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Modulation of Xenobiotic Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes by ToxCast Chemicals
ROTROFF, D., A. Beam, K. Freeman, S. Ferguson, E. LeCluyse, K. A. HOUCK, R. JUDSON, R. J. KAVLOCK, M. T. MARTIN, AND D. J. DIX. Modulation of Xenobiotic Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes by ToxCast Chemicals. Presented at Society of Toxicology Annual Meeting, Baltimore, MD, March 15 - 19, 2009.
Chemical potency and efficacy were determined relative to reference chemicals, and correlated with chronic hepatotoxicity endpoints from EPA’s ToxRefDB database. A preliminary analysis of the 5 nuclear receptor pathways at 48 hr was conducted based on expression of a representative gene for each pathway. Of the 320 ToxCast chemicals, 3 chemicals perturbed AhR (CYP1A1/2), 35 chemicals perturbed CAR (CYP2B6), 22 chemicals perturbed PXR (CYP3A4), 15 chemicals perturbed PPARa (HMGCS2), and 28 chemicals perturbed FXR (ABCB11) at ≥40% efficacy and <40 µM EC50. Many chemicals resulted in statistically significant responses at <40% efficacy.
ToxCast chemicals were assessed for induction or suppression of xenobiotic metabolizing enzyme and transporter gene expression using primary human hepatocytes. The mRNA levels of 14 target and 2 control genes were measured: ABCB1, ABCB11, ABCG2, SLCO1B1, CYP1A1, CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP3A4, UGT1A1, GSTA2, SULT2A1, HMGCS2, and control genes ACTB, GAPDH. These genes represent 5 nuclear receptor signaling pathways: AHR, CAR, PXR, PPARa and FXR. Gene expression was quantitatively measured by nuclease protection assays at 5 concentrations, 3 time points (6, 24, 48 hr), in 4 replicate wells. Hepatocytes from 2 male donors were isolated and cultured with 6 reference chemicals and 320 ToxCast phase I chemicals. CYP1A1/2 enzymatic activity and cell morphology were assessed in each well. Concentration-response curves were generated for 13,813 chemical, time and gene combinations. EC50 (effective concentration 50%) and Emax (effective maximum) values for gene expression determined modulation of nuclear receptor pathways by ToxCast chemical exposures.
Record Details:Record Type: DOCUMENT (PRESENTATION/ABSTRACT)
Organization:U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL CENTER FOR COMPUTATIONAL TOXICOLOGY