The whole zebrafish embryo model (ZFE) has proven its applicability in developmental toxicity testing. Since functional hepatocytes are already present from 36 h post fertilization onwards, whole ZFE have been proposed as an attractive alternative to mammalian in vivo models in hepatotoxicity testing. The goal of the present study is to further underpin the applicability of whole ZFE for hepatotoxicity testing by combining histopathology and next-generation sequencing-based gene expression profiling. To this aim, whole ZFE and adult zebrafish were exposed to a set of hepatotoxic reference compounds. Histopathology revealed compound and life-stage-specific effects indicative of toxic injury in livers of whole ZFE and adult zebrafish. Next-generation sequencing (NGS) was used to compare transcript profiles in pooled individual RNA samples of whole ZFE and livers of adult zebrafish. This revealed that hepatotoxicity-associated expression can be detected beyond the overall transcription noise in the whole embryo. In situ hybridization verified liver specificity of selected highly expressed markers in whole ZFE. Finally, cyclosporine A (CsA) was used as an illustrative case to support applicability of ZFE in hepatotoxicity testing by comparing CsA-induced gene expression between ZFE, in vivo mouse liver and HepaRG cells on the levels of single genes, pathways and transcription factors. While there was no clear overlap on single gene level between the whole ZFE and in vivo mouse liver, strong similarities were observed between whole ZFE and in vivo mouse liver in regulated pathways related to hepatotoxicity, as well as in relevant overrepresented transcription factors. In conclusion, both the use of NGS of pooled RNA extracts analysis combined with histopathology and traditional microarray in single case showed the potential to detect liver-related genes and processes within the transcriptome of a whole zebrafish embryo. This supports the applicability of the whole ZFE model for compound-induced hepatotoxicity screening.

Many chemicals can induce allergic contact dermatitis. Because evaluation of skin sensitizing potential by animal testing is prohibited for cosmetics, and screening of many chemicals is required within Registration, Evaluation, Authorisation and Restriction of Chemicals, urgent need exists for predictive in vitro assays to identify contact allergens. Keratinocytes (KC) are the first cells encountered when chemicals land on the skin. Therefore, KC form an important site of haptenization and their metabolism is likely to be important. Moreover, KC secrete mediators that affect processing and presentation of haptenized proteins by dendritic cells. To develop a KC-based in vitro assay to predict sensitizing potential of chemicals, in vitro exposure effects of eight contact sensitizers and six irritants on the KC cell line HaCaT were examined by gene profiling. Classifiers predictive of the class sensitizers or irritants were calculated, based on support vector machine (SVM) and random forest (RF) algorithms. Classifiers using high-ranking genes were 70% (SVM) and 62% (RF) accurate, based on three (SVM) and two to five (RF) features. Classifiers using oxidative stress pathway gene sets were 68-73% (SVM) and 69-71% (RF) accurate. Cross-validation showed that the top-3 of most discriminating genes added up to 13 genes and included oxidative stress gene HMOX1 irrespective of the chemical left out. Moreover, HMOX1 was the most significantly regulated gene. Gene Set Enrichment Analysis showed upregulation of "Keap1 dependent" and "oxidative stress" gene lists. In conclusion, KC expression profiling can identify contact sensitizers, providing opportunities for nonanimal testing for sensitizing potential. Moreover, our data suggest that contact sensitizers induce the oxidative stress pathway in KC.

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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.