Exposure to endocrine disrupting chemicals has 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. Here, 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 on 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 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. We identified a selection criterion based on 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.

Summary Sentence

The greater dynamic range of testosterone production in a primary rat Leydig cell assay permitted detection of chemical-induced testosterone inhibition that was not detected by the high-throughput screening format of the H295R steroidogenesis assay.