Expression Of Glutathione S-transferase Mu Research Articles

Reversal of High-Fat Diet-Induced Non-Alcoholic Fatty Liver Disease by Metformin Combined with PGG, an Inducer of Glycine N-Methyltransferase.

Nonalcoholic fatty liver disease (NAFLD) is a major cause of liver-related morbidities and mortality, and no effective drug treatment currently exists. We aimed to develop a novel treatment strategy to induce the expression of glycine N-methyltransferase (GNMT), which is an important enzyme regulating S-adenosylmethionine metabolism whose expression is downregulated in patients with NAFLD. Because 1,2,3,4,6-pentagalloyl glucose (PGG) is a GNMT inducer, and metformin was shown to upregulate liver mitochondrial GNMT protein expression, the effect of PGG and metformin was evaluated. Biochemical analysis, histopathological examination, immunohistochemical staining, reverse transcription-quantitative PCR (RT-qPCR), Western blotting (WB), proteomic analysis and Seahorse XF Cell Mito Stress Test were performed. The high-fat diet (HFD)-induced NAFLD mice were treated with PGG and metformin. Combination of PGG and metformin nearly completely reversed weight gain, elevation of serum aminotransferases, and hepatic steatosis and steatohepatitis. In addition, the downregulated GNMT expression in liver tissues of HFD-induced NAFLD mice was restored. The GNMT expression was further confirmed by RT-qPCR and WB analysis using both in vitro and in vivo systems. In addition, PGG treatment was shown to increase oxygen consumption rate (OCR) maximum capacity in a dose-dependent manner, and was capable of rescuing the suppression of mitochondrial OCR induced by metformin. Proteomic analysis identified increased expression of glutathione S-transferase mu 4 (GSTM4), heat shock protein 72 (HSP72), pyruvate carboxylase (PYC) and 40S ribosomal protein S28 (RS28) in the metformin plus PGG treatment group. Our findings show that GNMT expression plays an important role in the pathogenesis of NAFLD, and combination of an inducer of GNMT and metformin can be of therapeutic potential for patients with NAFLD.

Effect of Short‐Term and Long‐Term Exposure to Sucrose on Protein Expression and Oxidative Modification of Glutathione‐S‐Transferase Mu 1 (GSTM1) in Liver of Wild Type Mice

High dietary sugar intake is an environmental risk factor for the development of metabolic syndrome (MS), type 2 diabetes (T2D), and non‐alcoholic fatty liver disease (NAFLD). All three of these conditions are associated with oxidative stress and a high sugar diet has been shown to affect the protein expression and activity of antioxidant and detoxification enzymes including glutathione‐S‐transferase (GST). In this study, the effect of short‐term (ST) 2‐month and long‐term (LT) 8‐month sucrose exposure on the protein expression and oxidative modification of GST mu 1 (GSTM1) in liver of wild type mice was assessed. It was hypothesized that sucrose exposure would result in a decrease in GSTM1 protein expression and an increase in oxidative modification of GSTM1 as indexed by protein‐bound 4‐hydroxy‐2‐trans‐nonenal (HNE). It was determined that the protein expression of GSTM1 was significantly increased in the ST sucrose exposed mice, but significantly decreased in the LT control mice compared to the ST control mice. Additionally, GSTM1 was found to be significantly oxidatively modified by protein‐bound HNE in the LT sucrose exposed mice compared to the ST control mice, the ST sucrose exposed mice, and the LT control mice. These results suggest that a high sugar diet may impair the antioxidant status of the liver by oxidatively modifying detoxification enzymes such as GSTM1.

