Citation:

Woolard, E., G. Carswell, Steve Simmons, M. Babic, AND B. Chorley. Establishing a Cell-based Assay for Assessment of Cellular Metabolism on Chemical Toxicity. GEMS annual fall meeting, RTP, NC, November 07, 2017.

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 major drawback of current in vitro chemical testing is that many commonly used cell lines lack chemical metabolism. To help address this challenge, we are established a method for assessing the impact of cellular metabolism on chemical-based cellular toxicity. A commonly used human cell line with low endogenous metabolism (HEK298T) was engineered to overexpress cytochrome P450 monooxygenase (CYP) transgenes prevalent in human liver (CYP3A4, CYP2E1, CYP1A1, and CYP1A2). Each clone was mated to a unique DNA barcode. A cytotoxicity screen with individual clones was performed with 29 chemicals reported to be affected by CYP metabolism using CellTiter Glo (CTG, Promega). Of the 22 chemicals reported to be activated by CYPs, we measured only 6 that exhibited >10% cell number loss by one or more CYP overexpressing clones. Interestingly, 3 of the 7 chemicals that were reportedly detoxified by chemicals or inhibited CYP activity exhibited cytotoxic effects when compared to controls cells. Specific CYP subtype metabolism not represented in this pilot screen may account for differences from reported results. Concurrently, we are assessing higher throughput methods (multiplexed digital drop PCR and targeted DNA sequencing) that measure clone-associated barcodes that serve as surrogates of clone cell number (i.e., cytotoxicity). Such methods will allow us increase the number of CYPs assessed in a single measurement. Ultimately, these developed methods will provide a rapid chemical screen that is more biologically relevant and will advance chemical safety assessment. This abstract does not necessarily reflect US EPA policy.

Record Details: