基于双荧光素酶报告基因的DNA损伤与修复检测系统的建立与应用

doi:10.3969/j.issn.1004-616x.2013.06.013

Abstract

Abstract:

OBJECTIVE: Development of a screening system for DNA damage and repair based on dual luciferase assay in human HepG2 cells. METHODS：5 μmol/L CdCl2 was used to treat the plasmid pTK-RL in vitro. Plasmid pgadd153-luc and damaged pTK-RL were co-transfected into HepG2 cells and then the reconstituted HepG2 cells were treated with sixteen DNA-damaging agents and three non-genotoxic agents. The DNA damage and DNA repair abilities of HepG2 cells were measured by dual luciferase assay. Three organic extracts from different sites of soil (residential area，garbage dump and farmland) were evaluated by dual luciferase assay. DNA damage was detected by Comet assay. RESULTS：The dual luciferase assay could identify genotoxic and non-genotoxic agents. The three different soil samples had various levels of inducing DNA damage and decreasing DNA repair capacities in HepG2 cells following the order：residential area >garbage dump>farmland. CONCLUSION：In this study，the dual luciferase assay was developed to measure DNA damage and repair.

Key words: DNA damage, DNA repair, host cell reactivation assay, dual luciferase report gene, carcinogen

ZHANG Rong，NIU Yu-jie，FAN Long-gang，SHI Lei，WANG Qian，WANG Xiu-rong. Development and application of a screening system for DNA damage and repair based on dual luciferase assay in human HepG2 cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, doi: 10.3969/j.issn.1004-616x.2013.06.013.

References

[1] Collins AR，Ferguson LR. DNA repair as a biomarker[J]. Mutat Res，2012，736 (1/2)：2-4.

[2] Zhang R，Niu Y，Du H，et al. A stable and sensitive testing system for potential carcinogens based on DNA damage-induced gene expression in human HepG2 cell[J]. Toxicol In Vitro，2009，23 (1)：158-165.

[3] Thompson KL，Sistare FD. Selection of drugs to test the specificity of the Tg.AC assay by screening for induction of the gadd153 promoter in vitro[J]. Toxicol Sci，2003，74 (2)：260-270.

[4] Lin X，Gately DP，Hom D，et al. Quantification of tumor cell injury in vitro and in vivo using expression of green fluorescent protein under the control of the GADD153 promoter[J]. Int J Cancer，2001，91 (4)：555-562.

[5] Jia X，Zhu Y，Xiao W. A stable and sensitive genotoxic testing system based on DNA damage induced gene expression in Saccharomyces cerevisiae[J]. Mutat Res，2002，519 (1/2)： 83-92.

[6] Alexandrov K，Rojas M，Satarug S. The critical DNA damage by benzo(a) pyrene in lung tissues of smokers and approaches to preventing its formation[J]. Toxicol Lett，2010，198 (1)：63-68.

[7] Pitsikas P，Lee D，Rainbow AJ. Reduced host cell reactivation of oxidative DNA damage in human cells deficient in the mismatch repair gene hMSH2[J]. Mutagenesis，2007，22 (3)： 235-243.

[8] Decordier I，Loock KV，Kirsch-Volders M. Phenotyping for DNA repair capacity[J]. Mutat Res，2010，705 (2)：107-129.

[9] Nguyen TA，Slattery SD，Moon SH，et al. The oncogenic phosphatase WIP1 negatively regulates nucleotide excision repair[J]. DNA Repair ：Amst，2010，9 (7)：813-823.

[10] Liu X，Kramer JA，Swaffield JC，et al. Development of a highthroughput yeast-based assay for detection of metabolically activated genotoxins[J]. Mutat Res，2008，653 (1/2)：63-69.

[11] Wang D，Cai Z，Jiang G，et al. Determination of polybrominated diphenyl ethers in soil and sediment from an electronic waste recycling facility[J]. Chemosphere，2005，60 (6)：810-816.

[12] Shen C，Chen Y，Huang S，et al. Dioxin-like compounds in agricultural soils near e-waste recycling sites from Taizhou area，China：chemical and bioanalytical characterization[J]. Environ Int，2009，35 (1)：50-55.

