In vitro CYP-mediated drug metabolism in the zebrafish (embryo) using human reference compounds

Abstract

The increasing use of zebrafish embryos as an alternative model for toxicological and pharmacological studies necessitates a better understanding of xenobiotic biotransformation in this species. As cytochrome P450 enzymes (CYPs) play an essential role in this process, in vitro drug metabolism of four human CYP-specific substrates, i.e. dextromethorphan (DXM), diclofenac (DIC), testosterone (TST) and midazolam (MDZ) was investigated in adult male and female zebrafish, and in zebrafish embryos and larvae up to 120hours post-fertilization. Substrate depletion and production of their respective metabolites were measured using tandem quadrupole UPLC-MS/MS. Human liver microsomes were used as positive control. Adult zebrafish produced the two major human metabolites of DIC and DXM. For DIC the metabolite ratio was similar to that in man, whereas it was different for DXM. For TST, the major human metabolite could not be detected and MDZ was not metabolized. No sex-related differences were detected, except for the higher TST depletion rate in adult females. Zebrafish embryos and larvae showed no or only low biotransformation capacity. In conclusion, in vitro CYP-mediated drug metabolism in adult zebrafish shows differences compared to man and appears to be lacking in the early zebrafish life stages. As CYP-mediated drug metabolism in zebrafish may not be predictive for the one in man, we recommend including the zebrafish in metabolic stability testing of new compounds when considering non-clinical species for human risk assessment.

Zebrafish as Toxicological model for screening and recapitulate human diseases

“…Analysis of fluorescent intensity can be informative with regard to size or the number of hepatocytes [100] . However, has been described the inconvenient to work with the larvae, arguing that the CYP system, which plays an essential role in drug metabolism, is not yet fully developed in larvae and suggesting that some CYPs appear to be lacking in the early zebrafish life [101] . Furthermore, zebrafish embryos and larvae showed no or low biotransformation capacity of four human CYP-specific substrates, dextromethorphan, diclofenac, testosterone and midazolam [101] .…”

“…However, has been described the inconvenient to work with the larvae, arguing that the CYP system, which plays an essential role in drug metabolism, is not yet fully developed in larvae and suggesting that some CYPs appear to be lacking in the early zebrafish life [101] . Furthermore, zebrafish embryos and larvae showed no or low biotransformation capacity of four human CYP-specific substrates, dextromethorphan, diclofenac, testosterone and midazolam [101] . In contrast, has been reported the larva as a promising tool capable of distinguishing between hepatotoxic and non-hepatotoxic chemical analogues, implying that it may be applied as a screening model for DILI [102] .…”

Tools for studying the metabolism of new psychoactive substances for toxicological screening purposes – A comparative study using pooled human liver S9, HepaRG cells, and zebrafish larvae

“…In recent years, zebrafish (Danio rerio) larvae (embryos) have increasingly been discussed in the literature as a new in vivo tool (Howe et al, 2013;Saad et al, 2017;van Wijk et al, 2016). In contrast to adult zebrafish, larvae are not considered as animals until 120 hours post-fertilization (hpf) according to the European Directive 2010/63/EU but they already provide all benefits of an intact organism (EU, 2010;Saad et al, 2017). Thus, the use of zebrafish larvae in drug development is increasing e.g.…”

“…xenobiotics such as cytochrome P450 (CYP) oxygenases are identical (van Wijk et al, 2016) and zebrafish were already successfully used for metabolism studies of approved drugs (Kantae et al, 2016;Saad et al, 2017). So far, only one study has been published using zebrafish larvae in the NPS context; it was focused on distribution and toxicity studies of meta-chlorophenylpiperazine (mCPP) (Kirla et al, 2018).…”

Advancing the Zebrafish embryo test for endocrine disruptor screening using micro-injection: Ethinyl estradiol as a case study

“…The biotransformation genes—at least the set that we analyzed—showed increased transcription as of 72 hpf, which coincides with the development and activation of the hepatopancreas (Wilkins and Pack ), the major organ responsible for biotransformation. At 72 hpf, in vitro activity of some cytochrome P450s was also observed in zebrafish embryos (Saad et al ; Verbueken et al ). A similar decreasing pattern of internal doses, after a maximum of approximately 24 hpf, was also observed after aquatic exposure to benzocaine, phenacetin, metribuzin (Brox et al ), and benz[ a ]anthracene (Kuhnert et al ).…”