GSTA1 Research Articles

Comparative study of active and inactive hepatocarcinogens using a QSAR-based prediction model

In this study, we investigated global gene expression in primary rat hepatocytes treated with three active hepatocarcinogens (prednisolone, dehydroepiandrosterone and monocrotaline), three inactive hepatocarcinogens (hydrocortisone, glycyrrhetinic acid and lithocholic acid) and two unclassified chemicals (griseofulvin (possibly active) and prednisone (possibly inactive)). At 48 h after treatment with these eight chemicals, cells were harvested for RNA extraction. Global gene expression analyses were conducted using oligonucleotide microarrays to detect genes whose expression was altered. Differentially expressed genes (DEG) analysis, principal component analysis (PCA), gene set enrichment analysis (GSEA) and KEGG pathway analysis were also conducted. Seven-hundred and sixty genes whose expression was altered >1.2-fold (P<0.05; unpaired Welch’s t-test) were identified as DEGs for the active and inactive carcinogens. In PCA, prednisolone and dehydroepiandrosterone were located away from the inactive carcinogens. In contrast, monocrotaline was close to the inactive carcinogens. Hydrocortisone, glycyrrhetinic acid and lithocholic acid were separate from the active carcinogens. PCA identified griseofulvin as an active carcinogen and prednisone as an inactive carcinogen. GSEA detected several genes associated with hepatocarcinogenesis, such as glutathione S-transferase A2 and NADPH oxidase. KEGG pathway analysis showed that several pathways might be associated with hepatocarcinogenesis. Our results suggest that it may be feasible to differentiate active and inactive hepatocarcinogens by PCA, GSEA and KEGG pathway analysis.

GSTO2*N142D gene polymorphism associated with hypothyroidism in Italian patients

Hypothyroidism is a multifactorial endocrinal disease characterized by abnormally low thyroid hormone production. Thyroiditis is one of the primary causes of hypothyroidism, as it is an increasing level of inflammation in the thyroid gland that could be due to a failure of the anti-inflammatory response. Glutathione S-transferases are biomarkers of inflammation and oxidative stress. These phase II enzymes play a relevant role in detoxifying xenobiotic compounds. Particular attention has been focused on GSTA1, GSTM1, GSTO2, GSTP1, and GSTT1 genes to evaluate if GST gene polymorphisms are associated with hypothyroidism. We screened a case-control population (patients with hypothyroidism n=110, controls n=122) to analyze GST gene polymorphisms. GST SNPs were determined using the PCR-RFLP method, while GST null polymorphisms were determined using a Multiplex PCR. In this study, we found differences in genotype distribution between hypothyroid individuals and controls only for the GSTO2*N142D polymorphism. Logistic regression analysis, after adjustment for age and sex, confirmed this positive association (OR=4.56; 95% CI 1.22-17.00; p=0.009). The GSTO2 enzyme can catalyze several reactions important for countering oxidative stress: subjects with the D142 allele may have a deficiency in the antioxidant enzymatic system. A decrease in antioxidant capacity may trigger increased oxidative stress. Previous studies have highlighted the role of GST enzymes in inflammation disorders, but no data are available on their role in hypothyroidism. Our results suggest that GSTO2 could increase disease risk susceptibility and could act as a risk factor for hypothyroidism in Italian patients.

Glutathione S‐Transferase M1 Gene Polymorphism Is Associated with Susceptibility to Impaired Long‐term Allograft Outcomes in Renal Transplant Recipients

Despite improved post-transplantation care, progress in long-term kidney allograft survival of diabetic renal transplant recipients (pre-DM RTR) is worse than that of non-diabetic recipients (non-DM). We hypothesized that there are other potential risk factors, that predispose RTR to adverse renal allograft outcomes. A total of 323 transplant recipients who underwent renal transplantation between March 2000 and January 2008 were recruited. The composite end-point consisted of serum creatinine (SCr) doubling, graft failure, and death. Baseline clinical data were recorded, and polymerase chain reaction-restriction fragment length polymorphism measurements of interleukin (IL)-4, IL-10, IL-23, glutathione S-transferase (GST)A1, GSTM1, and GSTP1 polymorphisms were determined. The risk factors for developing the primary outcome were analyzed among these clinical and genetic factors. Within a mean follow-up of 71.1 ± 24 months, there were 43 (13.3 %) patients with the primary outcome. Stepwise multivariate Cox regression analysis was used to determine the risk factors for the primary outcome of RTR. Renal transplant recipients who possessed the GSTM1 null genotype had a 2.2-fold risk (95 % CI: 1.10-4.40; P = 0.026) of developing the primary outcome. Additionally, RTR that had DM before transplantation (aHR: 3.31; 95 % CI: 1.77-6.20; P = 0.0002) or changes in SCr 6 to 12 months after transplantation (aHR: 2.83; 95 % CI: 1.29-6.19; P = 0.0095) had an increased risk of developing the primary outcome. In addition to the adverse role played by DM, the GSTM1 null genotype also has an unfavorable influence on the long-term allograft outcome of RTR.

