Singlet Molecular Oxygen Induced Mutagenicity in a Mammalian Sv40‐based Shuttle Vector

Abstract

We have determined the deleterious effects of singlet oxygen (1O2), generated by thermal decomposition of the water-soluble endoperoxide 3,3'-(1,4-naphthylidene)dipropionate (NDPO2), on plasmid DNA. By following the electrophoretic mobility of DNA on agarose gels, we detected single and double strand breaks induced by treatment with NDPO2. The vector employed was a mammalian shuttle vector and the mutagenic consequences of these damages were investigated, using as mutation target the supF suppressor tRNA gene. A high increase of the mutation frequency, over the background, was observed in plasmids transfected in bacteria or after passage through mammalian cells. Trapping agents and quencher effects and other controls confirm the involvement of 1O2 in DNA damage and mutagenicity. These findings indicate that 1O2 can induce DNA lesions which are repaired by an error-prone process in prokaryotic and eukaryotic cells.

Novel XPG ( ERCC5 ) Mutations Affect DNA Repair and Cell Survival after Ultraviolet but not Oxidative Stress

“…Similar experiments were performed after exposure to oxidative stress induced by photosensitized MB. After exposure to visible light, the photosensitizer MB generates reactive oxygen species, primarily singlet oxygen, which in turn induces DNA damage, mainly 8‐hydroxy‐guanosine [Costa et al., ; Di Mascio et al., ; Epe et al., ]. Interestingly, at conditions under which XPCS1LV fibroblasts (XP‐G/CS cells) were sensitive to photosensitized MB treatment, the Brazilian XP‐G cells showed no sensitivity (Fig.…”

Mutation Spectrum Induced by Singlet Oxygen in Escherichia coli Deficient in Exonuclease III

“…M at 30 min after addition of 10 mM NDPO, (29). After treatment, the DNA samples were analyzed for the presence of DNA single-strand breaks by electrophoresis in agarose gels as described before (30) and then DNA was diluted to a concentration of 1 ng/kL before transfection into bacterial strains.…”

“…The NDP0,-treated pAC189 plasmid was analyzed for the presence of single-strand breaks by the distinction of covalent closed molecules (form I) and relaxed circles (form 11) in agarose gels (30). The data are presented in Table 1, and there is a dose-dependent increase in the number of breaks induced by '0,.…”

Plasmid DNA damage induced by singlet molecular oxygen released from the naphthalene endoperoxide DHPNO2 and photoactivated methylene blue

“…On assuming Poisson damage distribution, the average number of enzyme sensitive sites per plasmid is given by the expression X = -ln (1.4 x SC/(1.4 x SC + OC)), where SC and OC are the fluorescence of the supercoiled and relaxed forms, respectively, the factor 1.4 correcting for the difference in ethidium bromide binding to supercoiled compared to relaxed-DNA. 11,29 As a control group for linear-DNA (form III), plasmid was digested with BamHI enzyme (Promega, Southampton, UK) for 1 h at 37 °C. Note that pBluescript SK contains a unique BamHI restriction site.…”

“…7 It should be added that 1 O 2 is known to be mutagenic and genotoxic, [8][9][10] since it is able to react with DNA, thus leading to cell-killing and mutagenesis. In fact, DNA treated with different 1 O 2 sources was found to contain single-and double-strand breaks, 11 as well as producing base damage which may promote in vitro DNA synthesis arrest. [12][13][14] As a consequence of DNA lesions, 1 O 2 is proposed to be directly involved in degenerative processes such as cancer and aging.…”