title: Phenotypic and biomarker evaluation of zebrafish larvae as an alternative model to predict mammalian hepatotoxicity.
abstract: Zebrafish phenotypic assays have shown promise to assess human hepatotoxicity, though scoring of liver morphology remains subjective and difficult to standardize. Liver toxicity in zebrafish larvae at 5 days was assessed using gene expression as the biomarker approach, complementary to phenotypic analysis and analytical data on compound uptake. This approach aimed to contribute to improved hepatotoxicity prediction, with the goal of identifying biomarker(s) as a step towards the development of transgenic models for prioritization. Morphological effects of hepatotoxic compounds (acetaminophen, amiodarone, coumarin, methapyrilene and myclobutanil) and saccharin as the negative control were assessed after exposure in zebrafish larvae. The hepatotoxic compounds induced the expected zebrafish liver degeneration or changes in size, whereas saccharin did not have any phenotypic adverse effect. Analytical methods based on liquid chromatography-mass spectrometry were optimized to measure stability of selected compounds in exposure medium and internal concentration in larvae. All compounds were stable, except amiodarone for which precipitation was observed. There was a wide variation between the levels of compound in the zebrafish larvae with a higher uptake of amiodarone, methapyrilene and myclobutanil. Detection of hepatocyte markers (CP, CYP3A65, GC and TF) was accomplished by in situ hybridization of larvae to coumarin and myclobutanil and confirmed by real-time reverse transcription-quantitative polymerase chain reaction. Experiments showed decreased expression of all markers. Next, other liver-specific biomarkers (i.e. FABP10a and NR1H4) and apoptosis (i.e. CASP-3 A and TP53) or cytochrome P450-related (CYP2K19) and oxidoreductase activity-related (ZGC163022) genes, were screened. Links between basic mechanisms of liver injury and results of biomarker responses are described. Copyright © 2016 John Wiley & Sons, Ltd.
authors.affiliation: VITO NV, Applied Bio & Molecular Systems, Boeretang 200, B-2400, Mol, Belgium.
authors.forename: Sandra
authors.initials: S
authors.lastname: Verstraelen
authors.affiliation: GIGA-R, University of Liège, Avenue de l'Hopital 1, B34, B-4000, Liège, Belgium.
authors.forename: Bernard
authors.initials: B
authors.lastname: Peers
authors.affiliation: Toxicological Center, University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Belgium.
authors.forename: Walid
authors.initials: W
authors.lastname: Maho
authors.affiliation: VITO NV, Applied Bio & Molecular Systems, Boeretang 200, B-2400, Mol, Belgium.
authors.forename: Karen
authors.initials: K
authors.lastname: Hollanders
authors.affiliation: VITO NV, Applied Bio & Molecular Systems, Boeretang 200, B-2400, Mol, Belgium.|Epidemiology and Social Medicine, University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Belgium.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Remy
authors.affiliation: VITO NV, Applied Bio & Molecular Systems, Boeretang 200, B-2400, Mol, Belgium.
authors.forename: Pascale
authors.initials: P
authors.lastname: Berckmans
authors.affiliation: Toxicological Center, University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Belgium.
authors.forename: Adrian
authors.initials: A
authors.lastname: Covaci
authors.affiliation: VITO NV, Applied Bio & Molecular Systems, Boeretang 200, B-2400, Mol, Belgium.
authors.forename: Hilda
authors.initials: H
authors.lastname: Witters
mesh_terms: D000082:Acetaminophen / Q000633:toxicity; D000638:Amiodarone / Q000633:toxicity; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism; D002570:Ceruloplasmin / Q000235:genetics / Q000378:metabolism; D003374:Coumarins / Q000633:toxicity; D005260:Female; D005786:Gene Expression Regulation; D005819:Genetic Markers; D055106:Genome-Wide Association Study; D006801:Humans; D017403:In Situ Hybridization; D007814:Larva / Q000187:drug effects / Q000235:genetics; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008701:Methapyrilene / Q000633:toxicity; D009570:Nitriles / Q000633:toxicity; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D018675:Toxicity Tests; D014168:Transferrin / Q000235:genetics / Q000378:metabolism; D014230:Triazoles / Q000633:toxicity; D015027:Zebrafish / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D003374:Coumarins; D005819:Genetic Markers; D009570:Nitriles; D014168:Transferrin; D014230:Triazoles; D029961:Zebrafish Proteins; D000082:Acetaminophen; D008701:Methapyrilene; C030123:coumarin; C446685:systhane; D001189:Aryl Hydrocarbon Hydroxylases; C502466:cytochrome P-450 CYP3A65, zebrafish; D002570:Ceruloplasmin; D010089:Oxidoreductases, N-Demethylating; D000638:Amiodarone
keywords: Non-animal alternative assay; compound uptake; gene expression markers; hepatotoxicity; zebrafish larva