Genetic variability of glutathione S-transferase enzymes in human populations: Functional inter-ethnic differences in detoxification systems

Glutathione S-Transferase enzymes (GSTs) constitute the principal Phase II superfamily which plays a key role in cellular detoxification and in other biological processes. Studies of GSTs have revealed that genetic polymorphisms are present in these enzymes and that some of these are Loss-of-Function (LoF) variants, which affect enzymatic functions and are related to different aspects of human health.The aim of this study was to analyze functional genetic differences in GST enzymes among human populations. Attention was focused on LoF polymorphisms of GSTA1, GSTM1, GSTO1, GSTO2, GSTP1 and GSTT1 genes. These LoF variants were analyzed in 668 individuals belonging to six human groups with different ethnic backgrounds: Amhara and Oromo from Ethiopia; Colorado and Cayapa Amerindians and African Ecuadorians from Ecuador; and one sample from central Italy. The HapMap database was used to compare our data with reference populations and to analyze the haplotype and Linkage Disequilibrium diversity in different ethnic groups.Our results highlighted that ethnicity strongly affects the genetic variability of GST enzymes. In particular, GST haplotypes/variants with functional impact showed significant differences in human populations, according to their ethnic background. These data underline that human populations have different structures in detoxification genes, suggesting that these ethnic differences influence disease risk or response to drugs and therefore have implications for genetic association studies involving GST enzymes.In conclusion, our investigation provides data about the distribution of important LoF variants in GST genes in human populations. This information may be useful for designing and interpreting genetic association studies.

Glutathione S-transferase A1, M1, P1 and T1 null or low-activity genotypes are associated with enhanced oxidative damage among haemodialysis patients

Increased oxidative stress is a hallmark of end-stage renal disease (ESRD). Glutathione S-transferases (GST) are involved in the detoxification of xenobiotics and protection of oxidative damage. We hypothesized that genetic polymorphism in antioxidant enzymes GSTA1, GSTM1, GSTP1 and GSTT1 is more frequent in ESRD and modulates the degree of oxidative stress in these patients. GSTA1, GSTM1, GSTP1 and GSTT1 genotypes were determined in 199 ESRD patients and 199 age- and gender-matched controls. Markers of protein and lipid oxidative damage [thiol groups, carbonyl groups, advanced oxidative protein products, nitrotyrosine, malondialdehyde (MDA) and MDA adducts], together with total oxidant status and pro-oxidant-antioxidant balance were determined. Individual GST polymorphisms influence vulnerability to both protein and lipid oxidation, with GSTM1-null gene variant having the most pronounced effect. Furthermore, a strong combined effect of null/low-activity GSTM1, GSTT1, GSTA1 and GSTP1 genotypes in terms of susceptibility towards oxidative and carbonyl stress was found in ESRD patients. When patients were stratified according to GSTM1 and GSTT1, the highest oxidant damage was noted in those with the GSTM1-null/GSTT1-null genotype. The observed effect was even stronger in patients with the third low-activity GSTP1 or GSTA1 genotype. Finally, the level of oxidative and carbonyl stress was most pronounced in the subgroup of patients with all four null or low-activity GSTM1, GSTT1, GSTP1 and GSTA1 genotypes. According to the GST genotype, ESRD patients may be stratified in terms of the level of oxidative and carbonyl stress that might influence cardiovascular prognosis, but could also improve efforts towards individualization of antioxidant treatment.

