Fruits and vegetables or their natural constituents which increase detoxication enzymes and/or reduce activating enzymes are considered as good candidates to prevent chemically-induced carcinogenesis. In this study, rats were fed a diet supplemented with 20% onion powder for 9 days. Several cytochrome P450 (CYP)s enzymes (CYP 1A, 2B, 2E1, 3A), which are involved in carcinogen activation, were determined by measuring their enzyme activities using specific substrates. In addition, phase II enzymes activities such as UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST), involved in detoxication of carcinogens, were measured. Protein levels of CYPs and GST A1/A2, A3/A5, Ml, M2 and P1 were measured using antibodies in Western blots. Consumption of onion induced CYP 1A and CYP 2B activities while it decreased CYP 2E1 activity. This later modification was accompanied by a decrease of CYP 2E1 levels. The same dietary treatment caused a slight increase of the total GST activity. The relative proportions of GST subunits were modified. GST Al/A2 subunits were increased while GST A3/A5 and GST M2 subunits were decreased and GST M1 and P1 were not modified. Onion consumption also increased p-nitrophenol UGT activity. Taken together, these results suggest that the decrease of CYP 2E1 and the increase of phase II enzymes by onion can afford protection against some carcinogens, while the decrease of some GST subunits could increase the genotoxic effects of other chemicals. The modulating effect of onion could be ascribed to alk(en)yl polysulphides and/or glycosides of flavonols, which were identified in the onion powder.

Incomplete intestinal metaplasia (IM) is a precursor of stomach cancer. To identify risk factors of incomplete IM, a 2-stage survey was carried out in 1995 among 1,485 residents in Matzu, an area with highest mortality from stomach cancer in Taiwan. There were 312 study subjects including 174 men and 138 women sampled for the gastroendoscopic examination of IM. Information on personal and familial history of stomach cancer, cigarette smoking, alcohol consumption and intake frequency of various salted food items were obtained by personal interview based on a structured questionnaire. Blood samples were collected from each participant. Four biopsies per subject were taken from all subjects at gastroendoscopic examination to diagnose the status of IM pathologically. The Helicobacter pylori in biopsies was detected by the histomorphological or immunochemistry method, and antibodies against H. pylori in serum by the enzyme-linked immunosorbent assay. Plasma level of selenium was determined by atomic absorption spectrometry, plasma level of retinol, alpha-tocopherol, alpha-carotene, and beta-carotene by high performance liquid chromatography, genotypes of glutathione S-transferase (<em>GST</em>) <em>M1</em> and T1 and cytochrome P450 (CYP) 2E1 by polymerase chain reaction. The significant association between history of stomach cancer among first-degree relatives and incomplete IM was found (odds ratio [OR] = 2.50; 95% confidence interval [CI] = 1.15-5.43). There was no association between H. pylori infection and incomplete IM. Alcohol drinkers for >20 years had an elevated risk compared to non-drinkers (OR = 3.34; 95% CI = 1.19-9.39). No associations between incomplete IM and plasma levels of selenium, retinol, alpha-tocopherol, alpha-carotene and beta-carotene were found. Salted food including salted meat, dehydrated salted vegetables and raw salted seafood consumed at ages of </=15 and 16-30 years old was associated with an increased IM risk with OR ranging from 2-3. More striking associations between incomplete IM and salted food intake were observed among subjects with genotypes of <em>GST</em><em>M1</em> null, <em>GST</em>T1 non-null and CYP2E1 c1/c1. Our study suggests the importance of gene-environment interaction on the development of incomplete IM.

Evidence for a protective role of the glutathione biotransformation system in carcinogenesis is growing. However, most data on this system in relation to colorectal cancer originate from animal studies. Here we review the human data. In humans, a significant association was found between glutathione S-transferase (GST) activity in the mucosa along the gastrointestinal tract and the corresponding tumour incidence. Low activity was correlated with high tumour incidence and vice versa. Also, in normal colonic mucosa, GST activity is lower in patients at risk of colon cancer than in healthy controls and therefore interventions which increase the glutathione detoxification capacity may reduce cancer incidence. Consumption of vegetables and fruit is associated with a lower risk of colorectal cancer. Human intervention studies showed that (components from) vegetables induced colonic glutathione detoxification capacity. Such an effect could contribute to a lower colon cancer risk, but further data are needed. The human GSTs consist of four main classes--alpha (A), mu (M), pi (P) and theta (T)--each of which is divided into one or more isoforms. Functional polymorphisms are known for the GST genes M1, P1 and T1 and they all lead to less active enzymes compared to the wild-type gene products. However, studies that compared these GST polymorphisms in relation to colon cancer risk were not conclusive with respect to an increased or decreased risk of a particular genotype. Diet or medication can also influence the expression levels of specific isoenzymes and the effect of such interventions on cancer risk deserves more attention.

