Perturbation of Testicular Leydig Cell Proliferation and Gene Expression Analysis Using Sodium Arsenite

Recent research has established arsenic as an environmental carcinogen. Given arsenic\u27s wide spread distribution it has the potential to affect millions of people worldwide. On-going research is trying to establish the toxicities of this chemical. While much effort has been put forth in studying the effects of arsenic, its role in carcinogenesis remains unknown. The main goal of this study is to determine if arsenic exposure can produce an unstable environment in testicular Leydig cells. To examine this potential effect, we have designed two studies that will test short- term, as well as relatively long term exposure to arsenic in mouse testicular cells and its effect on cell proliferation and changes in gene expression. A mouse testicular Leydig cell line, TM3, derived from normal mouse testis will be used for both the proliferation and gene expression studies. The effect of arsenic on cell proliferation will be measured by cell proliferation assay, and gene expression changes will be determined by quantitative real-time PCR. The cells will be grown in culture medium containing 5% horse serum, 2.5% fetal bovine serum. Cell cultures will be switched to a serum-free medium prior to treatment with test 1 2 compound. Arsenic treatments will be carried out at 1pg, 10pg, 100pg, 1pg,and 10ng and 100ng per ml of sodium arsenite, along with a control for the proliferation studies. For the gene expression studies, only 1pg, 100pg, 10ng, 100ng per ml concentrations of sodium arsenite and a sodium arsenite-free control will be used. RNA will be isolated from the cells followed by analysis via quantitative RT-Pt.R, The hypothesis is that the sodium arsenite will cause cell proliferation in low dose treatments, and that there will be a significant decrease in the expression of DNA repair genes. This research is very important in that it may prove that there is a link between sodium arsenite and testicular cancer. It may also help in the understanding of the etiology of testicular cancer as wel