Glutathione S-transferase Isoenzymes Research Articles

Triazophos resistance mechanisms in the rice stem borer ( Chilo suppressalis Walker)

A field population of the rice stem borer ( Chilo suppressalis Walker) with 203.3-fold resistance to triazophos was collected. After 8-generation of continuous selection with triazophos in laboratory, resistance increased to 787.2-fold, and at the same time, the resistance to isocarbophos and methamidophos was also enhanced by 1.9- and 1.4-fold, respectively, implying some cross-resistance between triazophos and these two organophosphate insecticides. Resistance to abamectin was slightly enhanced by triazophos selection, and fipronil and methomyl decreased. Synergism experiments in vivo with TPP, PBO, and DEM were performed to gain a potential indication of roles of detoxicating enzymes in triazophos resistance. The synergism results revealed that TPP (SR, 1.92) and PBO (SR 1.63) had significant synergistic effects on triazophos in resistant rice borers. While DEM (SR 0.83) showed no effects. Assays of enzyme activity in vitro demonstrated that the resistant strain had higher activity of esterase and microsomal O-demethylase than the susceptible strain (1.20- and 1.30-fold, respectively). For glutathione S-transferase activity, no difference was found between the resistant and the susceptible strain when DCNB was used as substrate. However, 1.28-fold higher activity was observed in the resistant strain when CDNB was used. These results showed that esterase and microsomal- O-demethylase play some roles in the resistance. Some iso-enzyme of glutathione S-transferase may involve in the resistance to other insecticides, for this resistant strain was selected from a field population with multiple resistance background. Acetylcholinesterase as the triazophos target was also compared. The results revealed significant differences between the resistant and susceptible strain. The V max and K m of the enzyme in resistant strain was only 32 and 65% that in the susceptible strain, respectively. Inhibition tests in vitro showed that I 50 of triazophos on AChE of the resistant strain was 2.52-fold higher. Therefore, insensitive AChE may also involved in triazophos resistance mechanism of rice stem borer.

Glutathione Transferase Superfamily Behaves Like Storage Proteins for Dinitrosyl-Diglutathionyl-Iron Complex in Heterogeneous Systems

Electron paramagnetic resonance and kinetics experiments have been made to determine the formation, stability, and fate of the natural nitric oxide carrier, dinitrosyl-diglutathionyl-iron complex (DNDGIC), in heterogeneous systems approaching in vivo conditions. Both in human placenta and rat liver homogenates DNDGIC is formed spontaneously from GSH, S-nitroso-glutathione, and trace amounts of ferrous ions. DNDGIC is unstable in homogenates depleted of glutathione S-transferase (GST); an initial phase of rapid decomposition is followed by a slower decay, which is inversely proportional to the concentration. In the crude human placenta homogenate, GSTP1-1, which represents 90% of the cytosolic GST isoenzymes, is the preferential target for DNDGIC. It binds the complex almost stoichiometrically and stabilizes it for several hours (t1/2 = 8 h). In the presence of an excess of DNDGIC, negative cooperativity in GSTP1-1 opposes the complete loss of the usual detoxicating activity of this enzyme. In the rat liver homogenate, multiple endogenous GSTs (mainly Alpha and Mu class isoenzymes) bind the complex quantitatively and stabilize it (t1/2 = 4.5 h); negative cooperativity is also seen for these GSTs. Thus, the entire pool of cytosolic GSTs, with the exception of the Theta GST, represents a target for stoichiometric amounts of DNDGIC and may act as storage proteins for nitric oxide. These results confirm the existence of a cross-link between NO metabolism and the GST superfamily.

Polymorphisms in glutathione-S-transferase genes (GST-M1, GST-T1 and GST-P1) and susceptibility to prostate cancer among male smokers of the ATBC cancer prevention study.

Glutathione-S-transferase (GST) genes encode a family of detoxification enzymes that offer protection against endogenous and exogenous sources of reactive oxygen species (ROS). Germline variations in GST genes may alter the catalytic efficiency of GST isoenzymes leading to a potential increase in susceptibility to the genotoxic effects of ROS and electrophilic substances. A nested case-control study design was used to examine the association between the polymorphic GST genes and prostate cancer risk among Finnish male smokers of the ATBC Cancer Prevention Study. A case-case analysis was used to determine the association between these genetic polymorphisms and prostate cancer progression. Germline DNA was obtained from 206 prostate cancer cases and 194 controls frequency matched on age, intervention group and study clinic. Cases and controls were genotyped for three GST genes using MALDI-TOF mass spectrometry or multiplex polymerase chain reaction (PCR). Relative to the wild-type genotype, we observed a 36% reduction in prostate cancer risk associated with the GST-M1-null genotype (odds ratio (OR) 0.64, 95% confidence interval (CI) 0.43, 0.95). Unlike GST-M1, GST-T1-null (OR 0.74, 95% CI 0.42, 1.33) and GST-P1*B (OR 1.10, 95% CI 0.72, 1.69) were not strongly associated with prostate cancer risk. We did not observe any significant associations between the selected polymorphic GST genes and tumour grade or stage. In conclusion, we did not observe a direct association between polymorphic GST-T1 or GST-P1 and prostate cancer risk. Our observation of a relatively strong inverse association between the GST-M1-null genotype and prostate cancer risk needs to be confirmed in larger association studies.

Oxidative deoxyribonucleic acid damage in the eyes of glaucoma patients

Background Little is known about the molecular mechanisms responsible for the development of glaucoma, the leading cause of irreversible blindness worldwide. Some investigators have hypothesized that oxidative damage may be involved. We evaluated oxidative deoxyribonucleic acid (DNA) damage, in terms of 8-hydroxy-2′-deoxyguanosine (8-OH-dG), in the eyes of glaucoma patients. Methods Levels of 8-OH-dG were measured in the trabecular meshwork region from 42 patients with glaucoma and 45 controls of similar age and sex. Genotypes of glutathione S-transferase isoenzymes ( GSTM1 and GSTT1) were assessed by polymerase chain reaction in the same DNA samples. Results Levels of 8-OH-dG were significantly higher in glaucoma patients than in controls. Oxidative DNA damage in patients with glaucoma correlated significantly with intraocular pressure; in patients with primary open-angle glaucoma, it also correlated with visual field defects. GSTT1 was similar in the two groups, and had no effect on 8-OH-dG levels. Conversely, 8-OH-dG levels were significantly higher in GSTM1-null than in GSTM1-positive subjects. The GSTM1-null genotype was significantly more common in patients with primary open-angle glaucoma than in controls. Conclusion Oxidative DNA damage is significantly increased in the trabecular meshwork of glaucoma patients. GSTM1 gene deletion, which has been associated with an increased risk of cancer at various sites and molecular lesions in atherosclerosis, predisposes to more severe oxidative DNA damage in glaucoma patients. These findings may contribute to understanding the pathogenesis of glaucoma and may be useful in the prevention and treatment of this disease.

Inhibition of various glutathione S-transferase isoenzymes by RRR-α-tocopherol

The activity of human cytosolic glutathione S-transferases (GSTs) can positively or negatively be changed by various compounds. It is for instance known that RRR-α-tocopherol inhibits GST P1-1 [Haaften van R.I.M. et al. (2001) Alpha-tocopherol inhibits human glutathione S-transferase pi. BBRC 280, 631–633]. The effect of RRR-α-tocopherol on the other isoenzymes of GST in purified forms of the isoenzymes and in human liver cytosol (GST M and GST A) and lysate of human erythrocytes (GST P) is studied. It is found that all isoenzymes (purified enzymes and enzymes present in homogenates) are inhibited, in a concentration-dependent way, by RRR-α-tocopherol. GST P is in both cases inhibited with the highest potency compared to the other isoenzymes. It also appeared that the purified GST P1-1 isoenzyme is non-competitively inhibited by RRR-α-tocopherol. The IC 50 values of RRR-α-tocopherol for the purified isoenzymes of GST are much lower compared to the IC 50 values for human lysate and human liver cytosol. This is probably due to binding of RRR-α-tocopherol to proteins, e.g. albumin and hemoglobin, with higher affinity than to GST; so more RRR–α-tocopherol is needed to inhibit the enzyme. However, the inhibition of GSTs by RRR–α-tocopherol can still be of physiological relevance, because due to dermal application of cosmetic products very high concentrations vitamin E can be reached in the skin, where GST P1-1 is present. RRR-α-tocopherol might also be a good lead compound for the development of a new class of inhibitors of GST that can be used as adjuvant in cancer therapy.

