Development of a Convenient In Vivo Hepatotoxin Assay Using a Transgenic Zebrafish Line with Liver-Specific DsRed Expression

Abstract

Previously we have developed a transgenic zebrafish line (LiPan) with liver-specific red fluorescent protein (DsRed) expression under the fabp10a promoter. Since red fluorescence in the liver greatly facilitates the observation of liver in live LiPan fry, we envision that the LiPan zebrafish may provide a useful tool in analyses of hepatotoxicity based on changes of liver red fluorescence intensity and size. In this study, we first tested four well-established hepatotoxins (acetaminophen, aspirin, isoniazid and phenylbutazone) in LiPan fry and demonstrated that these hepatotoxins could significantly reduce both liver red fluorescence and liver size in a dosage-dependent manner, thus the two measurable parameters could be used as indicators of hepatotoxicity. We then tested the LiPan fry with nine other chemicals including environmental toxicants and human drugs. Three (mefenamic acid, lindane, and arsenate) behave like hepatotoxins in reduction of liver red fluorescence, while three others (17β-estradiol, TCDD [2,3,7,8-tetrachlorodibenzo-p-dioxin] and NDMA [N-nitrosodimethylamine]) caused increase of liver red fluorescence and the liver size. Ethanol and two other chemicals, amoxicillin (antibiotics) and chlorphenamine (pain killer) did not resulted in significant changes of liver red fluorescence and liver size. By quantitative RT-PCR analysis, we found that the changes of red fluorescence intensity caused by different chemicals correlated to the changes of endogenous fabp10a RNA expression, indicating that the measured hepatotoxicity was related to fatty acid transportation and metabolism. Finally we tested a mixture of four hepatotoxins and observed a significant reduction of red fluorescence in the liver at concentrations below the lowest effective concentrations of individual hepatotoxins, suggesting that the transgenic zebrafish assay is capable of reporting compound hepatotoxicity effect from chemical mixtures. Thus, the LiPan transgenic fry provide a rapid and convenient in vivo hepatotoxicity assay that should be applicable to high-throughput hepatotoxicity test in drug screening as well as in biomonitoring environmental toxicants.

Zebrafish: An Important Tool for Liver Disease Research

“…Screens to identify potentially hepatotoxic agents are of primary importance, and zebrafish provide an excellent, affordable system to identify hepatotoxic compounds. 119,130 The advantages of using zebrafish in toxicity studies include the ability to rapidly screen for the effects of multiple agents in an entire organism during different stages of development. Indeed, biotechnology companies have capitalized on the comparative cost benefit of using zebrafish to assess toxicity (for a review, see Driessen et al 119 and McGrath and Li 131 ).…”

Zebrafish as Toxicological model for screening and recapitulate human diseases

“…The availability of specific transgenic lines labeling the liver, such as fabp10:RFP, allows liver damage visualization after the treatment. Analysis of fluorescent intensity can be informative with regard to size or the number of hepatocytes [100] . However, has been described the inconvenient to work with the larvae, arguing that the CYP system, which plays an essential role in drug metabolism, is not yet fully developed in larvae and suggesting that some CYPs appear to be lacking in the early zebrafish life [101] .…”

Line-scan focal modulation microscopy

“…To demonstrate its potential, we conducted preliminary imaging experiments to visualize the constitutive expression of DsRed fluorescent protein in the liver of live 5-day-old zebrafish larvae of strain Tg (fabp10a:DsRed;elaA:egfp), a two-color transgenic zebrafish line (LiPan). 29,30 Zebrafish larvae were maintained in the fish facilities at the Department of Biological Sciences, National University of Singapore (NUS) according to the established protocols 31 and in compliance with Institutional Animal Care and Use Committee (IACUC) guidelines. Before imaging, a zebrafish was placed on a #0 glass-bottomed dish (MatTek, P35G-0-10-C) and positioned in the dorsal orientation.…”