Integrative Toxicological and Transcriptomic Assessment of Chemicals Associated with Environmental and Human Biomonitoring Studies and Their Mixtures in Zebrafish

By anchoring to the morphological effects of individual chemicals, we created mixtures of at least four components for two subclasses (diphenylmethanes and cyclopropanecarboxylic acid esters) in two formulations (equivalent proportion and equipotent). These two subclasses were selected due to a sufficient number of chemicals (four or more) having morphological effects. We then determined how the mixtures impacted zebrafish embryo development and the underlying transcriptional events.

The equipotent mixtures for both subclasses behaved synergistically, to differing degrees, while the equivalent proportion formulations did not. The diphenylmethane equivalent proportion mixture acted slightly antagonistically while its cyclopropanecarboxylic acid ester counterpart acted additively. The mixtures produced distinct transcriptional responses that were not the sum of the individual component responses as few differentially expressed genes (DEGs) were shared by all components and their respective mixtures within the same subclass.

DEGs unique to the mixture formulations indicate that mixture formulation influences toxicity and transcriptional responses, and the DEGs for each mixture were related to distinctly different processes. Our findings highlight the need for further experimentation prior to the implementation of a MAF as a default value, to better understand overall mixture behavior of chemicals of mixture risk concern and increase understanding of the influence that they have in different mixture formulations.