Sonja Suvakov

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

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

GSTA1, GSTM1, GSTP1, and GSTT1 polymorphisms and susceptibility to smoking-related bladder cancer: A case-control study

OBJECTIVES Glutathione S-transferases (GSTs) are a family of enzymes involved in detoxification. Genes encoding for GSTA1, GSTM1, GSTP1, and GSTT1 proteins are polymorphic, which can result in complete or partial loss of enzyme activity. Previous studies have associated polymorphisms of GSTA1, GSTM1, and GSTP1 genes with a higher risk of bladder cancer, but this is still controversial. Potential role of GSTA1 polymorphism in susceptibility to bladder cancer in Whites is lacking. We examined association between GSTA1, GSTM1, GSTP1, and GSTT1 gene variants and bladder cancer risk and evaluated whether they were modified by smoking. MATERIALS AND METHODS A hospital-based case-control study recruited 201 incidence cases and 122 age-matched controls. Deletion polymorphism of GSTM1 and GSTT1 was identified by polymerase chain reaction method. Single nucleotide polymorphism of GSTA1 and GSTP1 was identified by restriction fragment length polymorphism method. Uniconditional multivariate logistic regression was applied to model association between genetic polymorphisms and bladder cancer risk, as well as effect modification by smoking. RESULTS No significant difference was observed in the distributions of GSTM1, GSTT1, GSTA1, and GSTP1 gene variants between patients and controls. None of the examined polymorphisms was significantly associated with bladder cancer risk independently. The results of gene-smoking interaction analyses indicated a significant combined effect of smoking and all common GST polymorphisms tested (P for trend = 0.001). However, the most significant effect on bladder cancer risk was observed in smokers carrying lower activity GSTA1-AB/BB and GSTM-null genotype (OR = 3.5, P < 0.05) compared with GSTA1-AA and GSTM1-active non-smokers. Overall, the risk observed did not significantly differ with respect to quantity of cigarettes smoked. However, heavy smokers with GSTM1-null genotype had 2 times higher risk of bladder cancer than GSTM1-null light smokers (OR = 4.8 vs. OR = 2.0) when GSTM1-active non-smokers served as reference group. Smokers carrying both GSTM1-null and GSTA1-AB + BB genotypes exhibited the highest risk of bladder cancer (OR = 2.00, P = 0.123). CONCLUSIONS Null or low-activity genotypes of the GSTA1, GSTM1, GSTT1, and GSTP1 did not contribute independently towards the risk of bladder cancer in our patients. However, in association with smoking, both low activity GSTA1 and GSTM1-null genotype increase individual susceptibility to bladder cancer.

Glutathione S-Transferase T1, O1 and O2 Polymorphisms Are Associated with Survival in Muscle Invasive Bladder Cancer Patients

Objective To examine the association of six glutathione transferase (GST) gene polymorphisms (GSTT1, GSTP1/rs1695, GSTO1/rs4925, GSTO2/rs156697, GSTM1, GSTA1/rs3957357) with the survival of patients with muscle invasive bladder cancer and the genotype modifying effect on chemotherapy. Patients and Methods A total of 105 patients with muscle invasive bladder cancer were included in the study. The follow-up lasted 5 years. The effect of GSTs polymorphisms on predicting mortality was analyzed by the Cox proportional hazard models, while Kaplan-Meier analysis was performed to assess differences in survival. Results GSTT1 active, GSTO1 Asp140Asp or GSTO2 Asp142Asp genotypes were independent predictors of a higher risk of death among bladder cancer patients (HR = 2.5, P = 0.028; HR = 2.9, P = 0.022; HR = 3.9, P = 0.001; respectively) and significantly influenced the overall survival. There was no association between GSTP1, GSTM1 and GSTA1 gene variants with overall mortality. Only GSTO2 polymorphism showed a significant effect on the survival in the subgroup of patients who received chemotherapy (P = 0.006). Conclusion GSTT1 active genotype and GSTO1 Asp140Asp and GSTO2 Asp142Asp genotypes may have a prognostic/pharmacogenomic role in patients with muscle invasive bladder cancer.

