Mutagenesis, Vol. 17, No. 2, 105-109,
March 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Mutant frequencies and loss of heterozygosity induced by N-ethyl-N-nitrosourea in the thymidine kinase gene of L5178Y/TK+/–-3.7.2C mouse lymphoma cells
Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, FDA, Jefferson, AR 72079, USA and 1 National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC 27709, USA
N-ethyl-N-nitrosourea (ENU) is a potent monofunctional ethylating agent that has been found to be mutagenic in a wide variety of organisms from viruses to mammalian germ cells. To elucidate the mutagenicity of ENU at the Tk+/– locus of mouse lymphoma cells and to confirm the ability of the mouse lymphoma assay (MLA) to detect both point mutations and large DNA alterations, Tk+/– L5178Y cells were exposed to different doses of ENU. Treatment of the cells with ENU resulted in a linear dose response with mutant frequencies of up to 16-fold over control. Evaluation of mutant clone size showed that 36% of the 100 µg/ml ENU-induced clones (66% in control) were small colony mutants and 64% (34% in control) were large colony mutants. DNA isolated from mutants in the control culture and the 100 µg/ml ENU treatment group was analyzed for loss of heterozygosity (LOH) using allele-specific PCR. The majority of the small colony mutants, both ENU-treated (97%) and spontaneous (91%), lost the Tk1b allele. The percentage of allele loss in ENU-induced large colony mutants was distinctly different from that of the control. Twenty-three percent of ENU-induced large colony mutants lost their Tk1b alleles, whereas 73% of the large colony mutants from the control culture lost the allele (P < 0.001). Overall, 50% of the Tk mutants from the 100 µg/ml ENU-treated cultures (86% in control) showed LOH. Our data indicate that ENU is a potent mutagen in mouse lymphoma cells and that 100 µg/ml ENU induces equal numbers of point mutations and chromosomal mutations. This study serves to verify that the MLA detects both point mutations and chromosomal mutations.
2 To whom correspondence should be addressed. Tel: +1 870 543 7954; Fax +1 870 543 7682; Email: tchen{at}nctr.fda.gov
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