GSTA1 Research Articles

Hepatoprotective effects of ethanol extracts from Folium Syringae against acetaminophen-induced hepatotoxicity in vitro and in vivo

BackgroundThe leaves of Folium Syringae (FS) have been long used as a traditional Chinese folk medicine for their anti-inflammatory effect, utilized as an antibacterial and antiviral treatment. The purpose of this study was to investigate the potential hepatoprotective effects of FS on acetaminophen-induced hepatic injury in primary hepatocytes and mice. MethodsHepatocytes obtained by the inverse perfusion method were divided randomly into five groups. Prior to acetaminophen exposure, 3 different doses of FS ethanol extracts were given to hepatocytes and mice, respectively. Thereafter, transaminases, glutathione S-transferase A1 (GSTA1) and some hepatic indices were determined. ResultsFS ethanol extracts (200 μg/mL) pretreatment prevented all of the alterations, returning their levels to nearly those levels observed in the control group in vitro. Treatment with FS ethanol extracts (200 mg/kg) significantly reduced the toxicity induced by acetaminophen in vivo, which manifested as a decrease in transaminases, and the hepatoprotective effects of FS were similar to Silymarin (positive group). GSTA1 represented the same change trend as transaminases and hepatic indices, and at a dose of 100 μg/mL FS ethanol extracts in vitro and 100 mg/kg in vivo, GSTA1 content changed significantly (p < 0.01), but transaminases were insignificant (p > 0.05). ConclusionThe results of our investigation suggested that FS ethanol extracts possess significant protective effects against hepatotoxicity induced by acetaminophen both in vitro and in vivo. In addition, GSTA1 could be used as an indicator assessing the extents of hepatic injury, which is more sensitive than transaminases.

Astrocytic Expression of GSTA4 Is Associated to Dopaminergic Neuroprotection in a Rat 6-OHDA Model of Parkinson’s Disease

Idiopathic Parkinson’s disease (PD) is a complex disease caused by multiple, mainly unknown, genetic and environmental factors. The Ventral root avulsion 1 (Vra1) locus on rat chromosome 8 includes the Glutathione S-transferase alpha 4 (Gsta4) gene and has been identified in crosses between Dark Agouti (DA) and Piebald Virol Glaxo (PVG) rat strains as being associated to neurodegeneration after nerve and brain injury. The Gsta4 protein clears lipid peroxidation by-products, a process suggested to being implicated in PD. We therefore investigated whether PVG alleles in Vra1 are neuroprotective in a toxin-induced model of PD and if this effect is coupled to Gsta4. We performed unilateral 6-hydroxydopamine (6-OHDA) partial lesions in the striatum and compared the extent of neurodegeration in parental (DA) and congenic (DA.VRA1) rats. At 8 weeks after 6-OHDA lesion, DA.VRA1 rats displayed a higher density of remaining dopaminergic fibers in the dorsolateral striatum compared to DA rats (44% vs. 23%, p < 0.01), indicating that Vra1 alleles derived from the PVG strain protect dopaminergic neurons from 6-OHDA toxicity. Gsta4 gene expression levels in the striatum and midbrain were higher in DA.VRA1 congenic rats compared to DA at 2 days post-lesion (p < 0.05). The GSTA4 protein co-localized with astrocytic marker GFAP, but not with neuronal marker NeuN or microglial marker IBA1, suggesting astrocyte-specific expression. This is the first report on Vra1 protective effects on dopaminergic neurodegeneration and encourages further studies on Gsta4 in relation to PD susceptibility.

Glutathione S-transferase A1 mediates nicotine-induced lung cancer cell metastasis by promoting epithelial-mesenchymal transition.

The present study aimed to investigate the effect of glutathione S-transferase A1 (GSTA1) on lung cancer cell viability, invasion and adhesion in the presence of nicotine in vitro. Furthermore, the effect of GSTA1 on the epithelial-mesenchymal transition (EMT), a process strongly associated with lung cancer metastasis, was examined. Human lung carcinoma A549 cells were treated with various concentrations of nicotine (0.01, 0.1, 1 and 10 µM) and levels of GSTA1 mRNA and protein were measured by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. To knock down GSTA1 expression, GSTA1-small interfering RNA was transfected into A549 cells. Cell viability, invasion and adhesion abilities were determined by MTT, Transwell-Matrigel invasion and cell adhesion assays, respectively. The expression of the epithelial cell markers E-cadherin and keratin, and the mesenchymal cell markers vimentin and N-cadherin in A549 cells were examined by western blot analysis. The current study indicated that the expression of GSTA1 was increased in A549 cells following nicotine treatment. GSTA1 suppression inhibited the viability, invasion and adhesion of lung cancer cells. In addition, the increase in lung cancer cell viability, invasion and adhesion by nicotine was suppressed following GSTA1 knockdown. Furthermore, GSTA1 affected the expression of EMT markers in nicotine-treated or untreated lung cancer cells. Thus the present study demonstrates that GSTA1 promotes lung cancer cell invasion and adhesion and mediates the effect of nicotine on lung cancer cell metastasis in vitro. Furthermore, the results demonstrated that GSTA1 exerts its effect on lung cancer cell metastasis by promoting the EMT.