OBJECTIVE

Glutathione S-transferases (GSTs) are phase II metabolizing enzymes which catalyze the conjugation of glutathione (GSH) to electrophilic substrates and possess selenium-independent glutathione peroxidase activity. The GST enzyme family includes the cytosolic isoforms GST-alpha, mu (GSTM), pi (GSTP), theta (GSTT) and sigma (GSTS). GSTT1, P1 and M1 are polymorphic and altered polymorphic frequency of genes encoding these proteins has been suggested as a potential risk factor for the development of hematopoietic malignancies. Overexpression of GSTs has also been implicated in chemotherapeutic drug resistance. This study was undertaken to elucidate the potential functional relevance of these genetic polymorphisms in hematopoiesis.

METHODS

GST genotype of 14 hematopoietic cell lines was determined by polymerase- chain-reaction (PCR). Gene expression of GSTs in a cell line was detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) on TaqMan 7700 and by semi-quantitative RT-PCR. Cytosolic GST protein expression was detected by Western blot. GST conjugation activity was assayed using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate.

RESULTS

GSTP1 expression was higher than other GSTs in 13/14 cell lines and paralleled CDNB conjugation activity. GSTP1 and GSTM1 predominated in lymphoid lines whilst T1 expression was relatively greatest in erythroid lines but was absent in 7/12 non-null lines. GSTT2 was expressed in only 3/4 lines. The 3 cell lines which expressed GSTA1 were all erythroid.

CONCLUSIONS

Glutathione S-transerases showed differential lineage expression in hematopoietic cell lines. This implies a greater cytoprotective role for GSTT1 and GSTA1 in erythroid cells and GSTM1 in lymphoid cells. We postulate that inherited gene deletion of GSTT1 and M1 may produce increased genotoxic susceptibility for erythroid and lymphoid cell respectively, following exposure to xenobiotics that are substrates for these enzymes.

This study was designed to investigate the expression of different isoenzymes of glutathione transferase (GST) in the rat ovary and to follow possible changes in the pattern of expression during maturation and the different stages of the oestrus cycle. The GST subunits present in the rat ovary (as analyzed by HPLC of affinity-purified GSTs) were A3, A4, M1, M2, M3 and P1. The most abundant subunit in the ovary was A3, but the mu enzymes demonstrated the largest increase (3.5-fold) when the mature ovary was compared to the immature organ. An overall decrease in GST isoenzyme content during dioestrus was observed, but there were no other recurrent changes during the oestrus cycle. Treatment of immature rats with pregnant mare's serum gonadotropin clearly demonstrated that the mu and alpha isoenzymes are up-regulated by gonadotropin stimulation, i.e. a 4-6 fold increase was seen. This treatment also elevated the P1 subunit 3-fold. At present, it is only possible to speculate concerning the mechanism(s) underlying these variations in ovarian GST expression in connection with hormonal changes. The functional significance of these variations is not yet known.

Cytochrome P450 (CYP) 1A1, glutathione S-transferase (GST) M1, and GSTT1 gene polymorphisms have been shown to be associated with several diseases. In this study, CYP1A1 MspI, GSTM1 and GSTT1 gene polymorphisms were investigated in 134 Ovambo and 207 Mongolians, and the results were compared with those from previous studies. Using polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP) the frequency of CYP1A1 MspI mutation was determined. The multiplex PCR was used to determine the GSTM1 and GSTT1 polymorphism. The frequencies of wild-type, heterozygous variant and homozygous variant of the CYP1A1 MspI genotypes were 72.4%, 25.4% and 2.2%, and 22.7%, 55.6% and 21.7% in the Ovambos and Mongolians, respectively. The frequencies of GSTM1 (null) and GSTT1 (null) genotypes were 11.2% and 35.8%, and 46.4% and 25.6% in the Ovambos and Mongolians, respectively. The CYP1A1 MspI and GSTT1 (null) genotype distribution of the Ovambos was similar to that of African-Americans and some Caucasians. In contrast, the GSTM1 (null) genotype distribution was different from that of all other populations. Among Mongolians, the CYP1A1 MspI polymorphism showed the highest mutation frequencies, GSTM1 (null) was similar to that of East Asians, and GSTT1 (null) was different from that of almost all the Asians examined.