The Role of GSTM1 and GSTT1 Polymorphism in Patients with Renal Cell Carcinoma

The Role of GSTM1 and GSTT1 Polymorphism in Patients with Renal Cell Carcinoma Members of the glutathione S-transferase (GST) superfamily exhibit polymorphic expression. GSTs are investigated as biomarkers of risk for various cancers, including renal cell carcinoma (RCC). The aim of this study was to test the association between GSTM1 and GSTT1 polymorphism and susceptibility to RCC, independently or in conjunction with known risk factors. Genomic DNA was isolated from 182 controls and 76 patients with RCC. GSTM1 and GSTT1 genotypes were determined by multiplex PCR. Data obtained were analyzed with respect to RCC risk factors including smoking and occupational exposure. The frequency of GSTM1-null genotype was higher in patients with RCC (60.5%) compared to controls (47.2%). GSTT1-null genotype was found in 28.6% controls and 27.6% of cases. GSTM1-null individuals exhibit 1.9-fold increased risk of RCC (95% CI: 1.06-3.33). The presence of GSTT1 active genotype was associated with increased risk of RCC in occupationally exposed subjects when unexposed GSTT1-null subjects were used as a comparison group (OR: 2.48; 95% CI: 1.05-5.86). No association was found between the inactive form of GSTM1 and GSTT1 and smoking in RCC patients. In a Serbian cohort of patients, the presence of a GSTM1 active genotype is protective against RCC, whereas a GSTT1 active genotype increases RCC risk in occupationally exposed subjects. Uloga Polimorfizma Glutation S-Transferaza M1 i T1 kod Pacijenata sa Karcinomom Bubrežnog Parenhima Genetski polimorfizam je prisutan kod mnogih članova superfamilije glutation-S transferaza. U toku su istraživanja koja ispituju ulogu GST kao biomarkera za nastanak različitih karcinoma, uključujući karcinom bubrežnog parenhima (KBP). U ovoj studiji je ispitivana uloga GSTM1 i GSTT1 polimorfizma u nastanku KBP, nezavisno ili udruženo sa poznatim faktorima rizika za ovaj karcinom. DNK je izolovana iz krvi 182 kontrolna subjekta i 76 bolesnika sa KBP. Polimorfizam GSTM1 i GSTT1 je određivan metodom PCR-a. Dobijeni rezultati su analizirani u odnosu na faktore rizika za KBP, uključujući pušenje i profesionalnu izloženost. Učestalost GSTM1-nultog genotipa je bila viša kod bolesnika sa KBP (60,5%) nego kod kontrola (47,2%). Prisustvo GSTT1-nultog genotipa je utvrđeno kod 28,6% kontrola i 27,6% bolesnika sa KBP. Nosioci GSTM1-nultog genotipa imaju 1.9-puta veći rizik za KBP (95% CI: 1,06-3,33). Prisustvo GSTT1 aktivnog genotipa je udruženo sa povećanim rizikom za KBP kod profesionalno izloženih subjekata kada su kao referentna grupa uzeti neizloženi nosici GSTT1-nultog genotipa (OR: 2,48; 95% CI: 1,05-5,86). Nije otkrivena povezanost između nedostatka aktivne forme GSTM1 i GSTT1 i pušenja kod obolelih od KBP. Studija izvedena u Srbiji je pokazala da prisustvo GSTM1 aktivnog genotipa štiti od nastanka KBP, dok prisustvo GSTT1 aktivnog genotipa povećava rizik kod profesionalno izloženih osoba.

