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Objective: To construct the vectors of human glutathione S-transferase A1 (GSTA1), P1 (GSTP1), T1(GSTT1) genes and express in Escherichia coli (E. coli)., Methods: Human GSTA1, GSTP1 and GSTT1 gene whole length cDNAs were amplified by RT-PCR and then subcloned into pET-28a(+) vectors. The proteins were expressed in E. coli BL21(DE3). After purified by Ni2+ affinity chromatography, the enzymatic activities of GSTs were measured with 1-chloro-2,4 -dinitrobenzene (CDNB) as substrate., Results: The correct GSTA1, GSTP1 and GSTT1 genes were cloned. And soluble GSTA1, GSTT1, GSTP1 proteins were expressed in E.coli. After purification, GSTA1, GSTT1 and GSTP1 showed good enzymatic activities, which were 17.55, 0.02, 18.75 μmol·min-1·mg-1, respectively., Conclusion: The expression plasmids for GSTA1, GSTT1 and GSTP1 have been constructed and the recombinant proteins are expressed successfully.

Objectives: Glutathione S-transferase (GST) isoenzymes play important roles in resistance to cell apoptosis and carcinogenesis. We aimed to establish the relationship between GST expression and the prognosis of upper urinary tract urothelial carcinoma (UTT-UC) in Taiwan., Methods: This study retrospectively reviewed 46 patients with pathologically confirmed UUT-UC at Kaohsiung Medical University Hospital. In each patient, expression of GSTT1 and GSTP1 was compared between urothelial carcinoma and normal urothelial cells by Western blotting., Results: GSTP1 expression in the UUT-UC cells was significantly higher than that in normal urothelial cells (1.6 fold, p<0.001). Expression of GSTT1 was significantly associated with the invasiveness of the carcinoma (p=0.006)., Conclusions: In UUT-UC, GSTP1 might be a potential tumor marker, whereas high GSTT1 expression could be used as an indicator of cancer progression. This study is the first to demonstrate potential applications of different GST isoenzymes for biomolecular analysis of UUT-UCs in Taiwan.

Carcinogen detoxifying genes may be involved in pathogenesis of head and neck cancer (HNC). CYP1A1 is phase I enzyme that converts carcinogens into water soluble compounds which are easily excreted from body. GSTs constitute phase II detoxification enzymes that recognize these highly electrophilic compounds and detoxify them. Abnormal expression of these genes can potentially lead to cancer initiation. In present study, we analyzed protein expression of these genes in a total of 192 HNC patients and noncancerous healthy control serum samples screened for GSTs specific activity by ELISA. Furthermore, expression of these molecules was also determined in 49 HNC tissues/ adjacent control tissue by immunohistochemistry with specific antibodies. Mean serum GSTs specific activity was found to be 7.7 (+11.5)U/L in HNC patients and 11.4 (+7.5)U/L in controls. Significant decrease (P < 0.05) in GSTs specific activity was observed in HNC patients compared with controls (P < 0.001). Data for immunohistochemistry showed that CYP1A1 and GSTT1 was down expressed whereas GSTP1 was over expressed in HNC tissues compared with adjacent normal control tissues. Results of immunohistochemistry revealed 63 % HNC tissues had weak, 27 % moderate and 10 % strong staining for CYP1A1. For GSTT1, 27 % HNC tissues had no staining, 49 % weak staining, 16 % moderate and 8 % strong staining. Similarly for GSTP1, percentages for weak, moderate and strong staining were 6 %, 12 % and 82 % respectively. These reduced proteins observed in cancer patients highlight a potential breach on DNA repair mechanism when compared with control. Thus altered expression of these detoxifying molecules may collectively contribute to HNC development in Pakistani population.