[13] Alabi OA，Bakare AA，Xu X，et al. Comparative evaluation of environmental contamination and DNA damage induced by electronic-waste in Nigeria and China[J]. Sci Total Environ， 2012，423 (1)：62-72.

[1]	SUN Xin, LI Shuang, LU Xue, CAI Tianjing, LIU Ya, LIU Qingjie, ZHANG Wei. Effect of low-dose ionizing radiation on oxidative stress and DNA damage repair in human lymphocytoid cells [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2024, 36(2): 94-99.
[2]	ZHENG Yaqi, ZHAO Ziting, PAN Deyuan, LIU Luxin, XU Xiue, LIAO Liandi, WANG Shaohong, LI Enmin, XU Liyan. The expression of tonsoku-like DNA repair protein in esophageal carcinoma and its effect on the proliferation of esophageal squamous cell carcinoma [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2023, 35(6): 412-419.
[3]	LIU Xiangdong, ZHAO Zenglin, WANG Luanluan, ZHAN Rongkai, LIU Zengxiang, WU Xinyu, LIU Chenghu. Involvement of blood collections by time in combined chronic toxicity and carcinogenicity test for magnesium alloy materials [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2022, 34(2): 134-138.
[4]	ZHOU Changhui, HAN Tianjiao, HUANG Pengcheng, MA Jing, CHANG Yan. Validation study of comet assay in multiple organs of rats [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2020, 32(3): 233-237.
[5]	SHI Pan, LIN Chonghua, SHU Yifang, QU Jiaquan, SONG Shuliang. Role of mitochondrial citrate transporter SLC25A1 in radiosensitivity of human esophageal squamous cell carcinoma in vitro [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2020, 32(2): 132-138.
[6]	WANG Bingyu, ZHENG Kai, XU Xinyun, XIE Hongwei, YU Junhui, LONG Dingxin. PM_2.5 on DNA damage in human bronchial epithelial cells [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2019, 31(6): 469-473,497.
[7]	WANG Xun, LI Bin, LIU Liangyan, YANG Min, ZOU Yuliang. Health risk assessment of drinking water in the Longgang district of Shenzhen,China [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2019, 31(5): 359-362.
[8]	JIANG Pengtao, HU Zhifang, GAO Xingchun, GUO Na, ZHANG Dian, JIANG Fengliang. Involvement of p53 in regulation of DNA damage-induced cell death in MDA-MB-231 [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2018, 30(5): 349-353.
[9]	LI Hui, HU Chengjiu. Significance of expression of MDM2 mRNA and protein in skin scar carcinoma [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2018, 30(5): 365-367,373.
[10]	LIU Fang, WANG Zhuanzi, WEI Wei, LI Wenjian, DANG Bingrong. Total body irradiation of mice with carbon-ion caused damage to the hematopoietic system [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2018, 30(2): 109-113,119.
[11]	WANG Yuan, DENG Xiaofei. Effect of DNA glycosylase OGG1 against Helicobacter pylori-induced DNA damage in gastric epithelial cells [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2018, 30(2): 92-97,102.
[12]	CHEN Tingyu, SUN Jie, YANG Yu, ZHU Qiushuang, MIAO Zhi, ZHONG Tangwu, LU Chunfeng. The role of reactive oxygen species in isoniazid-induced DNA damage in L-02 cells and the protective effect of quercetin [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2016, 28(6): 472-476.
[13]	Bernd Kaina. DNA repair in cancer therapy with methylating anticancer drugs: improving therapeutic efficacy and reducing side effects [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2016, 28(5): 333-341,347.
[14]	LI Chen, QI Xuesong, LI Ning, FAN Li, GOU Qiao, LIU Jianxiang, TIAN Mei. UVA ultraviolet radiation on cellular injury to human keratinocytes [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2016, 28(5): 364-368.
[15]	LIU Ruixue, AN Xiufeng, FENG Li, LI Yongchao, ZHANG Bo. Mechanisms of natto lipopeptide-induced apoptosis in MCF-7 cells [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2015, 27(6): 459-462.

Development and application of a screening system for DNA damage and repair based on dual luciferase assay in human HepG2 cells

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments