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Xenobiotic metabolism in the zebrafish: a review of the spatiotemporal distribution, modulation and activity of Cytochrome P450 families 1 to 3

The zebrafish (Danio rerio) has been increasingly explored in pharmaceutical research as a promising alternative model for toxicological screens. This necessitates a thorough knowledge on the biotransformation processes for a correct interpretation of pharmacological and toxicological data. Physiologically, cytochrome P450 (CYP) enzymes, specifically CYP families 1-3, play a pivotal role in drug metabolism. And yet, information regarding activity of CYP, its isoforms, and conjugation enzymes in zebrafish is either scarce or conflicting. To account for this discrepancy, the available spatiotemporal, modulation and activity data on zebrafish CYP 1-3 families are reviewed in this paper and compared with human CYP data. The CYP genetic features and synteny are well characterized, as is their expression in different organ systems. Moreover, several substrates metabolized by humans also show metabolism in zebrafish, with other CYP isoforms possibly involved. Altogether, the five CYP1 members, 41 CYP2 members and five CYP3 members in zebrafish show distinct evolutionary and orthological similarities with humans.

Incubation at 32.5 °C and above causes malformations in the zebrafish embryo

Zebrafish embryos are increasingly used for developmental toxicity screening of candidate drugs and are occasionally co-incubated with a metabolic activation system at 32°C for 1, 2 or 4h, depending on their developmental stage. As this temperature is higher than the optimal temperature for zebrafish embryonic development (26-28.5°C), we investigated whether continuous incubation of zebrafish embryos from 2.5 until 96h post fertilization (hpf) at high temperatures (30.5-36.5°C) causes malformations. At 32.5°C tail malformations were observed as early as 24hpf, and these became even more prominent at 34.5 and 36.5°C. Cardiovascular and head malformations, edema and blood accumulations throughout the body were present at 36.5°C. Finally, temperatures higher than 28.5°C accelerated embryonic development except for 36.5°C, at which a lower hatching rate and hatching enzyme activity were observed. In conclusion, incubation of zebrafish embryos at 32.5°C and above from 2.5 until 96hpf causes malformations as early as 24hpf.

Ontogeny of CYP3A and P‐Glycoprotein in the Liver and the Small Intestine of the Göttingen Minipig: An Immunohistochemical Evaluation

Despite the increasing use of the minipig as a non‐rodent species in general and juvenile toxicity studies, knowledge on their biotransformation processes and their ontogeny is scarce. Such data are prerequisite for the correct interpretation of non‐clinical studies in this species. Therefore, the aim of our investigation was to immunohistochemically document the presence of the drug transporter P‐glycoprotein (Pgp) and the metabolizing cytochrome P450 (CYP) 3A subfamily in the livers (n = 115) and the small intestines (n = 74) of foetal, neonatal, juvenile and adult Göttingen minipigs. Pgp was expressed in the liver in all age groups, whereas its presence in the jejunum was detected from 86 days of gestation onwards. Low expression of CYP3A was detected in the jejunums and livers from foetal and neonatal piglets. During postnatal development, the immunoreactivity for CYP3A increased in both organs. A centrilobular pattern, with a more intense staining for CYP3A of the hepatocytes surrounding the central vein, was noticed in the postnatal livers. In conclusion, the presented data suggest that the intestinal and hepatic ontogeny of P‐glycoprotein and CYP3A in minipigs corresponds to that in man, in which a similar spatio‐temporal expression has been reported.

In vitro CYP1A activity in the zebrafish: temporal but low metabolite levels during organogenesis and lack of gender differences in the adult stage

The zebrafish (Danio rerio) is increasingly used as a screening model for acute, chronic and developmental toxicity. More specifically, the embryo is currently investigated as a replacement of in vivo developmental toxicity studies, although its biotransformation capacity remains a point of debate. As the cytochrome P450 1 (CYP1) family plays an important role in the biotransformation of several pollutants and drugs, a quantitative in vitro protocol was refined to assess gender- and age-related CYP1A activity in the zebrafish using the ethoxyresorufin-o-deethylase (EROD) assay. Microsomal protein fractions were prepared from livers of adult males and females, ovaries and whole embryo homogenates of different developmental stages. A large biological variation but no gender-related difference in CYP1A activity was observed in adult zebrafish. Embryos showed distinct temporal but low CYP1A activity during organogenesis. These in vitro data raise questions on the bioactivation capacity of zebrafish embryos in developmental toxicity studies.

