DNA-SEQUENCE CHANGES INDUCED BY 2 NITRIC-OXIDE DONOR DRUGS IN THE SUPF ASSAY

To refine our understanding of the mutational spectra one might expect on exposure of human cells to nitric oxide (NO), we have treated the plasmid pSP189 at pH 7.4 with two compounds that generate NO spontaneo usly in solution, and then sequenced the mutations found when the trea ted plasmid was transfected into human Ad293 cells and allowed to repl icate. G.C --> A.T transitions were the most abundant mutation observe d with these NO donor drugs, whereas in previous work, A.T --> G.C tra nsitions predominated when nitric oxide gas was bubbled through the pl asmid solution under otherwise identical conditions. A difference in r eactive intermediates formed in solution- versus gas-phase NO exposure was demonstrated by treating buffered 2,2'-azinobis(3-ethylbenzothiaz oline-6-sulfonate) (ABTS) or ferrocyanide, in the presence or absence of azide, aerobically with preformed solutions of NO, with solutions o f the two NO-releasing compounds, or with gaseous mixtures of equimola r NO/O-2 in air; oxidation of these substrates was extensive with the gas-phase NO source whether azide was present or not, while azide almo st completely quenched the oxidation pathway in the solution-phase rea ctions.