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

Abstract

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.

Differential uptake of and sensitivity to diphenhydramine in embryonic and larval zebrafish

“…For example, investigations of clofibric acid biotransformation [55], and a suite of other polar compounds [40], by developing zebrafish identified the presence of transformation products. Zebrafish embryos were also not capable of metabolizing benzyloxy-methylresorufin until 72 hpf, and these levels were greatly reduced from metabolite formation in adults [57]. For example, when comparing developing and adult zebrafish metabolism of benzophenone-2 and bisphenol S, larvae lacked the production of various metabolites and all capacity to eliminate parent and metabolites [56].…”

“…For example, when comparing developing and adult zebrafish metabolism of benzophenone-2 and bisphenol S, larvae lacked the production of various metabolites and all capacity to eliminate parent and metabolites [56]. Zebrafish embryos were also not capable of metabolizing benzyloxy-methylresorufin until 72 hpf, and these levels were greatly reduced from metabolite formation in adults [57].…”

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

“…This implies that proteratogenic compounds may lead to false negative results in developmental toxicity studies if zebrafish embryos do not have the capacity to bioactivate those compounds. The hypothesis has been tested by an earlier in vitro study in which intrinsic CYP activity was assessed in microsomes—artificial subcellular fractions of endoplasmic reticulum containing CYPs—from whole zebrafish embryo homogenates at different time points between 5 and 120 hpf by means of a fluorogenic non-specific CYP substrate, i.e., benzyloxy-methyl-resorufin (BOMR) [25]. Biotransformation of BOMR into the fluorescent metabolite, i.e., resorufin, is a measure for the CYP activity in the microsomes.…”

“…Since the liver and the intestine are the predominant sites for CYP-mediated drug metabolism (reviewed in [21]), intrinsic CYP activity was also assessed in liver microsomes prepared from adult female zebrafish as a reference for the embryos. In contrast to adults, zebrafish embryos showed no CYP-mediated metabolizing capacity in vitro during a major part of organogenesis, i.e., between 5 and 72 hpf, only poor CYP activity at 72 and 96 hpf and no CYP activity at 120 hpf [25]. Besides this in-house in vitro study, other research groups assessed (often after exposure to CYP inducers or inhibitors) CYP1 activity and, to a lesser extent, CYP3 activity during zebrafish organogenesis by using substrates that are specific for the respective CYP enzymes [26,27,28,29,30,31,32,33,34,35,36,37,38,39,40].…”

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 ).…”