Abstract
The zebrafish (Danio rerio) embryo is gaining interest as a bridging tool between in-vitro and in-vivo developmental toxicity studies. However, cytochrome P450 (CYP)-mediated drug metabolism in this model is still under debate. Therefore, we investigated the potential of zebrafish embryos and larvae to bioactivate two known anti-epileptics, carbamazepine (CBZ) and phenytoin (PHE), to carbamazepine-10,11-epoxide (E-CBZ) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH), respectively. First, zebrafish were exposed to CBZ, PHE, E-CBZ and HPPH from 5¼- to 120-h post fertilization (hpf) and morphologically evaluated. Second, the formations of E-CBZ and HPPH were assessed in culture medium and in whole-embryo extracts at different time points by targeted LC-MS. Finally, E-CBZ and HPPH formation was also assessed in adult zebrafish liver microsomes and compared with those of human, rat, and rabbit. The present study showed teratogenic effects for CBZ and PHE, but not for E-CBZ and HPPH. No HPPH was detected during organogenesis and E-CBZ was only formed at the end of organogenesis. E-CBZ and HPPH formation was also very low-to-negligible in adult zebrafish compared with the mammalian species. As such, other metabolic pathways than those of mammals are involved in the bioactivation of CBZ and PHE, or, these anti-epileptics are teratogens and do not require bioactivation in the zebrafish.
Highlights
In the last two decades, the field of toxicology has put a strong emphasis on the exploration and development of in-vitro models that accurately predict human toxicity.Developmental toxicity studies are assessing embryofetal death, reduced fetal growth and abnormal development following xenobiotic exposure in laboratory animals [1]
Since HPPH was not detected in the culture medium and embryos/larvae extracts exposed to 250 μM PHE at 120 hpf, a stage when the cytochrome P450 (CYP) enzymes should be active [19], we investigated whether PHE is metabolized by zebrafish, using adult zebrafish liver microsomes
We noted that the direct exposure of zebrafish embryos to E-CBZ and HPPH, which cause teratogenicity in mammals, did not cause morphological abnormalities, whereas the parent compounds, CBZ and PHE, induced teratogenicity, even from 48 hpf onwards
Summary
In the last two decades, the field of toxicology has put a strong emphasis on the exploration and development of in-vitro models that accurately predict human toxicity. Developmental toxicity studies are assessing embryofetal death, reduced fetal growth and abnormal development following xenobiotic exposure in laboratory animals [1]. Several initiatives have been set-up to reduce animal testing and to comply with the 3R’s principle. This has resulted in openings for alternatives to animal testing in the third revision of the ICHS5 guideline on the detection of toxicity to reproduction for human pharmaceuticals; as stated “A number of alternative in-vitro, ex-vivo, and non-mammalian in-vitro assays (alternative assays) have been developed to detect potential hazards to embryo-fetal development.
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