Glutathione S-transferase isoenzyme profile in non-tumor and tumor human kidney tissue.

Glutathione S-transferase (GST) isoenzyme profiles of non-tumor and tumor renal tissue of patients suffering from renal cell carcinoma (RCC) of the clear cell type were determined and compared to those of normal renal tissue. GST isoenzyme(s) were first separated on the basis of their affinity to glutathione sepharose 4B affinity column. Affinity-bound GSTs were further purified by anionic and cationic chromatofocusing. The results presented in this study show that non-tumor tissue distant from renal tumors and renal tumors have lower specific GST activity and a different isoenzyme profile than normal human kidney. Purification of normal kidney GSTs by affinity chromatography revealed the presence of two GST fractions: flow-through GST without affinity for glutathione affinity resin and GST fraction tightly bound to affinity matrix. In non-tumor kidney tissue of RCC patients, substantially less flow-through GST fraction was found, whereas renal tumors did not express flow-through GST at all. Isoelectric chromatofocusing indicated smaller numbers of GST isoenzymes in non-tumor and tumor kidney regions, with anionic forms dominating. It could be speculated that decreased expression of cationic GST isoenzymes (corresponding to class alpha) in non-tumor kidney tissue of RCC patients might be responsible for differences in sensitivity to specific carcinogens. The observations that RCCs are devoid of affinity flow-through GST and have small number of isoenzymes are further proof of low-level GST expression in RCC.

Exceptional activity of murine glutathione transferase A1-1 against (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-induced DNA damage in stably transfected cells.

We have shown previously that the alpha class murine glutathione transferase (GST) isoenzyme mGSTA1-1, unlike other mammalian class alpha GSTs, is highly efficient in catalyzing the glutathione (GSH) conjugation of (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the ultimate carcinogenic metabolite of benzo[a]pyrene. The present studies were undertaken to determine the efficacy of mGSTA1-1 in cellular protection against (+)-anti-BPDE-induced DNA damage in HepG2 cells stably transfected with mGSTA1 cDNA. Untransfected HepG2 cells, vector-transfected HepG2 cells (HepG2-vector), and cells transfected with mGSTA4 cDNA (HepG2-mGSTA4), an alpha class murine GST isoenzyme with low (+)-anti-BPDE-GSH conjugating activity, were used as controls for comparison. Intracellular GSH conjugation of (+)-anti-BPDE was significantly higher in mGSTA1-1-overexpressing HepG2 cells (HepG2-mGSTA1) than in HepG2-vector or HepG2-mGSTA4 cells. The formation of DNA-adducts of (+)-anti-BPDE, following a 10-, 20-, or 30-min exposure to 0.1, 0.5, or 1.0 microM [3H](+)-anti-BPDE, was reduced significantly in cells transfected with mGSTA1-1 compared with HepG2-vector or untransfected HepG2 cells. Consistent with the results with purified protein, overexpression of mGSTA4-4 had no effect on (+)-anti-BPDE-induced DNA damage. The results of the present study indicated that mGSTA1-1 was exceptionally effective in affording protection against (+)-anti-BPDE-induced DNA damage in a cellular system.

Glutathione S-transferase isoenzymes from Streptomyces griseus.

An inducible, cytosolic glutathione S-transferase (GST) was purified from Streptomyces griseus. GST isoenzymes with pI values of 6.8 and 7.9 used standard GST substrates including 1-chloro-2,4-dinitrobenzene. GST had subunit and native M(r)s of 24 and 48, respectively, and the N-terminal sequence SMILXYWDIIRGLPAH.

