R3 AM Ecotoxicogenomics of Emerging Chemical Issues
Thursday, 17 November 2005: 8:00 AM - 11:40 AM in Ballroom 3

(GRA-1117-816567) Effects of pharmaceuticals on gene expression and hormone production in H295R cell line using Q-R-T-PCR/ELISA.

Gracia, T¹, Jones, P¹, Hilscherova, K², Higley, E¹, Giesy, J¹, Newsted, J³, ¹ Zoology Department, National Food Safety and Toxicology Center, Michigan State University, E. Lansing, MI, USA² 2Research Centre for Environmental Chemistry and Ecotoxicology, Brno, Czech Republic³ Entrix Inc., E. Lansing, MI, USA

ABSTRACT- Pharmaceuticals can be released into the environment through many different pathways including excretion from human populations and large scale animal production facilities as well from the disposal of unused medications to drains. Usually these releases are discharged to wastewater and eventually to sewage treatment facilities (STF). STFs are not always effective in removing the active chemical from wastewater and frequently sludge from the STF is used as soil conditioner, thus together with other pollutants, pharmaceuticals find their way into the terrestrial and aquatic environments. Due to the nature of these substances, there is concern that they could disrupt endocrine processes through both receptor-mediated processes as well as indirect mechanisms. Here we use a method to evaluate the potential effects of pharmaceuticals on steroidogenesis using a steroid producing human adrenocortical carcinoma cell line (H295R). H295R cells were exposed for 48 hours to 15 commonly used and prescribed drugs and expression of 4 steroidogenic genes was measured by quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR). PCR product identities were confirmed by melting curve analysis, agarose gel electrophoresis, and nucleotide sequencing of the amplified sequences. In addition, using ELISA techniques, progesterone, testosterone, aldosterone and estradiol were measured in the media after the pharmaceutical exposure and compared to solvent control concentrations. Results showed that the expression of CYP11B2 was the most affected with up to ten fold induction being observed in cells treated with the macrolide tylosin and a ten fold decrease observed in cells treated with trimetropin. Ibuprofen reduced the expression of 3-HSD2 and CYP17 by 50% of control levels. The aromatase gene, CYP19, was only affected by doxycycline and trimetropin, with up to 3-fold increase in expression from controls. These results demonstrate the utility of the H295R assay system as a screen for chemicals that may affect steroidogenic systems.

Key words: Endocrine Disruptors, Bioassay, Pharmaceuticals, hormones