Mutational Specificity of l,3-Bis-(2-chloroethyl)-l-nitrosourea in a Chinese Hamster Ovary Cell Line1

The mutational specificity of the alkylating agent l-3-bis-(2-chloroethyl)-l-nitrosourea (BCNU) »as analyzed at the endogenous hemizygous (alenine phosphoribosyl transferase gene of the Chinese hamster ovary cell line D422. A 1-h treatment of the Chinese hamster ovary cells with 50 UM 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 muta tions; while G:C—A:T transitions, expected from miscoding of 06alkylguanine, represented only 16.3% (8 of 49) of the mutants recovered. This result was not anticipated, since Chinese hamster ovary cells are deficient in 06-alkylguanine-DNA alkyltransferase, which should ren der them especially sensitive to 06-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 BCNUinduced mutations are presented.