Competitive inhibition by inducer as a confounding factor in the use of the ethoxyresorufin-O-deethylase (EROD) assay to estimate exposure to dioxin-like compounds.
The ethoxyresorufin-O-deethylase (EROD) assay has been extensively used in whole animals and in cell culture as a biomarker of exposure to environmental contaminants such as dioxin-like compounds (DLCs). This paper addresses two controversial phenomena that arise when DLCs are examined by the EROD assay. Firstly, the maximum level of induced EROD activity varies with the identity of the inducing compound; secondly, the induced EROD activity reaches a concentration-dependent maximum level that is followed by an apparent reduction in activity when the concentration of inducer is further increased. These phenomena are completely explained by competitive inhibition of the EROD enzyme-substrate reaction by the dioxin-like compound. A kinetic model explains the biphasic appearance of EROD induction curves as a function of a compound's binding affinity with the Ah receptor (Kd) and its binding affinity to CYP 1A1 (Ki) which results in inhibition of the EROD enzyme-substrate reaction. These results limit the reliability of the information obtained from calibration curves of EROD activity versus concentration of a standard DLC such as 2,3,7,8-tetrachlorodibenzo-p-dioxin.
Petrulis
J R
JR
Toxicology Program, University of Guelph, Ont. Canada.
Bunce
N J
NJ
D000818:Animals; D001667:Binding, Competitive; D001681:Biological Assay / Q000379:methods; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism*; D004147:Dioxins / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D004781:Environmental Exposure; D004785:Environmental Pollutants / Q000633:toxicity*; D004790:Enzyme Induction / Q000187:drug effects; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D000072317:Polychlorinated Dibenzodioxins / Q000633:toxicity; D051381:Rats; D017207:Rats, Sprague-Dawley
D004147:Dioxins; D004785:Environmental Pollutants; D000072317:Polychlorinated Dibenzodioxins; D019363:Cytochrome P-450 CYP1A1