Glutathione S-transferase Isozymes Research Articles

Kırıkkale İlinde Spermophilus xanthoprymnus ve Meriones tristrami’de Glutatyon S-Transferaz-Alfa ve Glutatyon S-Transferaz-Pi Ekspresyon Düzeylerinin Yaşam Koşulları ve Doğal Habitat Farklılıkları Açısından İncelenmesi

Glutathione S-transferase (GST) is a multifunctional enzyme that provides homeostasis by catalyzing the first step in the formation of the end product mercapturic acid in the detoxification metabolic pathway. Being found in mammals, insects, fish, birds, annelids, molluscs, and many microorganisms, GST takes part the elimination of toxic substances taken into body by consuming food, and their transport by binding non-substrate ligands (e.g. heme and bilirubin) with GSH. In addition, it can prevent reactive electrophilic compounds from harming the body by covalent bonding similar compounds to each other. These xenobiotic acceptors affected by GST include nitrogen halogen compounds, organophosphates, and polycyclic aromatic hydrocarbons. Xenobiotics are oxygenated by this enzyme system, the next mechanism of oxygenated products is more oxygenation, and these products become more easily soluble in water. In this study, Glutathione S-Transferase was detected in the liver tissue of Spermophilus xanthoprymnus and Meriones tristrami and its characteristic features were determined. For this purpose, the animals were anesthetized with sodium pentobarbital and their liver tissues were harvested. After necessary preparations were completed, the samples were analyzed by using immunohistochemical staining method and the expressions of GST isozymes were determined. As a result, glutathione s-transferase-alpha and glutathione s-transferase-pi expression levels were found to differ in Spermophilus xanthoprymnus and Meriones tristrami samples obtained from different localities of Kırıkkale province. Differences in GST enzyme expression in these species indicate that both species differ in their detoxification capacity and response to xenobiotics.

Inhibitory Effect of Ascorbic Acid on Glutathione S-transferase from Human Erythrocytes

Background: Inhibition of certain specific glutathione S-transferase isozymes is associated with the efficacy of chemotherapy in the treatment of cancer patients. Objective: We aimed to determine whether ascorbic acid, which can be used as a drug and antioxidant food supplement, is a glutathione S-transferase isozyme inhibitor. Methods: For this purpose, glutathione S-transferase was first purified from human erythrocytes and the in vitro effect of ascorbic acid on enzyme activity was investigated. Then, kinetic studies were performed to evaluate the inhibition potential of ascorbic acid, which was determined to be an inhibitor of the enzyme. Results: The enzyme was purified 1286-fold with a yield of 81% and specific activity of 18.00 U/mg protein. The effects of ascorbic acid were tested on the in vitro enzyme activity of glutathione Stransferase and detected to be an inhibitor for the enzyme with 10 mM of IC50 value. Ki constants and inhibition type were also determined for the enzyme. Conclusion: This finding indicates that ascorbic acid can be an effective inhibitor of some GST isozymes increased in cancer cells. Abundant ascorbic acid intake may prevent cancer formation by strengthening the antioxidant defense system, on the other hand, it may increase the effectiveness of cancer treatment by inhibiting the chemotherapy-resistant GST enzyme.

Immunohistochemical approach to obesity disease in terms of expression levels of glutathione s-transferase (sigma, zeta, theta) isozymes

Objectives: Obesity is a complex multifactorial disease with recently increasing prevalence and incidence. Several studies have been conducted to explain the ethiology, pathophysiology, epidemiology, molecular and genetic mechanisms, and effective treatments of obesity. Glutathione S-transferase (GST) S1, GSTZ1, and GSTT1 are essential enzymes for oxidative stress and metabolism-related disorders. For this purpose, we aimed to reveal the role of GSTS1, GSTZ1, and GSTT1 in obesity. Methods: The gastric tissue samples were taken from the patients diagnosed with obesity who underwent bariatric surgery in Ankara Keçiören Training and Research Hospital General Surgery Clinic between 2017 and 2019. Immunostaining was performed on paraffin-embedded tissues to evaluate GSTS1, GSTZ1, and GSTT1 expressions. Laboratory data of the patients were recorded. All the results were analyzed statistically. Results: Weak GSTS1 expression was observed in 38.1% of tissues and moderate in 6.3%. 37.3% of the tissues presented weak GSTZ1 expression, and 11 (8.7%) displayed moderate. There were weak GSTT1 expressions in 7.1% of the tissues and moderate 0.8% of them. A positive and statistically significant correlation was observed between GSTS1 and GSTT1 expression levels ((r)=0.028, p = 0.010; p < 0.05). There were no significant differences between expression levels and gender, age, comorbidities, and medication usage (p > 0.05). Conclusions: GSTs, in particular GSTS1, GSTT1, and GSTZ1, might contribute to molecular mechanisms and the progression of obesity. In our study, GSTS1, GSTT1, and GSTZ1 were found to be moderately expressed in gastric tissues taken from obese patients. However, new studies using more samples and advanced techniques are needed to elucidate the relationship.

