Alteration of DNA by 5-(3-methyl-1- triazeno)imidazole-4-carboxamide (NSC-407347)

Abstract

5-(3-Methyl-1-triazeno)imidazole-4-carboxamide (NSC-407347, MTIC), an active intermediate in the metabolism of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (NSC-45388, DTIC), was investigated for its effect on DNA. When [ 3H]MTIC was added to an aqueous solution of calf thymus DNA, it was rapidly hydrolyzed, and less than 0.5 per cent of the added 3H was recovered in DNA as methyl groups attached to its base and phosphate constituents. It was estimated that the hydrolysis of phosphotriesters in methylated DNA released 0.5 per cent of total bound 3H of DNA in the experiment in vitro, and 5.7 per cent in vivo. When MTIC was added to tissue culture cells which had been prelabeled with [ 3H]thymidine, there was a large and immediate increase in acid-soluble radioactivity of both the cell media and the cells. At the same time, there was an accelerated drop in the specific activity of DNA, indicating that degeneration of DNA had occurred; MTIC at 10 −3 M had a greater effect than at 10 −5 M. Paper chromatographic studies showed that the acid-soluble radioactivity was predominantly in the form of thymidine. MTIC at 10 −4 M induced repair synthesis of DNA but inhibited semi-conservative replication. The extent of repair synthesis was greater at 30 min than after 5 min. Treatment of cells with MTIC at 10 −3 M irreversibly reduced the sedimentation of DNA in alkaline sucrose gradient. At 10 −5 M, the reduction was reversible, and a normal pattern was restored in 30 min. Comparison of these results with those of sedimentation analysis of DNA in formamide gradient at neutral pH indicated that single strand breaks which were apparent in alkaline sucrose were probably the result of exposure of alkali-labile lesions in DNA to high pH.