Assessment of the effects of chemicals on the expression of ten steroidogenic genes in the H295R cell line using real-time PCR.
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.
Hilscherova
Klara
K
Department of Zoology, National Food Safety and Toxicology Center, Center for Integrative Toxicology, Michigan State University, East Lansing 48824, USA.
Jones
Paul D
PD
Gracia
Tannia
T
Newsted
John L
JL
Zhang
Xiaowei
X
Sanderson
J T
JT
Yu
Richard M K
RM
Wu
Rudolf S S
RS
Giesy
John P
JP
D000302:Adrenal Cortex / Q000378:metabolism; D045744:Cell Line, Tumor; D002847:Chromatography, Agarose; D018076:DNA, Complementary / Q000096:biosynthesis / Q000302:isolation & purification; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004968:Estrogens, Non-Steroidal / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D010641:Phenotype; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013254:Steroid 17-alpha-Hydroxylase / Q000096:biosynthesis / Q000235:genetics; D013256:Steroids / Q000096:biosynthesis*; D000098682:Steroidogenic Acute Regulatory Protein
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D004968:Estrogens, Non-Steroidal; D010750:Phosphoproteins; D013256:Steroids; D000098682:Steroidogenic Acute Regulatory Protein; D012313:RNA; D013254:Steroid 17-alpha-Hydroxylase