Final Report: Validation of a Rapid Progestin-Based Endocrine Disruption Screening Assay

EPA 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
Phase: II
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)


Concerns regarding both the presence of endocrine disruptors in food, water, or other environmental media as well as the potential risk they pose to humans and wildlife have grown in recent years. In 1996, passage of the Food Quality Protection Act and Amendments to the Safe Drinking Water Act reflected these concerns and required the U.S. Environmental Protection Agency to develop a screening program, using appropriately validated test systems and other scientifically relevant information, to determine whether certain substances may have an endocrine effect in wildlife and humans.

The maturation of the amphibian oocyte represents the final stage of oogenesis that ultimately prepares the oocyte for fertilization. Oocyte maturation is marked morphologically by germinal vesicle breakdown (GVBD), and is induced by progesterone via an oocyte plasma membrane receptor, and androgens via a classical intercellular androgen receptor. Thus, maturation of the oocyte could potentially be disrupted by endocrine-disrupting chemicals (EDCs). Disruption of oocyte maturation events in Rana pipiens and Xenopus laevis by estradiol and a synthetic estrogen has been demonstrated previously. Thus, maturation of the oocyte could potentially be disrupted by EDCs. It has been previously hypothesized that progesterone-induced maturation of amphibian oocytes could be disrupted by environmental pollutants with anti-progestin activity.

Exposure to environmental contaminants can adversely affect individuals as well as meta-populations of amphibians. Most studies have focused on effects at the individual level. Extrapolating toxicological effects observed in individual specimens in the laboratory to effects at the meta-population level is extremely difficult and requires an adequate evaluation of adverse responses in the field. Negative effects at the meta-population level may be the result of multiple responses including lethal responses, sub-lethal responses, and modest changes in biochemical homeostasis. Sublethal responses include malformation, growth reduction, and developmental delay. Changes in biochemical homeostasis in amphibians resulting from perturbation of critical aspects of the endocrine system, such as reproductive hormonal pathways and the thyroid axis, also provide additional stress.

Currently, no standardized and well-validated alternative models exist for screening for progesterone-responsive EDCs. Thus, the specific goal of this research project was to validate and commercialize a routine EDC screening assay by developing the in vitro GVBD model using X. laevis oocytes. The GVBD model system for evaluating progestin-based EDCs must be well developed and properly validated. Therefore, the development and proper validation of this assay for use in evaluating EDCs in vitro must be performed before this assay can be successfully commercialized. The need to evaluate the impact of environmental contaminants on endocrine disruption is more imperative now then ever before and will continue to develop (fueled by discoveries of mal-developed limbs of frogs in the environment, incomplete metamorphosis [incompletely resorbed tails], disappearance of adult frogs, and skewed sex ratios [higher proportion of females]) until assays such as the models proposed are ready for widespread use. It should be noted, however, that the objective of the proposed research was not to replace existing in vivo mammalian model systems, but rather to augment the existing models. Also, the model system approach developed may be used with other amphibian species.

The primary objectives of Phase II were to: (1) validate the standard protocol developed for the Xenopus oocyte GVBD assay using known EDCs, suspected EDCs, compounds with unknown EDC activity, and materials with no apparent EDC activity; (2) prepare a Guidance Document regarding the performance of this potential Endocrine Disruptor Screening Program Tier 1 screening assay; and (3) continue efforts to commercialize the proposed assay system. These objects flowed directly as an extension of the Phase I objectives that were to develop and standardize the assay.

Summary/Accomplishments (Outputs/Outcomes):

Research 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 this research project was to validate and commercialize the X. laevis oocyte maturation GVBD model as a system for rapid evaluation of EDCs found in the workplace or the environment. Specifically, a 24-hour X. laevis assay modified from the original work of Pickford and Morris and Lui and Patino 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 progesterone 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 binding affinity of the toxicants to the OMPR, however, 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.

Of the 15 compounds tested during Phase II, 12 were found to be inhibitory and 3 were found to be stimulatory, with varying levels of potency. With the exception of copper, the compounds selected were expected to have some activity on GVBD, both positively and negatively, based on previous reports of reproductive toxicity. Additional studies with copper indicated that the stimulatory effect induced was the result of indirect synergistic effects on progesterone binding to the OMPR at the membrane level. Copper did not bind directly to the OMPR, but appeared to facilitate progesterone binding to the OMPR. The CAFO sample was tested as an unknown entity. The inhibitory nature of this sample subsequently was correlated to the estrogenic activity of the sample using E-SCREEN. Although the sample possessed moderate estrogenic activity, the strongly inhibitory response on GVBD could not be solely attributed to the estrogenic activity. From an estrogen equivalency standpoint, approximately 60 percent of the activity could be attributed to estrogenic substances. Further, the inhibitory activity was greater than that which could be attributed to competitive inhibition of progesterone binding to the OMPR. This would suggest that an alternative mechanism of action, such as inhibition of the endogenous androgen pathway, also could be involved. More work will be needed to identify other components acting to inhibit to GVBD process. The advantage of the 24-hour GVBD assay as an EDC screen is that it can be used effectively in toxicity characterization studies or in joint mixture assessments to evaluate interaction. Thus, this assay is well suited for identifying causative agents in complex mixtures, such as CAFO waste runoff.


Results from the present studies suggested that many of the test materials were capable of inhibiting or accelerating GVBD in vitro in a concentration-dependent manner with varying degrees of potency. Intra- and inter-laboratory variabilities were relatively low in testing performed at the two study sites. These results indicated that the technology developed in the present project was transferable to outside laboratories. Overall, results suggested that the Xenopus oocyte GVBD assay could be useful in high-throughput progestin/androgen screening.

Journal Articles on this Report : 3 Displayed | Download in RIS Format

Other project views: All 4 publications 3 publications in selected types All 3 journal articles
Type Citation Project Document Sources
Journal Article Fort DJ, Guiney PD, Weeks JA, Thomas JH, Rogers RL, Noll AM, Spaulding, CD. Effect of methoxychlor on various life stages of Xenopus laevis. Toxicological Sciences 2004; 81(2):454-466. 68D03044 (Final)
68D03024 (Final)
EPD04054 (Final)
not available
Journal Article Fort DJ, Thomas JH, Rogers RL. Effect of diverse xenobiotics on oocyte maturation in vitro in Xenopus. Journal of Applied Toxicology (in press, 2005). 68D03044 (Final)
not available
Journal Article Fort DJ, Rogers RL, Thomas JH, Noll AM, Guiney PD, Weeks JA. Inhibition of germinal vesicle breakdown (GVBD) in Xenopus oocytes in vitro by a series of substituted glycol ethers. Toxicological Sciences (in review, 2005). 68D03044 (Final)
not available

Supplemental Keywords:

endocrine-disrupting chemical, EDC, progestin, screening assay, in vitro, Endocrine Disruptor Screening Program, Tier II Tests, Xenopus laevis, germinal vesicle breakdown, GVBD, amphibian, reproduction, oocyte, development, growth, EPA, small business, SBIR,, RFA, Health, Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, POLLUTANTS/TOXICS, RESEARCH, Environmental Chemistry, Chemicals, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Monitoring/Modeling, Environmental Microbiology, Monitoring, Biochemistry, Environmental Monitoring, Biology, Endocrine Disruptors - Human Health, bioindicator, endocrine disruptor screening program, assays, EDCs, endocrine disrupting chemicals, animal model, Xenopus lifecycle model, developmental biology, animal models, biomonitoring, endocrine disruption screening assay

SBIR Phase I:

Development and Preliminary Validation of a Rapid Progestin-Based Endocrine Disruption Screening Assay  | Final Report