Enhanced soluble expression of glutathione S-transferase Mu from Rutilus kutum by co-expression with Hsp70 and introducing a novel inhibitor for its activity

One of the most critical challenges in recombinant protein production is the increment of protein solubility. The overexpression of glutathione S-transferases (GSTs) in cancers leads to the fast detoxification of drug substrates that is subsequently followed by increased drug resistance. In this study, the co-expression of Rutilus kutum Hsp70 (RkHsp70) with the mu-class GST (RkGST-μ) from the same source was analyzed to increase the solubility of aggregate-prone RkGST-μ. Based on the results, the co-expressed RkGST-μ cells showed substantially higher solubility and specific activity compared to control. The kinetic properties of the enzyme were measured and its optimum pH and temperature were observed at pH 8 and 35 °C. Furthermore, this study introduces ZM-093, a sulfamethoxazole-based azo with IC50 values of 24.45 μM (p < 0.0001), as a novel RkGST-μ inhibitor. Overall, the present study provides clear findings in support of using molecular chaperones to enhance the solubility of recombinant proteins.

Does maternal exposure to artificial food coloring additives increase oxidative stress in the skin of rats?

Glutathione-S-transferase (GST) and cytochrome P450 family 1 subfamily A polypeptide 1 (CYP1A1) metabolize and detoxify carcinogens, drugs, environmental pollutants, and reactive oxygen species. Changes of GST expression in tissues and gene mutations have been reported in association with many neoplastic skin diseases and dermatoses. Widely used artificial food coloring additives (AFCAs) also reported to effect primarily behavioral and cognitive function and cause neoplastic diseases and several inflammatory skin diseases. We aimed to identify the changes in expression of GSTs, CYP1A1, and vascular endothelial growth factor (VEGF) in rat skin which were maternally exposed AFCAs. A rat model was designed to evaluate the effects of maternal exposure of AFCAs on skin in rats. "No observable adverse effect levels" of commonly used AFCAs as a mixture were given to female rats before and during gestation. Immunohistochemical expression of GSTs, CYP1A1, and VEGF was evaluated in their offspring. CYP1A1, glutathione S-transferase pi (GSTP), glutathione S-transferase alpha (GSTA), glutathione S-transferase mu (GSTM), glutathione S-transferase theta (GSTT), and VEGF were expressed by epidermal keratinocytes, dermal fibroblasts, sebaceous glands, hair follicle, and subcutaneous striated muscle in the normal skin. CYP1A1, GSTA, and GSTT were expressed at all microanatomical sites of skin in varying degrees. The expressions of CYP1A1, GSTA, GSTT, and VEGF were decreased significantly, while GSTM expression on sebaceous gland and hair follicle was increased. Maternal exposure of AFCAs apparently effects expression of the CYP1A1, GSTs, and VEGF in the skin. This prominent change of expressions might play role in neoplastic and nonneoplastic skin diseases.

Glutathione S-transferase Mu 2-transduced mesenchymal stem cells ameliorated anti-glomerular basement membrane antibody-induced glomerulonephritis by inhibiting oxidation and inflammation

