James W DuMond | PubMed Health

PubMed2007

Genetic and epigenetic changes induced by chronic low dose exposure to arsenic of mouse testicular Leydig cells.

Arsenic is an important environmental carcinogen that affects millions of people worldwide through contaminated water supplies. Genotoxicity of arsenic has been a topic of controversy. Both genetic alterations (mutations) and epigenetic changes (methylation) have been shown to play a crucial role in environmental carcinogenesis. Chronic exposure to arsenic has been shown to induce malignant transformation of mammalian cells. However, the genetic aberrations induced by arsenic in this process are unclear. The purpose of this study was to determine if both lower (1 pg/ml) and higher concentrations (100 ng/ml) of arsenic induces either mutations or methylation changes that could lead to the development of genomic instability in TM3 cells, immortalized Leydig cells derived from normal mouse testis. Two independent exposure times were used in this study which resulted in cells of 33 and 100 generations in age. Arsenic-induced genetic and epigenetic changes were screened at a genome-wide level by random amplified polymorphic DNA (RAPD), also known as AP-PCR method with undigested DNA as well as DNA digested by the methylation sensitive isosizomeric restriction enzymes MSPI and HpaII and untreated controls. Changes in the DNA fingerprint of both, the restriction enzyme digested DNA (indicating methylation changes) as well as undigested DNA (indicating mutations) from arsenic-treated (low as well as high dose) samples were observed as compared to their controls. Thus, this study provides the first evidence at DNA sequence level for mutagenic potential of arsenic. Further characterization of these altered genomic regions is underway. The understanding of these genetic and epigenetic changes in arsenic-induced carcinogenesis will provide a basis for better interventional approaches in both the treatment and prevention of arsenic-induced cancer.

Animals↗

PubMed2006

Development of a self-proliferating Leydig cell line: a hyper-sensitive E-screening model.

The mechanisms of estrogenic endocrine disruption on the male reproductive tract are poorly understood. In order to examine estrogenic properties of xenobiotic chemicals on male tissues, we have developed a mouse Leydig cell line (TM3-SF) that self-proliferates under serum-free conditions. This cell line was derived from ATCC's cell line, TM3. The development of TM3-SF was accomplished over a 4-month period by a progressive serum starvation of the original TM3 cells. The newly established cell line was maintained under serum-free conditions for 20 passages prior to testing. Sensitivity of the TM3-SF cells to estrogens was assayed by cell proliferation studies. A total of four compounds, diethylstilbestrol (DES), 17beta-estradiol, 17alpha-estradiol, and Bis-phenol A, were tested. Significant increases in cell proliferation occurred at various concentrations ranging from 1 pg/ml to 100 ng/ml for all four compounds. The order of potency observed was DES > Bis-phenol A > 17beta-estradiol and > 17alpha-estradiol. In addition, we investigated the mechanism for the self-proliferative properties of TM3-SF. The results of these trials indicate that either inhibin or activin is a primary growth factor for this cell line as a 50% inhibition of growth was noted when cell cultures were exposed to the anti-betaa subunit of inhibin/activin. Furthermore, the addition of the anti-betaa subunit of inhibin/activin blocked the DES-induced proliferation of TM3-SF. We conclude that the growth of TM3-SF cells is estrogen sensitive and that either inhibin or activin is involved in the self-regulation of growth.

Activins↗