Objective: The immunohistochemical staining characteristics of glutathione S-transferase (GST) alpha (GSTA), pi (GSTP), mu (GSTM4), and theta (GSTT1) and P53 were investigated in laryngeal squamous cell carcinoma (LSCC) cases and normal laryngeal tissue from 46 patients. The relationships between expression of the GST isoenzymes and some clinicopathologic features were also examined., Patients and Methods: For immunohistochemical studies, tissues from 46 patients with LSCC at the Dr. Lütfi Kirdar Kartal Education and Research Hospital were used. The relationship between expression of the GST isoenzymes and P53 in normal and tumour tissue was analyzed using the Wilcoxon signed rank test. The correlation between GST isoenzymes and P53 and clinicopathologic data was also examined using the Spearman rank test., Results: When the normal and tumour tissues of these cases were compared according to their staining intensity and percentage of positive staining, GSTA expression in normal cells was significantly higher than in tumour cells, and GSTP and P53 expression was higher in tumour cells (p < .05). GSTM and GSTT1 expression was higher in normal cells; however, the statistical significance was low (p > .05). There was no correlation between P53 and GST expression in patients with LSCC. When the immunohistochemical results of GST isoenzymes and P53 were correlated with the clinical parameters, GSTA expression was increased in poorly differentiated laryngeal tumour, but GSTM4 and GSTT1 expression was decreased (p < .05)., Conclusion: According to these results, GST-A, -P, and T1 and P53 were important in the diagnosis of LSCC.

Human embryonic stem cells (hESCs) offer a potential unlimited source for functional human hepatocytes, since hESCs can differentiate into hepatocyte-like cells displaying a characteristic hepatic morphology and expressing several hepatic markers. These hepatocyte-like cells could be used in various human in vitro hepatocyte assays, e.g. as a test system for studying drug metabolism and drug-induced hepatotoxicity. Since the toxic effect of a compound is commonly dependent on biotransformation into metabolites, the presence of drug metabolising enzymes in potential test systems must be evaluated. We have investigated the presence of glutathione transferases (GSTs) in hepatocyte-like cells by immunocytochemistry and Western blotting. Results show that these cells have high levels of GSTA1-1, whereas GSTP1-1 is not present in most cases. GSTM1-1 is detected by immunocytochemistry but not by Western blotting. In addition, GST activity is detected in hepatocyte-like cells at levels comparable to human hepatocytes. These results indicate that the hepatocyte-like cells have characteristics that closely resemble those of human adult hepatocytes.

In rat liver, in addition to their intrinsic transferase activity, alpha-class GSTs have Se-independent glutathione peroxidase activity toward fatty acid hydroperoxides, cumene hydroperoxide and phospholipids hydroperoxides but not toward H(2)O(2.) We have previously shown that hepatic GST activity by these isoenzymes is significantly increased 24h after cadmium or manganese administration (Casalino et al., 2004). Here it is reported that Se-independent glutathione peroxidase activity by alpha-class GSTs is also stimulated in the liver of intoxicated rats. The stimulation is associated with a higher level of alpha-class GST proteins, whose induction is blocked by actinomycin D co-administration. The observed Se-independent glutathione peroxidase activity is due to alpha-class GST isoenzymes, as indicated by the studies with diethyldithiocarbamate which, at any concentration, equally inhibits both GST and Se-independent glutathione peroxidase and is an uncompetitive inhibitor of both enzymes. As for liver Se-GSPx, it is not at all affected under these toxic conditions. For comparison, we have evaluated the status of another important antioxidant enzyme, NAD(P)H:quinone reductase, 24h after cadmium or manganese administration. NQO1 too results strongly stimulated in the liver of the intoxicated rats. In these animals, a higher expression of Nrf2 protein is observed, actively translocated from the cytoplasm to the nucleus. The results with the transcription inhibitor, actinomycin D, and the effects on Nrf2 protein are the first clear indication that acute manganese intoxication, similarly to that of cadmium and other heavy metals, increases both the hepatic level of Nrf2 and its transfer from the cytoplasm to the nucleus where it actively regulates the induction of phase II enzymes.

Background: The glutathione S-transferases (GST) can metabolise endogenous and exogenous toxins and carcinogens by catalysing the conjugation of diverse electrophiles with reduced glutathione (GSH). Variations of GST enzyme activity could influence the susceptibility of developing cancers in certain areas of the gastrointestinal tract., Aims: The expression of the components of the glutathione system in the colon was investigated with respect to age, gender and localisation., Methods: Biopsies of macroscopically normal mucosa from both proximal and distal colon were collected from 208 patients (106 females, 102 males; mean age 61 years), who underwent colonoscopy for various clinical reasons. GSH content, total GST enzyme activity and the levels of the GST isoenzymes glutathione S-transferase P1 (GSTP1) and glutathione S-transferase M1 (GSTM1) were determined., Results: GST enzyme activity, GSH and GSTP1 levels decreased significantly from proximal to distal colon (GST activity: 264 vs. 244 nmol/min/mg protein, p < 0.001, GSH content: 32 vs. 30 nmol/mg protein, p = 0.022 and GSTP1 levels: 2.25 vs. 2.10 mug/mg protein, p < 0.001). In female patients there was a significant stepwise increase of GST-activities and GSTP1 levels from the age of under 50 years to over 70 years. Oral sex hormone substitution among female patients between 50 and 70 years suppressed GST-activities and GSTP1 content., Conclusions: The GSH-system in the colonic mucosa is expressed at a lower level in the distal colon (sigma) than in the colon transversum; whether this small difference translates into variations of incidence of colorectal cancer remains to be seen. Females express higher enzyme levels as they grow older, while in males no significant age effects were found. Elderly females might be better equipped with protective GSH-enzymes in the colon than males and this could contribute to the lower incidence of colorectal carcinomas in females.