IntroductionOxidative stress is implicated in tissue inflammation, and plays an important role in the pathogenesis of immune-mediated nephritis. Using the anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM-GN) mouse model, we found that increased expression of glutathione S-transferase Mu 2 (GSTM2) was related to reduced renal damage caused by anti-GBM antibodies. Furthermore, mesenchymal stem cell (MSC)-based therapy has shed light on the treatment of immune-mediated kidney diseases. The aim of this study was to investigate if MSCs could be utilized as vehicles to deliver the GSTM2 gene product into the kidney and to evaluate its potential therapeutic effect on anti-GBM-GN.MethodsThe human GSTM2 gene (hGSTM2) was transduced into mouse bone marrow-derived MSCs via a lentivirus vector to create a stable cell line (hGSTM2-MSC). The cultured hGSTM2-MSCs were treated with 0.5mM H2O2, and apoptotic cells were measured by terminal dUTP nick-end labeling (TUNEL) assay. The 129/svj mice, which were challenged with anti-GBM antibodies, were injected with 106 hGSTM2-MSCs via the tail vein. Expression of hGSTM2 and inflammatory cytokines in the kidney was assayed by quantitative PCR and western blotting. Renal function of mice was evaluated by monitoring proteinuria and levels of blood urea nitrogen (BUN), and renal pathological changes were analyzed by histochemistry. Immunohistochemical analysis was performed to measure inflammatory cell infiltration and renal cell apoptosis.ResultsMSCs transduced with hGSTM2 exhibited similar growth and differentiation properties to MSCs. hGSTM2-MSCs persistently expressed hGSTM2 and resisted H2O2-induced apoptosis. Upon injection into 129/svj mice, hGSTM2-MSCs migrated to the kidney and expressed hGSTM2. The anti-GBM-GN mice treated with hGSTM2-MSCs exhibited reduced proteinuria and BUN (58% and 59% reduction, respectively) and ameliorated renal pathological damage, compared with control mice. Mice injected with hGSTM2-MSCs showed alleviated renal inflammatory cell infiltration and reduced expression of chemokine (C-C motif) ligand 2 (CCL2), interleukin (IL)-1β and IL-6 (53%, 46% and 52% reduction, respectively), compared with controls. Moreover, hGSTM2-MSCs increased expression of renal superoxide dismutase and catalase, which may associate with detoxifying reactive oxygen species to prevent oxidative renal damage.ConclusionsOur data suggest that the enhanced protective effect of GSTM2-transduced MSCs against anti-GBM-GN might be associated with inhibition of oxidative stress-induced renal cell apoptosis and inflammation, through over-expression of hGSTM2 in mouse kidneys.

Characterization of the glutathione S-transferase-Mu ( GSTM) gene sequence and its expression in the hermaphroditic fish, Kryptolebias marmoratus as a function of development, gender type and chemical exposure

Endocrine-disrupting chemicals (EDCs) have been shown to disrupt the reproduction of natural populations of several aquatic organisms including fish. Previously, we showed that some EDCs modulated the expression of oncogenes and tumor suppression gene, p53 in the hermaphroditic killifish Kryptolebias marmoratus. Exposure to EDCs may affect reproductive tissues including ovotestis of K. marmoratus not only through the estrogenic action but also by oxidative stress. Glutathione S-transferases (GSTs) provide antioxidant defense against oxidative stress in all the vital tissues. In this study, firstly, the full sequence of a cDNA clone of GST-Mu ( GSTM) was identified by molecular cloning. The expression of GSTM was studied at different stages of development, and in the gonads of adult hermaphrodites and secondary males. Secondary males showed significantly lower expression of GSTM compared to hermaphrodites. When gonadal expression of GSTM was compared with other GSTs in the hermaphrodites, expression of GSTM was significantly higher than the others. Juvenile fish were exposed to three EDCs, bisphenol A (BPA), nonylphenol (NP) and octylphenol (OP) for 24 h and expression GSTM transcript was studied at different time intervals. BPA at 12 and 24 h caused upregulation of GSTM whereas OP showed similar effect at 12 h only. NP, on the other hand, caused significant downregulation at 12 as well as 24 h. When adult fish of both the genders were exposed to EDCs, except NP, the other EDCs caused significant upregulation of GSTM mRNA expression in gonads. Expression of GSTM in the gonads of secondary males was more pronounced than in the hermaphrodites. These findings indicate that the gonad of K. marmoratus has a robust antioxidant system. The gender-related differences in GSTM expression imply a differential sensitivity to EDCs in gender type.