Characterization of liver injury, oval cell proliferation and cholangiocarcinogenesis in glutathione S-transferase A3 knockout mice.

We recently generated glutathione S-transferase (GST) A3 knockout (KO) mice as a novel model to study the risk factors for liver cancer. GSTA3 KO mice are sensitive to the acute cytotoxic and genotoxic effects of aflatoxin B1 (AFB1), confirming the crucial role of GSTA3 in resistance to AFB1. We now report histopathological changes, tumor formation, biochemical changes and gender response following AFB1 treatment as well as the contribution of oxidative stress. Using a protocol of weekly 0.5 mg AFB1/kg administration, we observed extensive oval (liver stem) cell (OC) proliferation within 1-3 weeks followed by microvesicular lipidosis, megahepatocytes, nuclear inclusions, cholangiomas and small nodules. Male and female GSTA3 KO mice treated with 12 and 24 weekly AFB1 injections followed by a rest period of 12 and 6 months, respectively, all had grossly distorted livers with macro- and microscopic cysts, hepatocellular nodules, cholangiomas and cholangiocarcinomas and OC proliferation. We postulate that the prolonged AFB1 treatment leads to inhibition of hepatocyte proliferation, which is compensated by OC proliferation and eventually formation of cholangiocarcinoma (CCA). At low-dose AFB1, male KO mice showed less extensive acute liver injury, OC proliferation and AFB1-DNA adducts than female KO mice. There were no significant compensatory changes in KO mice GST subunits, GST enzymatic activity, epoxide hydrolase, or CYP1A2 and CYP3A11 levels. Finally, there was a modest increase in F2-isoprostane and isofuran in KO mice that confirmed putative GSTA3 hydroperoxidase activity in vivo for the first time.

Development of an assay for cellular efflux of pharmaceutically active agents and its relevance to understanding drug interactions.

Drug interactions may dictate the failure or success of a treatment. Patients undergoing hematopoietic stem cell transplantation (HSCT) are exposed to various types of drugs, and understanding how these drugs interact is of the utmost importance. The pharmacokinetics of busulfan, melphalan, and cyclophosphamide, drugs commonly used for HSCT, are known to be affected by a variety of other drugs with differing molecular structures. We hypothesized that these structurally unrelated drugs affect the transport of DNA-alkylating agents. To test this hypothesis, we developed a flow cytometry assay that used 5-carboxyfluorescein diacetate acetoxymethyl ester, which is cleaved by nonspecific intracellular esterases to 5-carboxyfluorescein (5-CF), a fluorescent ligand for the drug transporter MRP1. A decreased 5-CF efflux in the presence of a test compound suggests competitive inhibition. We demonstrated that chlorambucil, 4-hydroperoxycyclophosphamide, ketoconazole, ethacrynic acid, everolimus, and sirolimus strongly inhibited 5-CF efflux in lymphoma and leukemia cell lines. The efflux of these drugs partially depends on the glutathione (GSH) level, and their cytotoxicity is synergistic with inhibited GSH synthesis. This is consistent with the hypothesis that their GSH-conjugated products are ligands of a common cellular drug transporter. Our results may explain clinical observations on the effects of various drugs on the pharmacokinetics and pharmacodynamics of alkylating agents, and the assay may be used to deduce interaction mechanisms of drugs transported by a common system.

