Abstract
Cellular glutathione peroxidase (GSH-Pxl) is a nuclear encoded cytosolic antioxidant enzyme that reduces hydrogen peroxide and organic hydroperoxides with GSH as the hydrogen donor. However, it was found that the addition of commercial bovine glutathione peroxidase (GPxs) would decrease arsenite-induced micronucleus (MN) formation or genotoxicity in GPx deficient xrs-5 cells. And, the GPx inhibitor increase MN formation in CHO-Kl cells. To further determine whether GPx-1 plays a role to protect HF cells from arsenite-induced oxidative stress, GPx-1 antibodies were produced and used as probe for examining GPx-1 protein. The gpx-1 clning, GPx-1 expression, GPx-1 antibodies production and GPx-1 antibodies titration are as as followings: human GPx1-cDNA was subcloned into commercialy available pQE32 expression vector (Quiagen) and transfected into SG1009 E. coli. The transformed clone having the highest GPx expression was used to produce large quantity of GPx-1 protein as antigen. Antibodies to GPx-1 we produced from serum after several injections of GPx-1 antigen into rabbits. The results in Western blotting analysis showed that 1: 300 dilution of antibodies-containing serum could detect 12.5 ng GPx-1 antigen. In addition, GPx-1 in about 50μg of human fibroblast extract could be detected using same titration. To examine GPx-1 expression in human fibroblasts after arsenite or H2O2 treatment, GPx-1 antibodies of 3000-fild dilution and western blotting were used. The results showed a dose-dependent increase (form 1 to 3.3-fold) of GPx-1 expression in cells after treatment with 0-400μM aresenite for 4h or 0-160μM H2O2 for 30 min. More (2~3.4-fold( GPx-1 expression is found in cells after treatment with 2.5-5μM aresenite for 24h than those in untreated control. However, no GPx-1 expression was detect in cells treated with 10 or 20μM aresenite for 24h. Applied reverse transcription-coupled PCR analysis to examine gpx-1 mRNA in treated human fibroblasts, the results did not correlate with those of GPx-1 protein expression after same treatment protocols. The results showed a dose-dependent increase (form 1 to 1.9-fold) of GPx-1 mRNA transcription in cells after treatment with 0-400μM aresenite for 4h. Less (0.4-fold only) GPx-1 mRNA transcription from cells treated with aresenite for 24h at above concentra ion (0-20μM) were found than those from untreated control. In addition, no difference in GPx-1 mRNA transcription between sample from 0-160μM H2O2 treatment and untreated control.