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A non-destructive BFCOD assay for in vivo measurement of cytochrome P450 3A (CYP3A) enzyme activity in fish embryos and larvae

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Abstract

There is increasing interest in quantifying the exposure and effects of anthropogenic contaminants in fish. Determination of exposures in wild fish is routinely performed, but methods to investigate potential effects are less established. One of the most relevant approaches would be the use of in vivo assays, but existing assays are often limited to in vitro determination of enzyme activity. Many pharmaceuticals and some persistent pollutants activate, and are metabolized by cytochrome P4503A (CYP3A), which make it a relevant and desirable target for biomarker research. We altered the established 7-benzyloxy-4-trifluoromethylcoumarin-O-debenzylation (BFCOD) in vitro protocol for CYP3A activity determination, developing a rapid and inexpensive method to measure in vivo (and in ovo) CYP3A activity in two fish systems: Gulf killifish (Fundulus grandis) and zebrafish (Danio rerio) early life stages. Even with very low concentrations of 7-benzyloxy-4-trifluoromethyl coumarin (BFC, 0.06 µM or 20 µg/L), we were able to detect significant induction in CYP3A activity in embryos of F. grandis, as well as in larvae of D. rerio in response to benzo[a]pyrene (BaP) and fluoranthene (FL) exposures. Because of concerns regarding the possible contribution of CYP1A to BFCOD activity from previous research, we have used a CYP1A post-translational inhibitor (FL) in order to calculate the contribution of CYP1A to the BFCOD assay. We also dosed with benzo[k]fluoranthene (BkF) and showed significant induction of CYP1A activity, with no concurrent increase in CYP3A activity. In this paper, we have taken an established in vitro CYP3A activity assay, and utilized the reaction in a novel way to allow for the non-destructive determination of CYP3A. In summary, we describe a sensitive, cheap, fast and easy modified BFCOD assay for in ovo and in vivo determination of CYP3A activity for use in moderate throughput early-life-stage fish experiments.

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Acknowledgements

We would like to thank Dr. Crystal Usenko and Dr. Erika Abel for providing zebrafish embryos. We would also like to thank Benjamin Castellon, Chiyen Tseng, Lauren Waters, Willow Howard, Sarah Guberman, Kori Adair, and Erin Reynolds for their assistance with F. grandis aquaculture. Partial support was provided by the C. Gus Glasscock, Jr. Endowed Fund for Excellence in Environmental Sciences at Baylor University. This material is based upon work partially supported by the National Science Foundation Graduate Research Fellowship Program under Grant No 1356113 received by Alexis N. Carey.

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  1. Department of Environmental Science, Center for Reservoir and Aquatic Systems Research (CRASR), Baylor University, Waco, TX, 76798, USA

    Elias M. Oziolor, Alexis N. Carey & Cole W. Matson

  2. Institute for Biomedical Studies, Baylor University, Waco, TX, 76798, USA

    Elias M. Oziolor & Cole W. Matson

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  1. Elias M. Oziolor
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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.

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Oziolor, E.M., Carey, A.N. & Matson, C.W. A non-destructive BFCOD assay for in vivo measurement of cytochrome P450 3A (CYP3A) enzyme activity in fish embryos and larvae. Ecotoxicology 26, 809–819 (2017). https://doi.org/10.1007/s10646-017-1812-5

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