MON-258 Multiplexed Steroid Receptor BioAssays Detect Endocrine Disruptors Activity in Tap Water

Abstract Disclosure: F.J. Schaufele: XCellAssay, Inc. S. Ardeshna: None. A. Wood: None. Four trillion kg of industrial chemicals are manufactured worldwide each year. The US water supply is routinely surveyed by >95 tests for metals, pathogens, water treatment disinfectants and other entities prioritized as harmful. However, there are >100,000 mass-produced chemicals of unknown health consequence, any of which could individually, additively or synergistically impact physiologic function; Current water quality tests do not detect those 100,000 chemicals. One complementary solution is to monitor water quality using in vitro BioAssays that identify water samples containing chemicals at levels sufficient to alter the activities of physiologically responsive ‘Receptor’ proteins. To date, this approach is under evaluation by some water quality regulators for monitoring water sources, from stream to water treatment plant. We used BioAssays to examine tap water for Endocrine Disrupting Chemicals. 20 tap water samples were obtained from Northern California and examined with multiplexed BioAssays for any changes induced in the activity of three Nuclear Receptors (Glucocorticoid, Progesterone, Androgen). The BioAssays were extensively characterized for appropriate ligand specificity in dose response curves against a panel of 48 natural steroid and synthetic agonists or antagonists. Assay Specificity, Sensitivity and the fidelity of quantification were established in repeat studies against known concentrations of ligands. Since preliminary studies showed that half of the tap water samples contained microorganisms, 0.2 micron 96-well filter plates were identified that eliminated the microbial overgrowth while permitting quantitative recovery of all steroids examined. The resulting tap water filtrates were spiked with low concentrations of dexamethasone, progesterone and dihydrotestosterone (DHT) such that both agonist and antagonist actions could be monitored. Samples in 96-well plates were transferred to quadruplicate wells of a 384-well plate pre-seeded with stable, barcoded cell lines expressing the three multiplexed BioAssays. Samples were prepared and quantified in three independent studies. None of the 20 tap water samples showed reproducible agonist or antagonist effects on the Glucocorticoid or Progesterone Receptors. Three of the 20 samples showed (P<0.05) androgenic agonist activities, of unknown chemical origin, at ‘DHT-equivalent’ activity levels of 16.5 ± 1.4, 12.7 ± 2.2 and 7.8 ± 1.4 ng/L (ppB); for comparison, the published maximum ‘safe’ threshold for DHT is estimated at 4.5 to 11 ng/L The miniaturized processes developed may become a useful tool for wide-spread water monitoring of all known or unknown chemicals, or chemical combinations, that impact a high throughput, robotics-friendly, multiplexed BioAssay set. Presentation: Monday, July 14, 2025