Determination of the CYP1A-inducing potential of single substances, mixtures and extracts of samples in the micro-EROD assay with H4IIE cells.

Nature protocols 2015 Nov

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This protocol describes a quantitative and robust 96-well-plate-reader-based assay for the measurement of ethoxyresorufin-O-deethylase (EROD) activity using the rat hepatoma cell line H4IIE. The assay can be used to determine the cytochrome P450 subfamily 1A (CYP1A)-inducing potential of single substances, as well as of mixtures and extracts of samples. It is based on the aryl hydrocarbon receptor (AhR)-mediated induction of cytochrome P450 enzymes (subfamily 1A) in cells after exposure to dioxins and dioxin-like compounds. One enzymatic reaction catalyzed by CYP1A is the deethylation of the exogenous substrate 7-ethoxyresorufin to the fluorescent product resorufin, which is measured as EROD activity in the assay. The CYP1A-inducing potential of a sample can be reliably quantified by comparing the EROD activity with the concentration-response curve of the standard substance 2,3,7,8-tetrachlorodibenzo-p-dioxin, which can be detected at concentrations down to the picogram per liter range. A researcher familiar with the procedure can process up to 160 samples with four wells each within 3 d. The series described uses four plates with three concentrations per sample, which can be easily scaled to accommodate different sample sizes.

Citation

Andreas Schiwy, Markus Brinkmann, Ines Thiem, Gabriele Guder, Kerstin Winkens, Kathrin Eichbaum, Leonie Nüßer, Beat Thalmann, Sebastian Buchinger, Georg Reifferscheid, Thomas-Benjamin Seiler, Brigitte Thoms, Henner Hollert. Determination of the CYP1A-inducing potential of single substances, mixtures and extracts of samples in the micro-EROD assay with H4IIE cells. Nature protocols. 2015 Nov;10(11):1728-41