GSTA2 Promotes Liver Tumor Recurrence after Liver Transplantation through Regulating ROS Metabolism

Introduction: Tumor recurrence remains a major obstacle in hepatocellular carcinoma (HCC) recipients after liver transplantation. Our previous studies have indicated that elevated hepatic injury at early-phase after liver transplantation promotes late-phase tumor progression and invasion[1][2]. GSTA2 was identified to be significantly upregulated in early-phase of HCC recipients who developed with tumor recurrence in late-phase. We aimed to investigate the prognostic value of circulating GSTA2 in predicting tumor recurrence after liver transplantation and explore its underlying molecular mechanism. Methods: Post-transplantation plasma GSTA2 protein of 70 HCC recipients was examined by ELISA analysis. The prognostic value of plasma GSTA2 in predicting tumor recurrence and survival was examined by statistical analyses. The expression level of GSTA2 and ROS-associated genes was examined by real-time RT-PCR. Suppression of GSTA2 in HCC cell lines was established by lentiviral approach. The roles of GSTA2 in regulating HCC proliferation, metastasis and ROS metabolism were studied by in vitro and in vivo functional assays. The effect of GSTA2 in regulating other ROS-associated genes was studied by by RT2 Profiler PCR array. Results: Higher level of plasma GSTA2 at early-phase after liver transplantation significantly predicted tumor recurrence of HCC recipients (Sensitivity = 0.875; specificity = 0.481; P = 0.031) (Fig.1A). Higher level of plasma GSTA2 was also significantly associated with poor overall (P = 0.041) and disease-free survival (P = 0.024) of HCC recipients (Fig. 1B and 1C). The expression level of hepatic GSTA2 after liver transplantation was significantly correlated with ROS-associated genes such as GPX2 and SOD3. Suppression of GSTA2 in HCC cells significantly inhibited in vitro and in vivo proliferation and metastasis of HCC cells. Upregulation of GSTA2 could compensate H2O2-induced high ROS stress and damage in both normal liver and HCC cells. Alteration of GSTA2 expression in HCC cells could influence the expression level of several ROS-associated genes such as NCF1, SOD3, MT3, MBL2, ALB, TPO and GPX5. Conclusion: Post-transplantation circulating GSTA2 is a potential biomarker for predicting tumor recurrence and survival of HCC recipients after liver transplantation. Higher level of GSTA2 after liver transplantation could promote tumor recurrence through regulating ROS metabolism.

The sheep (Ovis aries) muscle proteome: Decoding the mechanisms of tolerance to Seasonal Weight Loss using label-free proteomics

Seasonal Weight Loss (SWL) is one of the most pressing issues in animal production in the tropics and Mediterranean. This work aims to characterize muscle proteome changes as a consequence of SWL in meat producing sheep, using a label-free proteomics approach. We compare three breeds: the Australian Merino (SWL susceptible), the Damara (SWL tolerant) and the Dorper (SWL intermediate tolerance). We identified 668 proteins of the sheep proteome, 95 with differential regulation. Also we observe that the more vulnerable to SWL a breed is, the more differential abundance proteins we find. Protein binding was the most frequently altered molecular function identified. We suggest 6 putative markers for restricted nutritional conditions independently of breed: ferritin heavy-chain; immunoglobulin V lambda chain; transgelin; fatty acid synthase; glutathione S-transferase A2; dihydrodiol dehydrogenase 3-like. Moreover, we suggest as related to SWL tolerance: S100-A10 Serpin A3-5-like and Catalase, subject however to necessary validation assays. The identification of SWL-tolerance related proteins using proteomics will lead to increased stock productivity of relevant interest to animal production, particularly if identified at the muscle level, the tissue of economic importance in meat production. Biological significanceSeasonal Weight Loss (SWL) is the most pressing issue in animal production in the tropics and the Mediterranean. To counter SWL, farmers often use animal breeds that have a natural ability to withstand pasture scarcity. Here we study the sheep muscle proteome at the muscle level, the tissue of economic importance in meat production. Furthermore, the identification of proteins that change their abundance in response to SWL using proteomics can contribute to increased stock productivity of relevant interest to animal production. We identified 668 proteins of the sheep proteome. We demonstrate that the following proteins are affected by restricted nutritional conditions: ferritin heavy chain; immunoglobulin V lambda chain; transgelin; fatty acid synthase; glutathione S-transferase A2; dihydrodiol dehydrogenase 3-like. Furthermore, S100-A10, Serpin A3-5-like and Catalase are proteins that changed their abundance in response to SWL. Nevertheless, it is important to highlight that Catalase values for the merino breed were close to significance and therefore catalase validation is of utmost importance.

Reduction of Aflatoxin B1 Toxicity by Lactobacillus plantarum C88: A Potential Probiotic Strain Isolated from Chinese Traditional Fermented Food "Tofu".