Differential scanning fluorometry (DSF), also referred to as fluorescence thermal shift, is emerging as a convenient method to evaluate the stabilizing effect of small molecules on proteins of interest. However, its use in the mechanism of action studies has received far less attention. Herein, the ability of DSF to report on inhibitor mode of action was evaluated using glutathione S-transferase (GST) as a model enzyme that utilizes two distinct substrates and is known to be subject to a range of inhibition modes. Detailed investigation of the propensity of small molecule inhibitors to protect GST from thermal denaturation revealed that compounds with different inhibition modes displayed distinct thermal shift signatures when tested in the presence or absence of the enzyme's native co-substrate glutathione (GSH). Glutathione-competitive inhibitors produced dose-dependent thermal shift trendlines that converged at high compound concentrations. Inhibitors acting via the formation of glutathione conjugates induced a very pronounced stabilizing effect toward the protein only when GSH was present. Lastly, compounds known to act as noncompetitive inhibitors exhibited parallel concentration-dependent trends. Similar effects were observed with human GST isozymes A1-1 and M1-1. The results illustrate the potential of DSF as a tool to differentiate diverse classes of inhibitors based on simple analysis of co-substrate dependency of protein stabilization.

The kinetics of spontaneous and human glutathione transferase catalysed formation of S-nitrosoglutathione (GSNO) from glutathione (GSH) and n-butyl- or amyl nitrite have been studied. At physiological pH and temperature, k2 values of 22.3 and 21.0 M-1.min-1 were obtained for n-butyl- and amyl nitrites, respectively. Rate enhancements, (kcat/Km x k2) x 10(-4), due to purified human GSH transferases A1-1, A2-2 and M1a-1a were, respectively, 7.00, 2.94 and 10.6 for n-butyl nitrite and 121, 3.92 and 34.5 for amyl nitrite. GSH transferase P1-1 showed no detectable catalysis of the formation of GSNO. The data suggest that the presence of GSTs A1-1, A2-2 or M1-1 contribute substantially to intracellular metabolism of alkyl nitrites to GSNO. The results may be significant with regard to the immunotoxicity of alkyl nitrites.

The environmental and biological monitoring of benzene exposure is crucial to prevent the toxic effects of this solvent in workers. The degree of correlation, however, between the two and of different biomarkers among them varies, particularly at low levels of exposure, depending on various factors, including variability in metabolizing enzymes and smoking habits. To investigate these further, a cohort of 28 petrochemical workers (6 smokers and 22 non smokers) was monitored throughout ten consecutive days, on two occasions, two years apart, by collecting in total 173 environmental and biological samples. The airborne benzene levels, the urinary t,t-muconic acid (t,t-MA) and S-phenylmercapturic acid (S-PMA) concentrations, and the glutathione S-transferases (GST) M1 and T1 genotypes were measured. S-PMA was the only metabolite statistically correlated with airborne benzene levels (r=0.447, P<0.0001), particularly in non smokers (r=0.667, P<0.0001), the smoking habit being the only variable influencing metabolite excretion. Finally, a reduced S-PMA excretion was found to be associated with the GSTT1, but not the GSTM1, null genotype. In conclusion, the results show that S-PMA, but not t,t-MA, is able to monitor exposure to low benzene concentrations and confirm that the GSTT1 null genotype has a significant influence on metabolite excretion. The influence of the GSTT1 null genotype, however, was low, even when studying each subject with several urine samples.

Glutathione S-transferases (GSTs) are a family of detoxification isozymes that protect cells by conjugating GSH to a variety of toxic compounds, and they may also play a role in the regulation of both cellular proliferation and apoptosis. We have previously shown that human GST P1-1, which is the most widely distributed extrahepatic isozyme, could be inactivated by the catechol estrogen metabolite 4-hydroxyequilenin (4-OHEN) in vitro [Chang, M., Shin, Y. G., van Breemen, R. B., Blond, S. Y., and Bolton, J. L. (2001) Biochemistry 40, 4811-4820]. In the present study, we found that 4-OHEN and another catechol estrogen, 4,17beta-hydroxyequilenin (4,17beta-OHEN), significantly decreased GSH levels and the activity of GST within minutes in both estrogen receptor (ER) negative (MDA-MB-231) and ER positive (S30) human breast cancer cells. In addition, 4-OHEN caused significant decreases in GST activity in nontransformed human breast epithelial cells (MCF-10A) but not in the human hepatoma HepG2 cells, which lack GST P1-1. We also showed that GSH partially protected the inactivation of GST P1-1 by 4-OHEN in vitro, and depletion of cellular GSH enhanced the 4-OHEN-induced inhibition of GST activity. In addition, 4-OHEN GSH conjugates contributed about 27% of the inactivation of GST P1-1 by 4-OEHN in vitro. Our in vitro kinetic inhibition experiments with 4-OHEN showed that GST P1-1 had a lower K(i) value (20.8 microM) compared to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 52.4 microM), P450 reductase (PR, 77.4 microM), pyruvate kinase (PK, 159 microM), glutathione reductase (GR, 230 microM), superoxide dismutase (SOD, 448 microM), catalase (562 microM), GST M1-1 (620 microM), thioredoxin reductase (TR, 694 microM), and glutathione peroxidase (GPX, 1410 microM). In contrast to the significant inhibition of total GST activity in these human breast cancer cells, 4-OHEN only slightly inhibited the cellular GAPDH activity, and other cellular enzymes including PR, PK, GR, SOD, catalase, TR, and GPX were resistant to 4-OHEN-induced inhibition. These data suggest that GST P1-1 may be a preferred protein target for equine catechol estrogens in vivo.

