You are here:
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. North Carolina Academy of Science 2018, NC, Raleigh, March 23 - 24, 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. This hinders the use and relevance of cell culture in high throughput chemical toxicity screening. To address this challenge, we engineered HEK293T cells to overexpress the cytochrome P450 monooxygenase (CYP) recombinant transgenes, which are commonly involved in chemical metabolism within the 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. Viability was assessed in the presence of four known cytotoxic chemicals (rotenone, fluazinam, galangin, and zoxamide) and DMSO vehicle control (p<0.05; two-tailed t-test). DNA was isolated from dosed cells and barcodes were quantified with 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 was observed. However, the predicted monotonic loss of DNA due to lower cell viability was not found. General cytotoxicity was further confirmed by CellTiter-Glo and CellTiter-Blue. Optimization of cell culture conditions will be necessary to refine this method as testing has shown that plate coating (i.e. polylysine and collagen) influences assay outcomes and may interfere with true DNA count. Future work will assess assay performance in the presence of CYP specific chemicals. Adapting this barcode method to high-throughput screening will improve characterization of CYP-mediated chemical toxicity. This abstract does not necessarily reflect US EPA policy.