An Evaluation of LH-Stimulated Testosterone Production by Highly Purified Rat Leydig Cells : A Complementary Screen for Steroidogenesis in the Testis
Citation:
Botteri, N., J. Suarez, S. Laws, AND G. Klinefelter. An Evaluation of LH-Stimulated Testosterone Production by Highly Purified Rat Leydig Cells : A Complementary Screen for Steroidogenesis in the Testis. Society of Toxicology, Baltimore, Maryland, March 12 - 16, 2017.
Impact/Purpose:
This work is relevant to CSS Task 1.3b - Milestone Title: Development of Putative AOPs for Reproductive Endpoints Based on Molecular Initiating Events Captured in High-Throughput Assay. Scientists involved in this task will evaluate a subset of chemicals evaluated in ToxCast using complimentary, moderate-throughput in vitro systems to help confirm the veracity of MIEs suggested by the HTP systems.
Description:
An Evaluation of LH-Stimulated Testosterone Production by Highly Purified Rat Leydig Cells: A Complementary Screen for Steroidogenesis in the Testis. 1Botteri, N., 2Suarez, J., 2Laws, S., 2Klinefelter, G.1Oak Ridge Institute for Science and Education, Oak Ridge, TN, 2 U.S. Environmental Protection Agency, ORD, NHEERL, TAD, RTP, NCThe H295R steroidogenesis assay uses an adrenocarcinoma cell line which fails to elicit LH mediated responses. This limits the assay’s ability to detect chemicals which disrupt LH-mediated Leydig cell responses in the testis. This study evaluated whether LH-stimulated T production by purified rat Leydig cells would be altered after exposure to chemicals that failed to decrease T production in the ToxCast H295R screen. Ten chemicals negative for T inhibition in the H295R screen, were selected based on alterations in upstream substrates (deoxycorticosterone, hydroxyprogesterone) expected to result in a decrease in T. Based on earlier work, simvastatin served as our positive control. Each chemical was tested over 6 concentrations ranging from 0.1 µM to 100 µM. Leydig cells were cultured overnight under maximal LH stimulation. A minimum of 3 replicate experiments were conducted for each format (24 and 96 well) and chemical tested; cell viability was assessed using a live/dead cytotoxicity kit. T data were excluded if viability was less than 80% of control. Initial evaluation using a 24-well Leydig cell assay confirmed that 4 of 10 chemicals produced significant (P < 0.05) concentration-related decreases in LH-stimulated T synthesis. To increase throughput and efficiency, we adopted a 96-well assay and observed that exposure to the same chemicals decreased T; in addition corticosterone decreased T. The lowest observed effective concentrations for simvastatin, metconazole, anilazine, hydroquinone and corticosterone were 0.1 µM, 1 µM, 3 µM, 10 µM, 100 µM, respectively. The 96-well assay data revealed decreases in T at lower concentrations (anilazine, metconazole). Perhaps greater sensitivity of the 96-well assay is attributed to the relative increase in T production by cells in this format compared to the 24 well format (1436 vs 738 ng T/106 Leydig cells). Using similar selection criteria, additional chemicals negative for T in the H295R cell assay will be screened using the 96-well Leydig cell assay. We will also determine chemical response with and without LH stimulation. The inherently greater sensitivity afforded by cells making over 1µg of T, together with the ability to capture LH-mediated alterations, makes the Leydig cell assay a reasonable complement to the H295R steroidogenic screen.This abstract does not reflect US EPA policy