Alpha-class glutathione S-transferases involved in the detoxification of aflatoxin B1 in ducklings.

The objective of this study was to identify the key glutathione S-transferase (GST) isozymes involved in the detoxification of Aflatoxin B1 (AFB1) in ducks' primary hepatocytes. The full-length cDNA encoding the 10 GST isozymes (GST, GST3, GSTM3, MGST1, MGST2, MGST3, GSTK1, GSTT1, GSTO1 and GSTZ1) were isolated/synthesized from ducks' liver and cloned into the pcDNA3.1(+) vector. The results showed that pcDNA3.1(+)-GSTs plasmids were successfully transferred into the ducks' primary hepatocytes and the mRNA of the 10 GST isozymes were overexpressed by 1.9-3274.7 times. Compared to the control, 75μg/L (IC30) or 150μg/L (IC50) AFB1 treatment reduced the cell viability by 30.0-50.0% and increased the LDH activity by 19.8-58.2% in the ducks' primary hepatocytes. Notably, the AFB1-induced changes in cell viability and LDH activity were mitigated by overexpression of GST and GST3. Compared to the cells treated with AFB1, exo-AFB1-8,9-epoxide (AFBO)-GSH, as the major detoxified product of AFB1, was increased in the cells overexpression of GST and GST3. Moreover, the sequences, phylogenetic and domain analysis revealed that the GST and GST3 were orthologous to Meleagris gallopavo GSTA3 and GSTA4. In conclusion, this study found that the ducks' GST and GST3 were orthologous to Meleagris gallopavo GSTA3 and GSTA4, which were involved in the detoxification of AFB1 in ducks' primary hepatocytes.

Mechanistic Study of Icaritin-Induced Inactivation of Cytochrome P450 2C9.

Icaritin (ICT) is a prenylflavonoid derivative that has been approved by National Medical Products Administration for the treatment of hepatocellular carcinoma. This study aims to evaluate the potential inhibitory effect of ICT against cytochrome P450 (CYP) enzymes and to elucidate the inactivation mechanisms. Results showed that ICT inactivated CYP2C9 in a time-, concentration-, and NADPH-dependent manner with Ki = 1.896 μM, Kinact = 0.02298 minutes-1, and Kinact/Ki = 12 minutes-1 mM-1, whereas the activities of other CYP isozymes was minimally affected. Additionally, the presence of CYP2C9 competitive inhibitor, sulfaphenazole, superoxide dismutase/catalase system, and GSH all protected CYP2C9 from ICT-induced activity loss. Moreover, the activity loss was neither recovered by washing the ICT-CYP2C9 preincubation mixture nor the addition of potassium ferricyanide. These results, collectively, implied the underlying inactivation mechanism involved the covalent binding of ICT to the apoprotein and/or the prosthetic heme of CYP2C9. Furthermore, an ICT-quinone methide (QM)-derived GSH adduct was identified, and human glutathione S-transferases (GST) isozymes GSTA1-1, GSTM1-1, and GSTP1-1 were shown to be substantially involved in the detoxification of ICT-QM. Interestingly, our systematic molecular modeling work predicted that ICT-QM was covalently bound to C216, a cysteine residue located in the F-G loop downstream of substrate recognition site (SRS) 2 in CYP2C9. The sequential molecular dynamics simulation confirmed the binding to C216 induced a conformational change in the active catalytic center of CYP2C9. Lastly, the potential risks of clinical drug-drug interactions triggered by ICT as a perpetrator were extrapolated. In summary, this work confirmed that ICT was an inactivator of CYP2C9. SIGNIFICANCE STATEMENT: This study is the first to report the time-dependent inhibition of CYP2C9 by icaritin (ICT) and the intrinsic molecular mechanism behind it. Experimental data indicated that the inactivation was via irreversible covalent binding of ICT-quinone methide to CYP2C9, while molecular modeling analysis provided additional evidence by predicting C216 as the key binding site which influenced the structural confirmation of CYP2C9's catalytic center. These findings suggest the potential of drug-drug interactions when ICT is co-administered with CYP2C9 substrates clinically.

