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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 microg/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 microg/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 &lt; 0.001). Overall, 50% of the Tk mutants from the 100 microg/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 microg/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.

mutagenesis

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