In this study, we investigated the potential of Lactobacillus plantarum isolated from Chinese traditional fermented foods to reduce the toxicity of aflatoxin B1 (AFB1), and its subsequent detoxification mechanism. Among all the investigated L. plantarum strains, L. plantarum C88 showed the strongest AFB1 binding capacity in vitro, and was orally administered to mice with liver oxidative damage induced by AFB1. In the therapy groups, the mice that received L. plantarum C88, especially heat-killed L. plantarum C88, after a single dose of AFB1 exposure, showed an increase in unabsorbed AFB1 in the feces. Moreover, the effects of L. plantarum C88 on the enzymes and non-enzymes antioxidant abilities in serum and liver, histological alterations of liver were assayed. The results indicated that compared to the control group, L. plantarum C88 alone administration induced significant increase of antioxidant capacity, but did not induce any significant changes in the histological picture. Compared to the mice that received AFB1 only, L. plantarum C88 treatment could weaken oxidative stress by enhancing the activity of antioxidant enzymes and elevating the expression of Glutathione S-transferase (GST) A3 through Nuclear factor erythroid (derived factor 2) related factor 2 (Nrf2) pathway. Furthermore, cytochrome P450 (CYP 450) 1A2 and CYP 3A4 expression was inhibited by L. plantarum C88, and urinary aflatoxin B1-N7-guanine (AFB-N7-guanine), a AFB1 metabolite formed by CYP 1A2 and CYP 3A4, was significantly reduced by the presence of viable L. plantarum C88. Meanwhile, the significant improvements were showed in histological pictures of the liver tissues in mice orally administered with viable L. plantarum C88. Collectively, L. plantarum C88 may alleviate AFB1 toxicity by increasing fecal AFB1 excretion, reversing deficits in antioxidant defense systems and regulating the metabolism of AFB1.

Differential representation of liver proteins in obese human subjects suggests novel biomarkers and promising targets for drug development in obesity

The proteome of liver biopsies from human obese (O) subjects has been compared to those of nonobese (NO) subjects using two-dimensional gel electrophoresis (2-DE). Differentially represented proteins were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS)-based peptide mass fingerprinting (PMF) and nanoflow-liquid chromatography coupled to electrospray-tandem mass spectrometry (nLC-ESI-MS/MS). Overall, 61 gene products common to all of the liver biopsies were identified within 65 spots, among which 25 ones were differently represented between O and NO subjects. In particular, over-representation of short-chain acyl-CoA dehydrogenase, Δ(3,5)-Δ(2,4)dienoyl-CoA isomerase, acetyl-CoA acetyltransferase, glyoxylate reductase/hydroxypyruvate reductase, fructose-biphosphate aldolase B, peroxiredoxin I, protein DJ-1, catalase, α- and β-hemoglobin subunits, 3-mercaptopyruvate S-transferase, calreticulin, aminoacylase 1, phenazine biosynthesis-like domain-containing protein and a form of fatty acid-binding protein, together with downrepresentation of glutamate dehydrogenase, glutathione S-transferase A1, S-adenosylmethionine synthase 1A and a form of apolipoprotein A-I, was associated with the obesity condition. Some of these metabolic enzymes and antioxidant proteins have already been identified as putative diagnostic markers of liver dysfunction in animal models of steatosis or obesity, suggesting additional investigations on their role in these syndromes. Their differential representation in human liver was suggestive of their consideration as obesity human biomarkers and for the development of novel antiobesity drugs.

Pharmacological Evaluation of Hepatoprotective Activity of AHPL/AYTAB/0613 Tablet in Carbon Tetrachloride-, Ethanol-, and Paracetamol-Induced Hepatotoxicity Models in Wistar Albino Rats.

