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The distribution, induction and isoenzyme profile of glutathione S-transferase and glutathione peroxidase in isolated rat liver parenchymal, Kupffer and endothelial cells.

ABSTRACT: The distribution and inducibility of cytosolic glutathione S-transferase (EC 2.5.1.18) and glutathione peroxidase (EC 1.11.1.19) activities in rat liver parenchymal, Kupffer and endothelial cells were studied. In untreated rats glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene and 4-hydroxynon-2-trans-enal as substrates was 1.7-2.2-fold higher in parenchymal cells than in Kupffer and endothelial cells, whereas total, selenium-dependent and non-selenium-dependent glutathione peroxidase activities were similar in all three cell types. Glutathione S-transferase isoenzymes in parenchymal and non-parenchymal cells isolated from untreated rats were separated by chromatofocusing in an f.p.l.c. system: all glutathione S-transferase isoenzymes observed in the sinusoidal lining cells were also detected in the parenchymal cells, whereas Kupffer and endothelial cells lacked several glutathione S-transferase isoenzymes present in parenchymal cells. At 5 days after administration of Arocolor 1254 glutathione S-transferase activity was only enhanced in parenchymal cells; furthermore, selenium-dependent glutathione peroxidase activity decreased in parenchymal and non-parenchymal cells. At 13 days after a single injection of Aroclor 1254 a strong induction of glutathione S-transferase had taken place in all three cell types, whereas selenium-dependent glutathione peroxidase activity remained unchanged (endothelial cells) or was depressed (parenchymal and Kupffer cells). Hence these results clearly establish that glutathione S-transferase and glutathione peroxidase are differentially regulated in rat liver parenchymal as well as non-parenchymal cells. The presence of glutathione peroxidase and several glutathione S-transferase isoenzymes capable of detoxifying a variety of compounds in Kupffer and endothelial cells might be crucial to protect the liver from damage by potentially hepatotoxic substances.

SUBMITTER: Steinberg P

PROVIDER: S-EPMC1133647 | biostudies-other | 1989 Dec

REPOSITORIES: biostudies-other

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Project description:BackgroundGlutathione (GSH) is one of the most important agents of the antioxidant defense system of the cell because, in conjunction with the enzymes glutathione peroxidase (GSH-Px) and glutathione S transferase pi (GSTpi), it plays a central role in the detoxification and biotransformation of chemotherapeutic drugs. This study evaluated the expression of GSH and the GSH-Px and GSTpi enzymes by immunohistochemistry in 30 canine mammary tumors, relating the clinicopathological parameters, clinical outcome and survival of the bitches. In an in vitro study, the expression of the genes glutamate cysteine ligase (GCLC) and glutathione synthetase (GSS) that synthesize GSH and GSH-Px gene were verified by qPCR and subjected to treatment with doxorubicin, to check the resistance of cancer cells to chemotherapy.ResultsThe immunohistochemical expression of GSH, GSH-Px and GSTpi was compared with the clinical and pathological characteristics and the clinical outcome in the bitches, including metastasis and death.The results showed that high immunoexpression of GSH was correlated to the absence of tumor ulceration and was present in dogs without metastasis (P < 0.05). There was significant correlation of survival with the increase of GSH (P < 0.05). The expression of the GSH-Px and GSTpi enzymes showed no statistically significant correlation with the analyzed variables (p > 0.05). The analysis of the relative expression of genes responsible for the synthesis of GSH (GCLC and GSS) and GSH-Px by quantitative PCR was done with cultured cells of 10 tumor fragments from dogs with mammary tumors.The culture cells showed a decrease in GCLC and GSS expression when compared with no treated cells (P < 0.05). High GSH immunoexpression was associated with better clinical outcomes.ConclusionTherefore, high expression of the GSH seems to play an important role in the clinical outcome of patients with mammary tumors and suggest its use as prognostic marker. The in vitro doxorubicin treatment significantly reduces the expression of GCLC and GSS genes so we can consider them to be candidates for predictive markers of therapeutic response in mammary cancer.

Project description:More attention has been recently directed toward glutathione peroxidase and s-transferase enzymes because of the great importance they hold with respect to their applications in the pharmaceutical field. This work was conducted to optimize the production and characterize glutathione peroxidase and glutathione s-transferase produced by Lactobacillus plantarum KU720558 using Plackett-Burman and Box-Behnken statistical designs. To assess the impact of the culture conditions on the microbial production of the enzymes, colorimetric methods were used. Following data analysis, the optimum conditions that enhanced the s-transferase yield were the De Man-Rogosa-Sharp (MRS) broth as a basal medium supplemented with 0.1% urea, 0.075% H2O2, 0.5% 1-butanol, 0.0125% amino acids, and 0.05% SDS at pH 6.0 and anaerobically incubated for 24 h at 40°C. The optimum s-transferase specific activity was 1789.5 U/mg of protein, which was ~12 times the activity of the basal medium. For peroxidase, the best medium composition was 0.17% urea, 0.025% bile salt, 7.5% Na Cl, 0.05% H2O2, 0.05% SDS, and 2% ethanol added to the MRS broth at pH 6.0 and anaerobically incubated for 24 h at 40°C. Furthermore, the optimum peroxidase specific activity was 612.5 U/mg of protein, indicating that its activity was 22 times higher than the activity recorded in the basal medium. After SDS-PAGE analysis, GST and GPx showed a single protein band of 25 and 18 kDa, respectively. They were able to retain their activities at an optimal temperature of 40°C for an hour and pH range 4-7. The 3D model of both enzymes was constructed showing helical structures, sheet and loops. Protein cavities were also detected to define druggable sites. GST model had two large pockets; 185Å3 and 71 Å3 with druggability score 0.5-0.8. For GPx, the pockets were relatively smaller, 71 Å3 and 32 Å3 with druggability score (0.65-0.66). Therefore, the present study showed that the consortium components as well as the stress-based conditions used could express both enzymes with enhanced productivity, recommending their application based on the obtained results.

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The distribution, induction and isoenzyme profile of glutathione S-transferase and glutathione peroxidase in isolated rat liver parenchymal, Kupffer and endothelial cells.

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