Characterization of Triptolide-Induced Hepatotoxicity by Imaging and Transcriptomics in a Novel Zebrafish Model.

Adriaan D Bastiaan Vliegenthart,John J Mullins,Jorge Del Pozo,Sarah Schneemann,Charlotte Buckley,Chunmin Wei,David J Webb,Cécile Berends,Carl Tucker,James W Dear,Carmelita M J De Potter

doi:10.1093/toxsci/kfx144

Abstract

Triptolide is a vine extract used in traditional Chinese medicines and associated with hepatotoxicity. In vitro data suggest that inhibition of RNA synthesis may be the mechanism of toxicity. For studying drug-induced liver injury the zebrafish has experimental, practical and financial advantages compared with rodents. The aim of this study was to explore the mechanism of triptolide toxicity using zebrafish as the model system. The effect of triptolide exposure on zebrafish larvae was determined with regard to mortality, histology, expression of liver specific microRNA-122 and liver volume. Fluorescent microscopy was used to track toxicity in the Tg(-2.8lfabp:GFP)as3 zebrafish line. Informed by microscopy, RNA-sequencing was used to explore the mechanism of toxicity. Triptolide exposure resulted in dose-dependent mortality, a reduction in the number of copies of microRNA-122 per larva, hepatocyte vacuolation, disarray and oncotic necrosis, and a reduction in liver volume. These findings were consistent across replicate experiments. Time-lapse imaging indicated the onset of injury was 6 h after the start of exposure, at which point, RNA-sequencing revealed that 88% of genes were down-regulated. Immune response associated genes were up-regulated in the triptolide-treated larvae including nitric oxide synthase. Inhibition of nitric oxide synthase increased mortality. Triptolide induces hepatotoxicity in zebrafish larvae. This represents a new model of drug-induced liver injury that complements rodents. RNA sequencing, guided by time-lapse microscopy, revealed early down-regulation of genes consistent with previous invitro studies, and facilitated the discovery of mechanistic inflammatory pathways.

Highlights

The effect of triptolide exposure on zebrafish larvae was determined with regard to mortality, histology, expression of liver specific microRNA-122 and liver volume
Triptolide-Induced Liver Toxicity in Zebrafish Larvae First, we determined whether TP induced liver injury in zebrafish
Out of 38 zebrafish larvae treated with TP (0.8 mM) only 3 did not have histological evidence of liver injury

Summary

Introduction

A single dose of TP that caused only mild toxicity in wild-type mice resulted in severe toxicity and death in cytochrome P450 gene deleted mice (Xue et al, 2011) These findings suggest that TP itself is toxic (not dependent on metabolism) and the higher degree of toxicity in the liver may be due to a higher TP distribution to this organ. It has been reported that TP can covalently bind to a subunit of the transcription factor II human complex (TFIIH) and cause inhibition of its DNA-dependent ATPase activity, which leads to the inhibition of RNA polymerase II mediated transcription (Titov et al, 2011) Another group confirmed that TP inhibited total RNA and mRNA de novo synthesis. These in vitro data suggest that inhibition of RNA synthesis may explain the pharmacology and toxicology of TP (Vispe et al, 2009)

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