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

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.

Antioxidants reduce reactive oxygen species but not embryotoxicity in the metabolic Danio rerio test (mDarT).

Mammalian liver microsomes are occasionally used as a metabolic activation system (MAS) to compensate for the low CYP-mediated bioactivation of drugs in zebrafish embryos, in the so-called mDarT. However, this MAS is embryotoxic and consequently zebrafish embryos are only exposed during a very limited developmental window. The main aim of this study was to try to reduce the embryotoxic properties of MAS in order to extend the exposure window in the mDarT. Removing the microsomes from the incubation medium prior to exposure of the zebrafish embryos did not reduce embryotoxicity. Free radicals (ROS) in the incubation medium were successfully reduced by antioxidants, but the medium remained embryotoxic. Single dosing of NADPH or omitting toxic components from the MAS preparation did also not reduce embryotoxicity. In conclusion, the exposure window in the mDarT could not be extended by reducing ROS levels, single dosing of NADPH or modifications of the MAS preparation.

In Vitro Biotransformation of Two Human CYP3A Probe Substrates and Their Inhibition during Early Zebrafish Development.

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.

Incubation at 32.5°C and above causes malformations in the zebrafish embryo.

Zebrafish embryos are increasingly used for developmental toxicity screening of candidate drugs and are occasionally co-incubated with a metabolic activation system at 32°C for 1, 2 or 4h, depending on their developmental stage. As this temperature is higher than the optimal temperature for zebrafish embryonic development (26-28.5°C), we investigated whether continuous incubation of zebrafish embryos from 2.5 until 96h post fertilization (hpf) at high temperatures (30.5-36.5°C) causes malformations. At 32.5°C tail malformations were observed as early as 24hpf, and these became even more prominent at 34.5 and 36.5°C. Cardiovascular and head malformations, edema and blood accumulations throughout the body were present at 36.5°C. Finally, temperatures higher than 28.5°C accelerated embryonic development except for 36.5°C, at which a lower hatching rate and hatching enzyme activity were observed. In conclusion, incubation of zebrafish embryos at 32.5°C and above from 2.5 until 96hpf causes malformations as early as 24hpf.