Naturally Occurring Variation in the Glutathione-S-Transferase 4 Gene Determines Neurodegeneration After Traumatic Brain Injury

Genetic factors are important for outcome after traumatic brain injury (TBI), although exact knowledge of relevant genes/pathways is still lacking. We here used an unbiased approach to define differentially activated pathways between the inbred DA and PVG rat strains. The results prompted us to study further if a naturally occurring genetic variation in glutathione-S-transferase alpha 4 (Gsta4) affects the outcome after TBI. Survival of neurons after experimental TBI is increased in PVG compared to the DA strain. Global expression profiling analysis shows the glutathione metabolism pathway to be the most regulated between the strains, with increased Gsta4 in PVG among top regulated transcripts. A congenic strain (R5) with a PVG genomic insert containing the Gsta4 gene on DA background displays a reversal of the strain pattern for Gsta4 expression and increased survival of neurons compared to DA. Gsta4 is known to effectively reduce 4-hydroxynonenal (4-HNE), a noxious by-product of lipid peroxidation. Immunostaining of 4-HNE was evident in both rat and human TBI. Intracerebral injection of 4-HNE resulted in neurodegeneration with increased levels of a marker for nerve injury in cerebrospinal fluid of DA compared to R5. These findings provide strong support for the notion that the inherent capability of coping with increased 4-HNE after TBI affects outcome in terms of nerve cell loss. A naturally occurring variation in Gsta4 expression in rats affects neurodegeneration after TBI. Further studies are needed to explore if genetic variability in Gsta4 can be associated to outcome also in human TBI.

The effect of menadione on glutathione S-transferase A1 (GSTA1): c-Jun N-terminal kinase (JNK) complex dissociation in human colonic adenocarcinoma Caco-2 cells

Glutathione S-transferases (GSTs) act as modulators of mitogen-activated protein kinase signal transduction pathways via a mechanism involving protein–protein interactions. We have demonstrated that GSTA1 forms complexes with JNK and modifies JNK activation during cellular stress, but the factors that influence complex association and dissociation are unknown. We hypothesized that menadione causes dissociation of GSTA1–JNK complexes, activates JNK, and the consequences of menadione exposure depend on GSTA1 expression. We demonstrate that menadione causes GSTA1–JNK dissociation and JNK activation in preconfluent Caco-2 cells, whereas postconfluent cells are resistant to this effect. Moreover, preconfluent cells are more sensitive than postconfluent cells to menadione-induced cytotoxicity. Activation of JNK is transient since removal of menadione causes GSTA1 to re-associate with JNK reducing cytotoxicity. Over-expression and knockdown of GSTA1 did not alter JNK activation by menadione or sensitivity to menadione-induced cytotoxicity. These results indicate that GSTA1–JNK complex integrity does not affect the ability of menadione to activate JNK. N-acetyl cysteine prevents GSH depletion and blocks menadione-induced complex dissociation, JNK activation and inhibits menadione-induced cytotoxicity. JNK activation and inhibits menadione-induced cytotoxicity. The data suggest that the mechanism of menadione-induced JNK activation involves the production of reactive oxygen species, likely superoxide anion, and intracellular GSH levels play an important role in preventing GSTA1–JNK complex dissociation, subsequent JNK activation and induction of cytotoxicity.

Glutathione S-transferase alpha 1 risk polymorphism and increased bilateral thalamus mean diffusivity in schizophrenia

Oxidative damage in brain cells is one of the factors hypothesized to be involved in the pathogenesis of schizophrenia. Glutathione S-transferase (GST) A1*B polymorphism, a genotype associated with a higher risk of oxidative damage, is associated with increased frequency of schizophrenia diagnosis. Thus, here we studied Glutathione S-transferase (GST) A1 polymorphism and diffusion tensor imaging-mean diffusivity (MD) data on deep grey matter brain structures in 56 patients with Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revised (DSM-IV-TR) schizophrenia. Clinical diagnosis and psychopathological symptom severity were assessed by using the Structured Clinical Interview for DSM-IV-TR (SCID-P) and the Scales for Assessment of Positive and Negative Symptoms (SAPS and SANS). Results confirmed that patients with schizophrenia who were carriers of the GSTA1 *B risk allele had an increased MD in bilateral thalami and increased severity of auditory and global hallucinations in comparison with non-B carriers. Thus, oxidative stress associated factors may be implicated in specific mechanisms of schizophrenia such as altered microstructure of the thalami and specific psychopathological features of auditory hallucinations.

