Stress Reporter Assay Ensemble as a Tool for Rapid Classification of Chemical Toxicants Authors: Steve Simmons, Ram Ramabhadran U.S. EPA/Office of Research and Development (ORD)/National Health and Environmental Effects Research Laboratory (NHEERL)/Neurotoxicology Division (NTD) Keywords: stress-response pathways, screening and prioritization, reporter assay, toxicant profiling, in vitro screening There is an increasing need for assays for the rapid and efficient assessment of toxicities of large numbers of environmental chemicals. To meet this need, we are developing cell-based reporter assays that measure the activation of key molecular stress pathways. We are using promoter regions from various stress-responsive genes such as heme oxygenase I (HO) and heat shock protein 70 (hsp70), as well as chimeric response elements for stress-response factors such as Nuclear Factor kappa B (NFkB) and Nuclear Factor E2-related factor (Nrf2) with the ultimate goal of building and characterizing a panel of reporter assays that can be multiplexed within the same cell line for use in high-throughput screening. Our preliminary work towards this goal with 10 putative stress-response reporters shows that several toxic compounds can activate subsets of these reporters in a dose-dependent manner. All tested toxicants activate and/or depress the activities of various stress reporters at different concentrations, indicating the differential sensitivities of the stress pathways reporters. Additionally, several stress reporter genes were stimulated by metal and non-metal stressors suggesting these genes respond to a broad spectrum of toxicants. The data presented will show that in vitro stress reporter assays can discriminate among toxic compounds at sub-lethal concentrations, as well as discriminate between highly toxic and moderately toxic compounds. Thus, stress reporter assays provide a quantitative method to assess cellular toxicity and also allow inference into mechanism(s) of toxicity. This is an abstract of a proposed presentation and does not necessarily reflect U.S. Environmental Protection Agency policy. Point of Contact: Steve Simmons Postdoctoral Fellow U.S. EPA/ORD/NHEERL/NTD 109 T.W. Alexander Drive Durham, NC 27711 919-541-1475 simmons.steve@epa.gov