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.
Highlights
Hepatotoxins are toxic chemicals that damage the liver or cause liver functional disorders
Human HepG2 cells have been popularly used with a capability of developing to a high content screening assay for screening a broad range of compounds [4] and the assay could be further improved by enhanced expression of major P450 enzymes [5]
While at the highest dose (25 mg/L), nearly 8% of surviving embryos/fry showed cardiac edema (Figure 1F), Quantitative analyses of red fluorescence intensity confirmed the dosagedependent reduction (Figure 2A, Table S2) and statistically significant reduction (8.9%) was observed at the concentration of 5 mg/L and 32% reduction at 25 mg/L. 2D measurement of liver size as covered by the red fluorescence in the LiPan fry were carried out and there was a similar reduction with the increase of acetaminophen concentrations (Figure 2A), and statistically significant reduction (13.0% and 18.0%) of liver size was observed at the concentrations of 20 and 25 mg/L, respectively
Summary
Hepatotoxins are toxic chemicals that damage the liver or cause liver functional disorders. It is crucial and important to develop rapid and convenient approaches for monitoring environmental contaminants and for detection of potential hepatotoxicity in drug candidates screening. To test hepatotoxicity of chemical compounds, a variety of assays have been developed. In addition to measurement of conventional liver enzyme activities in blood such as GGT (Gamma-glutamyltransferase), ALT (alanine aminotransferase) and AST (aspartate aminotransferase), which are released to blood due to liver damage [7], different types of hepatotoxins could be distinguished by screening different biomarkers; for example, a PCR array based assay is available to analyze the expression of 84 key genes implicated as potential biomarkers of liver toxicity [8]
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