Effects of arsenite treatment on DNA strand breaks, gene expression and DNA methylation of XRCC1, DNMT1 and DNMT3A in two human cell lines : lymphoblast (RPMI 1788) and embryonic kidney (HEK 293) | |
| Author | Sapkota, Ajaya |
| Call Number | AIT Thesis no.EV-12-26 |
| Subject(s) | Water--Purification--Arsenic removal Lymphoblastoid cell lines Arsenic waste DNA repair Arsenic--Toxicology |
| Note | A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Environmental Engineering and Management Inter - University Program on Environmental Toxicology, Technology and Management |
| Publisher | Asian Institute of Technology |
| Series Statement | Thesis ; no. EV-12-26 |
| Abstract | Arsenic is a ubiquitous metalloid found in air, water, soil and food. It has been classified as a known human carcinogen by International Agency for Research on Cancer (IARC). Exposure to arsenic compounds has been shown to associate with various diseases such as cardiovascular diseases, diabetes, and also increased risk for cancer of lungs, bladder, kidney and liver. Arsenic carcinogenesis involves both genetic and epigenetic mechanisms. Among them DNA damage and inhibition of DNA repair, and alteration in DNA methylation pattern are the most possible mechanisms of carcinogenesis due to toxic effect of inorganic arsenic compounds. Therefore, present study aimed to find the effects of short term arsenic treatment in terms of DNA damage, gene expression and DNA methylation in two cell lines and find a correlation between them. Two human cell lines, namely Human Embryonic Kidney cells (HEK 293, as a surrogate for kidney) and lymphoblast cells (RPMI 1788, as a surrogate for blood lymphocytes) were treated with sodium arsenite at four hours and various concentrations ranging from 1 –10 μM for HEK 293 and 1 –100 μM for RPMI 1788. DNA damage determined as DNA strand breaks, mRNA expression of DNA strand break repair gene, XRCC1, and promoter methylation of XRCC1, and expression and promoter methylation of DNA methyltransferase genes, DNMT1 and DNMT3A were determined. Comet analysis of DNA strand breaks showed that arsenite treatment caused a significant increase in DNA strand breaks with increasing concentration (p<0.05) in both cell types. Real time RT-PCR detection of expression of XRCC1 showed up-regulation in arsenite-treated HEK 293 cells, but down-regulation in RPMI 1788 cells. Promoter methylation f two DNA methyltransferase genes, namely DNMT1 and DNMT3A and a DNA repair gene, XRCC1, were performed using bisulfite conversion and pyrosequencing. Methylation level of XRCC1 in HEK 293 and RPMI 1788 cells was not affected by arsenite treatment. However, decreased level of promoter methylation of DNMT1 and DNMT3A was observed in arsenite-treated cells, but change was not significant. In DNMT genes, arsenite treatment significantly up-regulated the expression of DNMT1 and DNMT3A, in a dose-dependent manner, of both HEK 293 and RPMI 1788 cells. In conclusion, arsenite induced DNA strand breaks, altered XRCC1 expression as well as increased DNMT expression with slightly decreased promoter methylation of DNMT in both cell types. These results supported that lymphocytes could be used as surrogate cells for human biomonitoring of arsenic toxicity. However, HEK293 cells are more sensitive to arsenite than RPMI 1788. Therefore, if lymphocytes were used for monitoring arsenic induced toxicity in humans, affects felt in lymphocytes would clearly suggest that the organ, kidney, is experiencing greater level of toxicity. |
| Year | 2012 |
| Type | Thesis |
| School | School of Environment, Resources, and Development (SERD) |
| Department | Department of Energy and Climate Change (Former title: Department of Energy, Environment, and Climate Change (DEECC)) |
| Academic Program/FoS | Environmental Engineering and Management (EV) |
| Chairperson(s) | Panida Navasumrit,; |
| Examination Committee(s) | Shipin Oleg V.;Mathuros Ruchirawat;Jantamas Kanitwithayanun; |
| Scholarship Donor(s) | Ministry of Foreign Affairs, Norway; |
| Degree | Thesis (M.Sc.) - Asian Institute of Technology - Chulabhorn Research Institute - Mahidol University, 2012 |