U.S. Environmental Protection Agency Office of Research and Development Applying EcoToxChips to identify Potential Gene Expression Markers of Exposure to Glucocorticoid Receptor Agonists 1 ORISE Fellow, US EPA, Office of Research and Development, Great Lakes Toxicology and Ecology Division, Duluth, MN 2 US EPA, Great Lakes Toxicology and Ecology Division, Duluth, MN Introduction Principal Component Plot Conclusion Identification of Novel Transcriptomic Markers References • Among 375 genes evaluated, six genes were differentially expressed across more than one treatment and identified as biomarker candidates for GR-agonism. • The up-regulated genes were acsl5, scd, odc1, and gadd45ga. • The down-regulated genes were slc27a6 and dgat2. • These genes were all found to be sensitive at an environmentally relevant concentration of dexamethasone (40ng/L) and responded in the same direction across all treatments of dexamethasone and BDP. Furthermore, the magnitude of induction and/or down-regulation following dexamethasone was dose-dependent. • Results of this study provide additional candidate genes to consider as potential markers of in vivo exposure to GR-agonists in field studies employing caged fish. Jacob Collins1, Jenna Cavallin2, Alex Cole1, Kathleen Jensen2, Michael Kahl2, Kelvin Santana Rodriguez1, Ashley Kittelson1, Dan Villeneuve2 Glucocorticoid-mediated bioactivity has been detected in North American surface waters1. However, the bioavailability and in vivo potency of glucocorticoid receptor (GR) agonists in environmental mixtures remains undefined. • Dexamethasone (DEX) and Beclomethasone dipropionate (BDP) were used as positive controls related to glucocorticoid receptor (GR) agonism, • 40ng/L DEX was used an environmentally relevant concentration. • 18 µg/L of BDP was used as a positive control for glucocorticoid receptor agonism. • Initial selection of gene expression endpoints were based on mammalian genes known to be regulated by GR signaling: sgk1, nr3c1, nfkbiaa. Potential transcriptomic markers of GR activation were identified by the direction and magnitude of their Log2 fold change (FC). Targets that were determined to be viable were those that demonstrated a dose-dependent response. Preliminary Results 96 Hour Adult Fathead Minnow Study Test Conditions • 5-6 month old male fathead minnows (FHM) were distributed amongst 15 tanks with 3 replicate tanks per treatment. • Filtered and UV-treated Lake Superior Water (LSW) was utilized as control water. • A continuous flow through system of approximately 45mL/min was utilized. • The photoperiod was 16:8; light:dark. • Water temperature was kept at 25.3 ± 0.3°C. • FHM were fed bring shrimp twice daily. • Water conditions (temperature, pH, dissolved oxygen, conductivity, flow-rate, and survival) were monitored over the course of the test. Treatment Groups • LSW Control • Dex 40ng/L (0.1 nM) • Dex 0.40mg/L (1uM) • Dex 4.0mg/L (10uM) • BDP 17.8ug/L (34.2 nM) 1. Cavallin JE, Battaglin WA, Beihoffer J, Blackwell BR, Bradley PM, Cole AR, Ekman DR, Hofer RN, Kinsey J, Keteles K, Weissinger R, Winkelman DL, Villeneuve DL. Effects-Based Monitoring of Bioactive Chemicals Discharged to the Colorado River before and after a Municipal Wastewater Treatment Plant Replacement. Environ Sci Technol. 2021 Jan 19;55(2):974-984. doi: 10.1021/acs.est.0c05269. Epub 2020 Dec 29. PMID: 33373525; PMCID: PMC8135223. 2. Basu N, Crump D, Head J, Hickey G, Hogan N, Maguire S, Xia J, Hecker M. EcoToxChip: A next-generation toxicogenomics tool for chemical prioritization and environmental management. Environ Toxicol Chem. 2019 Feb;38(2):279-288. doi: 10.1002/etc.4309. PMID: 30693572. EcoToxChip Assay Procedure • RNA samples were converted to cDNA and combined with nuclease-free water and PCR master mix before distribution across a 96 well qPCR plate. • Samples from the 96 well plate were then transferred to a 384 well EcotoxxChip plate utilizing the Beckmen Coulter Biomek i5 • PCR analysis was perfor