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

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.

Refinement of the zebrafish embryo developmental toxicity assay

“…1 ). The metabolic activation protocol was developed and added to our ZEDTA, because we showed previously that zebrafish embryos have a low biotransformation capacity during a major part of organogenesis and, in contrast to mammals, they cannot rely on the maternal metabolic capacity due to their external development [4 , 5] . Consequently, test compounds that require bioactivation to exert their toxicity could be missed (and cause false negative results) in a standard ZEDTA.…”

Neurodevelopmental effects of natural and synthetic ligands of estrogen and progesterone receptors in zebrafish eleutheroembryos

“…Zebrafish embryos are metabolically competent and therefore it is likely that the natural steroids and the synthetic progestin used in our study are subjected to intensive metabolic activities (Le Fol et al, 2017;Verbueken et al, 2018). The metabolic pathways for E2, P4 and NOR are not known for zebrafish embryos and specific experiments should be conducted to determine the actual concentration of the 3 steroids within the brain of zebrafish embryos.…”

ahr2, But Not ahr1a or ahr1b, Is Required for Craniofacial and Fin Development and TCDD-dependent Cardiotoxicity in Zebrafish

“…To measure CYP1 enzyme activity in zebrafish embryos, a modified 7-ER-O-deethylase assay was used (Kais et al, 2017;Nacci et al, 1998;Otte et al, 2010;Verbueken et al, 2018). Embryos were treated from 1 to 3 dpf with 0.1% DMSO or 10 ng/ml TCDD, then exposed to 0.17% DMSO or 1.7 lM 7-ER for 1 h. Following exposure, tricaine was added to wells (0.02% final concentration) to anesthetize embryos for imaging, and embryos were manually positioned within the well in a single row.…”

“…To provide additional evidence that ahr2 acts independently of ahr1 receptors to mediate TCDD-induced toxicity, we assayed activity of CYP1A, a protein upregulated by AHR following TCDD exposure (Andreasen, Spitsbergen, et al, 2002;Otte et al, 2010;Verbueken et al, 2018). We hypothesized that ahr2 À/À embryos would demonstrate significantly reduced CYP1 activity, whereas ahr1 mutant embryos would demonstrate normal CYP1 activity due to retained function of ahr2.…”