Academic journal article Environmental Health Perspectives

Quantitative Comparisons of in Vitro Assays for Estrogenic Activities

Academic journal article Environmental Health Perspectives

Quantitative Comparisons of in Vitro Assays for Estrogenic Activities

Article excerpt

Substances that may act as estrogens show a broad chemical structural diversity. To thoroughly address the question of possible adverse estrogenic effects, reliable methods are needed to detect and identify the chemicals of these diverse structural classes. We compared three assays--in vitro estrogen receptor competitive binding assays (ER binding assays), yeast-based reporter gene assays (yeast assays), and the MCF-7 cell proliferation assay (E-SCREEN assay)--to determine their quantitative agreement in identifying structurally diverse estrogens. We examined assay performance for relative sensitivity, detection of active/inactive chemicals, and estrogen/antiestrogen activities. In this examination, we combined individual data sets in a specific, quantitative data mining exercise. Data sets for at least 29 chemicals from five laboratories were analyzed pair-wise by X-Y plots. The ER binding assay was a good predictor for the other two assay results when the antiestrogens were excluded ([r.sup.2] is 0.78 for the yeast assays and 0.85 for the E-SCREEN assays). Additionally, the examination strongly suggests that biologic information that is not apparent from any of the individual assays can be discovered by quantitative pair-wise comparisons among assays. Antiestrogens are identified as outliers in the ER binding/yeast assay, while complete antagonists are identified in the ER binding and E-SCREEN assays. Furthermore, the presence of outliers may be explained by different mechanisms that induce an endocrine response, different impurities in different batches of chemicals, different species sensitivity, or limitations of the assay techniques. Although these assays involve different levels of biologic complexity, the major conclusion is that they generally provided consistent information in quantitatively determining estrogenic activity for the five data sets examined. The results should provide guidance for expanded data mining examinations and the selection of appropriate assays to screen estrogenic endocrine disruptors. Key words. antiestrogens, chemical structure, data mining, endocrine disruptors, E-SCREEN assay, estrogens, estrogen receptor competitive binding assay, estrogen receptors, MCF-7 cell proliferation assay, species sensitivity, yeast-based reporter gene assay. Environ Health Perspect 108:723-729 (2000). [Online 26 June 2000] http://ehpnet1.niehs.nih.gov/docs/2000/108p723-729fang/abstract.html

Evidence that certain man-made chemicals have the ability to disrupt the endocrine systems of vertebrates by mimicking endogenous hormones has, in recent years, sparked intense international scientific discussion and debate (1). A growing national concern has resulted in legislation, including reauthorization of the Safe Drinking Water Act and passage of the 1996 Food Quality Protection Act, mandating that the U.S. Environmental Protection Agency (EPA) develop a screening program for endocrine-disrupting chemicals (EDCs) (2,3). Under this requirement, at least 15,000 existing chemicals will be experimentally evaluated for their potential to disrupt activities in the estrogen, androgen, and thyroid hormone systems. A high-through-put prescreen assay that uses a reporter gene system may be used to prioritize chemicals for screening (4). The battery of in vitro and short-term in vivo screening assays should optimize hazard identification and provide guidance for subsequent longer term, more definitive in vivo tests for toxicity (5).

Although endocrine disruption can result from a variety of biologic mechanisms, more data exist for estrogens than for the other classes of activity (6,7). Because in vivo bioassays are time consuming and labor intensive, a battery of in vitro and short-term in vivo assays are proposed to be used as a first screen for estrogenicity (4). Estrogens regulate the expression of specific genes and the secretion of certain hormones, and coordinate diverse processes such as cell proliferation, cell differentiation, and tissue organization through pleiotropic actions. …

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