Preliminary validation of high throughput screening methods for hepatotocity and genotoxicity in humanized HepaRG cells
10.7501/j.issn.1674-6376.2017.11.006
- VernacularTitle:人源HepaRG肝细胞毒性与遗传毒性高通量筛选方法的初步建立
- Author:
ruo Hai WEN
1
;
hui Zhi MAO
;
chao Xing GENG
;
Bo LI
;
Xue WANG
;
ying Zhi HUANG
Author Information
1. 中国食品药品检定研究院国家药物安全评价监测中心
- Keywords:
rhubarb anthraquinones;
HepaRG cell;
high throughput screening;
hepatotoxicity;
reactive oxygen species;
mitochondrial damage;
genotoxicity;
DNA breakage;
chromosome damage;
micronucleus assay;
single cell gel electrophoresis assay;
emodin;
aloe-emodin;
chrysophan
- From:
Drug Evaluation Research
2017;40(11):1550-1558
- CountryChina
- Language:Chinese
-
Abstract:
Objective To detect the hepatotoxicity biomarkers using normal human hepatocyte (HepaRG) and high-content screening,and to combine the micronucleus test and single cell gel electrophoresis to estalish a rapid screening platform for in vitro cytotoxitity and genotoxicity.Methods The effects of rhubarb anthraquinones (AQs) on the reactive oxygen species (ROS),intracellular Ca2+ concentration and mitochondrial membrane potential (MMP) in HepaRG cells were studied using appropriate fluorescent probes Hoechst33342、DCFH-DA、Fluo4-AM、Mito Tracker Red CMX Ros and high-content screening methods,and the potential genotoxiciy triggered by AQs were analyzed using the high-content based cytokinesis block micronucleus test and high throughput comet assay.Results The intracellular ROS level of HepaRG cells was significantly elevated by a 24 h treatment with Emodin (25.0 μg/mL),aloe-emodin (25.0 μg/mL) or chrysophanol (50.0 μg/mL),which are dose-concentration dependent (P ＜ 0.05 and 0.01);the intracellular Ca2+ increased and mitochondrial damage were observed with the treatment of aloe-emodin (25.0 μg/mL) and rhein (50.0 μg/mL,P ＜ 0.05 and 0.01).Comparing to control group,Emodin (25.0 μg/mL) induced an increased micronucleus rate (1.59％ ± 0.68 ％,P ＜ 0.01) and significantly higher percentage tail DNA and Olive tail moment (respectively 10.155％ ± 2.17％ and 0.510 ± 0.06,P ＜ 0.05 and 0.01) after 24 h;while the chrysophanol increased the micronucleus rate to 1.29％ ± 0.54％ (P ＜ 0.01) after 72 h.Conelnsion The results on the cytotoxicities and genotoxicities of AQs are consistent with the literatures.In this study,a rapid screening model for both hepatotoxicity and genotoxicity was successfully established,which will help with the early screening during the drug development stage.