Reversal of the drug-resistance phenotype in cancer cells usually involves the use of a chemomodulator that inhibits the function of a resistance-related protein. The aim of this study was to investigate the effects of MDR chemomodulators on human recombinant glutathione S-transferase (GSTs) activity. IC50 values for 15 MDR chemomodulators were determined using 1-chloro-dinitrobenzene (CDNB), cumene hydroproxide (CuOOH) and anticancer drugs as substrates. GSTs A1, P1 and M1 were inhibited by O6-benzylguanine (IC50s around 30 microM), GST P1-1 by sulphinpyrazone (IC50 = 66 microM), GST Al-1 by sulphasalazine, and camptothecin (34 and 74 microM respectively), and GST M1-1 by sulphasalazine, camptothecin and indomethacin (0.3, 29 and 30 microM respectively) using CDNB as a substrate. When ethacrynic acid (for GST P1-1), CuOOH (for A1-1) and 1,3-bis (2-chloroethyl)-1-nitrosourea (for GST M1-1) were used as substrates, these compounds did not significantly inhibit the GST isoforms. However, progesterone was a potent inhibitor of GST P1-1 (IC50 = 1.4 microM) with ethacrynic acid as substrate. These results suggest that the target of chemomodulators in vivo could be a specific resistance-related protein.

Geniposide, an iridoid glycoside isolated from the fruit of Gardenia jasminoides Ellis, has biological capabilities of detoxication, antioxidation, and anticarcinogenesis. We have recently found that geniposide possesses a potential for detoxication by inducing GST activity and the expression of GST M1 and GST M2 subunits. In this study, the signaling pathway of geniposide leading to the activation of GSH S-transferase (GST) was investigated. Primary cultured rat hepatocytes were treated with geniposide in the presence or absence of mitogen-activated protein kinase (MAPK) inhibitors and examined for GST activity, expression of GST M1 and M2 subunits, and protein levels of MAPK signaling proteins. Western blotting data demonstrated that geniposide induced increased protein levels of GST M1 and GST M2 (approximately 1.76- and 1.50-fold of control, respectively). The effect of geniposide on the increased protein levels of GST M1 and GST M2 was inhibited by the MEK-1 inhibitor PD98059, but not by other MAPK inhibitors. The GST M1 and GST M2 transcripts as determined by RT-PCR and GST activity were also inhibited concurrently by the MEK-1 inhibitor PD98059. The protein levels of up- and down-stream effectors of the MEK-1, including Ras, Raf, and Erk1/2, and the phosphorylation state of Erk1/2 were found to be induced by geniposide, indicating a two-phase influence of geniposide. The results suggest that geniposide induced GST activity and the expression of GST M1 and GST M2 acting through MEK-1 pathway by activating and increasing expression of Ras/Raf/MEK-1 signaling mediators.

Environmental tobacco smoke (ETS) exposure might increase the risk for childhood asthma, and we hypothesized the effect may be modified by the phase II genes NAD(P)H: quinone oxidoreductase 1 (NQO1) and glutathione S-transferase (GST) M1. To investigate the genetic and environmental associations with asthma, GSTM1 and NQO1 functional polymorphisms and ETS were analyzed in a two-staged cross-sectional study among elementary schoolchildren in Taiwan. Multiple logistic regression analysis revealed a significant association between the Ser allele of the NQO1 Pro187Ser polymorphism and asthma (OR=1.6, 95% CI 1.3-1.8). Although GSTM1 genotype itself was not significantly associated with asthma (OR=1.0, 95% CI 0.8-1.1), the GSTM1 genotype modified the association between the NQO1 polymorphism and asthma in children exposed to ETS (p=0.0002). The NQO1 gene might be involved in the development of asthma, especially in children carrying the GSTM1 null genotype who are exposed to ETS.

