ABSTRACT
Background and Objective: The aim of the present study was to evaluate oxidative stress byinvesting oxidatively damaged DNA AS Formamidopyrimidine DNA glycosylase (Fpg) -sensitive sites, glutathione peroxidase (GPx), superoxide dismutase (SOD) activities reduced glutathione (GSH) level and nitrite level as satble end product of in women receiving hormone replacement therapy (HRT). Materials and Methods: 127 healthy postmenopausal women receiving HRT and 25 healthy control postmenopausal women were included in this study. Women receiving HRT, comprised surgical menopausal women who underwent surgery for benign conditionsand received conjugated equine estrogen, 0.625 mg/day for 1year (group 1), 5 years (group 2) and more than 10 years (group 3), spontaneous postmenopausal women received conjugated equine estrogen, 0.625 (Premarin) mg/day and medroxyprogesterone acetate, 2.5 mg/day (Premelle) for 1 year (group 4), 5 years (group 5) and more than 5 years (group 6).We investigated in the present study the effects of HRT on nitrite level and GSH level, activities of SOD and GPx and oxidative damage to DNA by comet assays by measuring levels of Fpg-sensitive sites. Results: Although no significant differences were found in the SOD activities, in total group receiving HRT, increased DNA oxidation (P<0.001) together with an increased GPx activity (P<0.001) and nitrite level (P<0.001) as well as a decreased GSH level (P < 0.05) as compared with controls were observed. Conclusion: Estrogen alone or oestrogen in combination with progesterone and duration of use did not significantly alter the results. We evaluated that caused oxidative stress by investigating oxidative DNA damage as Fp-sensitive sites and GSH.NO levels in women receiving HRT.
Subject(s)
Analysis of Variance , Antioxidants/metabolism , Case-Control Studies , DNA Damage/drug effects , DNA-Formamidopyrimidine Glycosylase/metabolism , Dose-Response Relationship, Drug , Drug Administration Schedule , Estrogen Replacement Therapy/adverse effects , Estrogen Replacement Therapy/methods , Estrogens, Conjugated (USP)/administration & dosage , Estrogens, Conjugated (USP)/adverse effects , Follow-Up Studies , Hormone Replacement Therapy/adverse effects , Hormone Replacement Therapy/methods , Humans , Medroxyprogesterone Acetate/administration & dosage , Medroxyprogesterone Acetate/adverse effects , Middle Aged , Oxidation-Reduction/drug effects , Oxidative Stress/drug effects , Postmenopause/blood , Postmenopause/drug effects , Reference Values , Treatment OutcomeABSTRACT
<p><b>OBJECTIVE</b>To investigate the role of JWA gene in benzo (a) pyrene [B (a) P] induced DNA damage and repair effects in HeLa cells.</p><p><b>METHODS</b>The antisense JWA express vector (pEGFP-C1-asJWA) was constructed and stably transfected into HeLa cells. JWA deficient HeLa cells (asJWA-HeLa) was then screened and established. The general characteristics of asJWA-HeLa cells were investigated. DNA damage and repair cell culture model was conducted by treating the cells with 50 micromol/L B (a) P plus S9 for 3 hours and then the cells were maintained further 0, 1, 3, and 24 hours for DNA repairing. The damaged DNA was detected by single cell gel electrophoresis assay (comet assay).</p><p><b>RESULTS</b>JWA deficient HeLa cells (with a 31% of JWA protein expression as compared with the control) were obtained successfully. Compared with the empty vector transfected cells (C1-HeLa) and the untransfected HeLa cells, asJWA-HeLa cells were more sensitive to B (a) P exposure and with a delayed DNA repair process.</p><p><b>CONCLUSION</b>The JWA determined might function as a potential effective environmental responsive gene and actively participate the process of B (a) P exposure associated with intracellular signal pathways of DNA damage and repair.</p>
Subject(s)
Humans , Benzo(a)pyrene , Toxicity , DNA Damage , DNA Repair , DNA-Formamidopyrimidine Glycosylase , Genetics , Gene Expression , HeLa Cells , Heat-Shock Proteins , Genetics , Intracellular Signaling Peptides and Proteins , GeneticsABSTRACT
8-oxo-7,8-dihydroguanine (8-oxo-G) in DNA is a mutagenic adduct formed by reactive oxygen species. In Escherichia coli, 2,6-dihydroxy-5N-formamidopyrimidine (Fapy)-DNA glycosylase (Fpg) removes this mutagenic adduct from DNA. In this report, we demonstrate base excision repair (BER) synthesis of DNA containing 8-oxo-G with Fpg in vitro. Fpg cut the oligonucleotide at the site of 8-oxo-G, producing one nucleotide gap with 3' and 5' phosphate termini. Next, 3' phosphatase(s) in the supernatant obtained by precipitating a crude extract of E. coli with 40% ammonium sulfate, removed the 3' phosphate group at the gap, thus exposing the 3' hydroxyl group to prime DNA synthesis. DNA polymerase and DNA ligase then completed the repair. These results indicate the biological significance of the glycosylase and apurinic/ apyrimidinic (AP) lyase activities of Fpg, in concert with 3' phosphatase(s) to create an appropriately gapped substrate for efficient BER synthesis of DNA containing 8-oxo-G.
