Mutations induced by methylene blue plus light in single-stranded M13mp2.

Abstract

Reactive oxygen species are generated by a variety of cellular processes. These endogenously generated, reactive intermediates produce a multiplicity of DNA alterations and mutations and have been implicated in the pathogenesis of several human diseases. We report here that treatment of single-stranded M13mp2 bacteriophage DNA with methylene blue and white light generates increased levels of 8-hydroxydeoxyguanosine and that mutagenesis is both highly specific and dependent on the SOS response. Lesions produced block the progression of DNA synthesis one base preceding template guanines. In SOS-induced Escherichia coli, 97% of all methylene blue-induced mutations in the lacZa gene of M13mp2 DNA are single-base substitutions opposite template guanines. The most frequent mutations are G -+ C transversions. The G -+ T transversions expected from the presence of 8-hydroxydeoxyguanosine in the template strand occur, but at a lower frequency. Sequence data together with SOS dependency and the presence of replication blockage demonstrate that while 8-hydroxydeoxyguanosine may serve as an important marker to monitor oxygen-induced DNA damage in humans, it does not account for either the observed blockage to replication or the mutagenesis by methylene blue plus light in SOS-induced E. coli. Instead, an as yet unidenti lesion generated by active oxygen species is a more potent mutagenic event.In cells, oxygen is metabolized by a series of one-electron reductions. A result ofoxygen metabolism is the formation of free radicals and related species including hydroxyl radicals, hydrogen peroxide, superoxide ions, and singlet oxygen. These highly reactive molecules can damage many cellular macromolecules, including proteins, lipids, RNA, and DNA (1, 2), and the resultant deleterious effects have been hypothesized to contribute to the pathogenesis of many agerelated human diseases, including cancer, atherosclerosis, and joint diseases (3, 4). To elucidate the relationship of DNA damage by reactive oxygen species to the pathogenesis of diseases, methods are required to quantitate the different types of DNA alterations produced and to determine the mutagenic potential of each alteration. However, the multiplicity and complexity of reactions that generate reactive oxygen species in cells have complicated these analyses. First, there are multiple processes in cells that generate oxygen free radicals (2, 5), and these radicals undergo further reactions to form additional reactive species. Second, based on the multiplicity of nucleotide alterations produced by oxygen free radicals in vitro, it can be estimated that in DNA more than 100 different nucleotide adducts can be formed (6-9). Third, certain molecules that generate oxygen free radicals preferentially bind to specific nucleotide sequences in DNA (10, 11). Fourth, long-lived DNA damage and mutagenesis result from inadequate DNA repair and altered incorporation by DNAreplicating enzymes, respectively; these mechanisms, in eukaryotic cells, have yet to be characterized (12)....

p53 Mutations in non-small-cell lung cancers occurring in individuals without a past history of active smoking

“…Loeb and co-workers have clearly shown that while this tandem double mutation, a hallmark of damage to DNA by UV irradiation, is not produced by DNA polymerases or viral reverse transcriptases or by exposure to chemical carcinogens, it can be caused by oxidative damage to DNA Tkeshelashvili et al, 1993). G:C to C:G and G:C to T:A transversions, which were also found to be present at relatively high frequencies in the lung cancers of our non-smoker cohort, are also known to be induced by reactive oxygen species in certain circumstances (Moriya et al, 1991;McBride, 1992). The present findings suggest that oxygen radical species, which can be generated by various mechanisms, including normal cellular processes (Fridovich, 1983), might have a role in the development of lung cancers.…”

Tandem double CC-->TT mutations are produced by reactive oxygen species.

“…Recently, however, tandem double CC -+ TT mutations have been detected in forward mutation assays when using either Cu ions (12) or human neutrophils as a source of reactive oxygen species (13). The goal of this study was to determine whether oxygen free radicals were responsible for inducing this mutation and (15,27). Because of the rarity of CC -* TT mutations, we have elected to use SOS-induced cells to increase the sensitivity with which they can be detected.…”

“…Oxygen radicals cause a large number of modifications to cytosine residues, and single C -* T transitions are among the most common mutations produced by most (12,15) but not all (27) reactive oxygen species. Chemical modifications produced by reactive oxygen at cytosine residues include addition to the 5,6 double bond of the pyrimidine ring, formation of glycols, hydantoins, deamination products, and species with rearrangements of the pyrimidine ring (7,8).…”

The role of oxidative stress genes and effect of pH on methylene blue sensitized photooxidation ofEscherichia coli

“…All proteins, lipids, carbohydrates and nucleic acids are harmed by these radicals [7]. Moreover, it has been that various mutations occurred in DNA following the treatment with demonstrated methylene blue and light, as well as heredical diseases occurred in metabolic pathways [14].…”