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Functional toxicogenomic assessment of triclosan in human HepG2 cells using genome-wide CRISPR-Cas9 screen
Citation:
Xia, P., X. Zhang, Y. Xie, M. Guan, Dan Villeneuve, AND H. Yu. Functional toxicogenomic assessment of triclosan in human HepG2 cells using genome-wide CRISPR-Cas9 screen. ENVIRONMENTAL SCIENCE & TECHNOLOGY. American Chemical Society, Washington, DC, 50(19):10682-10692, (2016).
Impact/Purpose:
CRISPR-Cas9 technology is a cutting edge technique that allows for targeted gene knockout in human cells and other biological systems. Using this technology, libraries of cells in which tens of thousands of individual gene targets are knocked out can be employed to screen for genes that play a role in either conferring resistance or susceptibility to a given stressor. This technique has been effectively employed in drug development to associate chemicals with the pathways they interact with. However, to date, the approach has not been widely applied in mechanistic toxicology. The present paper represents one of the first attempts to apply this genome-wide knock out technology to infer potential mechanisms of toxicity. Specifically, the approach was applied to investigate genes whose presence confers resistance or susceptibility to the effects of the widely used antimicrobial, triclosan, when test at cytotoxic (e.g., at IC50 concentrations with regard to cell viability) or sublethal stress concentrations (e.g., IC10 or IC20). Results suggest interactions of triclosan with genes known play a role in obesity and breast cancer. Coupled with other orthogonal data, the approach offers novel lines of hypothesis generation and evidence that can support chemical screening and identification of adverse outcome pathways relevant to the toxic actions of specific chemicals. This research supports activities and goals under CSS Project 17.01, adverse outcome pathway discovery and development, and was the result of EPA’s collaboration with the Chinese Ministry of Science and Technology (MOST), project 3.1 Emerging Contaminants, subtask 3.1B - . Development and application of genome-wide mutant library of human cells as a basis for mechanistic classification of chemicals
Description:
Thousands of chemicals for which limited toxicological data are available are used and then detected in humans and the environment. Rapid and cost-effective approaches for assessing the toxicological properties of chemicals are needed. We used CRISPR-Cas9 functional genomic screening to identify potential molecular mechanism of a widely used antimicrobial triclosan (TCS) in HepG2 cells. Resistant genes (whose knockout gives potential resistance) at IC50 (50% Inhibition concentration of cell viability) were significantly enriched in adherens junction pathway, MAPK signaling pathway and PPAR signaling pathway, suggesting a potential molecular mechanism in TCS induced cytotoxicity. Evaluation of top-ranked resistant genes, FTO (encoding an mRNA demethylase) and MAP2K3 (a MAP kinase kinase family gene), revealed that their loss conferred resistance to TCS. In contrast, sensitive genes (whose knockout enhances potential sensitivity) at IC10 and IC20 were specifically enriched in pathways involved with immune responses, which was concordant with the transcriptomic profiling of TCS at concentrations
