In Vitro Studys Of Oxidative Damage Induced By Sodium Arsenite

Objective In order to study the cytotoxicity, proliferation inhibition and oxidative damage in A549 cells induced by sodium arsenite(NaAsO₂), by in vitro experiments, toxicology experimental ways and moleculer biology technique were used. Then, the present study was undertaken to explore the damage effect of NaAsO₂ against DNA and DNA damage repaire of A549 cells. The results of research could provide reliable evidence on the toxicity mechanism of NaAsO₂, and also provide more idea for exporing the correlation between pathogenesis of cancer and oxidative DNA damage induced by NaAsO₂.Methods To evaluate the dose-and-time-changes of A549 cells proliferation induced by sodium arsenite, the cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) test after A549 cells were treated with NaAsO₂ of control0,0.125,0.25,0.5,1.0,2.0,5.0,10.0,20.0,40.0,80.0,100.0μmol/L for exposure time of 12,24,48 and 72 hours, respectively, by observing the cell morphology. We calculated the noxious dose to human lung adenocarcinoma A549 cells of sodium arsenite, which was referenced for dose designing in follow-up experiments. In this research, clone formation inhibition test, proliferation doubling-time was used to observe the influence of NaAsO₂ on clone formation ability and proliferation ability. Then examined the chromosome damage and antioxidative activety of A549 cells poisoned with NaAsO₂ by micronucleus test in vitro and detection of antioxidative enzyme activety. To provide more evidence to the controversy on oxidative DNA damage and repair of cells exposed to NaAsO₂, comet assay was used to observe the oxidative damage and repair of A549 cells exposed to benzo(a)pyrene (BaP) and NaAsO₂, as well as the interaction of poisoning dose and order. At last, after A549 cells were treated with NaAsO₂ of control0,1,5,10,20μmol/L for 24hours, expression of hOGG1(human 8-oxoguanine DNA glyeosylasel) mRNA in A549 cells were detected by semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) and hOGGl protein was detected by West blot. We can analyse oxidative damage induced by NaAsO₂ and expression of damage repair gene hOGG1, and also provide more perfect reference for study of lung cancer therapeutic effect.Results The A549 cells proliferations were significally increased by NaAsO₂ of low concentration and markedly decreased by NaAsO₂ of high concentration with the dose-effect relation of a "U"curve. The MDA content were significally increased and the SOD and GSH-Px activities might be enhanced by sodiumarsenite of low concentration and decreased by of high concell tration with the dose-effect relation of a "U"curve. The rate of micronucleated cells increased after exposure to NaAsO₂. NaAsO₂ had no poisoning effect on DNA damage of A549 cell independently. However, DNA damage significally increased when combining with BaP. NaAsO₂ markedly restrain the DNA damage repair, and more severe DNA damage were observed when NaAsO₂ poisoned beforehand especially. NaAsO₂ effectively inhibited the expression of hOGGl gene in A549 cells with dose-effect relation.Conclusion NaAsO₂ had cytotoxicity and could usually made for oxidative damage and also damage repair gene hOGG1 observably. All results suggested that NaAsO₂ take on genetic toxicity, might by reducing damage repair and enhancing oxidative damage.