多细胞系胞质分裂阻滞微核细胞组学试验法的建立与应用

doi:10.3969/j.issn.1004-616x.2015.04.010

Abstract

Abstract: OBJECTIVE: This study attempted to establish the cytokinesis-block micronucleus cytomic test and to compare the susceptibilities of genotoxic biomarkers in a range of mammalian cell lines. The effects of TPL (triptolide) and DG(diammonium glycyrrhizinate) on CHL and MDCK were also investigated. METHODS: Method development: Cells were treated with mitomycin C in different concentrations for 6 h,followed by a CytoB (cytochalasins B) treatment covering around 1.5 cell doubling time. The cells were subsequently harvested,prepared on slides,stained and examined microscopically. The average CBPI (cytokinesis block proliferation index) and RI (replicative index) of each dose,and the Nec(necrosis rate permillage), Apop(apoptosis rate permillage),as well as the MN (micronucleus rate permillage), NBUD(nuclear bud rate permillage),NPB(nucleoplasmic bridge rate permillage) in binucleated cells were estimated. Method application: CHL was pretreated with DG for 24 h before treated with MMC for 6 h to evaluate the effect of DG on the genotoxicity induced by MMC. MDCK was treated with TPL (1 or 5 μg/mL) for 1 h as the potential protective effect of DG was also examined. RESULTS: Cytokinesis-block micronucleus cytomic test was successfully established in CHL,MDCK,L02 and Bhas 42 cell lines. The cell toxicity(CBPI,RI,Apop and Nec)- related indexes increased along with increasing MMC concentrations. The MN,NBUD and NPB also exhibited dose-dependent rising trend induced by MMC,however,the sensitivity for the above biomarkers varied for different cell lines. DG(10 and 100 μg/mL) significantly reduced MMC (0.2 μg/mL)-induced increment on MN,NBUD and NPB(P<0.05). TPL(5 μg/mL) treatment produced elevations of MN and NBUD in MDCK(P<0.05),which could be attenuated by DG (10 μg/mL) significantly(P<0.05). CONCLUSION: Cytokinesis-block micronucleus cytomic test made it possible to effectively evaluate multiple genetic toxicity indexes within a single slide. Our data clearly demonstrated that the genotoxic effects triggered by TPL were predominately caused by the DNA breakage or loss,while DG showed potent protective effects against the chromosome breakage in both CHL and MDCK cell lines and was able to reduce the toxicities induced by other agent.

Key words: cytokinesis-block micronucleus assay, micronucleus cytomic, MDCK, triptolide, diammonium glycyrrhizinate

CLC Number:

R394.6
R99

WEN Hairuo, DAN Mo, QI Naisong, GENG Xingchao, WANG Xue. Cytokinesis-block micronucleus cytomic test establishment in multiple mammalian cell lines[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2015, 27(4): 304-308.

