Sanne A.B. Hermsen

Relative embryotoxicity of two classes of chemicals in a modified zebrafish embryotoxicity test and comparison with their in vivo potencies

The zebrafish embryotoxicity test (ZET) is a fast and simple method to study chemical toxicity after exposure of the complete vertebrate embryo during embryogenesis in ovo. We developed a novel quantitative evaluation method to assess the development of the zebrafish embryo based on specific endpoints in time, the general morphology score (GMS) system. For teratogenic effects a separate scoring list was developed. The relative effects of eight glycol ethers and six 1,2,4-triazole anti-fungals were evaluated in this system and results were compared with in vivo developmental toxicity potencies. Methoxyacetic acid and ethoxyacetic acid appeared as the most potent glycol ether metabolites, inducing growth retardation and malformations. Other glycol ethers showed no developmental toxicity. Flusilazole appeared the most potent triazole, followed by hexaconazole, cyproconazole, triadimefon, myclobutanil and triticonazole, respectively. In general, the potency ranking of the compounds within their class in the ZET was comparable to their in vivo ranking. In conclusion, the ZET with the GMS system appears an efficient and useful test system for screening embryotoxic properties of chemicals within the classes of compounds tested. This alternative test method may also be useful for the detection of embryotoxic properties of other classes of chemicals.

Evaluation of an alternative in vitro test battery for detecting reproductive toxicants

The application of alternative methods in developmental and reproductive toxicology is challenging in view of the complexity of mechanisms involved. A battery of complementary test systems may provide a better prediction of developmental and reproductive toxicity than single assays. We tested twelve compounds with varying mechanisms of toxic action in an assay battery including 24 CALUX transcriptional activation assays, mouse cardiac embryonic stem cell test, ReProGlo assay, zebrafish embryotoxicity assay, and two CYP17 and two CYP19 activity assays. The battery correctly detected 11/12 compounds tested, with one false negative occurring, which could be explained by the absence of the specific mechanism of action of this compound in the battery. Toxicokinetic modeling revealed that toxic concentrations were in the range expected from in vivo reproductive toxicity data. This study illustrates added value of combining assays that contain complementary biological processes and mechanisms, increasing predictive value of the battery over individual assays.

Comparison of the mouse Embryonic Stem cell Test, the rat Whole Embryo Culture and the Zebrafish Embryotoxicity Test as alternative methods for developmental toxicity testing of six 1,2,4-triazoles

The relatively high experimental animal use in developmental toxicity testing has stimulated the search for alternatives that are less animal intensive. Three widely studied alternative assays are the mouse Embryonic Stem cell Test (EST), the Zebrafish Embryotoxicity Test (ZET) and the rat postimplantation Whole Embryo Culture (WEC). The goal of this study was to determine their efficacy in assessing the relative developmental toxicity of six 1,2,4-triazole compounds,(1) flusilazole, hexaconazole, cyproconazole, triadimefon, myclobutanil and triticonazole. For this purpose, we analyzed effects and relative potencies of the compounds in and among the alternative assays and compared the findings to their known in vivo developmental toxicity. Triazoles are antifungal agents used in agriculture and medicine, some of which are known to induce craniofacial and limb abnormalities in rodents. The WEC showed a general pattern of teratogenic effects, typical of exposure to triazoles, mainly consisting of reduction and fusion of the first and second branchial arches, which are in accordance with the craniofacial malformations reported after in vivo exposure. In the EST all triazole compounds inhibited cardiomyocyte differentiation concentration-dependently. Overall, the ZET gave the best correlation with the relative in vivo developmental toxicities of the tested compounds, closely followed by the EST. The relative potencies observed in the WEC showed the lowest correlation with the in vivo developmental toxicity data. These differences in the efficacy between the test systems might be due to differences in compound kinetics, in developmental stages represented and in the relative complexity of the alternative assays.

An abbreviated protocol for multilineage neural differentiation of murine embryonic stem cells and its perturbation by methyl mercury.

Alternative assays are highly desirable to reduce the extensive experimental animal use in developmental toxicity testing. In the present study, we developed an improved test system for assessing neurodevelopmental toxicity using differentiating embryonic stem cells. We advanced previously established methods by merging, modifying and abbreviating the original 20-day protocol into a more efficient 13-day neural differentiation protocol. Using morphological observation, immunocytochemistry, gene expression and flow cytometry, it was shown predominantly multiple lineages of neuroectodermal cells were formed in our protocol and to a lower extent, endodermal and mesodermal differentiated cell types. This abbreviated protocol should lead to an advanced screening method using morphology in combination with selected differentiation markers aimed at predicting neurodevelopmental toxicity. Finally, the assay was shown to express differential sensitivity to a model developmental neurotoxicant, methyl mercury.

Concentration-Response Analysis of Differential Gene Expression in the Zebrafish Embryotoxicity Test Following Flusilazole Exposure

The zebrafish embryotoxicity test (ZET) is considered a promising alternative model in predictive toxicology. Currently, morphological assessment of the embryo is the main readout for this assay. However, implementation of transcriptomics may help to detect more subtle effects, which may increase the sensitivity and predictability of the test. In this study, we tested a concentration response of flusilazole in the ZET. After exposure for 24 h postfertilization, microarray analysis revealed a number of processes to be regulated in a concentration-dependent way. We identified development related processes, retinol metabolism and transcription, as well as processes corresponding to the antifungal mechanism of action, steroid biosynthesis, and fatty acid metabolism, to be differentially regulated. Retinol metabolism and transcription were already significantly altered at concentrations that were not inducing morphological effects. Differential expression of genes related to steroid biosynthesis and fatty acid metabolism showed a concentration response similar to morphological response. An increase in concentration was also positively associated with an increase in magnitude of expression for individual genes within functional processes. Our study shows that transcriptomics analysis in the ZET is a more sensitive readout of compound-induced effects than morphological assessment. However, the interpretation of differential gene expression in terms of predicting morphological effects is not straightforward and requires further study.

