Biology subjects

Tagmount, A.

Publications and source records attributed to Tagmount, A..

5 recordsLinked to original sources

bioRxiv2026

Exposure Duration Shapes the Hepatic Response to GenX: Divergent Acute and Chronic Transcriptomic Profiles Reveal Non-Monotonic Dose Effects and Increased Sensitivity in Human Liver Spheroids

Hexafluoropropylene oxide dimer acid (GenX), a replacement for legacy per- and polyfluoroalkyl substances (PFAS), is increasingly detected in the environment, yet its chronic toxicity remains poorly characterized. Current safety assessments rely largely on short-term, high-dose studies that may not capture the biological consequences of long-term, low-dose exposure. To address this gap, we employed 3D human liver (HepG2/C3A) spheroids cultured in a continuously rotating bioreactor system (ClinoStar) to systematically evaluate dose- and time-dependent mRNA changes in response to GenX under environmentally relevant conditions. Spheroids were exposed to GenX (0.08-50 M, spanning environmentally relevant to mechanistically informative concentrations) for acute (4 days) and chronic (4 weeks) durations, followed by genome-wide TempO-Seq transcriptomic profiling and benchmark dose (BMD) modeling. GenX elicited pronounced non-monotonic mRNA changes in acute exposure conditions, with the greatest number of differentially expressed genes (DEGs) observed at an intermediate concentration (0.4 M). In contrast, chronic exposure exhibited a generally concentration-dependent increase in DEGs, except for the 10 M condition, indicating a more consistent dose-response relationship than acute exposure. Notably, acute and chronic exposures elicited qualitatively distinct mRNA changes with low concordance across matched concentrations, demonstrating that exposure duration was a major determinant of mRNA changes. Acute low-dose GenX exposure preferentially modulated mRNA encoding components of cell cycle-related pathways, whereas acute higher dose exposures suppress mRNA levels of the constituents of lipid metabolic pathways and increase expression of mRNA encoding proteins involved in stress- and toxicity-associated signaling. Chronic exposure revealed a different pattern of changes in mRNA expression not observed under acute exposure conditions, including suppression of cellular components involved in lipid-related pathways at the lowest concentration tested. At higher concentrations, mRNA levels of components of multiple metabolic pathways were altered. Benchmark dose modeling identified a significantly lower transcriptomic point of departure (tPOD) for chronic exposure as compared to acute exposure, suggesting increased cellular sensitivity to prolonged GenX exposure and supporting the relevance of chronic models for human exposure assessment. Collectively, these findings demonstrate that GenX elicits time-dependent and non-monotonic changes in mRNA levels of human liver (HepG2/C3A) spheroids, with distinct responses depending on the exposure duration and dose. This study, therefore, highlights the importance of incorporating chronic, human-relevant in vitro models and transcriptomic endpoints into PFAS risk assessment and suggests that conventional short-term assays may underestimate the biological impact of sustained low-dose exposure. Key message (Impact of the study)This study provides systematic comparisons of short term (4 day) versus longer term (4 weeks), environmentally relevant GenX exposure in human liver spheroids, revealing non-monotonic, time-dependent changes in mRNA levels encoding cellular components of lipid metabolism-related pathways with potential implications for appropriate dose and time exposure parameters for use in New Approach Methods to be applied in risk assessment.

pharmacology and toxicology↗

bioRxiv2025

Identification of Functional Genetic Components Modulating Toxicity Response to PFOS using Genome-wide CRISPR Screens in HepG2/C3A cells

Perfluorooctane sulfonate (PFOS) poses significant health and environmental risks due to its persistence and widespread use and has been linked to various adverse outcomes, such as liver toxicity. Although the molecular responses and toxicity effects of PFOS exposure have been extensively studied, considerable uncertainty remains regarding the causal mechanisms leading to PFOS-associated adverse effects. To help bridge this gap, we conducted CRISPR screens in HepG2/C3A human liver cells exposed to IC25 (170 {micro}M) of PFOS to identify genes and pathways influencing PFOS-induced cytotoxicity. Using a genome-wide CRISPR knockout library targeting 18,819 genes, we identified 340 candidate genes that modulate PFOS-induced cytotoxicity when genetically disrupted (189 gene disruptions increased sensitivity and 151 gene disruptions increased resistance). From these candidate genes, we individually disrupted two candidate genes, SLC6A9 which encodes the glycine transporter GlyT1, and CPSF2, and confirmed increased resistance to PFOS exposure. Further, molecular docking analysis predicts that PFOS directly binds to GlyT1 and functional inhibition of GlyT1 also increases resistance to PFOS exposure. Gene-Disease outcome association analysis using the Comparative Toxicogenomics Database (CTD) indicated an enrichment of candidate genes associated with cancer-related and liver disease phenotypes. KEGG and STRING enrichment analyses found over representation of several biological pathways including DNA damage response and cell cycle. Lastly, cross-species conservation analysis using the top two validated gene targets found that their pathways were highly conserved in several environmentally relevant species. These findings provide new mechanistic and functional insights into PFOS-induced cytotoxicity, highlight potential molecular targets for toxicity mitigation, and establish a foundation for cross-species toxicogenomic modeling of PFOS health effects.

pharmacology and toxicology↗

bioRxiv2025

Characterizing common loss-of-function genes and their potential utility in assessing population variability and chemical susceptibility

