Development of an in vitro chemical hazard screening method using human iPSCs with and without metabolic activation...

There is a growing need for new approach methodologies (NAMs) to screen the hazard of the increasing number of untested chemicals. Human induced pluripotent stem cells (h-iPSCs) represent a promising resource for developing such NAMs, provided their low metabolic potency is addressed. Here, we developed a proof-of-concept in vitro NAM combining h-iPSCs with pooled human liver S9 fractions (h-iPSCs-S9) to enable chemical hazard screening both with and without metabolic activation. Chlorinated paraffins (CPs) are mixtures of emerging contaminants whose regulation was delayed due to the lack of human-relevant data, and their effects in human cells remain poorly understood. Therefore, we used h-iPSCs-S9 to evaluate the hazard of short-, medium-, and long-chain CPs at human exposure levels (1-1000 μg/L). Three endpoints were assessed: cell viability; oxidative stress; and gene expression related to DNA repair systems, the peroxisome proliferator-activated receptor alpha-nuclear factor erythroid 2-related factor 2 (PPARα-Nrf2) axis, and xenobiotic metabolism-related nuclear receptors and cytochrome P450s. Following validation, h-iPSCs-S9 showed responses consistent with several CP-associated effects observed in vivo and discerned the toxic effects of the three congener groups. Three hypotheses were proposed, where CPs might (1) alter human DNA repair and promote damage tolerance, (2) induce oxidative stress by suppressing PPARα-Nrf2 signaling through antagonism, and (3) act as weak antagonists, rather than agonists, of human xenobiotic metabolism. Overall, this study presents h-iPSCs-S9 as a reproducible and scalable NAM fit for chemical hazard screening, and calls for further investigation of the toxic modes of action of CPs in human cells.