Validation of a Rapid Progestin-Based Endocrine Disruption Screening AssayEPA Contract Number: 68D03044
Title: Validation of a Rapid Progestin-Based Endocrine Disruption Screening Assay
Investigators: Fort, Douglas J.
Small Business: Fort Environmental Laboratories Inc.
EPA Contact: Manager, SBIR Program
Project Period: May 1, 2003 through April 30, 2005
Project Amount: $225,000
RFA: Small Business Innovation Research (SBIR) - Phase II (2002) Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , SBIR - Monitoring , Small Business Innovation Research (SBIR)
Fort Environmental Laboratories, Inc.'s Phase I research project resulted in the development and standardization of an assay that tests substances that might disturb reproductive and developmental processes in animals by interfering with the endocrine system. The primary goal of the proposed research was to validate and commercialize the Xenopus laevis oocyte maturation germinal vesicle breakdown (GVBD) model as a system for the rapid evaluation of endocrine-disrupting chemicals (EDCs) found in the workplace or the environment. Specifically, a 24-hour X. laevis assay modified from the original work of Pickford and Morris (Environmental Health Perspectives 1999;107(4):285-292) and Lui and Patino (Biology of Reproduction 1993;49:980-988) designed to evaluate progestin-active or anti-progestin EDCs in vitro was standardized and evaluated by conducting a preliminary validation study with a series of known EDCs, compounds found to be inactive, and chemicals with unknown activity.
The relative inhibitory potential of the toxicants study was ethinyl estradiol>>Aroclor 1260>atrazine>dieldrin. Testosterone and the confined animal feed operation (CAFO) waste complex mixture sample had a stimulatory effect on GVBD. Bisphenol A had no effect on GVBD, even at concentrations of 5,000 µM. The binding capacity of the toxicants to the oocyte membrane plasma receptor (OMPR) relative to progesterone was low. Interestingly, testosterone possessed some binding capacity to the OMPR, indicating that the OMPR may be a more precocious binding site than originally anticipated. However, the relative binding affinity of the toxicants to the OMPR was expressed as progesterone>>ethinyl estradiol (-)>testosterone (+)>atrazine (-)>Aroclor 1260 (-) >dieldrin (-)>CAFO sample (+)>bisphenol A (NE). The washout studies indicated that although the competitive binding affinity of ethinyl estradiol for the OMPR was the greatest of the test compounds evaluated in the present study, testosterone, dieldrin, and Aroclor 1260 were bound more tightly to the OMPR than ethinyl estradiol.
Because none of the currently developed high throughput EDC screening systems are capable of specifically screening for progesterone-active EDCs, the successful completion of the in vitro oocyte GVBD model development will provide the scientific community with a non-mammalian, cost-effective, rapid, and reliable method of screening EDCs. The ability to rapidly and cost-effectively screen for and evaluate the mechanisms of EDCs is an attractive alternative to the current laborious and expensive testing systems used today. Increasing concerns over the widespread finding of EDCs in the environment have dramatically increased the need for standardized assays, such as the X. laevis GVBD model, because few other progestin/antiprogestin-based in vitro assays are available today.