多遗传学终点的遗传毒性试验组合的建立

doi:10.3969/j.issn.1004-616x.2013.06.014

Carcinogenesis, Teratogenesis & Mutagenesis

Previous Articles Next Articles

Establishment of a new battery of toxicity tests for multiple genetic endpoints

MA Hua-zhi，SHI Chang^#，*，SHI Fu-jiang，SUN Yan-li，YU Yong-sheng，WU Chun-qi，WANG Quan-jun，DING Ri-gao

(Beijing Institute of Pharmacology and Toxicology, National Beijing Center for Drug Safety Evaluation and Research, Beijing 100850, China)

Received:2013-09-03 Revised:2013-09-16 Online:2013-11-30 Published:2013-11-30
Contact: SHI Chang，E-mail：shich08@hotmail.com

Abstract

Abstract:

OBJECTIVE: To establish Ames fluctuation test，in vitro micronucleus assay with Chinese hamster lung fibroblast cells (CHL) and mouse lymphoma thymidine kinase (tk+/-) locus mutation assay (MLA) as a new battery of methods to evaluate genotoxicity. METHODS：Ames fluctuation test：TA100 Salmonella strain was exposed to sodium azide (SA) without S9 and cyclophosphamide (CP) with S9 in 96-well plate. The number of positive wells was recorded and Chi-square test was performed to analyze statistical significance. In vitro micronucleus test：CHL cells were exposed to mitomycin C (MMC) without S9 for 4 h or 24 h，and CP with S9 for 4 h. The number of micronucleated cells in 2 000 cells was counted under oil lens to calculate micronucleus rate. Chi-square test was performed to analyze statistical significance. MLA：After spontaneous mutation having been eliminated，L5178Y 3.7.2C cells were exposed to MMC without S9 and CP with S9 for 3 h. Relative survival rate (RS)，relative suspension growth rate (RSG)，relative total growth rate (RTG)，plate efficiency (PE0，PE2)，mutation rate (MF) and the proportion of small colony (SC) were calculated. RESULTS：Both SA and CP showed statistically significant positive dose-dependently results without and with S9，respectively. Micronucleu s rates increased dose-dependently both in MMC (4 h，24 h，-S9) and CP (4 h，+S9) treated groups at different concentrations. MFs of L5178Y 3.7.2C cells exposed to different concentrations of MMC (4 h， 24 h，-S9) and CP (4 h，+S9) increased dose-dependently and more than twice compared to the vehicle control， while PE，RS，RSG and RTG decreased dose-dependently. CONCLUSION：These three new short-term assays could be used to detect genotoxicity more accurately and more extensively.

Key words: Ames fluctuation test, in vitro micronucleus assay, mouse lymphoma thymidine kinase locus mutation assay

MA Hua-zhi，SHI Chang，SHI Fu-jiang，SUN Yan-li，YU Yong-sheng，WU Chun-qi，WANG Quan-jun，DING Ri-gao. Establishment of a new battery of toxicity tests for multiple genetic endpoints[J]. Carcinogenesis, Teratogenesis & Mutagenesis, doi: 10.3969/j.issn.1004-616x.2013.06.014.

References

[1] Lynch AM，Sasaki JC，Elespuru R，et al. New and emerging technologies for genetic toxicity testing[J]. Environ Mol Mutagen， 2011，52 (3)：205-223.

[2] Reifferscheid G，Maes HM，Allner B，et al. International round- robin study on the Ames fluctuation test[J]. Environ Mol Mutagen， 2012，53 (3)：185-197.

[3] Takagi R，Suzuki Y，Seki Y，et al. Indium chloride-induced micronuclei in in vivo and in vitro experimental system[J]. J Occup Health，2011，53 (2)：102-109.

[4] Guo X，Verkler TL，Chen Y，et al. Mutagenicity of 11 cigarette smoke condensates in two versions of the mouse lymphoma assay[J]. Mutagenesis， 2011，26 (2)：273-281.

[5] Kamber M，Flückiger-Isler S，Engelhardt G，et al. Comparison of the Ames II and traditional Ames test responses with respect to mutagenicity，strain specificities，need for metabolism and correlation with rodent carcinogenicity[J]. Mutagenesis，2009， 24 (4)：359-366.

[6] Kirsch-Volders M，Plas G，Elhajouji A，et al. The in vitro MN assay in 2011：origin and fate，biological significance， protocols，high throughput methodologies and toxicological relevance[J]. Arch Toxicol， 2011，85 (8)：873-899.

[7] Doherty AT. The in vitro micronucleus assay[J]. Methods Mol Biol， 2012，817 (2)：121-141.

[8] Organization for Economic Cooperation and Development. Guideline for the testing of chemicals draft proposal for a new guideline 487：in vitro mammalian cell micronucleus test (MNvit)[S]. 2009.

[9] Shibai-Ogata A，Kakinuma C，Hioki T，et al. Evaluation of high-throughput screening for in vitro micronucleus test using fluorescence-based cell imaging[J]. Mutagenesis，2011，26 (6)： 709-719.

[10] Ogawa I，Furukawa S，Abe M，et al. Multi-endpoint genotoxic assay using L5178Y (Tk+/--3.7.2c) cells[J]. J Toxicol Sci， 2009， 34 (5)：547-553.

[11] Lloyd M，Kidd D. The mouse lymphoma assay[J]. Methods Mol Biol， 2012，817 (1)：35-54.

[12] Liviac D，Creus A，Marcos R. Mutagenic analysis of six disinfection by-products in the Tk gene of mouse lymphoma cells[J]. J Hazard Mater， 2011，190 (1/2/3)：1045-1052.

Establishment of a new battery of toxicity tests for multiple genetic endpoints

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 3

Recommended Articles

Metrics

Comments

[1]	CAO Yiyi, LIU Qian, XI Jing, MA Leilei, LIU Xiaojian, LUAN Yang. Comparative evaluation of mutagenicity of four aristolochic acids components using the Ames fluctuation test [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2016, 28(5): 398-402.
[2]	OU Hongmei，ZHOU Changhui，TU Honggang，HUANG Pengcheng，CHANG Yan. Establishment of flow cytometric in in vitro micronucleus assay [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2015, 27(1): 39-43.
[3]	CHEN Ying，LI Ying，GUAN Jun，WANG Zhong-hui，JIA Yu-ling，ZHOU Li，SUN Zu-yue*. Optimization of standardized Ames fluctuation test [J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2014, 26(3): 199-203.