Effects of dietary Nε-carboxymethyllysine on expression of the biotransformation enzyme, glutathione-S-transferase, in the rat

Nε-carboxymethyllysine (CML) is an advanced-glycation endproduct found in both heat-treated foods and in living organisms. In the experiments described below, we have evaluated the effects of CML on the expression of the detoxification enzyme, glutathione-S-transferase, in the rat and in intestinal cells in cell culture. In the first animal experiment, casein-linked CML was administered to Wistar rats at two pharmacological doses (110 and 300 mg CML/kg body weight/day) for 10 days. Phase II glutathione-S-transferase (GST) enzyme activity was analysed in the kidneys. In a second study, CML was administered in a diet supplemented with bread crust (25% w/w) such that rats received a moderate dose of 11 mg CML/kg body weight/day. The induction of GST isoenzymes was analysed in the kidneys. In vitro experiments were also performed to study the effects of casein-CML and bread crust on GST induction in the intestinal Caco-2 cell line.Both animal experiments revealed an inductive effect of casein-linked CML and of bread crust on GST activity in the kidneys. The results obtained from the cell culture experiments by enzyme activity analysis and Western blotting confirmed these inductive effects on the GST.In conclusion, dietary-advanced glycation products containing CML were shown to enhance the expression of GST in both animal models and in cell culture.

Expression of glutathione transferase isoenzymes in the human H295R adrenal cell line and the effect of forskolin.

In previous studies in our laboratory (L. Mankowitz, L. Staffas, M. Bakke, and J. Lund, Biochem J, 1995, 305, 111-118; L. Staffas, L. Mankowitz, M. Söderström, A. Blanck, I. Porsch-Hällström, C. Sundberg, B. Mannervik, B. Olin, J. Rydström, and J.W. DePierre, Biochem J, 1992, 286, 65-72) isoenzymes of GST, primarily of the mu class, have been shown to be downregulated by adrenocorticotropic hormone (ACTH) in rat and mouse adrenal cells. In the present investigation the human adrenal H295R cell line (W.E. Rainey, I.M. Bird, and J.I. Mason, Mol Cell Endocrinol, 1994, 100, 45-50) was examined in a similar manner. Analysis by reverse-phase HPLC revealed that these cells express four isoenzymes of GST, i.e., A1, A2, P1, and M4, as well as another unidentified protein that was retained by our affinity column (elution time of 32 min) and, thus, presumably binds glutathione. Among these forms, A1 was present at the highest level. Upon addition of forskolin (an activator of adenylate cyclase which has been shown previously to mimic the effect of ACTH on adrenal cells) to the culture medium, the level of A1 decreased approximately 70% by forskolin, whereas the levels of the other isoenzymes were slightly increased, and that of the unknown form doubled. Thus, the influence of ACTH on expression of GST isoenzymes in this human adrenal cell line differs from that in rat and mouse adrenal cells.

Purification and characterization of a novel glutathione S-transferase from Asaphis dichotoma

An isoenzyme of glutathione S-transferase (adGST) was purified from liver intestine of the seashell ( Asaphis dichotoma) by GST–Sepharose 4B affinity chromatography followed by reverse-phase HPLC. The enzyme has a p I value of 4.6 and is composed of two subunits each with a molecular weight of 23 kDa. It exhibits different catalytic activities toward the substrates 1-chloro-2,4-dinitrobenzene, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, ethacrynic acid, and p-nitrophenyl acetate and, fascinatingly, shows high activity toward CDNB. The amino acid composition of adGST was determined and found to be very similar to the Sloane squid GSTs. N-terminal analysis of the first 15 residues of adGST revealed that it has 73% sequence identity with the pig roundworm GSTs. The adGST shows characteristics similar to those of class sigma GSTs, as was indicated by its substrate specificity, N-terminal amino acid sequence, and amino acid composition.

Distribution of Components of the Glutathione Detoxification System Across the Human Placenta after Uncomplicated Vaginal Deliveries

The function of the glutathione-related detoxification system plays an important role to ensure an uncomplicated pregnancy outcome. This study was performed to investigate whether the components of the glutathione-related detoxification system are equally distributed among the different cotelydons in the human placenta. We measured glutathione, cysteine, glutathione S-transferase (GST) isoenzyme levels (GSTA1+A2, GSTP1, GSTM1 and GSTT1), enzyme activities of glutathione S-transferase and glutathione peroxidases, protein carbonyl levels, and antioxidant capacities at twelve different standardized positions in six placentae from healthy women after uncomplicated pregnancy and vaginal delivery. Data were statistically evaluated with a Friedman two-way ANOVA with Bonferroni correction. ‘Foetal’-side values were not significantly different from those at the ‘maternal’-side. Except for GSTA1+A2, no significant differences were found between different sampling sites indicating that the distribution of all parameters measured was homogenous throughout the placenta. Since levels of GSTA1+A2 were minor compared to those of GSTP1 and GSTT1, the clinical relevance of this heterogeneity may be limited. These results implicate that the location of sampling is not important as long as biopsies are taken from physiological cotelydons.

