Ninth International Symposium on Recent Advances in Environmental Health Research ORAL PRESENTATION
4|Oral
MOLECULAR AND CELLULAR MECHANISMS INVOLVED IN ARSENIC TOXICITY TO HUMAN HEPATOCELLULAR CARCINOMA (HEPG2) CELLS Erika T. Brown, Clement G. Yedjou, and Paul B. Tchounwou Molecular Toxicology Research Laboratory, NIH-Center for Environmental Health, College of Science, Engineering and Technology, Jackson State University, 1400 Lynch Street, P.O. Box 18540, Jackson, Mississippi, USA Abstract: Arsenic is a well-known toxic and carcinogenic agent associated with various human malignancies, including skin, lung, liver, kidney and bladder cancers. In the present studies, we used hepatocellular carcinoma (HepG2) cells as model to determine the molecular and cellular mechanisms involved in arsenic toxicity. To achieve this goal we studied cytotoxicity, oxidative stress, genotoxicity, apoptosis, as well as, transcription and signaling factors. Utilizing the MTT [3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl tetrasodium bromide] assay, we assessed the cytotoxicity of arsenic trioxide (ATO) to hepatocellular carcinoma (HepG2) cells, which revealed a significant decrease in cell viability and a LD50 of 8.58 Âľg/ml. To determine the role of oxidative stress in ATO toxicity, we performed the lipid peroxidation, glutathione peroxidase and catalase assays, respectively. The results of the lipid peroxidation showed a significant increase (p <0.05) of malondialdehyde levels with increasing arsenic trioxide concentrations. Results obtained from glutathione peroxidase and catalase assays showed a gradual decrease in antioxidant enzyme activity in ATO-treated cells as compared to the control. In order to determine genotoxic damage cause by arsenic trioxide in HepG2 cells, we performed the comet assay. Data of this assay demonstrated a significant dose-dependent increase in DNA damage, with respect to comet tail-length and tail moment, as a result of arsenic trioxide exposure. The transcriptional activation of stress genes and related proteins (p53, p21, p38 mapk, GADD45, Bcl-2 and cytochrome c) in arsenic trioxide treated HepG2 cells were assessed by western blot analysis. Data from the western blot analysis revealed an up-regulation of p53, p21, p38 mapk, GADD45, and cytochrome c expression, while there was a down-regulation of Bcl-2. Apoptotic biomarkers were measured both by flow cytometry analysis and DNA laddering assay. Flow cytometry data showed a strong dose-response relationship between ATO exposure and Annexin-V positive HepG2 cells. Similar, a statistically significant was recorded with regard to caspase 3 activity in HepG2 cells. These results were confirmed by data of the DNA laddering assay showing clear evidence of nucleosomal DNA fragmentation in ATO-treated cells. Taken together, our research demonstrated that oxidative stress may play a key role in the molecular mechanisms involved in arsenic induced toxicity and cellular damage. Keywords: Arsenic trioxide, HepG2 cells, oxidative stress, western blot analysis, apoptosis Acknowledgements: This research was financially supported by a grant from the National Institutes of Health (Grant No. 2G12RR013459-14), through the RCMI-Center for Environmental Health at Jackson State University.