Final Report: Development and Preliminary Validation of a Rapid Progestin-Based Endocrine Disruption Screening AssayEPA Contract Number: 68D02023
Title: Development and Preliminary 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: April 1, 2002 through September 1, 2002
Project Amount: $70,000
RFA: Small Business Innovation Research (SBIR) - Phase I (2002) RFA Text | Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , SBIR - Monitoring , Small Business Innovation Research (SBIR)
Concerns regarding the presence of endocrine disruptors in food, water, or other environmental media and the potential risk they pose to humans and wildlife have been growing in recent years. Passage in 1996 of the Food Quality Protection Act and Amendments to the Safe Drinking Water Act reflected these concerns and required the Environmental Protection Agency (EPA) to develop a screening program, using appropriate validated test systems and other scientifically relevant information, to determine whether certain substances may have an endocrine effect in wildlife and humans. The proposed work has resulted in the preliminary validation 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 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 (Environ. Health Perspect. 1999;107(4):285-292) and Lui and Patino (Biol. Reprod. 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 test materials included ethinyl estradiol, dieldrin, Bisphenol A, testosterone, Aroclor 1260, atrazine, and a stormwater runoff sample from a confined animal feed operation (CAFO). Range tests for each test material consisting of five logarithmically-separated test concentrations initially were used to determine the test concentration to be used in subsequent definitive tests. Each test consisted of cultured naked oocytes without progesterone (negative control), naked oocytes with 0.1 µM progesterone (positive control), and naked oocytes exposed to selected concentrations of the test material in the presence of 0.1 µM progesterone.
Because none of the currently developed high-throughput EDC screening systems are capable of specifically screening for progesterone-active EDCs, successful completion of the in vitro oocyte GVBD model development will provide the scientific community with a non-mammalian, cost-effective, rapid, 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.
Results from the present studies suggested that ethinyl estradiol and to a lesser extent, Aroclor 1260, atrazine, and dieldrin, were capable of inhibiting GVBD in a concentration-dependent manner. Testosterone and the CAFO waste complex mixture sample had a stimulatory effect on GVBD. The potency of the stimulatory effects of the CAFO sample were somewhat surprising and suggested that environmental mixtures are not only capable of inhibiting GVBD, but also are capable of inducing oocyte maturation. Bisphenol A had no effect on GVBD, even at concentrations of 5,000 µM, and thus possessed no EDC activity in this model. 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. The relative inhibitory potential of the toxicants study was: ethinyl estradiol>>Aroclor 1260>atrazine>dieldrin. 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.
Although many pesticides and some industrial chemicals have undergone extensive toxicological testing, this testing may have been inadequate to determine whether they interact with the endocrine system and whether additional testing is needed for EPA to assess and characterize both human health and ecological risk. Notwithstanding recognition that the scientific knowledge related to endocrine disruptors is still evolving, there is appropriate widespread agreement that the development of a screening and testing program is needed. By standardizing and validating the X. laevis GVBD model system for screening EDCs, Fort Environmental Laboratories, Inc. will provide the scientific community, chemical and pharmaceutical industries, appropriate regulatory agencies, and ultimately the public with a versatile short-term prescreening assay. Further, this model will enhance our understanding of the significance of the effects of EDCs on the reproductive systems of amphibians. The technical feasibility of the GVBD model for screening potential EDCs is high. Although more work will be required to validate the model and prepare the guidance document, the frog oocyte GVBD model appears to fit the criteria established for emerging Tier 1 screening assays that currently are being sought as potential additions to or replacements for the existing screening assay proposed for the Endocrine Disruptor Screening Program. The assays are relatively straightforward and could be commercialized following method standardization and validation. Results from the Phase I studies have shed light on an additional attribute of the GVBD model in that it is capable of broadly screening compounds with widespread endocrine activity, including estrogens, progestins, and androgens, which is desirable for Tier 1 screening tests. However, the use of selective receptor antagonists can be used to determine if the activity is mediated through the OMPR, the classical androgen receptor, or potentially both pathways. Although an extra 24 hours have been added to the test with the preculture phase, the test still can be completed within 48 hours, which is desirable for a screening test. Because the test is relatively inexpensive compared to many of the other proposed screening tests and many compounds can be tests simultaneously, the proposed oocyte GVBD model is ideal for high-throughput testing, which will be required for Tier 1 screening assays.