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

Nature Protocols volume 10, pages 1728–1741 (2015)Cite this article

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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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Figure 1: Summary of the micro-EROD bioassay with H4IIE cells.
Figure 2: Cleanup procedure for the removal of up to 1 g of fat content and unstable CYP1A inducers before analysis in the micro-EROD bioassay.
Figure 3: Layout for the exposure of H4IIE cells in 96-well tissue culture plates.
Figure 4: Specific EROD activity for TCDD with typical curve shape and parameters.
Figure 5: Example result from a pre–round robin test.

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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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  1. Andreas Schiwy and Markus Brinkmann: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Ecosystem Analysis, Institute for Environmental Research, Aachen Biology and Biotechnology (ABBt), Rheinisch-Westfälische Technische Hochschule Aachen (RWTH) Aachen University, Aachen, Germany

    Andreas Schiwy, Markus Brinkmann, Kerstin Winkens, Kathrin Eichbaum, Leonie Nüßer, Beat Thalmann, Thomas-Benjamin Seiler & Henner Hollert

  2. Food and Veterinary Institute Braunschweig/Hannover, Lower Saxony State Office for Consumer Protection and Food Safety (LAVES), Braunschweig, Germany

    Ines Thiem, Gabriele Guder & Brigitte Thoms

  3. Department G3: Biochemistry, Federal Institute of Hydrology (BFG), Ecotoxicology, Koblenz, Germany

    Sebastian Buchinger & Georg Reifferscheid

  4. College of Resources and Environmental Science, Chongqing University, Chongqing, China

    Henner Hollert

  5. College of Environmental Science and Engineering and State Key Laboratory of Pollution Control and Resource Reuse, Tongji University, Shanghai, China

    Henner Hollert

  6. State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, China

    Henner Hollert

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Contributions

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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Correspondence to Henner Hollert.

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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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