Cloning and expression of a novel Mu class murine glutathione transferase isoenzyme.

The present study describes the cDNA cloning, expression and characterization of a novel Mu class murine glutathione transferase (GST) isoenzyme. Screening of a cDNA library from the small intestine of a female A/J mouse using consensus probes derived from Mu class murine GST genes (mGSTM1-mGSTM5) resulted in the isolation of a full-length cDNA clone of a previously unknown Mu class GST gene (designated as mGSTM7). The choice of tissue was based on our previous identification in female A/J mouse small intestine of a potentially novel Mu class GST isoenzyme. The deduced amino acid sequence of mGSTM7, which comprises of 218 amino acid residues, exhibited about 67-78% identity with other Mu class murine GSTs. Recombinant mGSTM7-7 cross-reacted with anti-(GST Mu) antibodies, but not with anti-(GST Alpha) or anti-(GST Pi) antibodies. The pI and the reverse-phase-HPLC elution profile of recombinant mGSTM7-7 were different from those of other Mu class murine GSTs. The substrate specificity of mGSTM7-7 was also different compared with other Mu class murine GSTs. Interestingly, mGSTM7 had a higher identity with the human Mu class isoenzyme hGSTM4 (87% identity and 94% similarity in the amino acid sequence) than with any of the known mouse Mu class GSTs. Specific activities of recombinant mGSTM7-7 and human GSTM4-4 were comparable towards several substrates. For example, similar to hGSTM4-4, recombinant mGSTM7-7 was poorly active in catalysing the GSH conjugation of 1-chloro-2,4-dinitrobenzene and ethacrynic acid, and lacked activity towards 1,2-dichloro-4-nitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy)propane. These results suggested that hGSTM4-4 might be the human counterpart of mouse GSTM7-7. Reverse transcription-PCR analysis using mGSTM7-specific primers revealed that mGSTM7 is widely expressed in tissues of female A/J mice, including liver, forestomach, lung, kidney, colon and spleen.

Identification and characterization of GSTT3, a third murine Theta class glutathione transferase.

A novel Theta class glutathione transferase (GST) isoenzyme from mouse termed mGSTT3 has been identified by analysis of the expressed sequence tag database. The gene encoding mGSTT3 is clustered with the mGSTT1 and mGSTT2 genes on chromosome 10 and has an exon/intron structure that is similar to that of the other Theta class genes. mGSTT3 is expressed strongly in the liver and to a decreasing extent in the kidney and testis. Recombinant mGSTT3-3 expressed in Escherichia coli had a substrate-specificity profile that differed significantly from that of GSTT1-1 and GSTT2-2 isoenzymes. A molecular model of mGSTT3 suggested that, in comparison with GSTT2, a decrease in volume of the hydrophobic substrate-binding site and the loss of the sulphate-binding pocket prevents its use of the GSTT2 substrate 1-menaphthyl sulphate.

Polymorphism of glutathione S-transferases and susceptibility to acrylonitrile and dimethylsulfate in cases of intoxication

In this paper, the difficulties of genetic screening of occupationally exposed subjects for the evaluation of retrospective, and prospective, health risk assessments is illustrated with reference to glutathione S-transferase (GST) function. Individual differences in the magnitude and half-life of adduct levels, derived from background and occupational exposure, are observed largely independently of genetically determined conjugator status. During detoxification, GSTs play a critical role in providing protection against electrophiles and products of oxidative stress. GSTs are a superfamily of enzymes that may have broad and overlapping substrate specificities. Deficiencies of GST isoenzymes may be compensated by the presence of other isoforms and by the use of alternative metabolic pathways. This may be one reason for the abundance of controversial data on GST polymorphisms and adverse health effects.

