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.

Membrane Remodeling as a Key Player of the Hepatotoxicity Induced by Co-Exposure to Benzo[a]pyrene and Ethanol of Obese Zebrafish Larvae

“…From 5 days post-fertilization (dpf), the liver is indeed functional and expresses enzymes responsible for xenobiotic metabolism that resemble those expressed in humans like cytochrome P450 2E1 (CYP2E1) and alcohol dehydrogenase for alcohol or CYP1A for B[a]P [ 43 , 46 , 47 , 48 , 49 ]. Beside the numerous advantages of zebrafish larva, notably for assessing hepatotoxicant effects [ 50 , 51 , 52 , 53 ], this model has already been demonstrated as very useful for studying fatty liver diseases [ 54 , 55 , 56 , 57 , 58 , 59 ]. In addition, the suitability of this model for studying the involvement of membrane remodeling in chemical-induced liver toxicity has already been reported [ 33 ].…”

Cyclosporine exacerbates ketamine toxicity in zebrafish: Mechanistic studies on drug–drug interaction

“…Similar to human CYP3A, enhanced CYP3A65 transcription in the foregut of the zebrafish larvae in response to dexamethasone and the antibiotic rifampicin confirmed that zebrafish CYP3A65 is an ortholog of the human CYP3A gene (Chang et al, ; Tseng et al, ). CYP3A65 expression in zebrafish has been used as a hepatic biomarker (Al‐Habsi et al, ; Cunha et al, ; Verstraelen et al, ; Xia et al, ) and the zebrafish embryo model was shown to detect hepatotoxicants with higher specificity than the HepG2 cells (Jones et al, ).…”

Zebrafish larva as a reliable model forin vivoassessment of membrane remodeling involvement in the hepatotoxicity of chemical agents

“…They have a small size (1-4 mm) and are easy to maintain, making them cost-effective for an in vivo study. Thus, this model has been successfully chosen to develop a predictive tool for assessing chemical-induced hepatotoxicity with similar effects to those obtained in rodents or human (Sukardi et al, 2011;Hill et al, 2012;Driessen et al, 2013;He et al, 2013;Vliegenthart et al, 2014;Mesens et al, 2015;Verstraelen et al, 2016). Also, it has been increasingly used to study mechanisms of liver injury after acute exposure to a classic hepatotoxicant, alcohol (Passeri et al, 2009;Howarth et al, 2011;Tsedensodnom et al, 2013).…”