Expression of ovarian microsomal epoxide hydrolase and glutathione S-transferase during onset of VCD-induced ovotoxicity in B6C3F 1 mice

4-vinylcyclohexene diepoxide (VCD) specifically destroys small pre-antral follicles in the rodent ovary. VCD can be detoxified to an inactive tetrol by microsomal epoxide hydrolase (mEH), or by conjugation to glutathione (GSH) by glutathione S-transferase (GST). Formation of VCD-GSH adducts in the mouse ovary 4 h after VCD exposure (0.57 mmol/kg/day) has been demonstrated. Because the mouse ovary expresses both mEH and GST, expression of mEH and GST pi and mu during a time-course of VCD-induced ovotoxicity was evaluated in a neonatal mouse ovarian culture system. Ovaries from postnatal day 4 (PND4) B6C3F 1 mice were incubated with VCD (15 μM) for 2, 4, 6, 8, 10, 12, or 15 days. Following incubation, ovaries were histologically evaluated, or assessed for mRNA or protein expression. VCD did not cause follicle loss ( p > 0.05) on days 2, 4, or 6 of culture. At days 8, 10, 12, and 15, VCD reduced ( p < 0.05) both primordial and primary follicle numbers. Increased ( p < 0.05) expression of mEH, GST pi and GST mu mRNA was detected after 4 days of VCD exposure. This expression was reduced on days 6 and 8, when follicle loss was underway, but increased ( p < 0.05) after 10 days of exposure. mEH and GST pi proteins were elevated ( p < 0.05) following 8 days of VCD-exposure however there was no increase in GST mu protein. These findings suggest that with continuous exposure to VCD, increased expression of detoxification enzymes may participate in retarding the onset of follicle loss, but that this loss cannot ultimately be prevented.

Expression of xenobiotic and steroid hormone metabolizing enzymes in human breast carcinomas

The potential to metabolize endogenous and exogenous substances may influence breast cancer development and tumor growth. Therefore, the authors investigated the protein expression of Glutathione S-transferase (GST) isoforms and cytochrome P450 (CYP) known to be involved in the metabolism of steroid hormones and endogenous as well as exogenous carcinogens in breast cancer tissue to obtain new information on their possible role in tumor progression. Expression of GST pi, mu, alpha and CYP1A1/2, 1A2, 3A4/5, 1B1, 2E1 was assessed by immunohistochemistry for primary breast carcinomas of 393 patients from the German GENICA breast cancer collection. The percentages of positive tumors were 50.1 and 44.5% for GST mu and CYP2E1, and ranged from 13 to 24.7% for CYP1A2, GST pi, CYP1A1/2, CYP3A4/5, CYP1B1. GST alpha was expressed in 1.8% of tumors. The authors observed the following associations between strong protein expression and histopathological characteristics: GST expression was associated with a better tumor differentiation (GST mu, p = 0.018) and with reduced lymph node metastasis (GST pi, p = 0.02). In addition, GST mu expression was associated with a positive estrogen receptor and progesterone receptor status (p < 0.001). CYP3A4/5 expression was associated with a positive nodal status (p = 0.018). Expression of CYP1B1 was associated with poor tumor differentiation (p = 0.049). Our results demonstrate that the majority of breast carcinomas expressed xenobiotic and drug metabolizing enzymes. They particularly suggest that GST mu and pi expression may indicate a better prognosis and that strong CYP3A4/5 and CYP1B1 expression may be key features of nonfavourable prognosis.

Localization and characterization of glutathione- s-transferase isozymes alpha, mu, and pi within the mouse vomeronasal organ

The nasal cavity of vertebrates contains a variety of xenobiotic metabolizing enzymes that possess a broad range of substrate specificity ranging from metabolism of drugs, carcinogens, and steroid hormones, to dietary components and environmental pollutants. This investigation sought to localize the cellular expression and distribution of glutathione- s-transferase (GST) alpha, mu, and pi detoxifying enzymes, and to study GST activity toward different substrates in the mouse vomeronasal organ (VNO). Immunohistochemistry was used to identify GST alpha, mu and pi in the non-sensory and sensory layer of the VNO. Western blot analysis of cytosolic proteins revealed a qualitatively higher enzyme expression of GST alpha and mu in the main olfactory tissue (OE) in comparison to VNO tissue, whereas the GST pi isozyme was equally expressed in both. Total GST metabolism of 1-chloro-2, 4-dinitrobenzene (CDNB) revealed a higher activity level in the OE when compared to the VNO. In contrast, thin-layer chromatographic analysis of GST conjugation of the odorant, trans-2-hexenal ( t-hex) (10 mM) showed more conjugate formed per unit protein in the VNO than the OE. The analysis of GST expression and enzyme activity within the VNO parallels the reported localization of phase I metabolizing enzymes and suggests that GST isozymes play independent roles that characterize multiple processes within VNO chemosensitivity.

