Method validation of phosphorylated histone H2AX level detection using primary cultured hepatocytes in genotoxic agent screening
10.3867/j.issn.1000-3002.2016.02.008
- VernacularTitle:利用原代肝细胞模型检测细胞内磷酸化组蛋白H2AX表达筛选遗传毒物的方法验证
- Author:
Tao MENG
;
Panpan MIAO
;
Yuqing JI
;
Yong NIU
;
Ping BIN
;
Yufei DAI
;
Yuxin ZHENG
- Publication Type:Journal Article
- Keywords:
genotoxic agents;
hepatocytes;
primary culture;
histones;
flow cytometry
- From:
Chinese Journal of Pharmacology and Toxicology
2016;(2):135-143
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE To establish an in vitro test method and to evaluate the genotoxicity of chemicals using primary cultured mouse hepatocytes and the changes in phosphorylated histone H2AX(γH2AX)expression levels to provide a more reliable marker of the identification of genotoxicity. METHODS Hepatocytes were isolated from BALB/c mice by an improved two-step collagenase diges?tion method and then cultured in sandwich configuration. The primary cultured hepatocytes were treat?ed with various concentrations of four known genotoxic agents bleomycin(BLM),benzo(a)pyrene〔B (a)p〕,styrene and styrene-7,8-oxide(SO)within the range of 40 μmol · L-1 and two non-genotoxic agents azathioprine(Aza)and ciclosporin A(CsA)at different time points within 24 h. The cytotoxicity induced by these toxicants was assessed by CCK-8 assay. Then,the changes in γH2AX expression levels in treated cells were determined by flow cytometry. RESULTS The four genotoxic agents could be detected and two non-genotoxic agents could not be detected by this method. The γH2AX expression level was the highest when hepatocytes were exposed to BLM and SO for 3 h,or B(a)p and styrene for 6 h(P<0.01). The production of γH2AX was 25.67,18.36,12.43 and 14.25 for the four types of genotoxic agents,respectively,and was approximately 19,13,9 and 11 times that of the vehicle control group(P<0.01)at the optimum time point and concentration. There was a significant positive corre?lation between the indicated concentrations of genotoxic chemicals and γH2AX expression levels(P<0.01). In addition,the production ofγH2AX indicated no marked increase in two non-genotoxic agents such as Aza and CsA in comparison with the control group. CONCLUSION This test method can effec?tively distinguish genotoxic agents from non-genotoxic agents,and direct genotoxic agents from indirect genotoxic agents in the absence of S9. γH2AX might be a reliable marker for the identification of the potential genotoxicity of chemicals.