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

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.

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

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.

Gene transcription ontogeny of hypothalamic-pituitary-thyroid axis development in early-life stage fathead minnow and zebrafish

The hypothalamic-pituitary-thyroid (HPT) axis is known to play a crucial role in the development of teleost fish. However, knowledge of endogenous transcription profiles of thyroid-related genes in developing teleosts remains fragmented. We selected two model teleost species, the fathead minnow (Pimephales promelas) and the zebrafish (Danio rerio), to compare the gene transcription ontogeny of the HPT axis. Control organisms were sampled at several time points during embryonic and larval development until 33 days post-fertilization. Total RNA was extracted from pooled, whole fish, and thyroid-related mRNA expression was evaluated using quantitative polymerase chain reaction. Gene transcripts examined included: thyrotropin-releasing hormone receptor (trhr), thyroid-stimulating hormone receptor (tshr), sodium-iodide symporter (nis), thyroid peroxidase (tpo), thyroglobulin (tg), transthyretin (ttr), deiodinases 1, 2, 3a, and 3b (dio1, dio2, dio3a and 3b), and thyroid hormone receptors alpha and beta (thrα and β). A loess regression method was successful in identifying maxima and minima of transcriptional expression during early development of both species. Overall, we observed great similarities between the species, including maternal transfer, at least to some extent, of almost all transcripts (confirmed in unfertilized eggs), increasing expression of most transcripts during hatching and embryo-larval transition, and indications of a fully functional HPT axis in larvae. These data will aid in the development of hypotheses on the role of certain genes and pathways during development. Furthermore, this provides a background reference dataset for designing and interpreting targeted transcriptional expression studies both for fundamental research and for applications such as toxicology.

UPLC/MS MS data of testosterone metabolites in human and zebrafish liver microsomes and whole zebrafish larval microsomes

This article represents data regarding a study published in Toxicology in vitro entitled “ in vitro CYP-mediated drug metabolism in the zebrafish (embryo) using human reference compounds” (Saad et al., 2017) [1]. Data were acquired with ultra-performance liquid chromatography – accurate mass mass spectrometry (UPLC-amMS). A full spectrum scan was conducted for the testosterone (TST) metabolites from the microsomal stability assay in zebrafish and humans. The microsomal proteins were extracted from adult zebrafish male (MLM) and female (FLM) livers, whole body homogenates of 96 h post fertilization larvae (EM) and a pool of human liver microsomes from 50 donors (HLM). Data are expressed as the abundance from the extracted ion chromatogram of the metabolites.

Antioxidants reduce reactive oxygen species but not embryotoxicity in the metabolic Danio rerio test (mDarT)

Mammalian liver microsomes are occasionally used as a metabolic activation system (MAS) to compensate for the low CYP-mediated bioactivation of drugs in zebrafish embryos, in the so-called mDarT. However, this MAS is embryotoxic and consequently zebrafish embryos are only exposed during a very limited developmental window. The main aim of this study was to try to reduce the embryotoxic properties of MAS in order to extend the exposure window in the mDarT. Removing the microsomes from the incubation medium prior to exposure of the zebrafish embryos did not reduce embryotoxicity. Free radicals (ROS) in the incubation medium were successfully reduced by antioxidants, but the medium remained embryotoxic. Single dosing of NADPH or omitting toxic components from the MAS preparation did also not reduce embryotoxicity. In conclusion, the exposure window in the mDarT could not be extended by reducing ROS levels, single dosing of NADPH or modifications of the MAS preparation.