Implications of Glutathione-S-transferases in Mitogen-activated protein kinase pathway: Risk associated with anticancer drug resistance

Anticancer drug resistance is a perilous glitch to accomplish alleviation of disease in patients with cancer. The setback of drug resistance in chemotherapy is a weighty snag of present time. Hassle of carcinoma has ability to develop resistance that arises due to certain alterations in drug targets. In the course of anticancer administrations, development of targeted therapies anticipates a new arena to subdue the drug resistance. Exalted levels of certain glutathione transferase isozymes have been identified with malign transformations as well as with anticancer drug resistance. Glutathione-S-transferases are a large group of phase-II detoxification enzymes which catalyse the conjugation of reduced glutathione to a broad range of exogenous as well as endogenous compounds. The enzymes of this family are divided into two distinct subgroups namely: cytosolic enzymes and membrane bound microsomal enzymes. Human cytosolic glutathione-S-transferases are extremely polymorphic in nature and exist in many forms such as mu, pi, alpha, theta, omega and zeta. Among these enzymes, mu and pi classes take part in regulation of mitogen-activated protein kinase pathway in directing cellular responses with apoptosis signal-regulating kinase and c-Jun Nterminal kinase 1 protein interactions. In normal condition, these two protein are mainly activated in response to cellular stress. The implications of glutathione S-transferase in development of anticancer drug resistance have been observed in two distinct ways. The first way is through direct detoxification mechanism while the second way deals with the action of glutathione S-transferase (mu and pi forms) as inhibitor to mitogen-activated protein kinase pathway.

Short divalent ethacrynic amides as pro-inhibitors of glutathione S-transferase isozyme Mu and potent sensitisers of cisplatin-resistant ovarian cancers

The linking of ethacrynic acid with ethylenediamine and 1,4-butanediamine gave EDEA and BDEA, respectively, as membrane-permeable divalent pro-inhibitors of glutathione S-transferase (GST). Their divalent glutathione conjugates showed subnanomolar inhibition and divalence-binding to GSTmu (GSTM) (PDB: 5HWL) at ∼0.35 min−1. In cisplatin-resistant SK-OV-3, COC1, SGC7901 and A549 cells, GSTM activities probed by 15 nM BDEA or EDEA revealed 5-fold and 1.0-fold increases in cisplatin-resistant SK-OV-3 and COC1 cells, respectively, in comparison with the susceptible parental cells. Being tolerable by HEK293 and LO2 cells, BDEA at 0.2 μM sensitised resistant SK-OV-3 and COC1 cells by ∼3- and ∼5-folds, respectively, released cytochrome c and increased apoptosis; EDEA at 1.0 μM sensitised resistant SK-OV-3 and A549 cells by ∼5- and ∼7-fold, respectively. EDEA at 1.7 μg/g sensitised resistant SK-OV-3 cells to cisplatin at 3.3 μg/g in nude mouse xenograft model. BDEA and EDEA are promising leads for probing cellular GSTM and sensitising cisplatin-resistant ovarian cancers.

Implications of glutathione-S transferase P1 in MAPK signaling as a CRAF chaperone: In memory of Dr. Irving Listowsky.

Glutathione-S transferase P1 (GSTP1) is one of the glutathione-S transferase isozymes that belong to a family of phase II metabolic isozymes. The unique feature of GSTP1 compared with other GST isozymes is its relatively high expression in malignant tissues. Thus, clinically, GSTP1 serves as a tumor marker and as a refractory factor against certain types of anticancer drugs through its primary function as a detoxifying enzyme. Additionally, recent studies have identified a chaperone activity of GSTP1 involved in the regulation the function of various intracellular proteins, including factors of the growth signaling pathway. In this review, we will first describe the function of GSTP1 and then extend the details onto its role in the mitogen-activated protein kinase signal pathway, referring to the results of our recent study that proposed a novel autocrine signal loop formed by the CRAF/GSTP1 complex in mutated KRAS and BRAF cancers. Finally, the possibilities of new therapeutic approaches for these cancers by targeting this complex will be discussed.