Background:Hepatotoxicity ultimately leads to liver failure. Conventional treatment options for hepatotoxicity are limited and not safe.Objective:Formulation AHPL/AYTAB/0613 is developed to provide safer and effective hepatoprotective drug of natural origin. A study was conducted to evaluate hepatoprotective activity of AHPL/AYTAB/0613 (three dosages) in comparison with marketed formulations in carbon tetrachloride (CCl4), ethanol, and paracetamol-induced hepatotoxicity in Wistar albino rats.Materials and Methods:Three separate studies were conducted in models of CCl4, ethanol, and paracetamol-induced hepatotoxicity. Seven groups of animals were studied comparatively to evaluate the efficacy of AHPL/AYTAB/0613 in low, medium, and high dosage in comparison with silymarin and a marketed polyherbal formulation. The drugs were orally administered to rats for 10 days in CCl4 model and for 14 days in ethanol and paracetamol models. Animals were weighed periodically. After the study period, blood was tested for serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP), total bilirubin, and total protein levels. Liver tissue of sacrificed animals was examined histopathologically.Results:All the test formulations including all three dosages of AHPL/AYTAB/0613, significantly reduced levels of SGOT, SGPT, ALP, total bilirubin, in CCl4, ethanol, and paracetamol-induced hepatotoxicity models. There was significant increase in total protein level in all the tested formulations. All the test formulations effectively preserved the structural integrity of the hepatocellular membrane and liver cell architecture damaged by CCl4, ethanol, and paracetamol. When compared between groups, no statistically significant difference was observed. It can be concluded that AHPL/AYTAB/0613 possesses hepatoprotective activity in CCl4, ethanol, and paracetamol-induced hepatotoxicity in rats.Conclusion:AHPL/AYTAB/0613 can be effectively used as a hepatoprotective agent in the management of hepatitis caused due to various toxins.SUMMARYA polyherbal formulation AHPL/AYTAB/0613 containing Bhringaraja - Eclipta alba extract, Guduchi - Tinospora cordifolia extract, Daruharidra - Berberis aristata extract, Kakamachi - Solanum nigrum extract, Punarnava - Boerhaavia diffusa extract, Bhumyamalaki - Phyllanthus niruri extract, Kutaki - Picrorhiza kurroa extract, and Kalmegha - Andrograhis paniculata extract was assessed for its hepatoprotective activity. This activity was tested in carbon tetrachloride (CCl4), ethanol, and paracetamol-induced hepatotoxicity models in Wistar albino rats in comparison with two marketed formulations. It was observed that AHPL/AYTAB/0613 significantly reduced levels of serum glutamic-oxaloacetic transaminase, serum glutamic pyruvic transaminase, alkaline phosphatase, total bilirubin and also significantly increased total protein level. All the test formulations effectively preserved the structural integrity of the hepatocellular membrane and liver cell architecture damaged by CCl4, ethanol, and paracetamol. When compared between groups, no statistically significant difference was observed. Therefore, it was concluded that AHPL/AYTAB/0613 possesses hepatoprotective activity in CCl4, ethanol, and paracetamol-induced hepatotoxicity in rats.Abbreviations Used: CCl4: Carbon tetrachloride, SGOT: Serum glutamic-oxaloacetic transaminase, SGPT: Serum glutamic pyruvi transaminase, ALP: Alkaline phosphatase, UDCA: Ursodeoxycholic acid, US: United States, FDA: Food and Drug Administration, PBC: Primary biliary cirrhosis, GSTA1: Glutathione S-transferase A1, GSH: Glutathione, GPx: Glutathione peroxidase, GST: Glutathione S-transferases.

Genetic polymorphism of glutathione S-transferase A1 promoter in patient with colorectal cancer

Background: Novel allelic variants have been found in the glutathione S-transferase (GST) A1 gene. The former GSTA1 B allele is associated with low expression. Objective: The aim of this study was to determine the GSTA1 genotype in the Iranian patient with colorectal cancer and determine if there is any populational correlation between genotype and colorectal cancer in Kermanshah-Iran and Tehran-Iran individuals. Material and Methods: A total of 186 participants were included in the study, 52 Tehranian and 41 Kermanshahian patients with colorectal cancer, plus 52 Tehranian and 41 Kermanshahian healthy controls. GSTA1 genotype was determined by polymerase chain reaction-restriction fragment length polymorphism. Differences in genotype distributions between the different populational groups were evaluated us in Chi-squared test and a logistic regression was used to investigate the possible association of the genotypes with the disease in both populations. Odds ratio estimates were analyzed for allele frequencies. Results: The GSTA1 B/B genotype prevalence in patients with colorectal cancer (12.2%) was significantly higher than control group in Kermanshah (0.0%), No significant association of GSTA1 B/B genotype with the colorectal cancer was determined in Tehran subjects. No significant association of GSTA1 B/A genotype with the colorectal cancer was determined either population. Conclusion: These findings indicate that the GSTA1 B/B genotype in the Kermanshah population was related to colorectal cancer although there is no significant association between of GSTA1 B/B genotype and colorectal cancer in Tehran population. This study showed different results between populations.

The rs3957357C>T SNP in GSTA1 Is Associated with a Higher Risk of Occurrence of Hepatocellular Carcinoma in European Individuals.

