Through the consumption of food and water, the inhalation of air, and the physical contact with objects in the environment, many synthetic and natural compounds are constantly passing into our bodies. Some of these can affect the functioning of our endocrine system and, thus, lead to various adverse health effects. One such compound is bisphenol A, which has been partially replaced by other bisphenol analogs in the past decade due to the numerous attention-attracting studies that reported its adverse health effects. As a result, we are often exposed to mixtures of various bisphenols and many other toxins. Therefore, there is an increasing emphasis in toxicology on the assessment of mixtures, since it offers a more accurate evaluation of toxicity as the individual testing of compounds.
The objectives of our work were to test the estrogenic activity of bisphenols A, AF, C, F, S, and Z on the estrogen receptor α (ERα), to evaluate the estrogenic effects of two-component mixtures of bisphenol A with other bisphenols and to determine the estrogenic activity of the mixture of all six bisphenols. Additionally, we evaluated estrogenic activities of the products of bisphenols A, AF, and S after phase 1 or 2 metabolisms. To perform the assays, the hERα-HeLa-9903 cell line was used and measurements were made using luciferase assay. Before testing bisphenols, four control compounds were used to test the proper cell line functioning and a resazurin assay was performed to check for potential cytotoxicity of the tested bisphenols. Only a 25 μM mixture of all six bisphenols proved cytotoxic and was, therefore, omitted from the analysis.
All six of the tested bisphenols proved to be ERα agonists. The potency of their estrogenic activity decreased in the following order: bisphenol AF, Z, C, A, F, and S. From the measurements of the estrogenic potency of the individually tested bisphenols, the concentration addition model was calculated. The obtained results were compared to the measured estrogenic potency of the mixtures of bisphenols. The values matched very well, suggesting that the mixtures of bisphenols have an additive mode of action on the ERα. The bisphenol metabolism tests have shown that the most important mechanisms of metabolic detoxification of bisphenols are the enzymatic reactions of glucuronidation and sulfation. In the case of oxidative metabolism of bisphenols, however, the decrease of estrogenic activity was relatively low. This may be due to slower kinetics of the reactions or the formation of estrogen-active products.