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The Use of Purified Rat Leydig Cells Complements the H295R Screen to Detect Chemical-Induced Alterations in Testosterone Production
Citation:
Botteri Principato, N., J. Suarez, S. Laws, AND G. Klinefelter. The Use of Purified Rat Leydig Cells Complements the H295R Screen to Detect Chemical-Induced Alterations in Testosterone Production. BIOLOGY OF REPRODUCTION. Society for the Study of Reproduction, 98(2):239-249, (2018).
Impact/Purpose:
The U.S. Environmental Protection Agency’s (U.S. EPA) Endocrine Disruptor Screening Program is tasked with evaluating pesticides, chemicals, and environmental contaminants for potential endocrine disrupting effects that could be hazardous to human and wildlife health. Alterations in steroidogenic hormone action have been associated with developmental defects as well as dysfunction of the reproductive system [6, 7]. For decades, epidemiological data have been suggesting that semen quality has been declining. Compromised testosterone production in the fetal or adult testis can lead to abnormal male reproductive development and both quantitative and quality alterations in spermatogenesis, leading possibly to reduced fertility [11]. Research is needed to identify EDCs that have the potential to disrupt steroid hormone biosynthesis and to evaluate the risks these chemicals pose to the reproductive health of humans and wildlife. Despite the novel capabilities of the HTS H295R assay, it is limited in its ability to accurately predict chemical induced alterations in gonadal sex steroid hormones. Arguably, there is a need for sensitive and biologically relevant HTS gonadal based assays to directly measure chemical induced alterations in sex steroid hormone synthesis and provide a better prediction of potential impact on gonadal function. We proposed a complementary assay to screen for potential chemical induced alterations in testosterone production by the testis using a highly purified rat Leydig cell assay. Highly purified rat Leydig cells, resulting from a well-established multistep isolation procedure, produce hundreds of nanograms of testosterone upon stimulation by physiological levels of luteinizing hormone (LH). In addition to the large dynamic range, purified rat Leydig cells being LH responsive, permits the evaluation of biologically relevant LH stimulated alterations in testosterone production. Given these attributes, we hypothesized that a significant number of chemicals that were not detected as inhibitors of testosterone synthesis in the H295R assay would decrease testosterone production by LH stimulated Leydig cells, and therefore identified as probable EDCs. We examined 20 test chemicals that did not reduce testosterone in the HTS H295R assay and found that 85% of them 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.
Description:
Exposure to endocrine disrupting contaminants can compromise testosterone production and lead 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 utilized is a human adrenocortical carcinoma cell line, H295R, that does not synthesize gonadal steroids at the same level as the gonads, thus limiting assay sensitivity. Here, we propose a complementary assay to evaluate the potential for chemical induced alterations in testosterone production by the testis using a highly purified rat Leydig cell assay. 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 examined 20 test chemicals that did not reduce testosterone in the HTS H295R assay and found that 85% of them 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.
