ToxSci Advance Access originally published online on June 8, 2004
Toxicological Sciences 2004 81(1):78-89; doi:10.1093/toxsci/kfh191
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Toxicological Sciences vol. 81 no. 1 © Society of Toxicology 2004; all rights reserved.
Assessment of the Effects of Chemicals on the Expression of Ten Steroidogenic Genes in the H295R Cell Line Using Real-Time PCR
* Department of Zoology, National Food Safety and Toxicology Center, Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan 48824; ENTRIX Inc., East Lansing, Michigan 48864; Center for Coastal Pollution and Conservation City University of Hong Kong, Kowloon, Hong Kong, SAR China; and Institute for Risk Assessment Sciences (IRAS), University of Utrecht, 3508 TD Utrecht, Netherlands
Received May 7, 2004; accepted June 3, 2004
The potential for a variety of environmental contaminants to disturb endocrine function in wildlife and humans has been of recent concern. While much effort is being focused on the assessment of effects mediated through steroid hormone receptor–based mechanisms, there are potentially several other mechanisms that could lead to endocrine disruption. Recent studies have demonstrated that a variety of xenobiotics can alter the gene expression or activity of enzymes involved in steroidogenesis. By altering the production or catalytic activity of steroidogenic or steroid-catabolizing enzymes, these chemicals have the potential to alter the steroid balance in organisms. To assess the potential of chemicals to alter steroidogenesis, an assay system was developed using a human adrenocortical carcinoma cell line, the H295R cell line, which retains the ability to synthesize most of the important steroidogenic enzymes. Methods were developed, optimized, and validated to measure the expression of 10 genes involved in steroidogenesis by the use of real-time quantitative reverse transcriptase PCR. The effects of several model chemicals known to alter steroid metabolism, both inducers and inhibitors, were assessed. Similar expression patterns were observed for chemicals acting through common mechanisms of action. Time-course studies demonstrated distinct time-dependent expression profiles for chemicals able to modulate steroid metabolism. The assay, which allows simultaneous analysis of the expression of numerous steroidogenic enzymes, would be useful as a sensitive and integrative screen for the many effects of chemicals on steroidogenesis.
Key Words: steroidogenesis; bioassay; xenoestrogens; screening.
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