Glutathione S-transferases (GSTs) detoxify toxic molecules by conjugation with reduced glutathione and regulate cell signaling. Single nucleotide polymorphisms (SNPs) of GST genes have been suggested to affect GST functions and thus to increase the risk of human hepatocellular carcinoma (HCC). As GSTA1 is expressed in hepatocytes and the rs3957357C>T (TT) SNP is known to downregulate GSTA1 mRNA expression, the aims of this study were: (i) to explore the relationship between the TT SNP in GSTA1 and the occurrence of HCC; (ii) to measure GSTA1 mRNA expression in HCCs. For that purpose, we genotyped non-tumor-tissue-derived DNA from 48 HCC patients and white-blood-cell-derived DNA from 37 healthy individuals by restriction fragment length polymorphism (RFLP). In addition, expression of GSTA1 mRNA was assessed by real-time PCR in 18 matching pairs of HCCs and non-tumor livers. Survival analysis was performed on an annotated microarray dataset containing 247 HCC patients (GSE14520). The GSTA1 TT genotype was more frequent in HCC than in non-HCC patients (27% versus 5%, respectively), suggesting that individuals carrying this genotype could be associated with 2-fold higher risk of developing HCCs (odds ratio = 2.1; p = 0.02). Also, we found that GSTA1 mRNA expression was lower in HCCs than in non-tumor livers. HCCs expressing the highest GSTA1 mRNA levels were the smallest in size (R = -0.67; p = 0.007), expressed the highest levels of liver-enriched genes such as ALB (albumin, R = -0.67; p = 0.007) and COL18A1 (procollagen type XVIII, R = -0.50; p = 0.03) and showed the most favorable disease-free (OR = 0.54; p<0.001) and overall (OR = 0.56; p = 0.006) outcomes. Moreover, GSTA1 was found within a 263-gene network involved in well-differentiated hepatocyte functions. In conclusion, HCCs are characterized by two GSTA1 features: the TT SNP and reduced GSTA1 gene expression in a context of hepatocyte de-differentiation.

416 - Mitochondrial Oxidative Stress Induced by Downregulation of Antioxidant Enzymes Leads to Nuclear Protein Carbonylation by Retrograde Signaling in 3T3-L1 Adipocytes

Obesity-linked insulin resistance is mechanistically connected to local inflammation of adipose tissue, which produces a metabolic state characterized by oxidative stress and mitochondrial dysfunction. Antioxidants enzymes such as Glutathione S-transferase A4 (GSTA4), peroxiredoxin 3 (Prdx3) and glutathione peroxidase 4 (GPx4) expressions are selectively downregulated in adipose tissue of obese insulin-resistant mice and in human obesity-linked insulin resistance. Also, TNFα treatment of 3T3-L1 adipocytes resulted in decreased expression of GSTA4, GPx4, and Prdx3 and increased protein carbonylation. In addition, protein carbonylation is implicated as an initiating factor in mitochondrial dysfunction and ER-stress, providing a mechanistic connection between oxidative stress and metabolic disease. Histones are the primary components of chromatin and are notably susceptible to carbonylation because of their long lysine-rich tails. These modifications may have effects on histone code, leading to long lasting implications of human health, including insulin resistance. In this study GSTA4-Prdx3-GPx4-silenced 3T3-L1 adipocytes were evaluated for reactive oxygen species production (ROS), mitochondrial function and histones carbonylation. Downregulation of GSTA4, Prdx3 and GPx4 led to an significant increase in ROS, a significant increase in H3 and H4 histones carbonylation, and mitochondrial dysfunction. These results indicate that a downregulation of antioxidant enzymes in adipocytes leads to increased ROS production, mitochondrial dysfunction and nuclear protein carbonylation, and may contribute to the development of insulin resistance and type 2 diabetes.

Directed evolution of glutathione transferases towards a selective glutathione-binding site and improved oxidative stability

BackgroundGlutathione transferases (GSTs) are a family of detoxification enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic compounds. MethodsA library of alpha class GSTs was constructed by DNA shuffling using the DNA encoding the human glutathione transferase A1-1 (hGSTA1-1) and the rat glutathione transferase A1-1 (rGSTA1-1). ResultsActivity screening of the library allowed the selection of a chimeric enzyme variant (GSTD4) that displayed high affinity towards GSH and GSH-Sepharose affinity adsorbent, higher kcat/Km and improved thermal stability, compared to the parent enzymes. The crystal structures of the GSTD4 enzyme in free form and in complex with GSH were determined to 1.6Å and 2.3Å resolution, respectively. Analysis of the GSTD4 structure showed subtle conformational changes in the GSH-binding site and in electron-sharing network that may contribute to the increased GSH affinity. The shuffled variant GSTD4 was further optimized for improved oxidative stability employing site-saturation mutagenesis. The Cys112Ser mutation confers optimal oxidative stability and kinetic properties in the GSTD4 enzyme. ConclusionsDNA shuffling allowed the creation of a chimeric enzyme variant with improved properties, compared to the parent enzymes. X-ray crystallography shed light on how recombination of a specific segment from homologous GSTA1-1 together with point mutations gives rise to a new functionally competent enzyme with improved binding, catalytic properties and stability. General significanceSuch an engineered GST would be useful in biotechnology as affinity tool in affinity chromatography as well as a biocatalytic matrix for the construction of biochips or enzyme biosensors.

