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Updated: Oct 10, 2025
Small Molecule Screening and Toxicity Testing in Early-stage Zebrafish Larvae
Published on: March 7, 2025
Developmental Toxicity and Biotransformation of Two Anti-Epileptics in Zebrafish Embryos and Early Larvae
Chloé Bars1, Jente Hoyberghs1, Allan Valenzuela1
1Comparative Perinatal Development, Department of Veterinary Sciences, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium.
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.
Insights
Zebrafish embryos show teratogenic effects from carbamazepine (CBZ) and phenytoin (PHE), but do not significantly metabolize them into active epoxides. This suggests alternative pathways or direct teratogenicity in zebrafish developmental toxicity studies.
Area of Science:
- Developmental toxicology
- Pharmacology
- Comparative metabolism
Background:
- Zebrafish embryos are increasingly used for developmental toxicity testing, bridging in vitro and in vivo models.
- Cytochrome P450 (CYP)-mediated drug metabolism in zebrafish remains incompletely understood.
- Investigating the bioactivation of anti-epileptic drugs in zebrafish is crucial for model validation.
Purpose of the Study:
- To evaluate the potential of zebrafish embryos and larvae to bioactivate carbamazepine (CBZ) and phenytoin (PHE).
- To compare the metabolic capacity of zebrafish with mammalian models for these anti-epileptics.
- To assess the formation of carbamazepine-10,11-epoxide (E-CBZ) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) in zebrafish.
Main Methods:
- Zebrafish exposure to CBZ, PHE, E-CBZ, and HPPH from 5¼ to 120 hours post-fertilization (hpf).
- Morphological evaluation of zebrafish embryos and larvae.
- Targeted liquid chromatography-mass spectrometry (LC-MS) to quantify E-CBZ and HPPH in culture medium and whole-embryo extracts.
- Comparison of E-CBZ and HPPH formation in adult zebrafish liver microsomes versus human, rat, and rabbit microsomes.
Main Results:
- CBZ and PHE induced teratogenic effects in zebrafish, while E-CBZ and HPPH did not.
- HPPH was undetectable during organogenesis; E-CBZ formation was minimal and occurred late in organogenesis.
- Metabolism of CBZ and PHE to E-CBZ and HPPH was very low-to-negligible in adult zebrafish compared to mammals.
Conclusions:
- Zebrafish may utilize different metabolic pathways for CBZ and PHE bioactivation compared to mammals.
- The observed teratogenicity of CBZ and PHE in zebrafish might not require metabolic activation.
- Further research is needed to elucidate the specific metabolic pathways involved in anti-epileptic drug metabolism in zebrafish.
