Effects of per- and polyfluoroalkyl substances on molecular initiating events of cholestatic liver injury.

Per- and polyfluoroalkyl substances (PFAS) are abundantly used in a plethora of products with applications in daily life. As a result, PFAS are widely distributed in the environment, raising concerns regarding accumulation in humans. Recent data show impairment of bile acid metabolism by legacy PFAS, yet the potential of short-chain and alternative PFAS as well as the mechanistic basis of these cholestatic effects remain elusive. The present study relied on an in vitro test battery mechanistically anchored in an adverse outcome pathway network to assess the cholestatic potential of legacy, short-chain and alternative PFAS. Focus was put on the effects of PFAS on molecular initiating events, in particular transporter changes and bile canaliculi dynamics. Cultures of human HepaRG liver cells were exposed to 8 types of PFAS in 3 concentrations. Fluorescently labeled probes were used to investigate effects on the functionality of bile acid transporters. Effects at the transcriptional level were studied by means of quantitative reverse transcriptase polymerase chain reaction analysis. Bile canaliculi dynamics were monitored via in situ immunostaining and phase-contrast imaging. An integrated weight-of-evidence approach was applied to process the results of the in vitro test battery. PFAS inhibit the expression and activity of various bile acid transporters, including organic anion transporting polypeptides and multidrug resistance-associated proteins. Some PFAS inflicted morphological disruption of bile canaliculi. The highest overall cholestatic scores were observed for perfluorononanoic acid, perfluorooctane sulfonic acid and perfluorobutanoic acid, while C6 PFAS exhibited low cholestatic potential. This study shows the power of in vitro testing to elucidate mechanisms underlying hepatotoxicity induced by PFAS and, simultaneously, to quantify the cholestatic potential of these ubiquitous "forever chemicals".