Chemical class-specific gene expression changes in the zebrafish embryo after exposure to glycol ether alkoxy acids and 1,2,4-triazole antifungals.

The zebrafish embryotoxicity test (ZET) is an alternative test to predict embryotoxicity of substances based on morphological assessment. Implementing transcriptomics may increase sensitivity and objectivity of the test system. We applied the category approach to compare effects of compounds from two chemical classes, the glycol ethers and 1,2,4-triazoles, on the embryo. At 24h post fertilization, microarray analysis revealed several thousands of responsive genes after glycol ether exposure, whereas the triazoles significantly regulated only several hundreds of genes. Principal component analysis of the genes commonly regulated per chemical class demonstrated that the two classes can be distinguished. Gene set enrichment analysis showed that after glycol ether exposure mainly gene sets related to development were downregulated. After triazole exposure, gene sets corresponding to previously described mechanisms of action, such as glycolysis and fatty acid metabolism were regulated. Our results demonstrate that transcriptomics in the ZET provides a more sensitive endpoint than standard morphological assessment. In addition, information about mechanisms of action of substances may become available, thereby facilitating the extrapolation of findings to mammalian species including man.

Transcriptomic analysis in the developing zebrafish embryo after compound exposure: Individual gene expression and pathway regulation

The zebrafish embryotoxicity test is a promising alternative assay for developmental toxicity. Classically, morphological assessment of the embryos is applied to evaluate the effects of compound exposure. However, by applying differential gene expression analysis the sensitivity and predictability of the test may be increased. For defining gene expression signatures of developmental toxicity, we explored the possibility of using gene expression signatures of compound exposures based on commonly expressed individual genes as well as based on regulated gene pathways. Four developmental toxic compounds were tested in concentration-response design, caffeine, carbamazepine, retinoic acid and valproic acid, and two non-embryotoxic compounds, d-mannitol and saccharin, were included. With transcriptomic analyses we were able to identify commonly expressed genes, which were mostly development related, after exposure to the embryotoxicants. We also identified gene pathways regulated by the embryotoxicants, suggestive of their modes of action. Furthermore, whereas pathways may be regulated by all compounds, individual gene expression within these pathways can differ for each compound. Overall, the present study suggests that the use of individual gene expression signatures as well as pathway regulation may be useful starting points for defining gene biomarkers for predicting embryotoxicity.

Triazole-induced gene expression changes in the zebrafish embryo

The zebrafish embryo is considered to provide a promising alternative test model for developmental toxicity testing. Most systems use morphological assessment of the embryos, however, microarray analyses may increase sensitivity and predictability of the test by detecting more subtle and detailed responses. In this study, we investigated the possibility of relating gene expression profiles of structurally similar chemicals tested in a single concentration, to a complete transcriptomic concentration-response of flusilazole (FLU). We tested five other triazoles, hexaconazole (HEX), cyproconazole (CYP), triadimefon (TDF), myclobutanil (MYC), and triticonazole (TTC) at equipotent concentrations based on morphological evaluation. Results showed that every compound had a different degree of regulation within their anti-fungal and developmental toxicity pathways, steroid biosynthesis and retinol metabolism, respectively. Assuming that the ratio between these pathways is relevant for efficacy compared to developmental toxicity, we found TTC was more efficient and CYP was more toxic compared to the other triazoles. With the approach used in this study we demonstrated that gene expression data allow more comprehensive assessment of compound effects by discriminating relative potencies using these specific gene sets. The zebrafish embryo model can therefore be considered a useful vertebrate model providing information of relevant pathways related to anti-fungal mechanism of action and toxicological activity.

Zebrafish embryotoxicity test for developmental (neuro)toxicity: Demo case of an integrated screening approach system using anti-epileptic drugs

To improve the predictability of the zebrafish embryotoxicity test (ZET) for developmental (neuro)toxicity screening, we used a multiple-endpoints strategy, including morphology, motor activity (MA), histopathology and kinetics. The model compounds used were antiepileptic drugs (AEDs): valproic acid (VPA), carbamazepine (CBZ), ethosuximide (ETH) and levetiracetam (LEV). For VPA, histopathology was the most sensitive parameter, showing effects already at 60μM. For CBZ, morphology and MA were the most sensitive parameters, showing effects at 180μM. For ETH, all endpoints showed similar sensitivity (6.6mM), whereas MA was the most sensitive parameter for LEV (40mM). Inclusion of kinetics did not alter the absolute ranking of the compounds, but the relative potency was changed considerably. Taking all together, this demo-case study showed that inclusion of multiple-endpoints in ZET may increase the sensitivity of the assay, contribute to the elucidation of the mode of toxic action and to a better definition of the applicability domain of ZET.

Implementation of Transcriptomics in the Zebrafish Embryotoxicity Test

The zebrafish embryotoxicity test (ZET) is considered a promising alternative model in predictive developmental toxicity testing. The basic principle of this test entails the exposure of zebrafish embryos to a test compound and the subsequent morphological assessment of developmental and teratogenic effects. To increase the sensitivity and predictability of the test by detecting subtle and more detailed responses, implementation of transcriptomics analyses may be of great value. Transcriptomics analysis provides an objective tool that enables the profound evaluation of biological processes and provides the possibility to gain insight into specific physiological, pathological, or toxicological mechanisms in a high-throughput manner. This chapter gives an overview of the current implementation of transcriptomic analysis in the zebrafish embryotoxicity test for developmental toxicity testing and discusses future perspectives for this research.