Expression of rat liver glutathione-S-transferase GSTA5 in cell lines provides increased resistance to alkylating agents and toxic aldehydes

The glutathione-S-transferases (GST) are a major contributor to the eukaryotic cell's defences against chemical and oxidative stress. However, the role of individual GST isoenzymes in conferring resistance to xenobiotics has not been fully determined. We have examined the effect of the rat GSTA5 isoenzyme in the detoxication of alkylating agents and aldehydes by constructing a cell line in which it is stably expressed. The hamster fibroblast cell line V79 was transfected with a construct expressing GSTA5 from the CMV promoter. A stable clone (V79-GSTA5) was isolated after selecting for the neomycin phosphotransferase gene present on the introduced DNA. The cell line showed significantly increased levels of resistance towards the alkylating agents chorambucil and melphalan. Levels of resistance were 4–6-fold greater in V79-GSTA5 cells than in control cells. Increased levels of resistance were also observed towards the lipid peroxidation product acrolein (IC 50=80 μM compared with 17 μM in control cells). The V79-GSTA5 cells also showed a 4-fold increase in resistance to trans, trans muconaldehyde (IC 50=4 μM compared with l μM for control cells). GSTA5 did not protect against 4-hydroxynonenal, but it did provide greater levels of protection to hydrogen peroxide, with an IC 50 of 380 μM in V79-GSTA5 compared with 180 μM in control cells. In contrast, V79-GSTA5 cells were more sensitive to methyl glyoxal, suggesting that a methyl glyoxal–glutathione conjugate is more toxic that the parental compound. These data contribute towards the evaluation of the role of GSTA5 in the detoxication of these compounds.

Induction of cytosolic Glutathione S-transferases from Atlantic eel ( Anguilla anguilla) after intraperitoneal treatment with polychlorinated biphenyls

Glutathione S-transferases (GST) are phase II biotransformation enzymes influenced by exposure to foreign compounds and are proposed as biomarkers of environmental pollution. The hepatic and renal cytosolic GST isoenzymes from juvenile Atlantic eels were isolated and partially characterized. In both organs, three different GST isoenzymes were purified using HPLC and the final subcellular purified fraction resulted in enzymatic subunits of approximately 24 500 Da. Total GST activity was significantly higher towards the substrate 1-chloro-2,4-dinitrobenzene (CDNB) than the ethacrynic acid (ETHA). Induction of isoenzymes of GST was studied after intraperitoneal treatment with a single dose (25 mg/kg bw) of a polychlorinated biphenyls mixture. In the hepatic cytosol, an inductory effect on the major enzymatic isoform was observed when comparing treated with control eels. This was directly related to statistically significant ( P<0.05) differences in total GST activity toward the CDNB between treated and control groups, with an increase of 33.6% of GST total activity measured in the last subcellular purified samples. In the kidney cytosol, PCB treatment did not affect GST activity. Significant differences were observed when analyzing the effect of PCB treatment on the main GST subunit height (retention time of 27 min). These results indicate that hepatic GST profiles could be considered as a good tool in environmental studies as specific biomarkers of contamination.

Glutathione S‐transferase isoenzymes in the two‐spot ladybird, Adalia bipunctata (Coleoptera: Coccinellidae)

Isoenzymes of glutathione S-transferase (GST) in adult Adalia bipunctata, an aphidophagous predator, were studied. Cytosolic GST activity was studied in each beetle developmental stage. The highest activities towards both 1-chloro-2,4-dinitrobenzene (CDNB) and 2,4-dinitro-1-iodobenzene (DNIB) occurred in adults. The enzyme distribution was investigated in adults. While most of the enzymatic activity was found in the abdomen (40-50 and 34-63% respectively) using several concentrations of both CDNB and DNIB, significant differences were observed for the head and the thorax depending on the substrate. Activities were more abundant in the thorax with DNIB (37-47%) compared to the 13-19% obtained with CDNB. Some GST activity was also detected in the elytra. GSTs were purified by epoxy-activated Sepharose 6B affinity chromatography and applied to an HPLC column to determine the native molecular weight (69 kDa). Three isoenzymes were separated by chromatofocusing at pH ranges 7-4. Three bands with molecular mass from 23 to 26 kDa were visualised on SDS-PAGE. Their isoelectric points were 6.66, 6.36, and 6.21. The substrate specificities and the kinetic parameters (Vm and Km) of the isoenzymes showed large differences depending on the isoenzyme. Arch.

