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The Application of a Highly Purified Rat Leydig Cell Assay as a Complement to the H295R Steroidogenesis Assay for the Evaluation of Toxicant Induced Alterations in Testosterone Production
Citation:
Botteri, N., J. Suarez, S. Laws, AND G. Klinefelter. The Application of a Highly Purified Rat Leydig Cell Assay as a Complement to the H295R Steroidogenesis Assay for the Evaluation of Toxicant Induced Alterations in Testosterone Production. NC SOT, Durham, NC, October 30, 2017.
Impact/Purpose:
Poster presentation for NC Society of Toxicology Meeting. Poster previously presented at 2017 SOT Annual Meeting.
Description:
Exposure to endocrine disrupting chemicals have been associated with compromised testosterone production leading to abnormal male reproductive development and altered spermatogenesis. In vitro high throughput screening (HTS) assays are needed to evaluate risk to testosterone production, yet the main steroidogenesis assay currently utilized is a human adrenocortical carcinoma cell line, H295R, which does not synthesize gonadal steroids at the same level as the gonads, thus limiting assay sensitivity. We propose a complementary assay using a highly purified rat Leydig cell assay to evaluate the potential for chemical induced alterations in testosterone production by the testis. We evaluated a subset of chemicals that failed to decrease testosterone production in the HTS H295R assay. The chemicals examined fit into one of two categories based upon changes in substrates upstream of testosterone in the adrenal steroidogenic pathway (17α-hydroxyprogesterone and 11-deoxycorticosterone) that we predicted should have elicited a decrease in testosterone production. We found that 85% of the 20 test chemicals examined inhibited Leydig cell testosterone production in our assay. Importantly, we adopted a 96 well format to increase throughput and efficiency of the Leydig cell assay and identified a selection criterion based upon the AC50 values for 17α-hydroxyprogesterone and 11-deoxycorticosterone generated from the HTS H295R assay that will help prioritize chemicals for further testing in the Leydig cell screen. We hypothesize that the greater dynamic range of testosterone production and sensitivity of the Leydig cell assay permits the detection of small, yet significant, chemical induced changes not detected by the HTS H295R assay.