Glutathione-S-transferases (GST) play a central role in the inactivation of toxic drugs like cyclophosphamide (CP). These enzymes depict several polymorphisms with altered activity, and it has been shown that different polymorphisms influence the risk of malignancies and the outcome after chemotherapy. To prove the hypothesis that CP efficacy in children with nephrotic syndrome is influenced by polymorphic expression of GSTs, the genotype of 26 patients was analyzed and correlated with the outcome after CP treatment. All 26 children with steroid-sensitive nephrotic syndrome and frequent relapses or steroid dependency were treated with CP at a mean age of 6.7+/-4.0 years. CP was given in a dose of 2 mg/kg/day for 12+/-1 week. GST-M1, GST-P1 and GST-T1 polymorphisms were detected by PCR. In patients with GST-M1 null polymorphism, a significantly better rate of sustained remission was seen than in patients with the heterozygous or homozygous GST-M1 wildtype (0 versus 29%, P <0.01). In contrast, children with GST-P heterozygous or homozygous polymorphism had a significantly lower rate of sustained remission compared to homozygous wildtype (7 versus 38%, P <0.02). The GST-T1 genotype did not influence the outcome after CP treatment (P =0.32). Patients with the combination of GST-M1 null and GST-P1 wildtype did not relapse in 50%, compared to 6% in other children (P <0.01). We conclude that the polymorphic expression of GST-M1 and -P1 did significantly influence the long-term remission rate after CP treatment of steroid-sensitive nephrotic syndrome in children. Whereas GST-M1 null will increase cyclophosphamide efficacy, GST-P1 polymorphism seems to be related to enhanced susceptibility to further relapses.

BACKGROUND

Presbycusis is characterised by etiopathological changes in the cochlea of the inner ear due to genetic and environmental factors and has a serious impact on quality of life. The present study was aimed to evaluate the role of oxidant stress gene polymorphisms in the development of presbycusis.

METHODS

220 subjects with confirmed presbycusis from ENT specialists of MAA ENT hospital, Hyderabad, India from 2012 to 2014 were considered for the study. 270 age and sex matched controls were included in the study. Analysis of gene polymorphisms of SNPs cytochrome P450 1A1 (CYP1A1) 3801 T>C, 2455 A>G and 2453 A>C; glutathione S transferase (GST) T1 and M1; N-acetyl transferase (NAT2) 282 C>T and 857 G>A; uncoupled proteins (UCP1) (-3826) A>G and (UCP2) (866)G>A was carried out. Variations in the allelic and genotypic frequencies obtained were computed and analysed using appropriate statistical methods.

RESULTS

The results of the study indicated that CYP1A1 gene polymorphism at 2453 C>A (adjusted OR: 1.59, 95% CI: 1.01-2.87) and 2455 A>G (adjusted OR: 1.87, 95% CI: 1.07-3.37), double null genotype of GSTM1 and GSTT1 (adjusted OR: 8.88, 95% CI: 4.10-19.19), NAT2 gene at C282T (adjusted OR: 1.77, 95% CI: 1.02-3.11) and G590 A (adjusted OR: 1.83, 95% CI 1.20-3.63) and UCP2 (-866) G>A (adjusted OR: 12.39; 95% CI: 6.51-23.56) showed increased risk for presbycusis while CYP1A1 at 3801 T>C and UCP1 (-3286) A>G exhibited no association. The haplotype combinations of T-G-A of CYP1A1 at 3801, 2455 and 2453 positions as well as T-A of NAT2*6 at 282 and 590 positions were found to contribute significant risk for the onset of presbycusis.

CONCLUSIONS

Gene polymorphisms of CYP1A1 (A2455G, C2453A), NAT2*6 (C282T, G590 A), GST T1/M1 (double null genotype) and UCP2 (G-866 A) were found to contribute significant risk to presbycusis.

BACKGROUND

Oxidative stress plays key role in pathogenesis of Alzheimer's disease. Glutathione S-transferases (GSTs), a family of phase-II isoenzymes, play a critical role in providing protection against electrophiles and products of oxidative stress. Among different classes of GSTs, GSTM1 (Mu) and GSTT1 (theta) are found to be genetically deleted which results in decreased expression of the concerned enzyme. This study aims at preliminary analysis of the frequency of deletion of GSTM1 and GSTT1 and their association with late-onset Alzheimer's disease.