Synthesis and Study of 2-(Pyrrolesulfonylmethyl)-N-arylimines: A New Class of Inhibitors for Human Glutathione Transferase A1-1

Overexpression of human GSTA1-1 in tumor cells is part of MDR mechanisms. We report on the synthesis of 11 pyrrole derivatives as hGSTA1-1 inhibitors starting from 1-methyl-2-[(2-nitrobenzylsulfanyl]-1H-pyrrole. Molecular modeling revealed two locations in the enzyme H binding site: the catalytic primary one accommodating shorter and longer derivatives and the secondary one, where shorter derivatives can occupy. Derivative 9, displaying the highest inhibition and bearing a p-nitroarylimino moiety, and derivative 4, lacking this moiety, were studied kinetically. Derivative 9 binds (K(i(9)) = 71 ± 4 μM) at the primary site competitively vs CDNB. Derivative 4 binds (K(i(4)) = 135 ± 27 μM) at the primary and secondary sites, allowing the binding of a second molecule (4 or CDNB) leading to formation of unreactive and reactive complexes, respectively. The arylmethylsulfonylpyrrole core structure is a new pharmacophore for hGSTA1-1, whereas its derivative 9 may serve as a lead structure.

Glutathione S-transferase P1 c.313A > G polymorphism could be useful in the prediction of doxorubicin response in breast cancer patients

Identification of predicting factors for anthracyclines-based chemotherapy remains a clinical challenge. Glutathione S-transferase (GSTs) enzymes detoxify chemotherapy drugs and their metabolites. Several polymorphisms in GST genes result in reduced or no activity of the enzymes. Specifically, GSTM1 and GSTT1 genes are polymorphically deleted, the polymorphism GSTP1 c.313A>G (rs1695) determines the amino acid substitution Ile105Val, where the Val-containing enzyme has reduced activity. Also, GSTA1*B allele has reduced levels of GSTA1 enzyme. Several polymorphisms in GSTs have been associated with differences in survival for cancer patients treated with chemotherapy. We genotyped a total of five polymorphisms in GSTM1, GSTT1, GSTP1 and GSTA1 genes in 159 patients with locally advanced breast cancer, treated with single-agent doxorubicin or docetaxel (Taxotere). Gene expression microarrays were performed in 67 breast tumor samples. We correlate this data with treatment outcome. In multivariate analysis, patients homozygous GG for GSTP1 c.313A>G SNP had a lower risk of chemoresistance when treated with doxorubicin (odds ratio 0.106; confidence interval 0.012-0.898; P=0.040). No association was found in the docetaxel arm. Also, we found that GSTP1 expression varied significantly among breast cancer molecular subtypes. GSTP1 may constitute another tool contributing to individualized anthracycline-based therapy.

Chemoprotective activity of the isoflavones, genistein and daidzein on mutagenicity induced by direct and indirect mutagens in cultured HTC cells

Isoflavones are phenolic compounds widely distributed in plants and found in a high percentage in soybeans. They have important biological properties and are regarded as potential chemopreventive agents. The aim of this study was to verify the preventive effect of two soy isoflavones (genistein and daidzein) by a micronucleus assay, analysis of GST activity, and real-time RT-PCR analysis of GSTa2 gene expression. Mutagens of direct (doxorubicin) and indirect (2-aminoanthracene) DNA damage were used. Hepatoma cells (HTC) were treated with genistein or daidzein for 26h at noncytotoxic concentrations; 10μM when alone, and 0.1, 1.0 and 10μM when combined with genotoxic agents. The micronucleus test demonstrated that both isoflavones alone had no genotoxic effect. Genistein showed antimutagenic effects at 10μM with both direct and indirect DNA damage agents. On phase II enzyme regulation, the current study indicated an increase in total cytoplasmic GST activity in response to genistein and daidzein at 10μM supplementation. However, the mRNA levels of GSTa2 isozymes were not differentially modulated by genistein or daidzein. The results point to an in vitro antimutagenic activity of genistein against direct and indirect DNA damage-induced mutagenicity.