Impact of the GSTA1 Gene Polymorphism on Clinical Outcomes of HLA-Matched Sibling Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Myeloid Leukemia

Impact of the GSTA1 Gene Polymorphism on Clinical Outcomes of HLA-Matched Sibling Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Myeloid Leukemia

Up-regulation of glutathione-related genes, enzyme activities and transport proteins in human cervical cancer cells treated with doxorubicin.

Doxorubicin (DOX), one of the most effective anticancer drugs, acts in a variety of ways including DNA damage, enzyme inhibition and generation of reactive oxygen species. Glutathione (GSH) and glutathione-related enzymes including: glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferases (GST) may play a role in adaptive detoxification processes in response to the oxidative stress, thus contributing to drug resistance phenotype. In this study, we investigated effects of DOX treatment on expression and activity of GSH-related enzymes and multidrug resistance-associated proteins in cultured human cervical cancer cells displaying different resistance against this drug (HeLa and KB-V1). Determination of expression level of genes encoding GST isoforms and MRP proteins (GCS, GPX, GSR, GSTA1-3, GSTM1, GSTP1, ABCC1-3, MGST1-3) was performed using StellARray™ Technology. Enzymatic activities of GPX and GSR were measured using biochemical methods. Expression of MRP1 was examined by immunofluorescence microscopy. This study showed that native expression levels of GSTM1 and GSTA3 were markedly higher in KB-V1 cells (2000-fold and 200-fold) compared to HeLa cells. Resistant cells have also shown significantly elevated expression of GSTA1 and GSTA2 genes (200-fold and 50-fold) as a result of DOX treatment. In HeLa cells, exposure to DOX increased expression of all genes: GSTM1 (7-fold) and GSTA1-3 (550-fold, 150-fold and 300-fold). Exposure to DOX led to the slight increase of GCS expression as well as GPX activity in KB-V1 cells, while in HeLa cells it did not. Expression of ABCC1 (MRP1) was not increased in any of the tested cell lines. Our results indicate that expression of GSTM1 and GSTA1-3 genes is up-regulated by DOX treatment and suggest that activity of these genes may be associated with drug resistance of the tested cells. At the same time, involvement of MRP1 in DOX resistance in the given experimental conditions is unlikely.

Glutathione S-transferase A1 - a sensitive marker of alcoholic injury on primary hepatocytes.

The primary hepatocytes were extracted and purified from mice through improved Seglen two-step perfusion method. Ethanol-induced injury hepatocytes model in mice was used to investigate the importance of glutathione S-transferase A1 (GSTA1) in hepatocytes injury by comparison with other indicators, such as alanine aminotransferase, aspartate aminotransferase, malondialdehyde, glutathione and superoxide dismutase. The release of GSTA1 was demonstrated to be an earlier and more sensitive indicator of hepatocytes injury than other indicators. Significant increases in GSTA1 were detected at 2 h after ethanol exposure, while other indicators were undetected at this time. A markedly difference in other indicators were observed at 6 and 8 h. The release of GSTA1 was significantly increased at a concentration of 50 mmol/L ethanol, the lowest exposure concentration than that in other indicators. In contrast, other indicators release was not statistically significant until concentrations of 75 mmol/L and 100 mmol/L ethanol. These results suggest that GSTA1 can be detected at the early stage of low concentration ethanol exposure and that GSTA1 is more sensitive and reliable marker in ethanol-induced hepatic injury.

Expression levels of GSTA2 and APOD genes might be associated with carotenoid coloration in golden pheasant (Chrysolophus pictus) plumage.

