Evaluation of Metabolism of a Defined Pesticide Mixture through Multiple In Vitro Liver Models

Abstract

The evaluation of exposure to multiple contaminants in a mixture presents a number of challenges. For example, the characterization of chemical metabolism in a mixture setting remains a research area with critical knowledge gaps. Studies of chemical metabolism typically utilize suspension cultures of primary human hepatocytes; however, this model is not suitable for studies of more extended exposures and donor-to-donor variability in a metabolic capacity is unavoidable. To address this issue, we utilized several in vitro models based on human-induced pluripotent stem cell (iPSC)-derived hepatocytes (iHep) to characterize the metabolism of an equimolar (1 or 5 µM) mixture of 20 pesticides. We used iHep suspensions and 2D sandwich cultures, and a microphysiological system OrganoPlate® 2-lane 96 (MimetasTM) that also included endothelial cells and THP-1 cell-derived macrophages. When cell culture media were evaluated using gas and liquid chromatography coupled to tandem mass spectrometry methods, we found that the parent molecule concentrations diminished, consistent with metabolic activity. This effect was most pronounced in iHep suspensions with a 1 µM mixture, and was lowest in OrganoPlate® 2-lane 96 for both mixtures. Additionally, we used ion mobility spectrometry–mass spectrometry (IMS-MS) to screen for metabolite formation in these cultures. These analyses revealed the presence of five primary metabolites that allowed for a more comprehensive evaluation of chemical metabolism in vitro. These findings suggest that iHep-based suspension assays maintain higher metabolic activity compared to 2D sandwich and OrganoPlate® 2-lane 96 model. Moreover, this study illustrates that IMS-MS can characterize in vitro metabolite formation following exposure to mixtures of environmental contaminants.