For nearly 20 years, Dr. Simmons leads a research program dedicated to developing new in vitro high-throughput screening methods to identify human health hazards posed by environmental toxicants. He earned a B.S. in Biology from Lamar University (Texas) in 1999 and a Ph.D. in Molecular and Cellular Toxicology from North Carolina State University in 2006. Dr. Simmons first joined EPA in 2006 as a postdoctoral fellow where he developed high-throughput screening assays measuring chemical induction of cellular stress response pathways. He joined ORD’s National Health and Environmental Effects Research Laboratory (NHEERL) in 2008 as an investigator where he continued to develop in vitro screening assays in support of the ToxCast and Tox21 programs. In 2014, he joined the National Center for Computational Toxicology (NCCT) where has worked to add xenobiotic metabolism to ToxCast assays and to design new assays to identify disruptors of thyroid hormone signaling. In addition to assay development, his has also conducted several large high-throughput screening campaigns for endpoints ranging from cell stress and endocrine disruption pathways to mitochondrial disruption. In addition, Dr. Simmons has utilized his expertise in recombinant cloning, gene editing and viral vector techniques to manipulate expression of targeted genes as well as create and integrate novel genes to act as reporters of biological activity. Dr. Simmons has an extensive record of successful collaboration with the EPA and across the federal, non-profit, private, and academic sectors on a wide variety of toxicology and translational medicine projects.
Incorporation of Human CYP450 Metabolism into Endocrine Disruption Assays
Steven O. Simmons1, Daniel R. Hallinger1, Evan C. Brown2
1Office of Research and Development, U.S. Environmental Protection Agency, 2Oak Ridge Institute for Science Education.
Background and Purpose
The latest ToxCast strategic plan proposed transfecting chemically modified mRNAs encoding human cytochrome P450 (CYP) enzymes into cells to induce intracellular CYP expression and functional metabolic activity into cell-based assays. We screened > 2,000 ToxCast chemicals in an androgen receptor (AR) homodimerization assay (AR2) across 10 human liver CYP enzymes to identify anti-androgenic parent chemicals and their metabolites.
Methods
The AR2 assay (antagonist mode) was used to first screen 2,165 ToxCast chemical samples at an initial single-concentration using cells transfected with mRNAs encoding one of 10 human liver CYP450 enzymes. Negative (no metabolism) control cells were transfected with beta-galactosidase mRNA, or no mRNA. Active chemicals were then re-screened in concentration-response format. The effect size of normalized CYP activity was compared to no metabolism controls using the strictly standardized mean difference (SSMD) for each chemical sample by concentration by CYP enzyme combination (117,200 total).
Results
Initial testing identified 700 chemical samples as inactive across all conditions. Metabolic shifts in AR bioactivity were identified for 373 samples. CYP-induced deactivation (i.e., reduced AR inhibition) was observed for 245 samples while only 141 exhibited CYP-mediated bioactivation (i.e., increased AR inhibition). Most of the metabolically shifted bioactivity was enzyme-specific as 65% of deactivated and 74% of bioactivated samples were affected by only a single CYP enzyme. A disproportionate number of shifted samples were deactivated by CYP2C19 (40.7%) or bioactivated by CYP2E1 (25.7%), suggesting an outsized role for these two enzymes in metabolizing anti-androgenic chemicals.
Conclusion
Incorporation of xenobiotic metabolism into in vitro assays remains a critical challenge to the broader adoption of these alternative methods for regulatory purposes. This study highlights the inclusion of 10 CYP450 enzymes and their impact on an assay to measure human androgen receptor activity.
*This abstract does not necessarily represent US EPA policy.
