Abstract
At present, the zebrafish embryo is increasingly used as an alternative animal model to screen for developmental toxicity after exposure to xenobiotics. Since zebrafish embryos depend on their own drug-metabolizing capacity, knowledge of their intrinsic biotransformation is pivotal in order to correctly interpret the outcome of teratogenicity assays. Therefore, the aim of this in vitro study was to assess the activity of cytochrome P450 (CYP)—a group of drug-metabolizing enzymes—in microsomes from whole zebrafish embryos (ZEM) of 5, 24, 48, 72, 96 and 120 h post-fertilization (hpf) by means of a mammalian CYP substrate, i.e., benzyloxy-methyl-resorufin (BOMR). The same CYP activity assays were performed in adult zebrafish liver microsomes (ZLM) to serve as a reference for the embryos. In addition, activity assays with the human CYP3A4-specific Luciferin isopropyl acetal (Luciferin-IPA) as well as inhibition studies with ketoconazole and CYP3cide were carried out to identify CYP activity in ZLM. In the present study, biotransformation of BOMR was detected at 72 and 96 hpf; however, metabolite formation was low compared with ZLM. Furthermore, Luciferin-IPA was not metabolized by the zebrafish. In conclusion, the capacity of intrinsic biotransformation in zebrafish embryos appears to be lacking during a major part of organogenesis.
Highlights
The zebrafish (Danio rerio) embryo has emerged as an alternative animal model for developmental toxicity— called teratogenicity—screening of new drugs and environmental pollutants
cytochrome P450 (CYP) activity was assessed in adult zebrafish liver microsomes (ZLM) and in microsomes from whole zebrafish embryo homogenates (ZEM) of 5–120 hpf by means of the benzyloxy-methyl-resorufin (BOMR) assay
Since BOMR is a non-specific CYP substrate and clear spatio-temporal differences in CYP isoform expression have been reported [17], this could explain the lack of maternally derived CYP activity in our study. This in vitro study demonstrated that the non-specific CYP substrate BOMR was metabolized by adult zebrafish liver microsomes as well as by human liver microsomes
Summary
The zebrafish (Danio rerio) embryo has emerged as an alternative animal model for developmental toxicity— called teratogenicity—screening of new drugs and environmental pollutants (reviewed in [1,2,3,4]). Zebrafish embryos and larvae can be used in medium—or high—throughput screening because of their small size (0.5–4 mm) [6] and, as the embryos can be kept in small volumes (100 μL), only a small amount of compound is required (reviewed in [2]) The latter is very useful during early drug development when the availability of a new chemical entity (NCE)—as defined by the Food and Drug Administration (FDA) [7]—is still very low. The zebrafish liver and intestine—two important drug-metabolizing organs—develop late in organogenesis, i.e., between 72 and 96 hpf, which supports our hypothesis concerning the lack of intrinsic biotransformation by zebrafish embryos This hypothesis cannot be tested by just exposing zebrafish embryos to known mammalian proteratogens, as has been done previously [12], because this in vivo approach does not distinguish between teratogenic effects caused by the parent compound or by its metabolite. We used microsomes—subcellular fractions of endoplasmic reticulum containing cytochrome P450 (CYP) isoenzymes—from whole embryo homogenates and from adult zebrafish livers
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