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Identification of Functional Genetic Components modulating Cellular Toxicity Response to PFOS using Genome-wide CRISPR Screens

ABSTRACT: Per- and polyfluoroalkyl substances (PFAS) are a group of chemicals that pose significant health and environmental risks due to their persistence and widespread use. These substances, known for their strong chemical bonds, are linked to various health issues such as liver damage, developmental disorders, and immune dysfunction. However, the molecular mechanisms behind their toxicity are not fully understood. Although several studies have identified molecular responses to PFAS exposure, there remains considerable uncertainty about the underlying mechanisms leading to PFAS-associated health effects. To help bridge this gap, we conducted genome-wide CRISPR-based screens in HepG2/C3A human liver cells to identify genes and pathways influencing cellular toxicity upon PFAS exposure. We focused on perfluorooctane sulfonate (PFOS), a widely used and studied PFAS compound with known toxic effects, yet unclear molecular mechanisms underlying PFOS-induced adverse outcomes. Using a genome-wide CRISPR knockout library targeting 18819 genes, we identified 340 candidate genes which modulate the cellular toxicity of PFOS when disrupted. Pathway and enrichment analyses of the corresponding gene products suggest a role for cell cycle, DNA damage response, and Wnt/-catenin pathways in mediating PFOS cytotoxicity, while further gene-phenotype association analysis using the comparative toxicogenomics database indicated enrichment of gene products previously associated with liver disease and neoplasia. We carried out individual targeted disruption of two candidate genes, SLC6A9 and CPSF2, in HepG2/C3A to confirm their roles in resistance to PFOS exposure. Additionally, molecular docking analysis predicts that PFOS may bind directly to GlyT1, the SLC6A9 gene product. Lastly, cross-species relevance analysis using gene targets identified in our screens suggested that molecular pathways of PFOS toxicity are highly conversed in several environmentally relevant species.

ORGANISM(S): Homo sapiens

PROVIDER: GSE291529 | GEO | 2026/02/13

REPOSITORIES: GEO

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Project description:The potential effects of poly- and perfluoroalkyl substances (PFAS) are a recent human and environmental health concern. There is a persistent link between PFAS exposure and cancer, but the mechanisms are poorly understood. Although epidemiological evidence supporting PFAS exposure and cancer in general is conflicting, there is a relatively strong evidence linking PFAS and testicular germ cell tumors (TGCTs). However, there have been no mechanistic studies to date cancerning PFAS and TGCTs. In this report, the effects of the legacy PFAS, perfluorooctanesulfonic acid (PFOS) and the newer "clean energy" PFAS, lithium bis(trifluoromethylsulfonyl)imide (LiTFSi and called HQ-115) on TGCT tumorigenicity, survival, metabolism, and gene regulation were investigated. In vitro, proliferation and survival of both chemo-sensitive and -resistant TGCT cells were minimally affected by a wide concentration range of PFOS and HQ-115. However, both chemicals promoted the growth of TGCT cells in mouse xenografts at doses consistenet with human exposure but had minimal acute toxicity, as assessed by total body, kidney, and testis weight. PFOS, but not HQ-115, increased liver weight. Transcriptiomic alterations of PFOS-exposed normal mouse testes were dominated by cancer-related pathways and gene expression altersations associated with the H3K27me3 polycomb pathway and DNA methylation, epigenetic pathways were previously shown to be critical for the survival of TGCT cells after cisplatin-based chemotherapy. Similar pattersn of PFOS-mediated gene expression occured in PFOS-exposed cells in vitro. Metabolomic studies revealed that PFOS also altered metabolites associiated with steroid biosynthesis and fatty acid metabolism in TGCT cells, consistent with the proposed ability of PFAS to mimic fatty acied based ligands controlling lipid metabolism and the proposed role of PFAS as endocrine disrupters. Our data, this first cell and animal based study on PFAS in TGCTs, support a pro-tumorigenic effect of PFAS on TGCT biology and suggests epigenetic, metabolic, and endocrine disruption as potential mechanisms of action that are consistent with the non-mutagenic nature of the PFAS class.

Project description:Per- and polyfluoroalkyl substances (PFAS) are some of the most prominent organic contaminants in human blood that have the potential to disrupt biological processes and pathways in the liver. Although the toxicological implications from human exposure to perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) are well established, data on other, lesser-understood PFAS are limited. A challenge for regulatory authorities is to determine acceptable levels of human exposure to large, diverse classes of environmental contaminants. New approach methodologies (NAMs) that apply bioinformatics tools to interpret biological data are being increasingly considered to inform risk assessment when traditional toxicology methodologies are not amenable. The aim of the current investigation was to identify the biological responses relevant to PFAS mode of action as the concentration (benchmark dose) that these effects take place in order to utililze this information to inform/facilitate read-across for risk assessment of data-poor PFAS. A TempO-Seq platform (BioSpyder) measured gene expression changes in human liver microtissues (i.e., spheroids) after 1-day and 10-day exposures to increasing concentrations of PFAS. A bioinformatics framework was applied to 23 PFAS sub-classed into carboxylates (PFCAs), sulfonates (PFSAs), or PFAS precursors that were analyzed for the total altered transcripts, the concentration where effects take place, and identifying target genes of interest. 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 surpass liver-toxic transcriptomic thresholds established from previous studies than shorter chain PFAS. Longer-chain PFAS were also more potent, inducing transcriptional effects at lower concentrations. However, PFOS was the most potent PFAS and of all precursors, only PFOSA (a PFOS precursor) induced a response. The combined high-throughput transcriptomic and bioinformatics analyses revealed the capability of NAMs to assess the effects of PFAS in liver microtissues; such data improves our understanding of PFAS-related effects in humans and facilitates the use of read-across in human health risk assessment for other data-poor chemicals.

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Identification of Functional Genetic Components modulating Cellular Toxicity Response to PFOS using Genome-wide CRISPR Screens

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