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.
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 < 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.
Chen
Tao
T
Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, FDA, Jefferson, AR 72079, USA. tchen@nctr.fda.gov
Harrington-Brock
Karen
K
Moore
Martha M
MM
D000477:Alkylating Agents / Q000633:toxicity*; D000818:Animals; D001483:Base Sequence; D004252:DNA Mutational Analysis; D005038:Ethylnitrosourea / Q000633:toxicity*; D006579:Heterozygote; D007940:Leukemia L5178 / Q000201:enzymology* / Q000235:genetics*; D019656:Loss of Heterozygosity / Q000187:drug effects*; D051379:Mice; D018345:Mice, Knockout; D018895:Microsatellite Repeats; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009152:Mutagenicity Tests; D017354:Point Mutation; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013937:Thymidine Kinase / Q000235:genetics*; D014407:Tumor Cells, Cultured
D000477:Alkylating Agents; D013937:Thymidine Kinase; D005038:Ethylnitrosourea