The spectrum of mutations generated by passage of a hydrogen peroxide damaged shuttle vector plasmid through a mammalian host

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Abstract
Treatment of a plasmid shuttle vector (pZ189) with a combination of hydrogen peroxide and a ferric iron/EDTA complex prior to transfection and passage in simian (CV-1) cells increases the frequency of mutations at the supF locus by up to 60-fold over the spontaneous background. This increase in mutation frequency is abolished when the inhibitors desferrioxamine, superoxide dismutase, catalase or dimethyl sulfoxide are included in the initial reaction or when the iron/EDTA complex is omitted, a strong indication that the premutagenic damage arises as a result of direct attack by hydroxyl radical generated in a superoxide driven Fenton reaction. DNA sequence analysis of the mutated plasmids shows that 1) Deletions occuring in combination with base-substitutions arise in 22.5 percent of the induced mutants compared with only 3 percent of spontaneous mutants 2) Sixty percent of all induced deletion mutations involve the loss of a single base and 77 percent of these (20 out of 26) occur at two adenine-containing sites 3) The base-change spectrum of mutants arising in the treated plasmid population is marked by the predominance of mutants containing a single base-change and by an increase in changes at AT base pairs. These results provide direct information concerning the nature of mutations arising in mammalian cells as a result of hydroxyl radical mediated DNA damage.