The mRNA expression of placental glutathione S-transferase isoenzyme in hamster buccal-pouch carcinomas using reverse transcription–polymerase chain reaction

Placental glutathione S-transferase (GST-P) may facilitate cell proliferation and inhibit apoptosis, hence allowing for the expansion of a population of initiated tumor cells. The enhanced expression of GST-P at the protein level has been reported previously in chemically induced oral carcinomas in hamster buccal-pouch mucosa but the expression of GST-P at the mRNA level has not yet been demonstrated. The purpose of the present study was to assess the GST-P mRNA expression in 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal-pouch carcinomas using a reverse transcription-polymerase chain reaction (RT-PCR). Thirty-five outbred, young (6 weeks old), male, Syrian golden hamsters (Mesocricatus auratus) were randomly divided into one experimental group (15 animals), and two control groups (10 animals each). Bilateral pouches of a group of 15 animals of the experimental group were painted with a 0.5% DMBA solution three times a week for 12 weeks while each animal of one of the control groups was similarly treated with mineral oil. Another control group of 10 animals was untreated throughout the experiment. Areas of dysplasia and squamous-cell carcinomas with a 100% tumor incidence developed in all of the DMBA-treated buccal pouches. The mineral oil-treated and untreated pouches revealed no obvious changes. Placental glutathione S-transferase mRNA was demonstrated to be present amongst all the 12-week DMBA-treated hamster buccal-pouch mucosa animals, but not for the untreated animals or the animals for which the buccal pouch was treated with mineral oil. Multiple potential regulatory pathways including gene amplification, enhanced mRNA stability, chromosomal translocation/gene rearrangement, and hypomethylation of the promoter region can contribute to the overexpression of GST-P mRNA in DMBA-induced hamster buccal-pouch carcinomas. Further study is necessary to completely understand which candidate mechanism(s) will contribute principally to the increased GST-P mRNA expression in oral experimental carcinogenesis.

Differential expression of glutathione S-transferase isoenzymes in murine small intestine and colon

Glutathione (GSH) S-transferase (GST) isoenzymes of the small intestine and colon of female A/J mice have been purified and characterized to determine their interrelationships with other murine GSTs. Cytosolic GST activity in the small intestine was at least due to six isoenzymes with isoelectric points (p I) of 9.5, 9.3, 9.1, 8.5, 6.2 and 5.5. Small intestine isoenzymes with p I values of 9.5, 9.3, 8.5, and 6.2 were identical to the mGSTA1-1 (Alpha class), mGSTP1-1 (Pi class), mGSTM1-1 (Mu class) and mGSTA4-4 (Alpha class), respectively, of other A/J mouse tissues on the basis of their reverse-phase HPLC elution profile, immunological cross-reactivity and/or N-terminal region amino acid sequence. Even though GST9.1 of the small intestine cross-reacted with the antibodies raised against Pi class GST, reverse-phase HPLC and N-terminal amino acid sequence analyses suggested that this isoenzyme may be structurally different from mGSTP1-1 as well as mGSTP2-2. Likewise, despite immunological similarity with the Mu class GSTs, small intestine GST5.5 appeared to be different from other Mu class murine GSTs characterized previously. Cytosolic GST activity in the colon was mainly due to four isoenzymes with p I values of 9.8, 9.4, 6.6 and 5.8. While the identity of colon GST6.6 could not be established due to its low abundance, GST9.8, GST9.4 and GST5.8 were identical to mGSTP1-1, mGSTM1-1 and mGSTA4-4, respectively, of other A/J mouse tissues including the small intestine. Isoenzymes corresponding to small intestine GST9.1 and GST5.5 could not be detected in the colon. The results of the present study indicate that the small intestine of female A/J mice is better equipped for protection against toxic effects of electrophiles than colon.