Elucidation of dibenzo[a,l]pyrene and its metabolites as a mammary carcinogen: A comprehensive review

The general mechanism of cancer includes the metabolism of carcinogens to highly electrophilic metabolites capable of binding to DNA and other macromolecules, thereby initiating the cells. As the carcinogenesis mechanism is quite complex where diverse cellular mechanism(s) are involved in cancer promotion and progression, it is challenging to elucidate various underlying mechanisms. The intense research to study the diverse nature of cancer initiation and development with the associated risk factors and modulators has resulted in innumerable molecular and cellular markers specific to different cancer types. Almost all the exogenous compounds entering the cells are metabolized by enzymes of phase I and phase II. During biotransformation of any pro-carcinogens and other xenobiotics, the activation of phase I and suppression of phase II enzymes are required to exert their mutagenic, toxic, or carcinogenic effect. Metabolic activation, detoxification, cellular proliferation, programmed cell death, angiogenesis, and metastasis have been involved in target-specific pathways leading to oncogenic mechanisms elucidation. The interaction of parent xenobiotics with a particular target can either positively, negatively, or neutrally influence their respective cellular pathways. In the study, biotransformation by CYP450 isozymes, detoxification by GST (glutathione S-transferase) and NAT (N-acetyltransferase) isozymes, DNA adduction formation, and (dibenzo[a,l]pyrene) DBP-mediated cell proliferation have been comprehensively reviewed.

Molecular docking approach to elucidate metabolic detoxification pathway of polycyclic aromatic hydrocarbons

This study assessed the molecular interactions of (±)-anti-and-syn- dibenzo[a,l]pyrene-11,12-diol-13,14-epoxide (DBPDE), 7,12- dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide (DMBADE), N2- hydroxylated-PhIP(N2-OH-PhIP), (±)-anti-and-syn-benzo[a]pyrene-7,8-diol- 9,10-epoxide (BPDE) with various Glutathione S-transferase (GST) and N- acetyltransferase (NAT) isozymes. Our in-silico data revealed that GSTP1 (- 8.83 kcal/mol), showing more plausible binding as compared to GSTM1 (-8.74 kcal/mol) and GSTA1 (ΔG: -8.03 kcal/mol) against (-)-anti-DBPDE and (+)- syn-DBPDE. We also investigated the involvement of GST and NAT isozymes in the conjugation of DMBADE andN2-OH-PhIP as a control despite their preferred routes sulfonation and glucuronidation for detoxification. The findings exhibited feeble binding of different classes of GSTs with metabolites of DMBA and PhIP, as highlighted by their free energy of binding. The enzymatic activity of GSTM1 against the most potent diol-epoxide of benzo[a]pyrene (BP), (+)-anti-BPDE, and (+)-syn-BPDE followed by GSTP1 and GSTA1 has well documented. In addition, these findings provide new perspectives for most probable mechanistic details of the detoxification pathway of PAHs and xenobiotics useful in combination therapy for future ligand-based drug discovery and development.

Nrf2 activates antioxidant enzymes in the anoxia-tolerant red-eared slider turtle, Trachemys scripta elegans.

The freshwater red-eared slider turtle, Trachemys scripta elegans, experiences weeks to months of anoxia at the bottom of ice-locked bodies of water in the winter. While this introduces anoxia-reoxygenation cycles similar to the ischemia-reperfusion events that mammals experience, T. s. elegans does not suffer any apparent tissue damage. To survive prolonged anoxia and prevent cellular damage associated with reactive oxygen species, these turtles have developed numerous adaptions, including highly effective antioxidant defenses. Herein, we examined the subcellular localization and protein expression of nuclear factor erythroid-2-related factor 2 (Nrf2), a central transcription factor responsible for modulating cellular antioxidant responses, that was found to be upregulated and localized to the nucleus in anoxic turtles. Additionally, we examined protein levels of glutathione S-transferases (GSTs) and manganese superoxide dismutase (MnSOD) antioxidant enzymes in anoxic liver, kidney, heart, and skeletal muscle tissues. MnSOD levels were significantly higher in heart and muscle during anoxia, and the four GST isozymes (GSTK1, GSTT1, GSTP1, and GSTM3) were elevated in a tissue-specific manner during anoxia and/or aerobic recovery. Together, these results indicate that Nrf2 is likely involved in activating downstream antioxidant genes in response to anoxic stress. These results provide a possible Nrf2-mediated transcriptional mechanism that supports existing findings of enhanced antioxidant defenses that allow T. s. elegans to cope with anoxia-reoxygenation cycles, and subsequent oxidative stress.