References

[1] 曹佳,林真,余争平. 微核试验:原理、方法及其在人群监测和毒性评价中的应用[M]. 北京:军事医学科学出版社,2000:1-2.
[2] Benassi-Evans B,Fenech M. Chronic alcohol exposure induces genome damage measured using the cytokinesis-block micronucleus cytome assay and aneuploidy in human B lymphoblastoid cell lines[J]. Mutagenesis,2011,26(3):421-429.
[3] Oh SM,Kim HR,Park YJ,et al. Organic extracts of urban air pollution particulate matter (PM2.5)-induced genotoxicity and oxidative stress in human lung bronchial epithelial cells (BEAS-2B cells)[J]. Mutat Res,2011,723(2):142-151.
[4] Fenech M,Morley AA. Solutions to the kinetic problem in the micronucleus assay[J]. Cytobios,1985,43(172/173):233-246.
[5] Fenech M. The advantages and disadvantages of the cytokinesis-block micronucleus method[J]. Mutat Res,1997,392(1/2): 11-18.
[6] Fenech M. Cytokinesis-block micronucleus assay evolves into a “cytome” assay of chromosomal instability,miototic dysfunction and cell dead[J]. Mutat Res,2006,600(1/2):58-66.
[7] 向梦龙,敖琳,刘晋祎,等. 胞质分裂阻滞微核细胞组试验法的研究进展[J]. 癌变·畸变·突变,2012,24(3):241-244.
[8] Fenech M. Cytokinesis-block micronucleus cytome assay[J]. Nature Protocol,2007,2(5):1084-1104.
[9] 段化伟,郑玉新. 基于胞质分裂阻滞微核试验的细胞组学的进展和应用[J]. 中华预防医学杂志,2008,42(6):444-447.
[10] Gutiérrez-Enríquez S,Ramón YCT,Alonso C,et al. Ionizing radiation or mitomycin-induced micronuclei in lymphocytes of BRCA1 or BRCA2 mutation carriers[J]. Breast Cancer Res Treat,2011,127(3):611-622.
[11] Fenech M,Crott JW. Micronuclei,nucleoplasmic bridges and nuclear buds induced in folic acid deficient human lymphocytes-evidence for breakage-fusion-bridge cycles in the cytokinesis-block micronucleus assay[J]. Mutat Res,2002,504(1/2): 131-136.
[12] Gisselson D,Bjork J,Hoglund M,et al. Abnormal nuclear shape in solid tumors reflects mitotic instability[J]. Am J Pathol,2001,158(1):199-206.
[13] Gisselson D,Jonson T,Petersen A,et al. Telomere dysfunction triggers extensive DNA fragmentation and evolution of complex chromosome abnormalities in human malignant tumors[J]. Proc Natl Acad Sci US,2001,98(22):12683-12688.
[14] Satoh T,Yamamoto K,Miura KF,et al. Region-specific chromatin decondensation and micronucleus formation induced by 5-azacytidine in human TIG-7 cells[J]. Cytogenetic Genome Res,2004,104(1/4):289-294.
[15] Sasaki K,Mizusawa H,Ishidate M. Isolation and characterization of ras-transfected BALB/3T3 clone showing morphological transformation by 12-O-tetradecanoyl-phorbol-13-acetate[J]. Jpn J Cancer Res,1988,79(8):921-930.
[16] 曹易懿. 体外遗传毒性评价试验的优化[D]. 上海,上海交通大学生命科学技术学院,2012.
[17] 杨建一,高宝珍,宋春英,等. GTW对小鼠骨髓细胞微核和精子畸形的影响[J]. 生物技术通报,2006,5:113-116.
[18] 李佳,宋新波,余保林,等. 甘草抗氧化作用的研究进展[J]. 中国中医药杂志,2008,6(3):61-65.
[19] 李涯松,童培建,马红珍. 甘草对雷公藤治疗类风湿关节炎的减毒增效作用[J]. 中国中西医结合杂志,2006,26(12): 1117-1119.
[20] 夏成云,高月,周京国,等. 大戟配伍甘草对大鼠肝功能及肝脏微粒体中CYP3A2的影响[J]. 中国中医急症,2006, 15(9):1013-1015.

Cytokinesis-block micronucleus cytomic test establishment in multiple mammalian cell lines

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 3

Recommended Articles

Metrics

Comments

[1]	LU Yao, LIU Xinxia, XING Xiumei, ZHAO Zhiqiang, OU Xiaoyan, SU Xiaolin, SUN Yi, CHEN Jingli, SHEN Biling, JIANG Jun, HE Yun. DNA damage and telomere length changes in peripheral leukocytes from lead exposed workers [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2016, 28(3): 179-184.
[2]	LUO Yong-wei1, SHI Chang1, WU Chun-qi1, HU Zhong-hui1,YANG Bao-hua1, WANG Quan-jun1,ZHANG Yu-jie2, LIAO Ming-yang1,. The Reproductive Toxi-city of Antitumor Compound MC004 in Male Rat [J]. , 2009, 21(3): 218-221.
[3]	WU Xia-yu;WANG Xu;CAO Neng ;LIANG Zi-qing; WANG Xiao-yan; HUANG Yun-chao;XIA Xiao-ling. Methionine and Vitamin B12 Deficiency Induce Genome Instability in Human Lymphob-last Cell Line in Vitro [J]. , 2005, 17(6): 336-339.