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

Abstract

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 H 2 O 2 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 H 2 O 2 -induced mutants have been determined directly by sequencing a 150 bp fragment that includes the entire supF tRNA coding region. Deletions were observed in -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. IntroductionOxygen radicals appear to be involved in both the ageing process and in the initiation and progression of many human diseases, including cancer. A knowledge of the nature of mutations arising in DNA as a result of oxygen radical attack should provide important clues to understanding the molecular events that underlie these pathological changes. The characterization of the genotoxic action of the oxidizing agent, hydrogen peroxide, is particularly relevant in this respect for several reasons. In addition

Naturally occurring H-DNA-forming sequences are mutagenic in mammalian cells

“…The frequencies of different classes of H-DNA-induced mutations generated after replication in mammalian cells are displayed in Table 1. The majority of spontaneous mutations generated in the control plasmid in the mammalian cells were point mutations (40%) and deletions (50%), consistent with results of previous studies (27). As shown in Table 1, the mutation spectra of pGG32, pAG32, and pcMyc are very similar, even though the inserted H-DNA-forming sequences and the induced mutation frequencies differed for the three plasmids.…”

Expression of CuZn- and Mn-Superoxide Dismutase in Human Colorectal Neoplasms

“…Elevated levels may cause resistance to chemotherapeutic agents 40 on the one hand and higher effectivity of radiotherapy on the other. 41 Decreased levels may lead to increased in- cidence of mutations, 42 increased cell proliferation, 43,44 and can, in fact, be carcinogenic. 2,45 In conclusion, we have found mean decreased protein levels of both CuZn-SOD and Mn-SOD and total SOD activity in carcinomas.…”

Mitochondria, oxidative DNA damage, and aging

“…The mitochondrial polymerase gamma is no exception (32). 8-oxo-dG is not the most mutagenic lesion, based on the mutation spectrum resulting from oxidative stress, which favors G~A transitions (33,34). However, its biological relevance is demonstrated by the fact that enzymes for its repair and to prevent its incorporation into DNA are found in organisms ranging from E. coli (35) to human (36).…”