METHODS

In this study, association of the deletion type polymorphism of GST M1 and T1 as possible risk factors for dementia of Alzheimer's type was studied in 50 patients and 100 controls. Dementia was diagnosed by Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria, Mini Mental State Examination (MMSE) and Clinical Dementia Rating (CDR) scale. Genotyping was done by multiplex Polymerase Chain Reaction (PCR). Associations between null genotype of either GSTM1 and GSTT1 or both with Alzheimer's disease were analyzed by Chi-Square test.

RESULTS

Deletion of GSTT1 was found significantly associated with Alzheimer's disease (χ(2)=5.08, p=0.02*).

CONCLUSIONS

The odds of Alzheimer's disease in null GSTT1 is found to be increased by 2.47 times in comparison to positive GSTT1.

OBJECTIVE

The aim of this pilot study was to assess the association between polymorphisms in genes that encode for proteins involved in the pharmacokinetics/pharmacodynamics of glucocorticoids and the occurrence of respiratory distress syndrome (RDS) in preterm infants born to mothers treated with a complete course of betamethasone.

METHODS

Sixty-two preterm infants were enrolled. The C1236T, G2677T, and C3435T polymorphisms in the ABCB1 gene, BclI, N363S and ER22/23EK in the NR3C1 gene, I105V in the GST-P1 gene and GST-M1 and GST-T1 deletions were analyzed, and their association with the occurrence of RDS was assessed.

RESULTS

In univariate analysis, the heterozygous and homozygous presence of the I105V variant in the GST-P1 gene seemed to confer protection against the occurrence of RDS (P = 0.032), while no association for all other polymorphisms was observed. In multivariate analysis, selection from the reference model of independent variables based on AIC (Akaike information criteria) maintained three variables in the model: gestation, C3435T, and GST-P1 genotype.

CONCLUSIONS

Polymorphisms of the GST-P1 gene may influence the effect of antenatal steroids on the newborn lung.

One of the important pathways of resistance to anthracyclines is governed by elevated levels of glutathione (GSH) in cancer cells. Resistant cells having elevated levels of GSH show higher expression of multidrug-resistant protein (MRP); the activity of glutathione S-transferases (GSTs) group of enzymes have also been found to be higher in some drug-resistant cells. The general mechanism in this type of resistance seems to be the formation of conjugates enzymatically by GSTs, and subsequent efflux by active transport through MRP (MRP1-MRP9). MRPs act as drug efflux pump and can also co-transport drugs like doxorubicin (Dox) with GSH. Depletion of GSH in resistant neoplastic cells may possibly sensitize such cells, and thus overcome multidrug resistance (MDR). A number of resistance modifying agents (RMA) like DL-buthionine (S, R) sulfoxamine (BSO) and ethacrynic acid (EA) moderately modulate resistance by acting as a GSH-depleting agent. As most of the GSH-depleting agents have dose-related toxicity, development of non-toxic GSH-depleting agent has immense importance in overcoming MDR. The present study describes the resistance reversal potentiality of novel copper complex, viz., copper N-(2-hydroxy acetophenone) glycinate (CuNG) developed by us in Dox-resistant Ehrlich ascites carcinoma (EAC/Dox) cells. CuNG depletes GSH in resistant (EAC/Dox) cells possibly by forming conjugate with it. Depletion of GSH results in higher Dox accumulation that may lead to enhanced rate of apoptosis in EAC/Dox cells. In vivo studies with male Swiss albino mice bearing ascitic growth of EAC/Dox showed tremendous increase in life span (treated/control, T/C = 453%) for the treated group with apparent regression of tumor. Resistance to Dox in EAC/Dox cells is associated with over expression of GST-P1, GST-M1 (enzymes involved in phase II detoxification) and MRP1 (a transmembrane ATPase efflux pump for monoglutathionyl conjugates of xenobiotics). CuNG causes down regulation of all these three proteins in EAC/Dox cells. The effect of CuNG as RMA is better than BSO in many aspects.

BACKGROUND

Glutathione S-transferase (GST) T1 and M1 are detoxification enzymes which neutralize various carcinogenic compounds. Polymorphisms of the GSTT1 and GSTM1 genes which encode the enzymes could be associated with cancer risk.

METHODS

We investigated the association of GSTT1 and GSTM1 null polymorphisms with lung cancer risk in a tightly matched, considerably large sample in China. Genotyping was performed utilizing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and DNA sequencing, and associations were measured by using logistic regression analysis.