Association of GSTO1 and GSTO2 Polymorphism with Risk of End-Stage Renal Disease Development and Patient Survival

Summary Background: Oxidative stress in patients with end-stage renal disease (ESRD) is associated with long-term cardiovascular complications. The cytosolic family of glutathione S-transferases (GSTs) is involved in the detoxication of various toxic compounds and antioxidant protection. GST omega class members, GSTO1 and GSTO2 possess, unlike other GSTs, dehydroascorbate reductase and deglutathionylation activities. The aim of this study was to clarify the role of genetic polymorphisms of GSTO1 (rs4925) and GSTO2 (rs156697) as risk determinants for ESRD development, as well as in the survival of these patients. Methods: A total of 199 patients and 199 healthy subjects were included in the study and genotyped for both GSTO1 and GSTO2 polymorphism. Protein thiol and carbonyl groups as markers of protein oxidative damage were determined spectrophotometrically. Cox proportional hazard model and Kaplan-Meier analysis were performed to investigate the role of GSTO1 and GSTO2 genetic polymorphism on mortality of ESRD patients during the follow-up period (36 month). Results: Individuals carrying the variant GSTO2 GG genotype were at 2.45-fold higher risk of ESRD development compared to the wild type GSTO2 AA genotype (OR=2.45; 95%CI=1.18-5.07; p=0.016). The results of GSTO1/GSTO2 haplotype analysis showed that the haplotype combi - nation of GSTO1 (*A)/GSTO2 (*A) (GSTO1 variant/GSTO2 wild type allele) was protective for ESRD (OR=0.23 95%CI=0.12-0.44, p=0.001). Patients carrying at least one GSTO1 reference allele have shorter mean overall (Log rank=2.844, p =0.241) and cardiovascular survival probability (Log rank=4.211, p=0.122). Conclusions: GSTO polymorphisms have been shown to act as significant markers in assessing the risk of ESRD development and patients’ survival.

Antiepileptic Drugs Affect Protein, Lipid and DNA Oxidative Damage and Antioxidant Defense in Patients with Epilepsy

Summary Background: To get more insight into the effects of the most widely used antiepileptic drugs (AEDs) on the prooxidant/ antioxidant balance in epilepsy, a comparative analysis of the byproducts of oxidative damage and antioxidant de fense mechanisms was performed in patients with epilepsy treated with lamotrigine, carbamazepine and valproic acid. Methods: Byproducts of oxidative damage to proteins (reactive carbonyl derivatives, RCD and protein thiol groups, PSH), lipids (urinary isoprostanes, 8-epi-PGF2a) and DNA (urinary 8-hydroxy-2’-deoxyguanosine, 8-OHdG), as well as the activities of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPX) were measured in 60 patients with newly diagnosed seizure (at illness onset and after 6 months of treatment with lamotrigine, carbamazepine or valproic acid) and in 20 healthy controls. Results: In patients with epilepsy, RCD, urinary 8-epi-PGF2a and 8-OHdG, together with SOD and GPX activities were significantly increased, while P-SH were only slightly decreased. After 6 months of treatment with AEDs, a decrease was observed in RCD, urinary 8-epi-PGF2a and 8-OHdG to values slightly higher or similar to the control, while P-SH remained unchanged. A decrease was also observed in SOD and GPX activities, although they remained significantly in creased compared to controls. Conclusions: The results of this study have shown that treatments with lamotrigine, carbamazepine and valproic acid affect the prooxidant/antioxidant balance in patients with epilepsy. Kratak sadržaj Uvod: U pokušaju da se razjasne mehanizmi kojima naj cesce korisceni antiepilepticki lekovi (AEL) uticu na ravnotezu izmedu pro- i antioksidanasa u epilepsiji, u ovom radu je izvedena uporedna analiza pokazatelja oksidativnog o{te}enja i anti oksidantnih mehanizama za{tite kod bolesnika sa epilepsijom na terapiji lamotriginom, karbamazepinom ili valproi~ - nom kiselinom. Metode: Pokazatelji oksidativnog o{te}enja proteina (sa dr`aj karbonilnih grupa, RCD i koncentracija proteinskih tiol grupa plaz me, P-SH), lipida (urinarni izoprostani, 8-epi-PGF2a) i DNK (urinarni deoksiguanozin, 8-OHdG), kao i aktivnost anti oksidantnih enzima, superoksid dizmutaze (SOD) i glutation peroksidaze (GPX) odre|ivani su kod 60 bolesnika sa novo dijagnostikovanom epilepsijom (nakon prvog napada i posle 6 meseci, na terapiji lamotriginom, karbamazepinom ili valproi~nom kiselinom) i kod 20 zdravih osoba. Rezultati: Kod bolesnika sa epilepsijom, koncentracija RCD, 8-epi-PGF2a i 8-OHdG, kao i aktivnosti SOD i GPX su zna~ajno pove}ane, dok je koncentracija P-SH umereno smanjena. Nakon {estomese~ne terapije AEL, uo~eno je smanjenje koncentracije RCD, 8-epi-PGF2a i 8-OHdG do vrednosti sli~nih onim u kontrolnoj grupi, dok je koncentracija P-SH ostala neizmenjena. Zna~ajno smanjenje je uo~eno i u aktiv nosti SOD i GPX, mada vrednosti ostaju zna~ajno pove}ane u odnosu na kontrolu. Zaključak: Terapija lamotriginom, karbamazepinom i valproicnom kiselinom uti~e na ravnote`u izme|u prooksidanasa i antioksidanasa kod bolesnika sa epilepsijom.