Zonal expression and activity of glutathione S-transferase enzymes in the mouse olfactory mucosa

The rodent olfactory mucosa is characterized by a mosaic of gene expression that is exhibited among various cell types. Olfactory sensitivity in these animals is conveyed through odorant receptor families that are distinctly expressed within various subsets of the olfactory neuron population. Receptor neurons that express a particular class of odorant receptors exhibit bilaterally symmetric zones, which generally define their location within the nasal cavity. Less characterized are zonal expression profiles of proteins among non-neuronal cell types of the olfactory mucosa. In this study, we survey the expression of three glutathione S-transferase (GST) isozymes (alpha, mu, and pi) in the mouse olfactory mucosa and characterize the zonal expression of the mu isozyme. Immunohistochemistry and Western blot analysis of the GST mu isozyme reveal that the lateral olfactory turbinates I, Ib, II, IIb, and III display a greater intensity of expression for GST mu, in comparison to the dorsal and septal regions of the mucosa. GST alpha and pi isozymes do not display any distinct zonal organization in olfactory tissue of the adult mouse. When the general substrate 1-chloro-2-4-dinitrobenzene (CDNB) was used to assess GST activity within the olfactory tissue, the lateral turbinate regions displayed a higher level of activity when compared to dorsal or septal regions. Analysis of GST mu expression in prenatal and early postnatal olfactory tissue also reveals a zonal expression of the isozyme. We relate the significance of these findings to metabolic topography and olfactory chemosensory function.

Propylthiouracil alters the expression and activity of glutathione-dependent enzymes in the mouse olfactory mucosa

Propylthiouracil (PTU) is an anti-thyroid drug that reportedly can impair olfactory function in humans and mice. In the mouse, PTU treatment disrupts survival and differentiation of olfactory precursor neurons. While the mechanism responsible for this effect is not understood, it is suspected that these changes are consequent to localized toxicity due to PTU metabolism. In vitro and in vivo studies in other biological systems demonstrate that PTU can significantly alter glutathione S-transferase (GST) enzyme expression and activity. The localization of GST biotransformation enzymes in basal cells, sustentacular cells and Bowman’s glands of the olfactory mucosa suggests that these cells play a significant role in olfactory physiology. This study investigated the effects of PTU treatment, T 4 replacement therapy and thyroidectomy on GST expression, GST and glutathione peroxidase (GSH-PX) activity in mouse olfactory tissue. One month treatment with PTU revealed a significant decrease in expression of GST alpha (37%) as identified by Western blot analysis. In contrast, no change in GST mu expression was observed after 1 month of treatment. Concomitant treatment with T 4 caused a significant induction of GST alpha, and mu isozymes. GST enzyme activity significantly decreased after 1 month of PTU treatment (53%) and remained suppressed, despite the presence of exogenous T 4. GSH-PX activity significantly decreased after 1 month of PTU treatment (30%) and remained at control levels with T 4 supplementation. Thyroidectomy caused a 25% reduction in olfactory GST alpha expression. Total GST and GSH-PX activity were not altered in these animals. Supplementation with T 4 in thyroidectomized animals prevented the suppression of GST alpha expression. These results suggest that the combined action of localized PTU toxicity and altered levels of circulating thyroid hormone contribute to PTU-mediated abnormalities in the olfactory system.