Determining site occupancy of acetaminophen covalent binding to target proteins in vitro.

Acetaminophen (APAP)-related toxicity is caused by the formation of N-acetyl p-benzoquinone imine (NAPQI), a reactive metabolite able to covalently bind to protein thiols. A targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, using multiple reaction monitoring (MRM), was developed to measure APAP binding on selected target proteins, including glutathione S-transferases (GSTs). In vitro incubations with CYP3A4 were performed to form APAP in the presence of different proteins, including four purified GST isozymes. A custom alkylation agent was used to prepare heavy labeled modified protein containing a structural isomer of APAP on all cysteine residues for isotope dilution. APAP incubations were spiked with heavy labeled protein, digested with either trypsin or pepsin, followed by peptide fractionation by HPLC prior to LC-MRM analysis. Relative site occupancy on the protein-level was used for comparing levels of modification of different sites in target proteins, after validation of protein and peptide-level relative quantitation using human serum albumin as a model system. In total, seven modification sites were quantified, namely Cys115 and 174 in GSTM2, Cys15, 48 and 170 in GSTP1, and Cys50 in human MGST1 and rat MGST1. In addition, APAP site occupancies of three proteins from liver microsomes were also quantified by using heavily labeled microsomes spiked into APAP microsomal incubations. A novel approach employing an isotope-labeled alkylation reagent was used to determine site occupancies on multiple protein thiols.

The activities of GST isozymes in stomach tissues of female obese patients

Abstract Objectives Obesity has become an important public health problem because of its increasing prevalence and relation with many diseases and mortality. Studies have shown its association with oxidative stress. In this study, the effect of obesity on total amount of thiol and some glutathione S-transferase (GST) isozymes were investigated which could serve as an important criteria in dose adjustment of some certain drugs in obese. Methods The gastric tissues removed by gastrectomy operation from 29 morbid obese female patients were analysed for thiol levels and activities of total GST, GSTT1-1 and GSTM1-1. Patients were grouped according to age, presence of hypertension and/or diabetes, and family history. Results The average total thiol was 131.22 (±7.74) nmol/mg protein with no significant differences in between the groups. GSTT1 specific activities were about 20% higher in four groups: with ages over 35 years old, with hypertension, without diabetes and finally without family history, with respect to other groups. The differences between total GST and GSTM1 activity levels of experimental groups were not significant. Conclusions This is the first study to compare activities of GST isozymes and total thiol content in the stomach tissues of obese female patients accompanying some common metabolic disorders, age and family history.

Identification of 3-Bromo-1-Ethyl-1H-Indole as a Potent Anticancer Agent with Promising Inhibitory Effects on GST Isozymes.

Indole-based heterocyclic compounds play important roles in pharmaceutical chemistry due to their unexpected biological and pharmacological properties. Herein, we describe novel biological properties (antioxidant, antimicrobial and anti-cancer) of 3- bromo-1-ethyl-1H-indole (BEI) structure. BEI was synthesized from 1-Methyl-2-phenylindole and N-bromosuccinimide and was characterized by using 1H and 13C NMR. Cytotoxicity was determined by MTT assay. Apoptosis analysis of BEI was determined by Arthur™ image-based Cytometer. Different methods were applied to assess the antioxidant activity of BEI. Molecular docking studies were conducted to determine the interactions of bonding between GST isozymes and BEI. According to the antioxidant and antimicrobial activity assays, BEI compound showed reduced total antioxidant activity compared to the Trolox standard, whereas it showed moderate antimicrobial activity against Aspergillus niger and Phytophora eryhtrospora. Notably, the BEI compound demonstrated substantial selective cytotoxicity for the first time towards cancer cell lines, and there existed a significant decrease in the percentage of live cells treated with BEI, in comparison to the control ones. Interestingly, BEI exhibited a promising glutathione S-transferase isozymes inhibition. The results of this study suggest that BEI seems to be a promising molecule to be used in the design of new anti-cancer agents that provide superiority to present commercial anti-cancer drugs.