Upregulated glutathione transferase omega-1 correlates with progression of urinary bladder carcinoma

ABSTRACT Objectives: Newly discovered glutathione transferase omega 1 (GSTO1-1) plays an important role in the glutathionylation cycle, a significant mechanism of protein function regulation. GSTO1-1 expression pattern has not been studied in transitional cell carcinoma (TCC), as yet. Methods: A total of 56 TCC tumor and corresponding non-tumor specimens were investigated. Glutathione content and thioltransferase activity were measured spectrophotometrically. Protein-glutathione mixed disulfides were measured fluorimetrically. GSTO1-1 expression was determined by immunoblot and qPCR. Immunoprecipitation with GSTO1-1 antibody was followed by immunoblot using anti-GSTO1, GSTP1, c-Jun, JNK, Akt, phospho-Akt, and ASK1 antibody, while for the total S-glutathionylation levels non-reducing electrophoresis was performed. Results: The contents of reduced glutathione and thioltransferase activity were significantly increased in tumor compared to non-tumor tissue. The increased GSTO1 expression in tumor tissue showed clear correlation with grade and stage. However, decreased total protein glutathionylation level in tumor compared to non-tumor samples was found. Immunoprecipitation has shown an association of GSTO1-1 with GSTP1, Akt, phospho-Akt, and ASK1 proteins. Conclusions: GSTO1 deglutathionylase activity suggests its potential important role in redox perturbations present in TCC. Increased GSTO1-1 expression might contribute to TCC development and/or progression supporting the notion that GSTO1-1 may be a promising novel cancer target.

Associations of GSTM1*0 and GSTA1*A genotypes with the risk of cardiovascular death among hemodialyses patients.

BackgroundThe presence of glutathione transferase (GST) M1 null genotype (GSTM1-null) in end-stage renal disease (ESRD) patients is associated with lower overall survival rate in comparison to those with GSTM1-active variants. We examined association between GSTM1 and GSTT1 deletion polymorphisms as well as SNPs in GSTA1/rs3957357 and GSTP1/rs1695 genes with overall and cause-specific cardiovascular mortality in ESRD patients.MethodsTotal of 199 patients undergoing hemodialysis were included in the study. Median value of time elapsed from dialysis initiation until the death, or the end of follow-up was 8 ± 5 years. The effect of GSTM1, GSTT1, GSTP1 and GSTA1 gene polymorphisms on predicting overall and specific cardiovascular outcomes (myocardial infarction, MI or stroke) was analyzed using Cox regression model, and differences in survival were determined by Kaplan-Meier.ResultsGSTM1-null genotype in ESRD patients was found to be independent predictor of overall and cardiovascular mortality. However, after false discovery rate and Bonferroni corrections this effect was lost. The borderline effect modification by wild-type GSTA1*A/*A genotype on associations between GSTM1-null and analyzed outcomes was found only for death from stroke. Homozygous carriers of combined GSTM1*0/GSTA1*A genotype exhibited significantly shorter time to death of stroke or MI in comparison with carriers of either GSTM1-active or at least one GSTA1*B gene variant. The best survival rate regarding cardiovascular outcome was found for ESRD patients with combined GSTM1-active and mutant GSTA1*B/*B genotype.ConclusionsCombined GSTM1*0/GSTA1*A genotypes might be considered as genetic markers for cardiovascular death risk in ESRD patients, which may permit targeting of preventive and early intervention.