Dietary Zinc Deficiency Decreases Glutathione S-Transferase Expression in the Rat Olfactory Epithelium

Zinc deficiency leads to olfactory and gustatory dysfunction, but little is known about the underlying molecular mechanism of this phenomenon. We examined the effect of dietary zinc deficiency on the rat olfactory epithelium. Immunoreactivities of glutathione S-transferase (GST) mu, neuron-specific enolase (NSE) and proliferating cell nuclear antigen (PCNA), and in situ hybridization of GST mu mRNA in the olfactory epithelia were examined under different dietary zinc intake conditions. Adult male rats were fed a zinc-deficient (ZD) diet (0.5 mg zinc/kg diet), whereas control rats, including pair-fed (PF) and zinc-adequate (ad libitum consumption, AL) groups, were fed a zinc-adequate diet (58 mg zinc/kg diet) for 7 wk. We also examined the effect of zinc replacement (ZR) by subsequently feeding half of the ZD group a zinc-adequate diet for 5 wk after the initial 7-wk deprivation. No significant differences in immunoreactivity for NSE in olfactory epithelial receptor cells or for PCNA in basal cells were noted among groups. Intense GST mu immunoreactivity and hybridization signals were observed in olfactory supporting cells of AL, PF and ZR groups, but very minimal or no such signal was noted in ZD rats. Our findings indicated that zinc deficiency reduces GST mu expression in the supporting cells of rat olfactory epithelia but does not affect receptor cell proliferation or maintenance.

Electronmicroscopic analysis with antibodies to putative pheromone receptor on rat vomeronasal organ

The nasal cavity of vertebrates contains a variety of xenobiotic metabolizing enzymes that possess a broad range of substrate specificity ranging from metabolism of drugs, carcinogens, and steroid hormones, to dietary components and environmental pollutants. This investigation sought to localize the cellular expression and distribution of glutathione-s-transferase (GST) alpha, mu, and pi detoxifying enzymes, and to study GST activity toward different substrates in the mouse vomeronasal organ (VNO). Immunohistochemistry was used to identify GST alpha, mu and pi in the non-sensory and sensory layer of the VNO. Western blot analysis of cytosolic proteins revealed a qualitatively higher enzyme expression of GST alpha and mu in the main olfactory tissue (OE) in comparison to VNO tissue, whereas the GST pi isozyme was equally expressed in both. Total GST metabolism of 1-chloro-2, 4-dinitrobenzene (CDNB) revealed a higher activity level in the OE when compared to the VNO. In contrast, thin-layer chromatographic analysis of GST conjugation of the odorant, trans-2-hexenal (t-hex) (10 mM) showed more conjugate formed per unit protein in the VNO than the OE. The analysis of GST expression and enzyme activity within the VNO parallels the reported localization of phase I metabolizing enzymes and suggests that GST isozymes play independent roles that characterize multiple processes within VNO chemosensitivity.

Glutathione and Related Enzymes in Tumor Progression and Metastases of Human Melanoma

We have shown previously that overexpression of p-170 glycoprotein-mediated multidrug resistance plays only a minor role in conferring chemoresistance to human melanoma cells. In addition to membrane transporters like p-170, metabolizing enzyme systems have been implicated in altered drug sensitivity. Recently, glutathione and associated enzymes have been associated with resistance to alkylating substances, particularly in gastrointestinal and gynecologic cancers. In this study, we investigated whether increased levels of glutathione and related enzymes may play a role in chemoresistance in melanoma. Levels of glutathione, glutathione S-transferase (GST), glutathione reductase, and gamma-glutamyl transpeptidase were analyzed in melanoma and non-melanoma cell lines. In addition, 18 melanoma metastases derived from skin and lymph nodes were examined. Levels of gamma-glutamyl transpeptidase were statistically different in cells derived from melanocytic tumors compared with non-melanoma cell lines and normal cells. In addition, GST levels in metastases derived from skin or lymph nodes were significantly lower than those in permanent cell lines. However, levels of glutathione and related enzymes in metastases and cell lines fluctuated over a wide range, up to 40-fold, regardless of treatment status or origin of metastases. In a second part of the study, the expression of GST isoenzymes alpha, mu, and pi was studied by immunohistology in 10 benign nevi, 29 primary melanomas, and 39 melanoma metastases before and during chemotherapy. Expression of GST isoenzymes was increased with tumor progression, and GST pi was the strongest isoform expressed. However, no correlation was found between GST levels by immunohistochemistry and the course of tumor progression, between GST levels in metastases obtained before or during chemotherapy, or between GST levels and clinical response. These data suggest that alterations in glutathione metabolism and the expression of GST do not play a major role in resistance to chemotherapeutic drugs in melanoma.

