An evaluation on combination effects of phenolic endocrine disruptors by estrogen receptor binding assay

Phenolic compounds are widely distributed in the natural environment, typically existing as a mixture at the nanomole or micromole per liter level. Among the phenolic compounds, 4-nonylphenol, 4-t-octylphenol, bisphenol A and 2,4-dichlorophenol attract the most concern due to their abundance and risks in the natural environment. The former three are known as endocrine disruptors causing feminization in various organisms, whereas the latter requires further clarification for its estrogenic effect. This study aims to evaluate the combination effects of these chemicals with estrogen receptor binding as an endpoint. An ELISA based receptor binding assay was employed to avoid radioactive pollution in the traditional assay. The results showed that all these chemicals could bind with estrogen receptor with a relative binding affinity of bisphenol A>4-t-octylphenol>4-nonylphenol>2,4-dichlorophenol. The four chemicals were further mixed in two ways, at an equipotent ratio and at an equal environmental level ratio, and their combination effects on receptor binding were evaluated with both the toxicity units method and concentration addition model. The resulting effects of both mixtures showed an antagonistic mode, which was assumed to be a general mode of action with estrogen receptor binding assay due to competitive ligand binding on receptors.