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Measuring cytochrome P450 activity in aquatic invertebrates: a critical evaluation of in vitro and in vivo methods

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Abstract

The first step in xenobiotic detoxification in aquatic invertebrates is mainly governed by the cytochrome P450 mixed function oxidase system. The ability to measure cytochrome P450 activity provides an important tool to understand macroinvertebrates’ responses to chemical stressors. However, measurements of P450 activity in small aquatic invertebrates have had variable success and a well characterized assay is not yet available. The general lack of success has been scarcely investigated and it is therefore the focus of the present work. In particular, the suitability of the substrate selected for the assay, the sensitivity of the assay and the possible inhibition/attenuation of enzymatic activity caused by endogenous substances were investigated. 7-ethoxycoumarin-O-dealkylation activity of Daphnia magna, Chironomus riparius larvae and Hyalella azteca was assessed in vivo and in vitro and possible inhibition of enzymatic activity by macroinvertebrates homogenate was investigated. Activities of D. magna and C. riparius larvae measured in vivo were 1.37 ± 0.08 and 2.2 ± 0.2 pmol h−1 organism−1, respectively, while activity of H. azteca could not be detected. In vitro activity could be measured in C. riparius larvae only (500–1000 pmol h−1 mg microsomal protein−1). The optimization of the in vitro assay has been especially long and resource consuming and particularly for D. magna, substances that inhibited cytochrome P450 activity seemed to be released during tissue homogenization preventing activity measurements in vitro. We therefore recommend testing the P450 inhibition potential of homogenate preparations prior to any investigation of P450 activity in vitro in macroinvertebrates.

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Abbreviations

ACN:

Acetonitrile

BSA:

Bovine serum albumin

DTT:

DL-dithiothreitol

ECOD:

7-ethoxycoumarin-O-dealkylation

EDTA:

Ethylenediaminetetraacetic acid disodium salt dihydrate

EROD:

7-ethoxyresorufin-O-dealkylation

K2HPO4·3H2O:

Potassium phosphate dibasic trihydrate

KH2PO4 :

Potassium dihydrogen phosphate

MFO:

Mixed function oxidase

MgCl2·6H2O:

Magnesium chloride hexahydrate

NADPH:

ß-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate

PMSF:

Phenylmethanesulfonyl fluoride

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Acknowledgments

The authors thank laboratory technician Anja Weibell for taking good care of the organisms’ cultures and Post Doc Tomas Laursen for his suggestions on the cytochrome P450 in vitro assay as well as for solving practical issues.

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  1. Andreas Kretschmann

    Present address: Department of Pharmacy, Analytical Biosciences, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark

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  1. Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg, Denmark

    Michele Gottardi, Andreas Kretschmann & Nina Cedergreen

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  1. Michele Gottardi
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Gottardi, M., Kretschmann, A. & Cedergreen, N. Measuring cytochrome P450 activity in aquatic invertebrates: a critical evaluation of in vitro and in vivo methods. Ecotoxicology 25, 419–430 (2016). https://doi.org/10.1007/s10646-015-1600-z

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