This article reports the first absolute quantitative analysis of expression patterns of murine transcripts (Gsta1/2, Gsta3, Gsta4, Gstm1, Gstm2, Gstm3, Gsto1, Gstp1/2, Gstt1, Gstt2) coding for most glutathione S-transferases (GSTs) of alpha, mu, omega, pi, and theta classes. We examine how the steady-state numbers of transcripts are modulated in association with: three animal organs (liver, kidney, and lung) where extensive detoxification occurs; two species (Mus musculus and Mus spretus) representing common laboratory and aboriginal mice; and two genetic and animal living conditions (wild-derived inbred animals and free-living mice). Moreover, quantitations performed examine how the pulmonary steady-state Gst mRNA amounts are affected in M. musculus by paraquat (a superoxide generator), and in M. spretus by dwelling at a polluted area. The results point to complex tissue-, species-, and life condition-dependent expression of the investigated transcripts. Among others, they show: i) the ubiquity of most transcripts, except Gstm3 mRNA that was virtually absent or at very low amounts (< or = 0.001 molecules/pg) in kidney and lung of M. spretus; ii) unique expression profiles for each transcript and mouse organ examined; iii) outstanding species-specific differences in basal amounts of most Gst mRNAs, this effect being most apparent in the case of Gsta1/2, Gsta3, Gstm2, Gsto1, Gstt1, and Gstt2; iv) paraquat-induced upregulation of most Gst mRNAs, with the notable exception of those coding for theta class GSTs; v) a tendency for mice dwelling at a wildlife reserve of having lower and more homogeneous Gsta3 mRNA levels than those collected in an anthropogenic environment.

Atrazine is one of the most widely used herbicides in the United States and has been detected, occasionally, at low levels in drinking water sources. The biotransformation of atrazine in humans has not been fully characterized. Rodent studies suggest Phase I-dominated biotransformation with minor Phase II-mediated biotransformation by glutathione S-transferase(s) (GST). In human urine, mercapturates of atrazine are significant metabolites, yet the specific GST form(s) responsible for glutathione (GSH) conjugation have not been identified. Using recombinant alpha, mu, pi and theta class human GSTs, we demonstrated that only hGSTP1-1 displays significant activity toward atrazine (7.1 nmol/min/mg protein). We also confirmed that mouse GST Pi (pi) protein is responsible for the GSH-dependent biotransformation of atrazine in mouse liver; recombinant mGSTP1-1 had a specific activity of 7.3-nmol/min/mg protein. Furthermore, cytosolic fractions from mouse and human liver conjugated atrazine with glutathione at rates of 282.3 and 3.0 pmol/min/mg, respectively. Docking studies of the atrazine-GST conjugate in the hGSTP1-1 substrate-binding site were used to elucidate a basis for the dramatic difference in activity between mouse GSTP1-1 and GSTP2-2 (7.14 versus 0.02 nmol/min/mg protein, respectively). The inactivity of mGSTP2-2 appears to be attributable to an indirect structural disruption of the G-site by Pro12. Possible effects of the hGSTP1 polymorphisms were investigated. No significant differences in catalytic-specific activity were noted among purified proteins corresponding to the four hGSTP1 variants: hGSTP1(*)A (most common form), hGSTP1(*)B (Ile105Val), hGSTP1(*)C (Ile105Val, Ala114Val), and hGSTP1(*)D (Ala114Val). Overall, this work supports a physiological role for GSTs in atrazine biotransformation and indicates a novel diagnostic substrate for human and mouse GSTP1-1 proteins.