Glutathione S-transferase genes and the risk of recurrent miscarriage in Italian women

To investigate the role of glutathione S-transferases (GSTs) in the pathogenesis of recurrent miscarriage (RM). Genetic association study. University of Rome, Tor Vergata and San Giovanni Calibita, Fatebenefratelli Hospital. One hundred twenty-one women with RM and 113 women without pregnancy complications. Genomic DNA extracted from buccal cells and screening of positive/null genotypes of GSTM1 and GSTT1 genes and single nucleotide polymorphisms of GSTA1, GSTO2, and GSTP1 genes. Occurrence of GST polymorphisms. Women with at least one GSTA1*-69T allele are more frequent in the RM group than in the control group: 67% vs. 48%, respectively. Significant outcomes were obtained considering different genetic models: codominant, dominant, and log-additive. In addition, the combined analysis suggests that GSTA1 and GSTM1 variants have a significant interaction in RM risk. Our study highlighted a significant association between the GSTA1 gene and an increased risk of RM. In particular, the -69T allele in the GSTA1 gene may be considered as a predisposing factor of RM.

Effect of phytochemicals on phase II enzyme expression in infant human primary skin fibroblast cells

Phase II metabolising enzymes enable the metabolism and excretion of potentially harmful substances in adults, but to date it is unclear whether dietary phytochemicals can induce phase II enzymes differently between adults and infants. We investigated the expression of phase II enzymes in an in vitro model of primary skin fibroblasts at three different developmental stages, 1month, 2 years and adult, to examine potential differences in age-related phase II enzymes in response to different phytochemicals (5-20μm) including sulphoraphane, quercetin and catechin. Following phytochemical treatment, a significant increase in mRNA of glutathione S-transferase A1 (GSTA1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) was observed, with the most marked increases seen in response to sulphoraphane (3-10-fold for GSTA1, P=0·001, and 6-35-fold for NQO1, P=0·001-0·017). Catechin also induced 3-5-fold changes in NQO1 transcription, whereas quercetin had less effect on NQO1 mRNA induction in infant cells. Moreover, NQO1 protein levels were significantly increased in 2-year-old and adult cell models in response to sulphoraphane treatment. These results suggest that metabolic plasticity and response to xenobiotics may be different in infants and adults; and therefore the inclusion of phytochemicals in the infant diet may modulate their induction of phase II metabolism, thereby providing increased protection from potentially harmful xenobiotics in later life.

Nuclear factor erythroid 2-like 2 (Nrf2) expression in end-stage liver disease

The transcription factor Nrf2, encoded by NFE2L2 gene is a key regulator of cellular defense against oxidative and electrophilic stress, also governing the expression of many phase II detoxification enzymes. Nrf2 is negatively regulated by KEAP1 protein. Recent studies have shown that Nrf2 might also constitute an important mediator of inflammatory processes. In the current study the expression of Nrf2 in livers from patients with end-stage liver disease has been investigated. Surgical specimens were obtained from explanted livers of 24 patients with end-stage liver disease of different etiology. Control samples were obtained from nontumoral liver tissue from 6 patients with metastatic liver tumors. Nrf2 expression was evaluated by means of qRT-PCR, Western-blot and immunohistochemical staining. KEAP1 gene expression was investigated at mRNA level. The expression of the NFE2L2 gene was decreased in all groups of end-stage liver disease samples as compared with the controls (mean 0.470±1.20 of the value observed in the control samples, p=0.003). Decreased values of NFE2L2/KEAP1 mRNA ratio were also observed in end-stage liver disease groups (0.60±0.24 of the value observed in the control samples, p=0.019). The results were generally confirmed in Western-blot and immunohistochemical analysis of Nrf2 protein. Different expression pattern of Nrf2 regulated genes in end-stage liver disease samples were observed: glutamate-cysteine ligase (GCLC) and glutathione-S-transferase A1 (GSTA1) were significantly down-regulated in most liver disease groups, whereas heme oxidase 1 (HMOX1) and NAD(P)H dehydrogenase [quinone] 1 (NQO1) were not significantly suppressed. Treatment of HepG2 cells with pro-inflammatory cytokines resulted in significant decrease of GSTA1, NFE2L2 and GCLC expression, while the exposure had no significant influence on KEAP1, HMOX1, and NQO1 mRNA levels. Nrf2 deficiency may be one of the factors underlying impaired liver function in detoxification processes. It remains to be established in further studies if the observed decrease of Nrf2 expression is just a result of liver cirrhosis or is primary, playing a role in disease pathogenesis.

