From mRNA Expression of Drug Disposition Genes to In Vivo Assessment of CYP-Mediated Biotransformation during Zebrafish Embryonic and Larval Development.

Evy Verbueken,Steven J Van Cruchten,Jelena Periz-Stanacev,Dries Knapen,Anna Tochwin,Waleed F A Marei,Ellen D G Michiels,Lucia Vergauwen,Chris J Van Ginneken,Evelyn Stinckens,Jonathan S Ball,Chloé Bars,Isabelle J Gabriëls

doi:10.3390/ijms19123976

Abstract

The zebrafish (Danio rerio) embryo is currently explored as an alternative for developmental toxicity testing. As maternal metabolism is lacking in this model, knowledge of the disposition of xenobiotics during zebrafish organogenesis is pivotal in order to correctly interpret the outcome of teratogenicity assays. Therefore, the aim of this study was to assess cytochrome P450 (CYP) activity in zebrafish embryos and larvae until 14 d post-fertilization (dpf) by using a non-specific CYP substrate, i.e., benzyloxy-methyl-resorufin (BOMR) and a CYP1-specific substrate, i.e., 7-ethoxyresorufin (ER). Moreover, the constitutive mRNA expression of CYP1A, CYP1B1, CYP1C1, CYP1C2, CYP2K6, CYP3A65, CYP3C1, phase II enzymes uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) and sulfotransferase 1st1 (SULT1ST1), and an ATP-binding cassette (ABC) drug transporter, i.e., abcb4, was assessed during zebrafish development until 32 dpf by means of quantitative PCR (qPCR). The present study showed that trancripts and/or the activity of these proteins involved in disposition of xenobiotics are generally low to undetectable before 72 h post-fertilization (hpf), which has to be taken into account in teratogenicity testing. Full capacity appears to be reached by the end of organogenesis (i.e., 120 hpf), although CYP1—except CYP1A—and SULT1ST1 were shown to be already mature in early embryonic development.

Highlights

The thalidomide tragedy in the late fifties and early sixties resulted in the obligatory use of a second, non-rodent, animal species in developmental toxicity studies
The present study contributes to a better understanding of the ontogeny of metabolism and transport of xenobiotics in the zebrafish, and suggests that, in general, the disposition of xenobiotics in zebrafish embryos is immature during a major part of the organogenesis period, i.e., before 72 hpf
This may lead to false negative results in the case of proteratogens, whereas the teratogenic potential might increase in the case of teratogens since immature biotransformation might result in a higher internal concentration of the teratogenic parent compound

Summary

Introduction

The thalidomide tragedy in the late fifties and early sixties resulted in the obligatory use of a second, non-rodent, animal species in developmental toxicity studies. The zebrafish embryo developmental toxicity assay (ZEDTA) considers the physiological parameters of a whole organism together with the advantages of an in vitro model. Due to these benefits, several pharmaceutical companies and contract research organizations (CROs) have already adopted the ZEDTA as an early screening method to reduce the number of compounds that need to be tested in a mammalian model (reviewed in [7]). The FET (chemicals) uses exposure windows between 1.5 and 96 hpf [10], whereas the ZEDTA (pharmaceuticals) commonly uses exposure windows between 4 and 120 hpf to ensure that the entire zebrafish organogenesis period is covered [6,8,9,13,14]

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Conclusion