GSTM1 genotype is an independent prognostic factor in clear cell renal cell carcinoma

PURPOSE Owing to dual functionality of cytosolic glutathione S-transferases (GSTs), they might affect both the development and the progression of renal cell carcinoma (RCC). However, the data on the prognostic value of GST polymorphism in patients with RCC are scarce. Hence, we evaluated the effect of GST gene variants on both the risk of RCC development and the postoperative prognosis in patients with clear cell RCC (ccRCC). METHODS GST genotypes were determined in 305 patients with RCC and 326 matched controls, whereas the overall survival was evaluated in patients with ccRCC only. The presence of GSTM1:ASK1 protein-protein interaction in ccRCC tissue samples was analyzed by methods of immunoprecipitation and immunoblot. RESULTS We noted an increased risk of RCC development in carriers of GSTM1-null and GSTP1-variant genotype (P<0.05). On the contrary, survival analysis indicated shorter overall survival for patients with ccRCC with GSTM1-active genotype (P = 0.026). Furthermore, patients with ccRCC with GSTM1-active genotype had significantly higher hazard ratio (P<0.05), in analyzed regression models, compared with the carriers of GSTM1-null genotype. Finally, the presence of GSTM1:ASK1 protein-protein interaction was found in all RCC tissue samples studied. CONCLUSIONS Carriers of GSTM1-null and GSTP1-variant genotypes are in increased risk of RCC development. On the contrary, GSTM1-null genotype is associated with favorable postoperative prognosis in ccRCC. The possible molecular mechanism underlying the role of GSTM1 protein in RCC progression might be the presence of GSTM1:ASK1 protein-protein interaction. Hence, determination of GSTM1-genotype might serve as a valuable indicator in both RCC risk assessment and postoperative prognosis.

Association of glutathione-S-transferase gene polymorphism and lipoprotein subclasses in hemodialysis patients.

OBJECTIVES End-stage renal disease (ESRD) is characterized by profound dyslipidemia and enhanced oxidative stress. The patients also show evidence of exhausted and/or deficient anti-oxidative defense enzymes, one of them being glutathione-S-transferase (GST). This study investigates relationship between GST gene polymorphism and low-density lipoprotein (LDL) and high-density lipoprotein (HDL) subclasses in ESRD. DESIGN AND METHODS GSTM1, T1, and P1 genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism in 160 patients undergoing hemodialysis. LDL and HDL subclasses were separated by gradient gel electrophoresis and biochemical parameters were measured by routine laboratory methods. RESULTS GSTM1-positive patients had higher proportion of small, dense LDL III particles than those with GSTM1-null genotype (P<0.05). Similarly, GSTP1-Ile/Ile patients had higher proportion of LDL III (P<0.05), but more HDL 2b and less HDL 3a particles than GSTP1-Ile/Val and Val/Val carriers (P<0.05). LDL subclass distribution in smokers with GSTM1-null genotype was shifted towards smaller particles, as compared to GSTM1-positive and GSTM1-null non-smokers. Smokers with GSTP1-Ile/Val and Val/Val genotypes had smaller LDL size than their non-smoking counterparts (P<0.05). Both smokers and non-smokers with GSTP1 Ile/Ile genotype had more LDL III particles than non-smokers carrying Val allele. Non-smokers with GSTP1 Ile/Ile genotype had more HDL 2b subclasses than non-smokers with GSTP1-Ile/Val and Val/Val (P<0.05), but less HDL 3a particles than smokers with GSTP1-Ile/Val and Val/Val genotypes (P<0.05). GSTT1 gene polymorphism had no effect on lipoprotein subclass distributions. CONCLUSIONS Our results demonstrate significant associations between low activity GST genotypes and proatherogenic lipoprotein particles in hemodialysis patients which might further increase their cardiovascular disease risk.