Mechanisms of arsenite-induced carcinogenesis

Inorganic arsenic is an environmental contaminant found in the American Southwest, Mexico, Chile, India and Taiwan and is associated with skin cancer, bladder cancer, Blackfoot disease, and Reynaud's syndrome. We used sodium arsenite to treat Normal Human Epidermal Keratinocyte (NHEK) as an in vitro system to study the effects of arsenic. Gene expression alteration was studied using cDNA microarray. We created expression profiles for arsenite-treated NHEKs and further studied the most affected gene: cyclin D. Next, in NHEK treated with submicromolar sodium arsenite (200, 400 and 800 nM), we observed increases of cell growth within 3 days. We also employed neutral red for viable staining on cell growth studies. The results showed similar growth stimulation on arsenite-treated cells. Flow cytometry was used for arsenite-treated NHEKs stained with propidium iodide. Analysis of cell subpopulations in the G1 and G2 phases of the cell cycle showed that in arsenite-treated cultures, more cells were entering G2/S compartments than in untreated controls.;We then performed Quantitative Polymerase Chain Reaction (Real time PCR) to examine cyclin D1 transcription in arsenite-treated NHEK cells. Our results showed that arsenite-treated NHEK exhibits a 3-fold increase in cyclin D1 transcription on day 3. Protein expression of cyclin D1 was examined in western blot analysis. Our results showed that 400 nM arsenite caused a time dependent induction of cyclin D1 protein expression. Results of electrophoretic mobility shift assays (EMSA) showed that arsenite also stimulated binding of the transcription factors, AP1 and CREBP to their respective binding motifs within 3 days. This supports a mechanism of oncogenesis based on persistent upregulation of D-type cyclins leading to a concomitant loss of G1/S checkpoint control.;The second part of my research focuses on the mitochondrial DNA (mtDNA) deletions and ROS (reactive oxygen species) caused by sub-micromolar arsenite in NHEK cells. Ultraviolet B (UVB) radiation was employed to observe the synergistic effect on NHEKs treated with arsenite. Our results showed that arsenite markedly enhanced mtDNA deletions and deletions were formed in a UVB dose-dependent manner in arsenite treated NHEKs. Reactive oxygen species (ROS) formation, as measured by oxidation of 5', 6'-carboxy-2', 7'-dichlorodihydrofluorescein diacetate (DC-FDA) indicated that UVB may enhance ROS within 2 hours but, surprisingly, arsenite was found to bring about a reduction in UVB-irradiated NHEKs after 24 hours.