Comparative effect assessment of bisphenol analogs by high content analysis in zebrafish (Danio rerio) embryos

Bisphenol A (BPA), a plastic additive, has been banned from certain products, particularly because of its endocrine-disrupting properties. As a result, substitutes such as bisphenol E (BPE), bisphenol G (BPG), bisphenol AP (BPAP), and bisphenol Z (BPZ) were developed, which may be equally or even more bioactive. In this study, a comparative and comprehensive assessment was conducted using a high-content screening (HCS) assay, with zebrafish (Danio rerio) embryo, with a focus on non-endocrine (neuro)developmental effects. In contrast to previous analyses, the comparison was based on an automated, quantitative image and video analysis approach, which reduces observer bias and increases the reproducibility of results. Zebrafish embryos were exposed to 5 concentrations of each bisphenol, with predicted baseline toxicity as the highest concentration, followed by a 2-fold serial dilution. The (neuro)developmental effects were quantified by using behavior endpoints, heart rate assessment and quantification of deviation of morphological endpoints from controls. For all endpoints, concentration-response modeling was applied to derive effect concentrations. The specificity of responses was estimated by applying the baseline toxicity and calculating effect ratios to observed mortality. The effect profile of bisphenols was compared with two presumable baseline toxicants (Diphenylamine and Methylaniline). Based on the detailed assessment of BPA and its 4 substitutes, a screening approach was developed to prioritize further BPA analogues for detailed assessment. Our results show that the absence of the posterior swimbladder was the most sensitive endpoint for all the tested bisphenols, particularly for BPE. Comparison to baseline toxicity indicated that the developmental effects were in the range of predicted baseline toxicity for all bisphenols.