Inter-individual and population variability in susceptibility to chemical exposures confounds determination of threshold exposure levels to protect the most vulnerable. Current risk assessment frameworks, in the absence of empiric chemical-specific data, generally recommend default or probabilistic adjustment factors to account for such variability. We present an experimental approach to incorporate common genetic variants potentially impacting population-level differences in toxicant susceptibility into human cell-based models for any cellular apical endpoint of interest. We focus on the genes with the most common aggregate loss-of-function (LoF) alleles in the gnomAD v3.0 data which we designated as the PopVarLoF set. Unexpectedly, enrichment analysis of these genes found significant overrepresentation of gene products playing important functional roles in toxicology. Interrogation of GWAS and PheWAS databases found that these genes are associated with diverse metabolic phenotypes consistent with the relevance of the PopVarLoF set in studying variability of toxicant response in human populations. We further characterized the PopVarLoF set by developing custom lentiviral CRISPR knockout libraries targeting the PopVarLoF genes to assess their functional essentiality in the HepG2/C3A cell line. Functional disruption of 14 of the PopVarLoF genes ([~]1 %) without toxicant exposure resulted in significant growth defects in this cell line, consistent with the majority of PopVarLoF gene products having non-essential roles. The development of human cell-based toxicity assays or other NAMs which include the empiric assessment of common genetic sources of population variability in susceptibility to chemical exposure could contribute to more robust risk assessment which protects vulnerable populations while reducing uncertainty. Impact statementWe characterize common loss of function genetic variants which could impact toxicant susceptibility and describe an approach to incorporate them into NAMs to enable empiric estimates of the contribution of genetic variability to diverse toxicity endpoints.

pharmacology and toxicology↗

bioRxiv2025

Physiologically Relevant 3D CRISPR Screening Enhances Mechanistic Insight into Chemical Toxicity Compared to 2D Screening

Many omics-based approaches in toxicology research primarily rely on correlative data, often lacking functional relationships or causal links between genotypes and phenotypes. CRISPR-based approaches can overcome this limitation by establishing direct causal connections between genes and toxicological phenotypes. Moreover, CRISPR screens enable scalable and systematic interrogation of gene function and associated mechanisms following chemical exposure, predominantly using in vitro models. In line with the paradigm of new approach methodologies (NAMs) in toxicology research, CRISPR screens hold promise to provide an in vitro cell-based functional toxicogenomics approach. One of the main limitations of conventional in vitro assays is their compromised physiological relevance to humans due to their inability to fully recapitulate in vivo physiology. To improve the functional and physiological relevance of the toxicogenomics approach, we developed a 3D CRISPR screening system using HepG2/C3A spheroids generated and cultivated in a continuously rotating bioreactor (ClinoStar). We first performed time-course 3D CRISPR screens to identify genes that confer growth disadvantage or advantage, influencing spheroid development compared to 2D cultures. We then applied this approach to a chemical toxicity study using doxorubicin, comparing the performance of the 3D and 2D systems in identifying chemical-specific mechanisms. The results showed that the 3D system captured more candidate genetic determinants and biological pathways related to DNA damage processes--a known toxicity mechanism of doxorubicin--demonstrating improved performance in identifying chemical-specific pathways over the 2D counterpart. In our screens, we employed custom CRISPR sgRNA libraries representing common human loss-of-function genetic variants (mean allele frequency > 0.1% in all individuals catalogued in the genome aggregation database), which potentially affect toxicity responses. By comparing our CRISPR screen results with previously reported genetic associations for doxorubicin response, we found that the 3D system identified more known associated genes than the 2D system. Together, the 3D CRISPR screening system demonstrated its feasibility and utility for physiologically relevant functional toxicogenomics. This platform enables in vitro NAMs, by providing a scalable and effective approach to identify causal genetic determinants and biological pathways that modulate chemical-induced toxicity.

pharmacology and toxicology↗

bioRxiv2021

Genome-scale CRISPR Screens Identify Host Factors that Promote Human Coronavirus Infection

The COVID-19 pandemic has resulted in 153 million infections and 3.2 million deaths as of May 2021. While effective vaccines are being administered globally, there is still a great need for antiviral therapies as potentially antigenically distinct SARS-CoV-2 variants continue to emerge across the globe. Viruses require host factors at every step in their life cycle, representing a rich pool of candidate targets for antiviral drug design. To identify host factors that promote SARS-CoV-2 infection with potential for broad-spectrum activity across the coronavirus family, we performed genome-scale CRISPR knockout screens in two cell lines (Vero E6 and HEK293T ectopically expressing ACE2) with SARS-CoV-2 and the common cold-causing human coronavirus OC43. While we identified multiple genes and functional pathways that have been previously reported to promote human coronavirus replication, we also identified a substantial number of novel genes and pathways. Of note, host factors involved in cell cycle regulation were enriched in our screens as were several key components of the programmed mRNA decay pathway. Finally, we identified novel candidate antiviral compounds targeting a number of factors revealed by our screens. Overall, our studies substantiate and expand the growing body of literature focused on understanding key human coronavirus-host cell interactions and exploit that knowledge for rational antiviral drug development. One Sentence SummaryGenome-wide CRISPR screens identified host factors that promote human coronavirus infection, revealing novel antiviral drug targets.

microbiology↗