Glutathione S-transferase enzyme activity and protein expression in patients with recurrent tonsillitis and idiopathic tonsillar hypertrophy.

The palatine tonsil is a significant part of the secondary immune system. Tonsillitis and idiopathic tonsillar hypertrophy (ITH) are the most common pathologies of this component. Although there are studies on their pathogenesis, there is insufficient study of the role of antioxidant agents. Glutathione S-transferase (GST) isozymes contribute to the antioxidation reactions in the tissue via the glutathione pathway. The purpose in this study was to reveal the levels of the GST enzyme activity and protein expression of GSTP1 and GSTA1 isozymes in patients with tonsillitis and tonsil hypertrophy, and to investigate their role in the pathogenesis of these diseases. Sixteen patients with recurrent tonsillitis and 5 patients with ITH and were included in the study. Cytosolic extracts were prepared from post-tonsillectomy tissues of both patient groups and GST enzyme activities were measured. The expression of GSTP1 was found to be significantly higher than GSTA1 in tissue samples of patients with ITH and recurrent tonsillitis (P<0.001). Increased GST activity and GSTP1 isozyme expression were shown in patients with recurrent tonsillitis compared to the idiopathic tonsillar hypertrophy study group. There was a positive correlation between the expressions of GSTP1 (P=0.040; r=0.47). Increased GST activity and GSTP1 isozymes were demonstrated histologically in the pathogenesis of ITH and recurrent tonsillitis. We believe that the data of changes in antioxidant capacity, obtained from studies with more extensive and larger samples, would support our findings.

Benzyl isothiocyanate attenuates the hydrogen peroxide-induced interleukin-13 expression through glutathione S-transferase P induction in T lymphocytic leukemia cells.

We investigated the effect of benzyl isothiocyanate (BITC) on the hydrogen peroxide-induced gene expression of a T-helper-2 cytokine, interleukin (IL)-13, in T lymphocytic leukemia Jurkat cells. The 24-h pretreatment of BITC significantly inhibited the IL-13 expression enhanced by hydrogen peroxide. Although the BITC pretreatment did not change the enhanced level of the phosphorylated c-Jun N-terminal kinase (JNK), it significantly inhibited the nuclear translocation of c-Jun induced by hydrogen peroxide. BITC also increased the protein expression of glutathione S-transferase (GST) isozymes, GSTP1/2, as well as the total GST activity. A GSTP1/2-specific inhibitor, 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX), significantly counteracted the inhibitory effect of BITC on the hydrogen peroxide-enhanced IL-13 upregulation as well as the c-Jun nuclear translocation. Taken together, these results suggested that BITC inhibits the oxidative stress-mediated IL-13 mRNA expression, possibly through interference of the c-Jun phosphorylation by GSTP.

Reduced Expression of Glutathione S-Transferase α 4 Promotes Vascular Neointimal Hyperplasia in CKD

Neointima formation is the leading cause of arteriovenous fistula (AVF) failure. We have shown that CKD accelerates this process by transforming the vascular smooth muscle cells (SMCs) lining the AVF from a contractile to the synthetic phenotype. However, the underlying mechanisms affecting this transformation are not clear. Previous studies have shown that the α-class glutathione transferase isozymes have an important role in regulating 4-hydroxynonenal (4-HNE)-mediated proliferative signaling of cells. Here, using both the loss- and gain-of-function approaches, we investigated the role of glutathione S-transferase α4 (GSTA4) in modulating cellular 4-HNE levels for the transformation and proliferation of SMCs. Compared with non-CKD controls, mice with CKD had downregulated expression of GSTA4 at the mRNA and protein levels, with concomitant increase in 4-HNE in arteries and veins. This effect was associated with upregulated phosphorylation of MAPK signaling pathway proteins in proliferating SMCs. Overexpressing GSTA4 blocked 4-HNE-induced SMC proliferation. Additionally, inhibitors of MAPK signaling inhibited the 4-HNE-induced responses. Compared with wild-type mice, mice lacking GSTA4 exhibited increased CKD-induced neointima formation in AVF. Transient expression of an activated form of GSTA4, achieved using a combined Tet-On/Cre induction system in mice, lowered levels of 4-HNE and reduced the proliferation of SMCs. Together, these results demonstrate the critical role of GSTA4 in blocking CKD-induced neointima formation and AVF failure.

