Mutagenesis by hydrogen peroxide treatment of mammalian cells: a molecular analysis

Hydrogen peroxide is an oxidizing agent which can be generated intracellularly either during normal metabolism or by treatment with external agents including solar UV radiation. Simian cells (CV-1) transfected with the SV40-based shuttle vector plasmid pZ189 have been treated with H2O2 and then incubated to allow repair and replication of the plasmid. The frequency of mutations at the supF locus of the recovered plasmid increases by a factor of up to four over the spontaneous value. The nucleotide changes associated with 100 spontaneous and 100 H2O2-induced mutants have been determined directly by sequencing a 150 bp fragment that includes the entire supF tRNA coding region. Deletions were observed in approximately 45% of both the spontaneous and induced mutants, whereas single or multiple base changes arose in 68 and 57% of the induced and spontaneous mutants respectively. The spectrum of induced mutations is characterized by (i) the occurrence of deletions associated with base changes (16% of all mutants analysed) and (ii) small deletions of 3 bp and less (51% of all deletion mutants sequenced). Sixty-five per cent (15 out of 23) of all small deletions (spontaneous and induced) are associated with runs of between two and five identical bases and eight of them arise at a mutational 'hotspot' region of five cytosines between bp 172 and 176. The majority (19 out of 30) of completely sequenced deletions observed in the spontaneous spectrum contain either (i) small (2-10 bp) direct repeat sequences that lie immediately outside one deletion terminus and immediately inside the second deletion terminus or (ii) small (2-3 bp) inverted repeat sequences lying immediately inside the two deletion termini. Most deletions that we have observed are therefore likely to arise as a consequence of specific aspects of DNA structure.