| Arsenic is obiquire element,which exists in environment and has been used in industry and in agriculture. Arsenic is a metalloid. Human can exposed to arsenic mainly by ingestion and breath,and absorption from skin.There are about 100 millions people lived in arsenic exposed area,including 5.6-14.66 million Chinese people. Aproximately 1.59 million people had been identified to expose to arsenic within the last 5 years. Extensive studies have shown that arsenic can be considered as a major environmental contaminant and may associated with the occurrence of adverse health effects,such as skin alteration,peripheral vascular disease and cancer,with skin,lung and bladder. WHO has listed arsenic as an important chemical for environmental control.However,the mechanism for carcinogenesis induced by arsenic is still unclear. Several mode of arsenic action had been proposed by previous studies that involved the arsenic effect on DNA repair,DNA damage,DNA methalytion,and gene mutation. Alteration in redox status by arsenic and its effect on signal transduction has been suggested to relate to cell proliferation and cause cancer finally. But there was still no satisfied result about mechanism of carconigenesis induced by arsenic.Current study tried to use cellular and molecular biology technique for the establishment of a arsenic induced cell transformation model and identify the relationship between redox status caused by arsenic exposure and cell transformation. The role of redox gene expression and alteration of proto-oncogene and anti-oncogene of cell exposed to arsenic in cell transformation has been studied.Objective:1. To measure arsenic cytetoxicity and determine thein NIH 3T3 cell. To establish a cell transformation model induced by low dose and long time exposure with arsenic. 2. To investigate the arsenic effect on glutathione (GSH) level and related gene expression in NIH 3T3 cells treated by arsenic both in short time as well as long time,and its role in arsenic induced cell transformation. 3. Study on the effect of short-time and long-time treatment with arsenic on thioredoxin and thioredoxin reductase gene expression,and its role on cell transformation induced by long time and low dose exposure to arsenic,the interaction between arsenic and TPA and HiOi was also investigated. 4. To investigate the arsenic effect on proto-oncogene c-jun c-fos,and anti-oncogene p53 gene expression in cells treated by arsenic,and its role in arsenic induced cell transformation. To provide the evidence for the mechanism of arsenic induced cell transformation.Methods:1. Cellular toxicity was measured by using neutral red uptake assay. A minor modification of the neutral red absorption spectrophotometric test was used to measure cell viability. 0.1 uM and 0.5uM As(III) were selected as the doses for the induction of cell transformation. Cells were recovered in normal medium once a week. After 10 weeks,cells treated with arsenic were measured the cellular toxicity,and morphology changes were observed. Cell transformation was conformed by Anchorage-independent growth. 2. Cell extracts were obtained after each treatment time point for enzyme activity assay. Glutathione concentration was determined by using Clarke method;Glutathione transferase (GST) activity was determined spectrophotometrically at 340nm by assessing the formation of conjugated glutathione and CDNB;Gluthione reductase (GR) was assayed using protocols described by Styblo and Thomas. The enzyme activity was determined by following the rate of decrease in NAPDH absorbance due to reduction of GSSG. 3. Whole cell extracts were prepared at each time point,and then the protein concentration wasdetermined using BioRad protein assay kit. Protein expression of Glutathione transferase (GST),hemeoxygenase (HO-1),c-jun,c-fos and p53 were determined by SDS-Polyacrymide Gel Electrophoresis and Western Blot Assay. 4. Primers for GR,GPx,TRX,TR-1,TR-2,p53 were designed according to the specific sequences of each gene,and the each probe was synthesized using 1...
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