Protective effect of aqueous extract of Phyllanthus fraternus against bromobenzene induced changes on cytosolic glutathione S-transferase isozymes in rat liver

The aim of this study was to investigate beneficial effect of aqueous extract of Phyllanthus fraternus (AEPF) on bromobenzene (BB) induced changes on cytosolic glutathione S-transferase (GST) isozymes in rat liver. Administration of BB significantly decreased the activity of GST, however, prior administration of AEPF prevented the BB induced decrease in GST activity. Further the cytosolic GSTs were purified from 3 groups of animals (control, BB and AEPF+BB administered) and resolved into three protein bands on SDS-PAGE. Densitometric analysis showed a significant decrease in BB group compared to control. Further, 2D PAGE analysis resolved these proteins into 8 bands which were identified as five isozymes of alpha, two of Mu and one of theta by MALDI-TOF MS and also observed decreased levels of isozymes in BB group. However, on prior administration of AEPF significantly prevented the BB induced decrease in GSTs and restored to normal levels.

Characterization of glutathione S-transferases in the detoxification of metolachlor in two maize cultivars of differing herbicide tolerance

Glutathione S-transferases (GSTs) have been widely studied in relation to their role in herbicide tolerance and detoxification. However, a detailed characterization of GSTs from herbicide tolerant and sensitive maize cultivars is still lacking. In this study, we determined the mechanism of differential tolerance between two maize cultivars which had 4-fold difference tolerance to metolachlor. The metabolism rate of metolachlor was more rapid in the tolerant cultivar (Zea mays L. cv Nongda86) than the susceptible one (Zea mays L. cv Zhengda958). Addition of the GST inhibitor ethacrynic acid reduced the metabolism of metolachlor indicating the involvement of GSTs in the differential detoxification of metolachlor. The expression profiles of 32 GST isozymes were measured using quantitative RT-PCR. The results showed the expression of GST genes were slightly up-regulated in Nongda86, but severely inhibited in Zhengdan958 24h after metolachlor treatment. The genes GSTI, GSTIII, GSTIV, GST5, GST6 and GST7, which can detoxify chloroacetanilide herbicides, were all expressed higher in Nongda86 compared to Zhendgan958. The result of GST activity was consistent with the gene expression profiles. Collectively, higher-level expression of GST genes, leading to higher GST activity and faster herbicide detoxification, appears to be responsible for the difference in tolerance to metolachlor in two maize cultivars.

Analysis of differentially expressed proteins between the spinetoram-susceptible and -resistant strains of Plutella xylostella (L.)

Plutella xylostella (L.), a worldwide vegetable pest, has developed resistance to spinetoram, which was previously effective for the control of P. xylostella (L.). The insecticidal resistance mechanism is essential to develop effective resistance management strategies. To explore the spinetoram resistance mechanism, a comparative proteomics approach was used to investigate the proteomic differences between the spinetoram-susceptible strain (SS) and spinetoram-resistant strain (RS) of P. xylostella (L.). Approximately 280 protein spots were detected on each SDS-PAGE gel. Of these, 19 proteins were successfully identified by MALDI-TOF-MS.·There were 6 significantly down-regulated spots and 13 up-regulated spots in RS, which showed significantly difference compared to that in SS. Based on the gene ontology(GO) system and KEGG database, the 19 identified proteins were classified into 6 groups including metabolisms, signal transduction, chaperones, transcriptional, protein synthesis, structural protein. Meanwhile, the expression profiles of 5 resistant related protein were further analysed by qRT-PCR. The results showed that 60S acidic ribosomal protein P2, glutathione S-transferase isozyme 3 and glutathione S-transferase delta were significantly up-regulated, while phosphoglycerate mutase and receptor for activated protein kinase C homolog were significantly down-regulated. The expression tendency of mRNA was in accordance with which of protein. This study provided evidences that spinetoram induces proteomic changes in P. xylostella (L.), and it is contributed to help us understand the resistance mechanism of P. xylostella (L.) to spinetoram.