RESULTS

We found that the null genotypes of both polymorphisms resulted in an increased lung cancer risk, with an odds ratio (OR) of 1.57 (95% confidence interval (CI) 1.23-2.00) for GSTT1 and OR 1.87 (95% CI 1.46-2.39) for GSTM1 (p < 0.01). Significant associations remained after stratification by histopathology (p < 0.01) and smoking status (p < 0.05). When gender-stratified association was performed, we found that the GSTT1 null genotype resulted in an increased risk among males (adjusted OR 2.95, 95% CI 2.07-4.20; p < 0.01) but not females (p>> 0.05), while the GSTM1 null genotype resulted in an increased risk among females (adjusted OR 2.18, 95% CI 1.54-3.10; p < 0.01) but not males (p < 0.05).

CONCLUSIONS

Polymorphisms in GSTT1 and GSTM1 are associated with the risk of lung cancer in a gender-specific manner.

1. Xenobiotic-metabolizing enzymes constitute an important line of defence against a variety of carcinogens. Many are polymorphic, constituting the basis for the wide interindividual variation in metabolic capacity and possibly a source of variation in the susceptibility to chemical-induced carcinogenesis. The aim of the present study was to determine the frequencies of important allelic variants in the N-acetyltransferase 2 (NAT2) and glutathione S-transferase (GST) genes in the Iranian population and compare them with frequencies in other ethnic populations. 2. Genotyping was performed in a total of 229 unrelated healthy subjects (119 men, 110 women) for NAT2 and 170 unrelated healthy subjects (89 men, 81 women) for GST from the general Tehran population. A combination of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) was applied for typing of NAT2 polymorphisms. Detection of GSTM1 and GSTT1 null alleles was performed simultaneously using a multiplex PCR assay. 3. The frequencies of specific NAT2 alleles were 0.299, 0.314, 0.380, 0.007 and 0.000 for 4 (wild-type), 5 (C481T, M1), 6 (G590A, M2), 7 (G857A, M3) and 14 (G191A, M4), respectively. The most prevalent genotypes were NAT2 5/6 (29.70%) and 4/6 (21.40%). The GSTM1- and GSTT1-null alleles were detected in 44.7 and 21.2% of subjects, respectively. 4. We found that Iranians resemble Indians with regard to allelic frequencies of the tested variants of NAT2. The predominance of slow (49.36%) and intermediate (41.47%) acetylation status compared with wild-type rapid acetylation status (9.17%) in the study group suggests the significant prevalence of the slow acetylator (SA) phenotypes in the Iranian population. Our data confirmed that Iranians are similar to other Caucasian populations in the frequency of both GSTM1- and GSTT1-null alleles.

Recent studies indicate that the prevalence of reflux esophagitis (RE) in China is increasing. RE is one of the most common esophageal complications associated with gastroesophageal reflux disease (GERD) and RE-Barrett's esophagus-esophageal adenocarcinoma (EAC) sequence has been considered as an histogenesis model for EAC in Western countries. RE is only present in a subset of patients with GERD, suggesting an altered susceptibility to RE may exist in these GERD individuals. However, the genetic changes related with high susceptibility to RE is largely unknown. The polymorphisms in glutathione S-transferases (GSTs) T1, M1 and P1 have been reported with high susceptibity to esophageal cancer in Chinese people. The present case-control study was thus undertaken to characterize the genetic polymorphisms of GSTs and their correlation with susceptibility to RE. One hundred and nine patients with RE, 97 patients with nonerosive reflux disease (NERD) and 97 normal controls were recruited in this study. All the subjects were from Beijing, China, and received endoscopic examination and questionnaires for RE. Genomic DNA was extracted from the lymphocytes of peripheral blood for each subject. Genotypes of the GSTM1 and GSTT1 genes were analyzed by a multiplex PCR method. A->>G polymorphism of codon 104 of the GSTP1 gene was detected using PCR-based restriction fragment length polymorphisms (RFLP). The variant GSTP1 genotypes (*A/*Bomicron*B/*B) was found with a high frequency in the case with RE (40%), and followed by NERD (25%) and normal control (22%). The differences were statistically significant (P < 0.05). The risk for RE increased 2.42-fold [odds ratio (OR); 95% confidence interval (95% CI), 2.42 (1.22-4.80)] in the subjects with variant GSTP1 genotype. The subjects with positive variant GSTP1 genotypes and negative H. pylori infection showed increasing tendency for risk of RE [OR (95% CI), 2.67 (1.06-6.70)]. However, the subjects with GSTT1 and GSTM1 polymorphisms did not show any correlation with high risk for RE or NERD. No significant interactions were identified between the variant GSTs and cigarette smoking, or alcohol drinking and subtype of RE. The present result suggests that GSTP1 genetic polymorphism may be one of the high susceptibility factors involved in the mechanisms of RE. H. pylori infection may play a protective role against RE.

