Abstract
This protocol describes how to use cytochrome P450–dependent monooxygenase (CYP)-expressing cell lines in toxicity testing of chemicals in vitro. Selected cells amenable to permanently grow in culture are genetically manipulated to stably express single CYP enzymes originating from any species of interest. This expression can be characterized by, for example, determining CYP mRNA content, CYP protein level (western blotting or in situ immunofluorescence) and CYP-mediated enzyme activity (substrate conversion assays). These cells can be used to determine substrate specificities and species differences, e.g., in the bioactivation of drugs. Once constructed, CYP-expressing cells can serve as a straightforward and reliable tool in toxicity testing and the corresponding assays could be adapted for high-throughput analysis. Using these cells, enzyme assays can be performed in a matter of hours. This protocol is exemplified with V79 fibroblasts from Chinese hamster (Cricetulus griseus), modified to express human cytochrome P450 1B1 (CYP1B1). These cells are characterized for their CYP1B1-linked properties by in situ immunofluorescence and their activity in the 7-ethoxyresorufin-O-deethylase enzyme assay. This is followed by an assay showing metabolic activation of the polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene by CYP1B1, along with the toxicological endpoints of cytotoxicity and micronucleus formation.
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Acknowledgements
A.L. gratefully acknowledges the help and advice of former colleagues at the Universities of Mainz, Germany (K.-L. Platt and A. Seidel), and Munich, Germany (H. Greim, J. Doehmer, A. Schneider, V. Soballa, W. Schober and N. Krebsfänger), as well as colleagues at the Cancer Center of Purdue University, Indiana, USA (W.M. Baird, S.L. Ralston, S.L. Coffing, B. Mahadevan and V. Melendez-Colon). Most of the work upon which this protocol is based was performed in the laboratories of J. Doehmer (Munich) and W.M. Baird (Purdue).
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A.L. produced the compounds and its enantiomerically pure derivatives, contributed to the characterization of the transgenic V79 cell line and the design and performance of the assays described and conducted the DNA binding and adduct analyses; R.L. and E.F. provided additional experimental data, and T.T. produced the manuscript.
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Landsiedel, R., Fabian, E., Tralau, T. et al. Chemical toxicity testing in vitro using cytochrome P450–expressing cell lines, such as human CYP1B1. Nat Protoc 6, 677–688 (2011). https://doi.org/10.1038/nprot.2011.316
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DOI: https://doi.org/10.1038/nprot.2011.316
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