Molecular Pharmacology, Vol 18, 497-502, Copyright © 1980 by the American Society for Pharmacology and Experimental Therapeutics

Comparative Effects of Methyl- and Ethylnitrosourea on DNA Directing Cell-Free DNA-Dependent Synthesis of -Galactosidase

¹ Laboratory of Experimental Pathology, National Cancer Institute, Bethesda, Maryland 20205

An in vitro DNA-directed system for synthesis of -galactosidase was used to study the effects of methylnitrosourea (MNU) and ethylnitrosourea (ENU) on function of the DNA template. Both MNU and ENU inhibited formation of -galactosidase activity when added to the complete system at the beginning of incubation; this inhibition increased with increasing concentrations of either MNU or ENU. When template DNA was exposed to MNU or ENU before use in the protein synthesis system, -galactosidase synthesis was greatly reduced. Under comparable conditions, MNU was more inhibitory than ENU. Incubation of DNA with MNU or ENU resulted in DNA alkylation, which increased linearly with the concentration of nitrosourea over the range 0-1% alkylated nucleotides. MNU was shown to act both as an alkylating agent and, presumably via its degradation product NCO^-, as a carbamoylating agent, but alkylation was far more extensive than carbamoylation after 10 min at 37°C and pH 8.0. The degree of inhibition of -galactosidase synthesis was directly related to the extent of total alkylation, irrespective of whether MNU or ENU was allowed to react with template DNA. No difference in genotoxicity between these two agents was observed which could be ascribed to differences in sites of alkylation. Carbamoylation of DNA by exposure to KNCO did not inhibit synthesis of -galactosidase, although KNCO was highly inhibitory when added to the complete system.

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ACKNOWLEDGMENT The authors would like to thank Mrs. Maxine Bellman for her excellent help in typing the manuscript.