Arsenic contamination of drinking water is a real public health problem in certain areas of South-East Asia where chronic exposure has been correlated to higher rates of lung, skin, bladder, kidney, and liver cancer. Although arsenic carcinogenicity is well established, the mechanism by which it induces cancer is poorly understood. Recent evidence suggests that oxidative stress could be a possible mechanism for the carcinogenic effects of arsenic. Selenium, in the form of selenocysteine, is necessary for the activity of several enzymes with a role in the defense against reactive oxygen species (ROS), primarily thioredoxin reductases (TrxR) and glutathione peroxidases (Gpx). One of the key enzymes in the incorporation of Se into selenoproteins is selenophosphate synthetase (SPS). SPS catalyzes the activation of Se to selenophosphate, and is the first step in the pathway of selenoprotein biosynthesis. SPS contains a conserved vicinal dithiol motif (CXC) within a region of amino acids that have been predicted to be a selenium binding site. Our hypothesis is that arsenite inhibits new selenoprotein synthesis, thus indirectly increasing the level of ROS. In this study we have developed a spectrophotometric assay for SPS. Using this assay, we have determined that arsenite inhibits SPS activity. Kinetic analysis of this inhibition showed that arsenite, a trivalent form of arsenic, acts as a competitive inhibitor with the substrate, sodium selenide. This inhibition of SPS could represent a potential molecular mechanism for oxidative stress induced upon arsenite treatment of human cell lines in culture. To further study the effects of trivalent arsenicals at a cellular level we decided to use a human keratinocyte cell line, HaCaT as a cell culture model. Our study showed that although arsenite does not alter cell proliferation or protein synthesis, it specifically inhibits new selenoprotein synthesis. However, short term or long term exposure of HaCaT cells to arsenite failed to result in changes to Gpx and TrxR levels. Since the radioisotope selenium used in labeling studies is selenite, these results indicate that an alternate source of selenium may bypass the inhibitory effects of arsenite. Future studies will focus on studying the effects of arsenicals on keratinocytes cultured in a defined medium allowing a better control of the selenium source.
Master of Science (M.S.)
Burnett College of Biomedical Sciences
Molecular Biology and Microbiology
Length of Campus-only Access
Masters Thesis (Open Access)
Konate, Fanta, "The Effects Of Arsenic On Selenoprotein Biosythesis" (2005). Electronic Theses and Dissertations, 2004-2019. 6100.