Abstract
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.
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Acknowledgements
This protocol was applied within the §64-LFGB working group 'Wirkungsbezogene Analytik' (effect directed analysis) for a pre–round robin test. The authors acknowledge the federal office of Consumer Protection and Food Safety (BVL) for their support in this project. The protocol was used and further adopted in context of the project 'DioRAMA—Assessment of the dioxin-like activity in sediments and fish for sediment evaluation' that received funds from the German Federal Ministry of Transport and Digital Infrastructure. The authors acknowledge the German National Academic Foundation ('Studienstiftung des deutschen Volkes') for a personal scholarship granted to M.B. H.H. was supported by the Chinese 111 Program (College of Environmental Science and Engineering and Key Laboratory of Yangze Water environment, Ministry of Education, Tongji University).
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All authors contributed extensively to the work presented in this paper, read and edited it, and gave their final approval for publication. A.S. and M.B. have contributed equally to the work and share first authorship. A.S. has adopted the protocol from an initial version of I.T., G.G. and B.T., and established it together with K.W., L.N. and K.E. in our laboratory. M.B. and A.S. wrote the manuscript and compiled the protocol. T.-B.S. B.T., S.B., G.R. and H.H. gave technical support and conceptual advice.
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Schiwy, A., Brinkmann, M., Thiem, I. et al. Determination of the CYP1A-inducing potential of single substances, mixtures and extracts of samples in the micro-EROD assay with H4IIE cells. Nat Protoc 10, 1728–1741 (2015). https://doi.org/10.1038/nprot.2015.108
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DOI: https://doi.org/10.1038/nprot.2015.108
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