The mutational specificity of the alkylating agent 1-3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) was analyzed at the endogenous hemizygous adenine phosphoribosyl transferase gene of the Chinese hamster ovary cell line D422. A 1-h treatment of the Chinese hamster ovary cells with 50 µm BCNU resulted in a toxicity level of 62% and induced mutation at this target with a frequency of 32.1 mutants/106 survivors (6-fold above background). Analysis of 49 BCNU-induced mutants at the DNA sequence level revealed that BCNU induced primarily base substitutions. The predominant BCNU-induced mutations were G:C→T:A transversions, which comprised 51% (25 of 49) of the mutations; while G:C→A:T transitions, expected from miscoding of O6-alkylguanine, represented only 16.3% (8 of 49) of the mutants recovered. This result was not anticipated, since Chinese hamster ovary cells are deficient in O6-alkylguanine-DNA alkyltransferase, which should render them especially sensitive to O6-alkylguanine-mediated mutations. It was also notable that two “hotspots” for BCNU-induced G:C→T:A transversions were observed, which involved different surrounding DNA sequences but similar helix parameters when analyzed by an application of Calladine's Rules. Possible mechanisms for the observed BCNU-induced mutations are presented.


Supported in part by National Institute of Environmental Health Services Research Grant R01 ESO5540 awarded to W. D. S.; a research grant from the Cuyahoga County Unit, Ohio Division of the American Cancer Society, awarded to M. L. V.; a research grant from Glaxo, Inc., to W. D. S. and M. L. V.; and a Cancer Center core grant to the Ireland Cancer Center (P30CA43703).

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