ToxSci Advance Access first published online on February 23, 2009
This version published online on February 25, 2009
Toxicological Sciences, doi:10.1093/toxsci/kfp037
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Published by Oxford University Press 2009.
The Mouse Lymphoma Assay detects recombination, deletion, and aneuploidy
1 Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research/FDA, 3900 NCTR Rd, Jefferson, AR, USA 72079 2 Cytogenetics Laboratory, Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR, USA 72205 3 College of Life Science and Technology, Shanghai Jiao Tong University, Shanghai, China 4 Division of Genetics and Mutagenesis, National Institute of Health Sciences, Tokyo, Japan 158-8501
* To whom correspondence should be addressed at (current address): Office of New Drugs, Center for Drug Evaluation and Research, Food and Drug Administration, HFD-540, 10903 New Hampshire Ave, Silver Spring, MD 20993. Phone 301-796-0852, E-mail: jianyong.wang{at}fda.hhs.gov
Received October 31, 2008; revision received January 26, 2009; accepted January 29, 2009
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Abstract |
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The mouse lymphoma assay (MLA) uses the thymidine kinase (Tk) gene of the L5178Y/Tk-3.7.2C mouse lymphoma cell line as a reporter gene to evaluate the mutagenicity of chemicals and physical agents. The MLA is recommended by both the US FDA and the US EPA as the preferred in vitro mammalian cell mutation assay for genetic toxicology screening because it detects a wide range of genetic alterations, including both point mutations and chromosomal mutations. However, the specific types of chromosomal mutations that can be detected by the MLA need further clarification. For this purpose, three chemicals, including two clastogens and an aneugen (3'-azido-3'-deoxythymidine, mitomycin C and taxol), were used to induce Tk mutants. Loss of heterozygosity (LOH) analysis was used to select mutants that could be informative as to whether they resulted from deletion, mitotic recombination or aneuploidy. A combination of additional methods, G-banding analysis, chromosome painting, and a real-time PCR method to detect the copy number of the Tk gene was then used to provide a detailed analysis. LOH involving at least 25% of chromosome 11, a normal karyotype and a Tk copy number of 2 would indicate that the mutant resulted from recombination, whereas LOH combined with a karyotypically visible deletion of chromosome 11 and a Tk copy number of 1 would indicate a deletion. Aneuploidy was confirmed using G-banding combined with chromosome painting analysis for mutants showing LOH at every microsatellite marker on chromosome 11. From this analysis, it is clear that mouse lymphoma Tk mutants can result from recombination, deletion and aneuploidy.
Key Words: Mouse Lymphoma Assay; Mutation type; Loss of heterozygosity; Cytogenetics; Copy number; 3'-Azido-3'-deoxythymidine; Mitomycin C; Taxol; Thymidine Kinase.
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