GSTM1 null genotype as risk factor for late-onset Alzheimer's disease in Italian patients

Alzheimer's disease (AD) is the most common form of dementia in the elderly. The causes of AD are very complex but there is general agreement about the existence of a link between Alzheimer's disease and oxidative stress. The Glutathione S-transferases (GSTs) act to detoxify products of oxidation that cause damage to macromolecules. Particular attention has been focused on GST genes because polymorphisms are important determinants of disease risk.To evaluate if GSTA1, GSTM1, GSTP1, and GSTT1 genes are associated with LOAD we screened a case–control population (n=311).Differences in genotype distributions between AD patients and controls were found only for the GSTM1 null genotype (P<0.001). In addition, a logistic regression analysis also conferred a positive association between the GSTM1 null genotype and LOAD after adjustment for age and gender (OR=2.09; 95%CI=1.31–3.35).The GSTM1 enzyme detoxifies substances such as exogenous and endogenous metabolites and plays a regulatory role in cellular signaling. Previous studies have highlighted that GSTM1 has a role in neurodegenerative disorders, but no data have associated the GSTM1 gene with AD risk. Our outcome suggests that the GSTM1 null genotype is a risk factor for AD in Italian patients.

Polymorphisms of glutathione S-transferases M1, T1, P1 and A1 genes in the Tunisian population: An intra and interethnic comparative approach

Genetic polymorphisms in glutathione S-transferases (GSTs) genes might influence the detoxification activities of the enzymes predisposing individuals to cancer risk. Owing to the presence of these genetic variants, inter-individual and ethnic differences in GSTs detoxification capacity have been observed in various populations. Therefore, the present study was performed to determine the prevalence GSTM1*0/*0, GSTT1*0/*0, GSTP1 Ile105Val, and GSTA1*A/*B polymorphisms in 154 healthy individuals from South Tunisia, and to compare them with those observed in North and Centre Tunisian populations and other ethnic groups. GSTM1 and GSTT1 polymorphisms were analyzed by a Multiplex-PCR approach, whereas GSTP1 and GSTA1 polymorphisms were examined by PCR-RFLP. The frequencies of GSTM1*0/*0 and GSTT1*0/*0 genotypes were 53.9% and 27.9%, respectively. The genotype distribution of GSTP1 was 47.4% (Ile/Ile), 40.9% (Ile/Val), and 11.7% (Val/Val). For GSTA1, the genotype distribution was 24.7% (*A/*A), 53.9% (*A/*B), and 21.4% (*B/*B). The combined genotypes distribution of GSTM1, GSTT1, GSTP1 and GSTA1 polymorphisms showed that thirty one of the 36 possible genotypes were present in our population; eight of them have a frequency greater than 5%. To the best of our knowledge, this is the first report of GSTs polymorphisms in South Tunisian population. Our findings demonstrate the impact of ethnicity and reveal a characteristic pattern for Tunisian population. The molecular studies in these enzymes provide basis for further epidemiological investigations in the population where these functional polymorphisms alter therapeutic response and act as susceptibility markers for various clinical conditions.

Computational Evidence for the Catalytic Mechanism of Human Glutathione S-Transferase A3-3: A QM/MM Investigation

A Quantum Mechanics/Molecular Mechanics (QM/MM) computational investigation of the catalytic mechanism of the human glutathione transferase A3-3 (hGSTA3-3) has been carried out. The results demonstrate that the isomerization reaction is concerted, but highly asynchronous: in the first reaction phase the glutathione (GSH) negative sulfur (thiolate) acts as a base and deprotonates carbon C4 of the substrate Delta(5)-androstene-3,17-dione (Delta(5)-AD); in the second reaction phase the hydroxyl proton of the tyrosine fragment Y9 is AD). The initial state of the enzyme is subsequently restored by transferred to C6 affording the Delta(4)-androstene-3,17-dione product (Delta(4)-transferring a proton from the GSH sulfur to the tyrosine negative oxygen. There is no evidence for a genuine stepwise mechanism involving the formation of a real dienolate intermediate as suggested in previous papers. Furthermore, our computations have evidenced that, when we consider the whole process (including the restoring of the enzyme), GSH behaves as a base/acid catalyst (as hypothesized by some authors), but it requires the participation of the tyrosine Y9 acting as a proton shuttle. A fingerprint has been used to rank the electrostatic effects on the catalysis of the various residues surrounding the active site. This analysis highlights the role played by the arginine residue R15 in stabilizing the initial complex in agreement with previous suggestions based on-crystal structures.

