PARENT SESSION

WP2 Chemical and Biological Analysis of Endocrine Disrupting Compounds
255 Portland Ballroom
1:20 PM - 4:40 PM, Wednesday

() Quantitative assessment of chemical effects on steroidogenic enzymes in H295R cell line using Q-RT- PCR.

Gracia, T¹, Hilscherova, K¹, Zhang, X¹, Jones, P¹, Sanderson, J², Giesy, J¹, Newsted, J³, ¹ MICHIGAN STATE UNIVERSITY, East Lansing, Michigan, USA² Institute for Risk Assessment Sciences , University of Utrecht, Utrecht, Utrecht, The Netherlands³ ENTRIX Inc., East Lansing, Michigan, USA

ABSTRACT- Endocrine disruption may not solely be produced by receptor-mediated processes but may also occur through indirect mechanisms. Here we report the development of a method for the evaluation of potential effects of environmental chemicals on steroidogenic enzymes. Gene expression was measured by quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) in a steroid producing human adrenocortical carcinoma cell line (H295R). PCR assays were developed and optimized for 12 target enzymes. PCR product identities were confirmed by melting curve analysis, agarose gel electrophoresis, and nucleotide sequencing of the amplified sequences. H295R cells were exposed to 4 known inducers and 7 known inhibitors of steroidogenesis and after 24 hours gene expression was quantified by Q-RT-PCR. Inducers of steroidogenesis altered the expression of several enzymes. Expression of CYP11B2 and 3-HSD was increased in excess of 10 fold by treatment with 8-Br-cAMP or forskolin. Phorbol-12-myristate 13-acetate increased CYP19 expression over 10-fold but decreased CYP17 expression by almost 90%. Among the inhibitors, spironolactone generally decreased gene expression but induced CYP19 expression almost 10-fold. Daidzein, aminoglutethimide and spironolactone significantly reduced the expression of Steroidogenic Acute Regulatory protein. The assay system developed shows considerable potential for the determination of altered expression profiles specific for various chemicals.

Key words: Bioassay, Screening, Steroidogenesis, Xenoestrogens