Expression of GST-mu transferase in breast cancer patients and healthy controls.

The expression of glutathione S-transferase mu was measured by a qualitative immunoenzymatic assay in the blood samples of 108 women (63 breast cancer patients and 45 healthy controls) in order to analyze the relationships of GST-mu phenotype and smoking habits with tumor characteristics of breast cancer patients, such as tumor extension, nodal status, hormone receptor status and DNA content by flow cytometry. GST-mu was expressed in 53/108 (49%) of cases without any significant difference between healthy or neoplastic subjects, smokers or non-smokers, pre or post-menopausal, younger or older subjects. Moreover, the percentages of the GST-mu phenotype did not differ significantly in patients with different ER and PgR tumor status, tumor extension or nodal status. By contrast, aneuploid DNA tumor content was shown to be significantly associated with GST-mu expression (24% and 76% GST-mu positive, respectively, in diploid and aneuploid cases; p < 0.003). The biological meaning of this association remains to be interpreted.

Immunohistochemical demonstration of glutathione S-transferases in primary human breast carcinomas.

The expression of cytosolic glutathione S-transferase (GST) isoenzymes has been assessed in a series of 74 primary human breast carcinomas using an immunohistochemical method. GST pi was detected in sections from all 74 tumours; it was expressed by non-epithelial (stromal and inflammatory) cells in 62 tumours (84 per cent), but by tumour epithelium in only 35 (47 per cent). Non-neoplastic mammary epithelium was uniformly positive for GST pi. Expression of GST alpha and mu was observed in 19 and 42 per cent of the tumours, respectively, and was largely confined to the neoplastic component. Lack of staining of tumour epithelium for GST pi was significantly associated with poorer tumour differentiation (higher grade). There was no association between expression of any of the three isoenzymes and either menopausal status or expression of c-erbB-2 oncogene protein product. Immunohistochemistry is a useful method for the investigation of expression and cellular localization of GSTs within tumours; such data are needed to improve our understanding of the role of these enzymes in neoplasia and in resistance to cytotoxic drug therapy.

Immunohistochemical localization of glutathione S-transferases α μ and π in normal tissue and carcinomas from human colon

Analysis of the heterogeneity of glutathione S-transferase (GST) isozyme expression was carried out by immunohistochemical evaluation of human colon biopsy tissue from 30 patients. Using polyclonal antibodies specific for the GST alpha, mu and pi families of isozymes, an increased expression of pi was found in 21/30 carcinoma specimens compared to their pair-matched controls. This isozyme was the most prevalent in all colon samples. GST mu was expressed at reduced levels in 20/30 carcinoma specimens when compared to normal. GST alpha showed no consistent change. Analysis of the immunostaining in different cell types showed that the highest intensity stain for all isozymes was in the columnar epithelial cells. These cells were primarily responsible for the proportional changes in GST pi and mu between carcinoma and normal tissues. In addition, goblet (crypt), endothelial and muscle cells stained positively. In the lamina propria, lymphocytes and phagocytes stained positively, while fibroblasts, plasma cells and leukocytes were negative. Endocrine cells were also negative. The differential expression of GST pi and mu, confirming biochemical data, supports the potential utility of GST pi as a carcinoma marker.