High-throughput transcriptomics and benchmark concentration modeling for potency ranking of per- and polyfluoroalkyl substances (PFAS) in exposed human liver cell spheroids

Abstract

Per- and polyfluoroalkyl substances (PFAS) are some of the most prominent organic contaminants in human blood. Although the toxicological implications from human exposure to perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) are well established, data on lesser-understood PFAS are limited. New approach methodologies (NAMs) that apply bioinformatic tools to high-throughput data are being increasingly considered to inform risk assessment for data-poor chemicals. The aim of this investigation was to identify biological response potencies (i.e., benchmark concentrations: BMCs) following PFAS exposures to inform read-across for risk assessment of data-poor PFAS. Gene expression changes were measured in primary human liver cell microtissues (i.e., 3D spheroids) after 1-day and 10-day exposures to increasing concentrations of 23 PFAS. The cells were treated with four subgroups of PFAS: carboxylates (PFCAs), sulfonates (PFSAs), fluorotelomers, and sulfonamides. An established pipeline to identify differentially expressed genes and transcriptomic BMCs was applied. We found that both PFCAs and PFSAs exhibited a trend toward increased transcriptional changes with carbon chain-length. Specifically, longer-chain compounds (7 to 10 carbons) were more likely to induce changes in gene expression, and have lower transcriptional BMCs. The combined high-throughput transcriptomic and bioinformatic analyses supports the capability of NAMs to efficiently assess the effects of PFAS in liver microtissues. The data enable potency ranking of PFAS for human liver cell spheroid cytotoxicity and transcriptional changes, and assessment of in vitro transcriptomic points of departure. These data improve our understanding of the health effects of PFAS and will be used to inform read-across for human health risk assessment.

Internal Relative Potency Factors for the Risk Assessment of Mixtures of Per- and Polyfluoroalkyl Substances (PFAS) in Human Biomonitoring

“… 27 , 139 Wishing to have a better understanding of the risk associated with cumulative exposure to PFAS, we encourage researchers to focus on identifying the unknown fraction of PFAS in human blood and to subsequently investigate the toxicokinetics and toxicity of these substances. This investigation could be done by performing in vivo animal experiments, but researchers may also employ other sources, such as in vitro studies and accompanying quantitative in vitro to in vivo extrapolation (QIVIVE) methods, 140 , 141 or human epidemiological studies. Such data would allow researchers to extend the number of internal RPFs and to verify the animal toxicity data-derived RPFs with alternative resources.…”

Internal Relative Potency Factors Based on Immunotoxicity for the Risk Assessment of Mixtures of Per- and Polyfluoroalkyl Substances (PFAS) in Human Biomonitoring

“…For practical application of the RPF method for PFAS, we furthermore stimulate scientific debate to work towards agreement of RPFs for PFAS. Several efforts have been made since 2018 on deriving RPFs for PFAS, such as RPFs based on cytotoxicity and intracellular reactive oxygen species formation in human HepG2 cells in vitro (Amstutz et al, 2022), based on gene expression in human HepaRG cells (Louisse et al in preparation) and in human liver spheroids in vitro (Reardon et al, 2020); based on thyroid hormone disruption potential in vitro (Behnisch et al, 2021); and based on several systemic toxicity effects in vivo (Goodrum et al, 2021;Bil et al 2022b;Rietjens et al, 2021;Zeilmaker et al, 2018). The RPF analyses performed in these publications do not necessarily adhere to the same methodological approaches and criteria, for instance on ensuring parallel curve fitting (Bil et al 2022b).…”

The use of in vitro methods in assessing human health risks associated with short‐chain perfluoroalkyl and polyfluoroalkyl substances (PFAS)

“…The use of well‐characterized cell lines offers a wide array of possibilities for manipulating experimental variables to obtain a multitude of endpoints in a timely manner. The utility of screening methods employing HTS methods has already been demonstrated for PFAS chemicals (Reardon et al, 2020; Rowan‐Carroll et al, 2021; Singh & Hsieh, 2021). While not inclusive of all assays, endpoints, and cell types, Table 2 provides some examples of approaches that can be and are being used for assessing the human health effects of short‐chain PFAS.…”