Ontogeny of in vitro Cytochrome P450-mediated drug metabolism in zebrafish
13 december 2017
Campus Drie Eiken, gebouw O, Aula O1 - Universiteitsplein 1 - 2610 Antwerpen (Wilrijk) (route: UAntwerpen, Campus Drie Eiken
17 - 19 uur
Paul Cos en Steven Van Cruchten
Doctoraatsverdediging Moayad Saad - Departement Farmaceutische Wetenschappen
In this research, we investigated potential differences and similarities in CYP-related drug metabolism between zebrafish and man, as well as the ontogeny of CYP activity in zebrafish.
To this end, we compared in vitro alternative models for teratogenicity studies, including the zebrafish embryo in Chapter 1. This comparison showed the importance of the zebrafish embryo as a promising alternative model. Additionally, we compiled all available zebrafish CYP data regarding spatiotemporal gene expression, protein levels and intrinsic activity. Chapter 2 describes in detail our research hypotheses and objectives. The first step in this research was to optimise our experimental design including tissues collection, protein fractions’ preparation and microsomal protein stability assay. In Chapter 3 we assessed CYP1 activity in adult zebrafish and zebrafish embryos using the EROD assay. The aim was first to establish standard experimental conditions that minimize any confounding factors in EROD activity. Afterwards, any potential gender differences in liver CYP1 activity in adult zebrafish and the ontogeny of CYP1 activity during organogenesis were assessed. Our EROD data showed a lack in CYP1 activity in zebrafish embryos compared to adults. No sex-related differences were detected.
CYP2 and 3–related activity was tested in Chapter 4 using human CYP-specific substrates. Despite the fact that the major metabolites of diclofenac showed a very similar ratio in zebrafish compared to humans, dextromethorphan showed differences in its metabolite ratio for these species. Testosterone was metabolized in both species but into totally different metabolites. Surprisingly, midazolam was not metabolized at all in zebrafish. The last two substrates show that toxicity studies in zebrafish need to be interpreted with caution for human risk assessment when pharmacokinetics play a key role. Moreover, zebrafish embryo showed no or very low CYP activity during the early developmental stages. In Chapter 4 we also showed that testosterone was metabolized in both species. However, the main human metabolite was not detected in zebrafish. Therefore, a sensitive LC-MS/MS method was applied on testosterone microsomal stability assay samples to compare the metabolites in both species (in Chapter 5). Apparently, zebrafish and humans do not share the same main metabolites for testosterone.
Finally, we discussed our research results in Chapter 6 and compared them to the available data from other conventionally used animals in toxicity and pharmacological studies. In conclusion, significant differences in biotransformation of xenobiotics can be expected between humans and zebrafish, which must be taken in consideration during human risk assessment.