Localization Of Glutathione S-transferase Research Articles

Role of Glutathione-S transferase Pi (GSTπ) during the treatment of doxorubicin and the plant extract Phyllanthus urinaria L. in H9c2 cells

Doxorubicin (DOX) is widely used to treat many types of cancer but frequently causes cardiotoxicity [1]. Our previous study has shown that an extract from Phyllanthus urinaria (PU) protected against DOX-induced cytotoxicity in H9c2 cells [2]. In this study, we examined the mechanism of cytoprotection in association with the expression and localization of glutathione-S transferase in H9c2 cells. The expression of antioxidant enzymes including glutamylcysteine synthetase (GCS), MnSOD, and CuZnSOD was also investigated. We found that among three major GST subtypes GST Pi (GSTP) is predominantly expressed in H9c2 cells. While no significant alterations in the enzyme activities, the cytoprotective effect of PU treatment appeared to involve nuclear localization of GSTP. Using RNA interference technique to suppress GSTP expression provided evidence that DOX was highly accumulated in nuclei and apoptosis was increased as evaluated by TUNEL assay. In conclusion, PU may be used as an alternative resource of antioxidants with distinctive mechanisms of action that may be suitable for specific type of oxidative insults. Acknowledgements: Srinakharinwirot University Research Fund; The Matsumae International Foundation

Identification and localization of glutathione S-transferase as a potential target enzyme in Brugia species.

Brugia filarial nematodes are pathogenic lymphatic-dwelling parasites that, like other helminths, may modify the host's defense mechanisms by a major detoxification process involving glutathione-binding proteins such as glutathione S-transferases (GSTs). In the present study, soluble extracts of third-stage larvae, adult male and female worms, microfilariae of either B. pahangi or B. malayi or the adult worm excretory-secretory products of B. malayi were used to determine GST activity. These extracts and affinity-purified fractions of B. pahangi adult worms had a specific enzymatic activity when 1-chloro-2,4-dinitrobenzene was used as a substrate. The observance of this enzyme in all life cycle stages of Brugia spp. demonstrates its ubiquitous nature. Lavage of intraperitoneally infected jirds, but not that of uninfected jirds, also showed increased enzymatic activity, suggesting that GST is secreted in vivo. Soluble proteins of both Brugia spp. were strongly recognized by antibodies in sera from rabbits immunized with affinity-purified native GST of Onchocerca volvulus. Immunohistochemical studies localized these proteins in adult worms, demonstrating cross-reactivity between the GST of these two filarial nematodes. The effect of this enzyme on the motility and viability of adult worms, microfilariae, and larvae was tested in vitro using a battery of known GST inhibitors. Of all those tested, ethacrynic acid, N-ethylmalemide, 4-nitropyridine-oxide, or 1-chloro-2,4-dinitrobenzene at micromolar concentrations reduced the viability and motility of microfilariae, third-stage larvae, and adult worms. These results suggest that Brugia GSTs are major metabolic enzymes and may play an important role in the parasite's survival.

Immunohistochemical Detection of Glutathione S-transferase P in Postnatal Rat Liver after Administration of Dexamethasone.

The localization of glutathione S-transferase P (GST-P) in postnatal rat liver was examined by an immunohistochemical technique. Dexamethasone (DEX) was injected intraperitoneally into postnatal rats at a dose of 2 μg/g body weight daily for 4 days. GST-P was detected only in the cytoplasm of epithelial cells of the interlobular bile ducts in the livers of 8-day-old rats that did not receive DEX-administration. In contrast, dispersed-positive hepatocytes were observed in the liver lobules of rats that were administered DEX from 4 days after birth. In these rats, GST-P was found in the cytoplasm of those hepatocytes and epithelial cells of the interlobular bile ducts. GST-P was observed only in the cytoplasm of epithelial cells of the interlobular bile ducts in the livers of 13-day-old rats that did not receive DEX-administration and those that began DEX-administration at 9 days. The immunoreactivity was stronger compared to the 8-day-old rats. These results indicate that DEX promotes the appearance of hepatocytes that contain the GST-P protein in early postnatal rat liver.

