MUTATIONS INDUCED BY DACARBAZINE ACTIVATED WITH CYTOCHROME-P-450

The mutagenicity of the antitumor drug dacarbazine (DTIC) is due to al kylation of cellular DNA by metabolites resulting from the metabolism of this drug by the mixed function oxidase system. In the present stud y, we used an in vitro shuttle vector assay to study the base and sequ ence specificity of mutagenesis by DTIC. The shuttle vector plasmid pS P189 was treated with DTIC (1-2.5 mM) in vitro in a reconstituted cyto chrome P-450 system at 37 degrees C for either 30 or 60 min. SupF tRNA gene insert contained in the plasmid was sequenced after replication of the drug-treated plasmid in human Ad 293 cells followed by amplific ation in indicator bacteria. Mutagenesis of DTIC in this system was de pendent upon the presence of the cytochrome P-450 reconstituted system and NADPH. Mutations induced by DTIC included single base substitutio ns (35%), single base deletions (30.5%), single base insertions (19.4% ) and large deletions (13.8%). Among the substitutions, transversions and transitions were in the ratio of 1:0.7. Base pairs 108 and 127 in the SupF tRNA of the pSP189 were identified as mutational hot spots.