-SG and MLP-SG are efficiently transported by MRP1 with similar V(max) although the K(m) for CHB-SG (0.37 microm) is significantly lower than for MLP-SG (1.1 microM). These results indicate that MRP1 is required for GSTA1-1-mediated resistance to CHB in order to relieve potent product inhibition of the enzyme by intracellular CHB-SG formed. The kinetic properties of MRP1 are well suited to eliminate CHB-SG at pharmacologically relevant concentrations. For MLP detoxification, where product inhibition of GSTA1-1 is less important, GSTA1-1 does not confer resistance because of the relatively poorer catalytic efficiency of MLP-SG formation. Similar analyses can be useful for predicting the pharmacological and toxicological consequences of MRP and GST expression on cellular sensitivity to various other electrophilic xenobiotics.     

Alpha-Class Glutathione S-Transferases in Wild Turkeys (Meleagris gallopavo): Characterization and Role in Resistance to the Carcinogenic Mycotoxin Aflatoxin B1

Domestic turkeys (Meleagris gallopavo) are one of the most susceptible animals known to the toxic effects of the mycotoxin aflatoxin B₁ (AFB₁), a potent human hepatocarcinogen, and universal maize contaminant. We have demonstrated that such susceptibility is associated with the inability of hepatic glutathione S-transferases (GSTs) to detoxify the reactive electrophilic metabolite exo-AFB₁-8,9-epoxide (AFBO). Unlike their domestic counterparts, wild turkeys, which are relatively AFB₁-resistant, possess hepatic GST-mediated AFBO conjugating activity_. Here, we characterized the molecular and functional properties of hepatic alpha-class GSTs (GSTAs) from wild and domestic turkeys to shed light on the differences in resistance between these closely related strains. Six alpha-class GST genes (GSTA) amplified from wild turkeys (Eastern and Rio Grande subspecies), heritage breed turkeys (Royal Palm) and modern domestic (Nicholas strain) turkeys were sequenced, and catalytic activities of heterologously-expressed recombinant enzymes determined. Alpha-class identity was affirmed by conserved GST domains and four signature motifs. All GSTAs contained single nucleotide polymorphisms (SNPs) in their coding regions: GSTA1.1 (5 SNPs), GSTA1.2 (7), GSTA1.3 (3), GSTA2 (3), GSTA3 (1) and GSTA4 (2). E. coli-expressed GSTAs possessed varying activities toward GST substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA), cumene hydroperoxide (CHP). As predicted by their relative resistance, livers from domestic turkeys lacked detectable GST-mediated AFBO detoxification activity, whereas those from wild and heritage birds possessed this critical activity, suggesting that intensive breeding and selection resulted in loss of AFB₁-protective alleles during domestication. Our observation that recombinant tGSTAs detoxify AFBO, whereas their hepatic forms do not, implies that the hepatic forms of these enzymes are down-regulated, silenced, or otherwise modified by one or more mechanisms. These data may inform of possible molecular mechanisms of resistance to AFB₁, and may also have the benefit of identifying genetic markers which could be used to enhance AFB₁ resistance in modern domestic strains.     

Impact of the genes UGT1A1, GSTT1, GSTM1, GSTA1, GSTP1 and NAT2 on acute alcohol-toxic hepatitis

Alcohol metabolism causes cellular damage by changing the redox status of cells. In this study, we investigated the relationship between genetic markers in genes coding for enzymes involved in cellular redox stabilization and their potential role in the clinical outcome of acute alcohol-induced hepatitis. Study subjects comprised 60 patients with acute alcohol-induced hepatitis. The control group consisted of 122 healthy non-related individuals. Eight genetic markers of the genes UGT1A1, GSTA1, GSTP1, NAT2, GSTT1 and GSTM1 were genotyped. GSTT1 null genotype was identified as a risk allele for alcohol-toxic hepatitis progression (OR 2.146, P=0.013). It was also found to correlate negatively with the level of prothrombin (β= ?11.05, P=0.037) and positively with hyaluronic acid (β=170.4, P=0.014). NAT2 gene alleles rs1799929 and rs1799930 showed opposing associations with the activity of the biochemical markers γ-glutamyltransferase and alkaline phosphatase; rs1799929 was negatively correlated with γ-glutamyltransferase (β=?261.3, P=0.018) and alkaline phosphatase (β= ?270.5, P=0.032), whereas rs1799930 was positively correlated with Γ-glutamyltransferase (β=325.8, P=0.011) and alkaline phosphatase (β=374.8, P=0.011). Enzymes of the glutathione S-transferase family and NAT2 enzyme play an important role in the detoxification process in the liver and demonstrate an impact on the clinical outcome of acute alcohol-induced hepatitis.     

Functional promiscuity correlates with conformational heterogeneity in A-class glutathione S-transferases

The structurally related glutathione S-transferase isoforms GSTA1-1 and GSTA4-4 differ greatly in their relative catalytic promiscuity. GSTA1-1 is a highly promiscuous detoxification enzyme. In contrast, GSTA4-4 exhibits selectivity for congeners of the lipid peroxidation product 4-hydroxynonenal. The contribution of protein dynamics to promiscuity has not been studied. Therefore, hydrogen/deuterium exchange mass spectrometry (H/DX) and fluorescence lifetime distribution analysis were performed with glutathione S-transferases A1-1 and A4-4. Differences in local dynamics of the C-terminal helix were evident as expected on the basis of previous studies. However, H/DX demonstrated significantly greater solvent accessibility throughout most of the GSTA1-1 sequence compared with GSTA4-4. A Phe-111/Tyr-217 aromatic-aromatic interaction in A4-4, which is not present in A1-1, was hypothesized to increase core packing. "Swap" mutants that eliminate this interaction from A4-4 or incorporate it into A1-1 yield H/DX behavior that is intermediate between the wild type templates. In addition, the single Trp-21 residue of each isoform was exploited to probe the conformational heterogeneity at the intrasubunit domain-domain interface. Excited state fluorescence lifetime distribution analysis indicates that this core residue is more conformationally heterogeneous in GSTA1-1 than in GSTA4-4, and this correlates with greater stability toward urea denaturation for GSTA4-4. The fluorescence distribution and urea sensitivity of the mutant proteins were intermediate between the wild type templates. The results suggest that the differences in protein dynamics of these homologs are global. The results suggest also the possible importance of extensive conformational plasticity to achieve high levels of functional promiscuity, possibly at the cost of stability.