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Optimization of DNA Barcode Method to Assess Altered Chemical Toxicity due to CYP-mediated Metabolism.
Citation:
Woolard, E., G. Carswell, Steve Simmons, AND B. Chorley. Optimization of DNA Barcode Method to Assess Altered Chemical Toxicity due to CYP-mediated Metabolism. SOT 57th Annual meeting, San Antonio, TX, March 11 - 15, 2018.
Impact/Purpose:
A primary criticism of in vitro toxicity testing is the lack of xenobiotic metabolism of the cell models commonly used; such test methods are only capable of testing the toxicity of the parent compound. Therefore, the generation of toxic metabolites or alternatively detoxification of parent compounds cannot be observed using these models. While detoxification is important, bioactivation (generation of toxic metabolites) is of greater importance from a toxicity testing perspective because missing bioactivation generates false negatives. Most toxic metabolites are generated by Phase I enzymatic oxidation reactions mediated primarily by cytochrome P450 monooxygenases (CYPs) from families CYP1, CYP2 and CYP3. Of the 57 human CYP genes, 23 (40%) belong to these three families. Many of these CYP enzymes are known to catalyze the formation of toxic metabolites of environmental chemicals. This task aims to generate human cells that stably express a transgene encoding single human CYP enzyme with an associated DNA barcode and then use multiplexed pools of these cells to identify CYP(s) that confer sensitivity or resistance to environmental chemical exposure. Multiplexed pools of these cells could also be used to identify CYP(s) that enhance the endocrine disrupting activity or stress response profile of environmental chemicals.
Description:
A drawback of current in vitro chemical testing is that many commonly used cell lines lack chemical metabolism. To address this challenge, we present a method for assessing the impact of cellular metabolism on chemical-based cellular toxicity. A cell line with low endogenous metabolism (HEK293T) was engineered to overexpress cytochrome P450 monooxygenase (CYP) recombinant transgenes prevalent in human liver (CYP1A1, CYP1A2, CYP2E1, and CYP3A4). Each of the five clones (four CYPs and one empty vector control) were mated to unique DNA barcodes and used as surrogates of cell viability that could be distinguished in a mixed clonal culture by PCR. Four known cytotoxic chemicals (rotenone, fluazinam, galangin, and zoxamide) were evaluated. We evaluated general cytotoxicity on individual clones using CellTiter-Glo (CTG) and CellTiter-Blue (CTB). Toxicity was observed with both assays for all chemicals tested (p<0.05 vs. DMSO controls; two-tailed t-test), however CTG results did not agree with observable cell loss after rotenone (9% vs. ~60% loss) and galangin (12% vs. ~65% loss) exposures. Morphological characteristics indicated different necrotic, apoptotic, or a combination of cell death mechanisms mediated by these chemicals, partially explaining assay discrepancies. To assess the cytotoxic impact on individual clones, DNA was isolated from dosed cells and barcodes were quantified using equal volumes by digital droplet PCR (ddPCR) in a mixed clonal culture. A strong correlation (Pearson’s r = 0.29–0.93, x̄ = 0.69) between total DNA concentration and individual DNA barcode counts were observed, suggesting barcode counts were representative of DNA isolated. However, the predicted monotonic loss of DNA due to lower cell viability was not observed, suggesting these measurements were not representative of individual clonal toxicity. We speculate this discrepancy is the result of excess DNA retained in the extracellular matrix which may be influenced by the mechanism of cell death and/or plating matrix. Future optimization of cell culture conditions and efficient washing of DNA associated with non-viable clonal cells will be necessary to refine this method. The overall goal will be to adapt this barcode method as a high-throughput screen to rapidly assess CYP-mediated chemical toxicity. This abstract does not necessarily reflect US EPA policy.