Glutathione S-transferase (GST), as antioxidant enzyme, protects tissue from oxidative damage typical for many pathologic conditions as type 1 diabetes (T1D) and its chronic complications. The aim of the study was to compare the prevalence of GST T1/M1 gene polymorphisms between diabetic adolescents with (CAN+) and without (CAN-) cardiovascular autonomic neuropathy. Forty-six subjects with T1D at the age 15-19 years were enrolled. CAN was diagnosed in 19 patients (41.3%) based on standard cardiovascular tests. GST M1 null genotype was more prevalent in CAN+subjects but this was not statistically significant (OR=1.889, 0.61-6.55, p>0.05). GST T1 wild genotype nearly 5-fold increased the risk of CAN (OR=4.952, 1.13-21.739, p<0.05). Regarding genotype combination, GST T1/M1 wild/null genotype was significantly more frequent in CAN+compared to the CAN- subjects (OR=3.96, 1.024-15.302, p<0.05). No significant difference was found in any biochemical parameters between CAN+and CAN- subgroups. Multivariable logistic regression showed that none of the biochemical parameters estimated was considered a risk factor for CAN, however GST T1 wild and GST T1/M1 wild/null represented a risk factor for CAN development (OR=2.227, 1.079-4.587, p<0.05 and OR=1.990, 1.026-3.859, p<0.05, respectively). GST T1 wild allele and GST T1/M1 wild/null genotype can be considered as risk factors for CAN in Slovak adolescents with T1D.

To examine the association between genetic polymorphisms of glutathione S-transferase (GST) M1 and T1 and susceptibility to esophageal cancer, a multiplex polymerase chain reaction method was used to detect the presence or absence of the GSTM1 and GSTT1 genes in genomic DNA isolated from surgically removed esophageal tissues or scraped esophageal cells from cases with cancer (n = 45), cases with severe hyperplasia (n = 45), and sex/age matched normal controls (n = 45) from a high risk area, Linxian, China. Results showed that the frequency of the GSTM1-null genotype in cancer cases (44.4%) or hyperplasia cases (44.4%) was not significantly different from that in controls (46.7%). Similarly, no statistically significant differences were observed in the frequency of GSTT1-null genotype in cancer cases (40.0%) or hyperplasia cases (37.8%) when compared with the controlled population (51.1%). However, the frequency of combined GSTM1-nonnull and GSTT1-nonnull genotypes in cases with cancer (40.0%) and cases with hyperplasia (37.8%) showed a significant increase compared to that in controls (22.2%). Persons with both GSTM1 and GSTT1 positive genotypes had 4-fold risk in developing esophageal cancer (odds ratio, OR = 4.20; 95% confidence interval, CI = 1.23-14.36) and 2.6-fold risk for hyperplasia (OR = 2.64; 95% CI = 0.84-8.30), respectively. These results suggest that combined GSTM1-nonnull and GSTT1-nonnull genotypes may act as risk factor in the development of esophageal cancer in Linxian population.

The glutathione transferase (GST) isoenzyme profile was determined in two human tumor cell lines, U1690 derived from a small cell lung cancer and U1810 derived from a non-small cell lung cancer. U1810 cells are 3.2-fold more resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) than are U1690 cells, a finding ascribable in part to the expression of O6-alkylguanine-DNA alkyltransferase activity in the U1810 cells. GST P1-1 and GST A1-1 were determined quantitatively by enzyme-linked immunoassay and were found to be 1.3- and 15-fold higher in the cytosol fraction of U1690 cells as compared to U1810 cells, respectively. The higher BCNU resistance in U1810 cells can, therefore, not be correlated with the expression of these isoenzymes. However, sodium dodecyl sulfate/polyacrylamide gel electrophoresis in combination with immunoblot analysis demonstrated a class Mu GST, which was identified as GST M3-3 on the basis of electrophoretic mobility and cross-reaction with anti-rat GST 3-3 antibodies. This isoenzyme was detectable in U1810 cells but not in U1690 cells. Studies with purified human GST A1-1, GST M1-1, GST M3-3, and GST P1-1 demonstrated that GST M3-3, but not the other isoenzymes, catalyzed the denitrosation of BCNU. Such inactivation of BCNU has previously been demonstrated with rat class Mu GSTs (M. T. Smith et al., Cancer Res., 49: 2621-2625, 1989) but not with any human GST. These findings suggest that GST M3-3 contributes to BCNU resistance in the U1810 cells.