Application of the ethoxyresorufin-O-deethylase (EROD) assay to mixtures of halogenated aromatic compounds

The ethoxyresorufin-O-deethylase (EROD) assay monitors the induction of the xenobiotic-metabolizing enzyme cytochrome P-450 (CYP) 1A1 and is a widely used biomarker for exposure of wildlife to substances that bind the aryl hy drocarbon (Ah) receptor. in this work the induction of EROD activity by sin gle compounds and binary mixtures in primary rat hepatocytes was compared w ith the predictions of a kinetic model involving mixtures of inducers. The inducing agents were also examined for their ability to activate the Ah rec eptor to its DNA-binding form and for their ability to act as competitive i nhibitors for CYP 1A1. Xenobiotics that failed to activate the Ah receptor did not induce EROD activity. Competitive inhibition for CYP 1A1 between th e xenobiotic and 7-ethoxyresorufin caused EROD activity to fall at high xen obiotic concentrations. Competition for a limited number of Ah receptor sit es depressed the EROD activity of a strong inducer such as 2,3,7,8-tetrachl orodibenzo-p-dioxin at high concentrations of a weak inducer. Application o f the kinetic model to the example of a mixture of low concentrations of di benzo-p-dioxins and much higher concentrations of polychlorinated biphenyls indicated that EROD assays often seriously underestimate the true potency of an environmental sample. Hence the EROD assay cannot be used for determi ning dioxin equivalent concentrations using the toxic equivalence factor ap proach. (C) 2001 by John Wiley & Sons, Inc.