Developmental Toxicity and Biotransformation of Two Anti-Epileptics in Zebrafish Embryos and Early Larvae.

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 5»- 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.

Biological variability hampers the use of skeletal staining methods in zebrafish embryo developmental toxicity assays.

Zebrafish embryo assays are used by pharmaceutical and chemical companies as new approach methodologies (NAMs) in developmental toxicity screening. Despite an overall high concordance of zebrafish embryo assays with in vivo mammalian studies, false negative and false positive results have been reported. False negative results in risk assessment models are of particular concern for human safety, as developmental anomalies may be missed. Interestingly, for several chemicals and drugs that were reported to be false negative in zebrafish, skeletal findings were noted in the in vivo studies. As the number of skeletal endpoints assessed in zebrafish is very limited compared to the in vivo mammalian studies, the aim of this study was to investigate whether the sensitivity could be increased by including a skeletal staining method. Three staining methods were tested on zebrafish embryos that were exposed to four teratogens that caused skeletal anomalies in rats and/or rabbits and were false negative in zebrafish embryo assays. These methods included a fixed alizarin red-alcian blue staining, a calcein staining, and a live alizarin red staining. The results showed a high variability in staining intensity of larvae exposed to mammalian skeletal teratogens, as well as variability between control larvae originating from the same clutch of zebrafish. Hence, biological variability in (onset of) bone development in zebrafish hampers the detection of (subtle) treatment-related bone effects that are not picked-up by gross morphology. In conclusion, the used skeletal staining methods did not increase the sensitivity of zebrafish embryo developmental toxicity assays.

DMSO Concentrations up to 1% are Safe to be Used in the Zebrafish Embryo Developmental Toxicity Assay.

Dimethyl sulfoxide (DMSO) is a popular solvent for developmental toxicity testing of chemicals and pharmaceuticals in zebrafish embryos. In general, it is recommended to keep the final DMSO concentration as low as possible for zebrafish embryos, preferably not exceeding 100 µL/L (0.01%). However, higher concentrations of DMSO are often required to dissolve compounds in an aqueous medium. The aim of this study was to determine the highest concentration of DMSO that can be safely used in our standardized Zebrafish Embryo Developmental Toxicity Assay (ZEDTA). In the first part of this study, zebrafish embryos were exposed to different concentrations (0-2%) of DMSO. No increase in lethality or malformations was observed when using DMSO concentrations up to 1%. In a follow-up experiment, we assessed whether compounds that cause no developmental toxicity in the ZEDTA remain negative when dissolved in 1% DMSO, as false positive results due to physiological disturbances by DMSO should be avoided. To this end, zebrafish embryos were exposed to ascorbic acid and hydrochlorothiazide dissolved in 1% DMSO. Negative control groups were also included. No significant increase in malformations or lethality was observed in any of the groups. In conclusion, DMSO concentrations up to 1% can be safely used to dissolve compounds in the ZEDTA.

Refinement of the zebrafish embryo developmental toxicity assay.

Several pharmaceutical and chemical companies are using the zebrafish embryo as an alternative to animal testing for early detection of developmental toxicants. Unfortunately, the protocol of this zebrafish embryo assay varies between labs, resulting in discordant data for identical compounds. The assay also has some limitations, such as low biotransformation capacity and fewer morphological endpoints in comparison with the in vivo mammalian developmental toxicity studies. Consequently, there is a need to standardize and further optimize the assay for developmental toxicity testing. We developed a Zebrafish Embryo Developmental Toxicity Assay (ZEDTA) that can be extended with a metabolic activation system and/or skeletal staining to increase its sensitivity. As such, the ZEDTA can be used as a modular system depending on the compound of interest.•Our protocol is customized with a metabolic activation system for test compounds, using human liver microsomes. This system ensures exposure of zebrafish embryos to metabolites that are relevant for human risk and safety assessment. As human liver microsomes are toxic for the zebrafish embryo, we developed a preincubation system with an ultracentrifugation and subsequent dilution step.•Additionally, we developed a skeletal staining protocol that can be added to the ZEDTA modular system. Our live Alizarin Red staining method detects several bone structures in 5-day old zebrafish larvae in a consistent manner.