Immunohistochemical demonstration of epithelial glutathione S‐transferase isoenzymes in normal, benign, premalignant and malignant human oral mucosa

The expression and localization of glutathione S-transferase (GST) isoenzymes in the epithelium of normal oral mucosa (n = 9), overlying reactive fibrous hyperplasia (n = 9), and of potentially malignant [leukoplakia (n = 25), submucous fibrosis (n = 12), verrucous hyperplasia (n = 16)] and malignant [squamous cell carcinoma (n = 36), verrucous carcinoma (n = 13)] oral lesions were examined immunohistochemically using polyclonal antibodies raised against GST isoenzymes (alpha, mu and pi) with the standard avidin-biotin-peroxidase complex (ABC) method. GST alpha, mu and pi were almost completely absent in the epithelium of normal oral mucosa and overlying benign fibrous tissues. GST alpha staining was cytoplasmic and focally positive, while GST mu staining was similar to but weaker than that seen for GST alpha. GST pi showed both cytoplasmic and nuclear staining and was expressed in 60% of leukoplakias with mild dysplasia (n = 15), 80% of leukoplakias with moderate to severe dysplasia (n = 10). 75% of submucous fibrosis samples (n = 12), 75% of verrucous hyperplasias (n = 16), 77% of verrucous carcinomas (n = 13), 81% of well-differentiated squamous cell carcinomas (n = 26) and 70% of moderate- to poorly-differentiated squamous cell carcinomas (n = 10). In addition, GST pi expression was independent of the state of differentiation of oral cancers. Since GST pi was significantly over-expressed in the oral premalignant and malignant lesions, the kinetics of GST pi-positive cells and the value of GST pi as a tumor marker in oral carcinogenesis need further investigation.

Immunohistochemical localization, purification, and characterization of human urinary bladder glutathione S-transferases

This study describes immunohistochemical localization, purification and characterization of glutathione S-transferase (GST) of human urinary bladder. Even though all the three major classes of isoenzymes (α, μ, and π) were expressed in human bladder, more than 90% of total GST activity was accounted for by a π class anionic form. Human bladder α, μ, and π class GSTs were immunologically related to respective isoenzymes of other human tissues. GST π was present in all 13 samples analyzed, whereas GST α and μ were detected in nine and eleven samples, respectively. GST α of human bladder appeared to be unique, because unlike this class of GSTs of other human tissues, bladder enzyme had lower affinity for GSH linked to epoxy-activated Sepharose 6B affinity resin. Immunohistochemical staining indicated localization of GST α in epithelial surface cells, underlying submucosa and smooth muscle, whereas μ and π class isoenzymes were predominantly distributed in epithelial surface cells. These results suggest that human bladder GSTs may play an important role in providing protection against xenobiotics because epithelium is considered a target for several carcinogens and all the three classes of isoenzymes are expressed in these cells.

正常および実験的ラット停留精巣におけるグルタチオンS-トランスフェラーゼの免疫組織化学的研究

Using the antibody for glutathione S-transferase (GST) purified from human kidney, normal testes and experimental cryptorchid testes from newborn to 20-week-old rats were immunohistochemically stained by the peroxidase antiperoxidase (PAP) method. The cryptorchidism was surgically created at 1 week of age. The localization of GST was particularly examined by light microscopy, and the amount of Leydig cells was measured by a stereological method. 1. Leydig cells in the normal and cryptorchid testes showed strong GST activity at all ages. The amount of these cells in normal testes increased from 4 to 8 weeks of age and then slightly decreased, whereas in cryptorchid testes it was significantly larger than in the normal testes at 20 weeks of age, indicating hyperplasia of Leydig cells. 2. In the normal and cryptorchid testes, degenerating primary spermatocytes with GST activity appeared in the seminiferous tubules at 2 to 4 weeks of age. In the cryptorchid testis, degenerating germ cells with GST activity were also found in the regressing seminiferous tubules after 4 weeks of age. It is possible that GST acts as a detoxification system in the degenerating germ cells. 3. The PAP staining of GST in the rat testes is considered to be useful method for evaluating metabolic function of the spermatogenic cells and the distribution and amount of Leydig cells. 4. Experimental cryptorchidism showed that germ cells become sensitive after 4 weeks of age.

Immunochemical localization of glutathione S-transferases in rat lung.

Glutathione (GSH) S-transferases are a family of enzymes involved in the detoxication of foreign compounds or xenobiotics. Since lung is constantly exposed to environmental pollutants and oxidative stress, the six forms of GSH S-transferases reported to be present in rat lung may play an important role in the detoxication processes. Present studies using immunocytochemical techniques indicate that significant amounts of GSH S-transferases are associated with bronchioles and alveoli. It is suggested that these GSH S-transferases may be present in bronchiolar Clara cells, alveolar endothelia and Type II cells. All the five subunits Ya, Ya′, Yb, Yb′, and Yc which in their binary combinations give rise to the different forms of GSH S-transferases are present in bronchiolar and alveolar tissue.