Carotenoids, which generate yellow, orange, and red colors, are crucial pigments in avian plumage. Investigations into genes associated with carotenoidbased coloration in avian species are important; however, such research is difficult because carotenoids cannot be synthetized in vertebrates as they are only derived from dietary sources. Here, the golden pheasant (Chrysolophus pictus) was used as a model in analysis of candidate gene expression profiles implicated in carotenoid binding and deposition. Using mass and Raman spectrometry to confirm the presence of carotenoids in golden pheasant feathers, we found C40H54O and C40H56O2 in feathers with yellow to red colors, and in the rachis of iridescent feathers. The global gene expression profiles in golden pheasant skins were analyzed by RNA-seq and all six carotenoid binding candidate genes sequenced were studied by realtime PCR. StAR4, GSTA2, Scarb1, and APOD in feather follicles showed different expressions in red breast and orange nape feathers compared with that of iridescent mantle feathers. Further comparison of golden pheasant yellow rump and Lady Amherst's pheasant (Chrysolophus amherstiae) white nape feathers suggested that GSTA2 and APOD played a potential role in carotenoid-based coloration in golden pheasant.

Impact of CYP2E1, GSTA1 and GSTP1 gene variants on serum alpha glutathione S-transferase level in patients undergoing anaesthesia.

BackgroundThe serum glutathione S-transferase alpha (α-GST) concentration has been used as a marker of hepatic condition. After sevoflurane anaesthesia a mild impairment of hepatocellular integrity was observed. Genetic polymorphisms in CYP2E1, GSTA1 and GSTP1 genes, affecting enzymes activity, may possibly influence the hepatotoxic effect of sevoflurane. The aim of this study was to assess the influence of genetic polymorphism of CYP2E1, GSTA1 and GSTP1 genes on serum α-GST level in 86 unrelated patients representing ASA physical status I-II, undergoing laryngological surgery under general anaesthesia with sevoflurane.MethodsThe serum samples from three perioperative time points were analyzed using ELISA. Genetic variants were detected by pyrosequencing and sequencing. Finally, the statistical associations between serum α-GST concentration and analyzed alleles of CYP2E1, GSTP1 and GSTA1 genes were estimated.ResultsThe allele GSTA1*B (−567G, −69T, −52A) frequency was 0.43, whereas the alleles c.313G and c.341T of GSTP1 were identified with frequencies of 0.28 and 0.1 respectively. The -1053T allele of the CYP2E1 gene was observed with 0.01 frequency. We found serum α-GST concentrations in homozygous changes c.313A>G and c.341C>T of the GSTP1 gene significantly higher at the end of anaesthesia as compared with the levels at pre-anaesthetic and 24 h post-anaesthetic time points. Moreover, GSTA1 wild type genotype was associated with increased α-GST concentration at 24 h after the end of anaesthesia.ConclusionsGSTP1 gene polymorphism has an impact on the perioperative serum α-GST concentration in patients undergoing sevoflurane anaesthesia. A similar association, although not statistically significant exists between GSTA1 gene variants and perioperative serum α-GST level.

Antiseptic effect of sea cucumber (Holothuria atra) against multi-organ failure induced by sepsis: Molecular and histopathological study.

Sepsis is a systemic inflammatory response to infection and severe sepsis patients can develop acute lung and liver injury. The aim of the present study was to evaluate the efficacy of Holothuria atra methanolic body wall extract (HaE), as an antioxidant and anti-inflammatory agent against induced sepsis in a cecal ligation and puncture (CLP) rat model. In total, 30 males albino rats were divided into three groups (n=10 each) as follows: Sham (Sh), which was used as negative control; sepsis (Se), which was used as a positive control and was subjected to CLP surgery; and Ho, which was subjected to CLP and fed with 200 mg/kg (body weight) of HaE, once daily for 7 days. Subsequently, the expression of various genes was detected by polymerase chain reaction, while liver and lung tissues were examined by immunohistochemistry. The expression of Caspase-3 was significantly reduced in liver and lung tissues in the Ho group, while the expression levels of Gsta2, Cat and Sod1 genes were slightly reduced in the Ho group, when compared with the Se group. In addition, expression levels of tumor necrosis factor, interferon-γ, liver interleukin (IL)1b and lung IL1a were reduced in the Ho group compared with the Se group. Furthermore, histopathological changes were observed in liver tissues of the Se group, including congestion of hepatoportal blood vessel and focal hepatic necrosis, while lung tissues showed marked edema, hemorrhage and alveolar septal thickening. The Ho group showed apparent normal hepatic parenchyma and slight interstitial pneumonia. Immunohistochemical staining of caspase-3 in liver and lung tissues showed no expression in the Sh group, strong expression in the Se group and moderate expression in the Ho group. In conclusion, HaE demonstrated beneficial effect against induced sepsis, which may be attributed to its antioxidant and antiapoptotic activities.