Citation:

Farmer, W., A. Murr, E. Lachendro, AND T. Stoker. Linking In Vitro Cyclooxygenase Inhibition to Disruption of Ovulation and Decreased PGE2 Production in Gonadotropin-Primed Immature Rats. Triangle Consortium of Reproductive Biology, RTP, NC, February 25, 2017.

Impact/Purpose:

Research poster presentation describing the effects of chemicals on cyclooxygenase inhibition and the subsequent effect on PGE2 production and ovulation in a gonadotropin-primed immature female rat model (both in vivo and in vitro assessments). This work was conducted to complete an adverse outcome pathway (AOP) for the CSS Vertebrate Reproduction Task.

Description:

Linking In Vitro Cyclooxygenase Inhibition to Disruption of Ovulation and Decreased PGE2 Production in Gonadotropin-Primed Immature Rats1,2Farmer, WT, 2Murr, AS, 1,2Lachendro, E, 2Stoker, TE.1Oak Ridge Institute for Science and Education, US DOE, Oak Ridge, TN 37831; 2Endocrine Toxicology Branch, TAD, NHEERL, ORD, U.S. EPA, RTP, NC.Cyclooxygenase 2 (COX2) is an enzyme required for synthesis of prostaglandin E2 (PGE2), which regulates key ovulatory events. Several chemicals have been identified as COX2 inhibitors in two of the U.S. EPA ToxCast high throughput (HTP) assays. These putative COX2 inhibitors may disrupt ovulation and thereby support predictive modeling of female reproductive adverse outcome pathways (AOPs). We used data from the HTP assays to select a known COX2 inhibitor, Celecoxib (CXB, IC50 of 0.15 μM) and two putative COX2 inhibitors, Butylparaben (BPN, IC50 of 10 µM) and tert-Butylhydroquinone (HQN, IC50 of 5.3 μM), to evaluate their effects on ovulation and ovarian follicle production of PGE2 in a gonadotropin stimulated immature female rat model. BPN is bactericidal and is commonly found as an additive in cosmetics, medicine suspensions and food. HQN is currently in use as a food preservative. In the first experiment, we examined the effect of these putative COX2 inhibitors on ovulation. To stimulate follicle production, we administered pregnant mare serum gonadotropin (PMSG 20IU) to post-natal day 24 female rats. After 48 hours, females were gavaged with either vehicle (methylcellulose, sesame oil), CXB (50 mg/kg), BPN (125, 250, 500 mg/kg), or HQN (105 mg/kg). Ovulation was induced 3 hours later by a subcutaneous injection of human chorionic gonadotropin (hCG 10IU). CXB females exhibited a significant reduction in the number of oocytes ovulated (p < 0.05) compared to controls. BPN females showed a dose-dependent decrease in number of oocytes ovulated with the highest doses exhibiting a significant reduction (250, P <0.05; 500, P <0.01). HQN did not inhibit ovulation. In the second experiment, we examined the effect of COX2 inhibitors on PGE2 production from cultured ovarian follicles. Preovulatory follicles were collected from PMSG treated females and cultured (3 per well) for 24 hours in M199 (unstimulated control), M199 + hCG (stimulated control) or M199 + hCG + a COX2 inhibitor (CXB 1uM, 10uM; BPN 25uM, 50uM; HQN 10uM, 20uM). Media samples were collected at 0, 4, 8, and 24 hours for analysis of PGE2 or progesterone (P4) production. M199 alone did not stimulate production of PGE2 or P4. CXB and all putative COX2 inhibitors significantly decreased PGE2 production from follicles in a dose and time dependent manner compared to hCG stimulated controls. P4 stimulated by hCG was similar in all treatment groups as compared to M199 + hCG, suggesting these toxicants specifically inhibit PGE2 production. Therefore, COX2 inhibition by environmental chemicals can be linked to adverse effects on ovulation using this animal model. Additional dose response data on PGE2 production in future work will help in the development of a quantitative AOP for PGE2 inhibition and reduced ovulatory quotient. This abstract does not necessarily reflect U.S.EPA policy.

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