Subject(s)
DNA Glycosylases/metabolism , DNA Repair , DNA, Bacterial/chemistry , DNA-Formamidopyrimidine Glycosylase/metabolism , Escherichia coli/enzymology , Guanine/analogs & derivativesABSTRACT
<p><b>OBJECTIVE</b>To confirm that arsenic (As) induces oxidative DNA damage in phytohemagglutinin (PHA)-stimulated and unstimulated human lymphocytes.</p><p><b>METHODS</b>The alkaline comet assay combined with specific enzyme (Formamidopyrimidine-DNA glycosylase, FPG) digestion was used to measure As-induced base damage.</p><p><b>RESULTS</b>The enzyme-sensitive sites were readily detected with the alkaline comet assay after the cells were treated with 10 micromol As for 2 hours. The repair patterns observed for FPG-created DNA single strand breaks (SSBs) in As-treated cells were comparable to those in hydrogen peroxide (H(2)O(2))-treated cells. The enzyme-created SSBs, As-induced base damage, were more significantly revealed in PHA-stimulated lymphocytes. About 63% and 68% of SSBs induced by As and H(2)O(2), respectively, were repaired in PHA-stimulated lymphocytes by 2-hour repair incubation, but about 34% and 43%, respectively, were repaired in unstimulated cells. About 40% and 49% of base damage induced by As and H(2)O(2), respectively, were repaired in PHA-stimulated lymphocytes, but about 19% and 21 %, respectively, were repaired in unstimulated cells.</p><p><b>CONCLUSIONS</b>As induces oxidative DNA damage in human lymphocytes within micromolar concentrations. Like the damage induced by H(2)O(2), As-induced DNA damage was more slowly repaired in unstimulated lymphocytes.</p>
Subject(s)
Adult , Humans , Arsenic , Pharmacology , DNA Damage , DNA Repair , DNA, Single-Stranded , DNA-Formamidopyrimidine Glycosylase , Electrophoresis , Methods , Hydrogen Peroxide , Pharmacology , Lymphocytes , N-Glycosyl Hydrolases , Oxidation-Reduction , Phytohemagglutinins , PharmacologyABSTRACT
To elucidate role of the three enzymes in hepatocarcinogenesis, hMTH1, hOGG1 and hMYH, mRNA expression were examined by using RT/semi-quantitative real-time PCR and 8-O-HdG levels was studied by HPLC/ECD in HCC and non-tumorous liver tissue of 21 patients with hepatocellular carcinoma (HCC). It was found that the 8-OHdG level in non-tumourous liver tissue was significantly higher than in HCC tissue (P = 0.006), and this was correlated with the degree of inflammation. The hMTH1 expression in HCC tissue was significantly higher than in non-tumorous liver tissue (P = 0.014). Inversely, The hMYH alpha expression was significantly increased (P = 0.039) in non-tumorous liver tissue. No difference was seen in hOGG1 expression in non-tumorous liver and HCC tissue. A significant linear correlation between hMTH1 and hOGG1 expression was found both in HCC tissue (r = 0.809, P < 0.001) and in non-tumorous liver tissue (r = 0.883, P < 0.001). Our findings suggested a reactive rather than pathogenic role of the DNA repair enzymes in the hepatocarcinogenesis.