Camel Milk Modulates the Expression of Aryl Hydrocarbon Receptor-Regulated Genes,Cyp1a1, Nqo1, andGsta1, in Murine hepatoma Hepa 1c1c7 Cells

There is a traditional belief in the Middle East that camel milk may aid in prevention and treatment of numerous cases of cancer yet, the exact mechanism was not investigated. Therefore, we examined the ability of camel milk to modulate the expression of a well-known cancer-activating gene, Cytochrome P450 1a1 (Cyp1a1), and cancer-protective genes, NAD(P)H:quinone oxidoreductase 1 (Nqo1) and glutathione S-transferase a1 (Gsta1), in murine hepatoma Hepa 1c1c7 cell line. Our results showed that camel milk significantly inhibited the induction of Cyp1a1 gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent Cyp1a1 inducer and known carcinogenic chemical, at mRNA, protein, and activity levels in a concentration-dependent manner. In addition, camel milk significantly decreased the xenobiotic responsive element (XRE)-dependent luciferase activity, suggesting a transcriptional mechanism is involved. Furthermore, this inhibitory effect of camel milk was associated with a proportional increase in heme oxygenase 1. On the other hand, camel milk significantly induced Nqo1 and Gsta1 mRNA expression level in a concentration-dependent fashion. The RNA synthesis inhibitor, actinomycin D, completely blocked the induction of Nqo1 mRNA by camel milk suggesting the requirement of de novo RNA synthesis through a transcriptional mechanism. In conclusion, camel milk modulates the expression of Cyp1a1, Nqo1, and Gsta1 at the transcriptional and posttranscriptional levels.

Expression of NRF2 and NRF2-modulated genes in peripheral blood leukocytes of bladder cancer males

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is an oxidant-responsive transcription factor involved in induction of antioxidant genes. We assessed NRF2 and selected NRF2-modulated gene expression: glutathione S-transferase A1 and P1 (GSTA1 and GSTP1), mitochondrial superoxide dismutase (SOD2) in blood leukocytes of 51 bladder cancer patients and 90 control males. A significant up-regulation of SOD2 expression (P=0.002) was observed in leukocytes of patients. NRF2 expression was positively correlated with GSTP1 and with SOD2 mRNA level, both in patients and controls. These data suggest disturbances in SOD2 transcription in circulating blood leukocytes of males with bladder cancer. Moreover, concomitant constitutive expression of NRF2 and its target genes may suggest important role of NRF2 transcription factor in positive regulation of antioxidant genes, resulted in enhanced cytoprotection in human peripheral blood leukocytes.

Analysis of DNA variations in GSTA and GSTM gene clusters based on the results of genome-wide data from three Russian populations taken as an example

BackgroundExtensive genome-wide analyses of many human populations, using microarrays containing hundreds of thousands of single-nucleotide polymorphisms, have provided us with abundant information about global genomic diversity. However, these data can also be used to analyze local variability in individual genomic regions. In this study, we analyzed the variability in two genomic regions carrying the genes of the GSTA and GSTM subfamilies, located on different chromosomes.ResultsAnalysis of the polymorphisms in GSTA and GSTM gene clusters showed similarities in their allelic and haplotype diversities. These patterns were similar in three Russian populations and the CEU population of European origin. There were statistically significant differences in all the haploblocks of both the GSTM and GSTA regions when the Russian populations were compared with populations from China and Japan. Most haploblocks also differed between the Russians and Nigerians from Yoruba, but, some of them had similar allelic frequencies. Special attention was paid to SNP rs4986947 from the intron of the GSTA4 gene, which is represented in apes by an A nucleotide. In the Asian and African samples, it was represented only by a G allele, and both allelic variants (G/A) occurred in the Russian and European populations.ConclusionsThe results obtained suggest the presence of common features in the evolutionary histories of the GSTA and GSTM gene regions, and that African subpopulations were involved differently in the formation of the European and Asian human lineages.