Influence of the antioxidant N-acetylcysteine and its metabolites on damage induced by bleomycin in PM2 bacteriophage DNA

Abstract

Bleomycin is considered to be a useful model compound for studying environmental carcinogenesis, due to its broad spectrum of DNA damaging properties. In addition, bleomycin is a useful antitumor drug because of its cytotoxic properties. To investigate the influence of the antioxidant N-acetylcysteine and its metabolites glutathione and cysteine on bleomycin-induced DNA damage and more importantly to gain insight into the biological relevance of such damage, PM2 DNA was exposed to Cu(2+)-bleomycin in the presence and absence of the thiols N-acetylcysteine, glutathione and cysteine. It was found that the presence of these thiols led to a considerable enhancement of bleomycin-induced single- and double-strand breaks and a concomitant decrease in the biological activity of PM2 DNA in a dose-dependent way. A similar observation was made when ascorbic acid was used. Bleomycin showed no DNA damaging activity when PM2 DNA was pretreated with the strong Fe ion chelator desferal and its activity was strongly inhibited by the addition of Cu2+ ions or under hypoxic (N2) conditions. Cu(2+)-bleomycin under our conditions is not active by itself, but most probably after binding to DNA exchanges Cu2+ for Fe3+ bound to DNA. Fe(3+)-bleomycin is then reduced to Fe(2+)-bleomycin, a process potentiated by the added antioxidants, and subsequently activated by O2. The contribution to biological inactivation of bleomycin alone or in the presence of ascorbic acid is only approximately 15%. The contribution to lethality in the presence of thiols is higher. These results indicate that ascorbic acid only enhances the DNA damaging properties of bleomycin, whereas the thiol compounds in addition influence the type of DNA damage. The remainder of the biological inactivation is probably caused by double damage, such as single-strand breaks with closely opposed alkali-labile sites or base damage.

Comparison of bleomycin and radiation in the G2 assay of chromatid breaks

“…In this way, it can make another break in close vicinity to the first break. As measured in a plasmid DNA model system, bleomycin induces dsb more efficiently than radiation; the ratio between double-and single-strand breaks when induced by bleomycin is 1:9, whereas for radiation the ratio is 1:100 (Cloos et al 1996).…”

“…The breaks and gaps of 50 metaphases per slide were scored separately under oil immersion, 12506 magnification (Zeiss microscope, Jena, Germany). Criteria for scoring breaks and gaps were published by Cloos et al (1996). To assess the G2 block, the mitotic index was also determined as a percentage by scoring 2000 cells.…”

“…Moreover, it was shown that elevated bleomycin sensitivity was not an independent risk factor but was only relevant as a marker for an increased risk of developing cancer in exposed individuals, i.e. smokers (Cloos et al 1996). In contrast, chromosomal radiation sensitivity was shown to be an independent risk factor for breast cancer .…”

Microarray Analysis of Bleomycin-Exposed Lymphoblastoid Cells for Identifying Cancer Susceptibility Genes

“…The question arose whether bleomycin-induced chromatid break sensitivity is linked with cancer predisposition in the same way as radiation-induced sensitivity. Although bleomycin is called a ''radiomimetic'' agent, it differs from radiation in the way the damage is induced (15). Despite the differences, a good correlation was found when bleomycin-and radiation-induced b/c values were compared in the mutagen sensitivity test of the same cells (16), suggesting that a kind of similar mechanism is underlying these damage-sensitive phenotypes.…”

Human Lymphoblastoid Proteome Analysis Reveals a Role for the Inhibitor of Acetyltransferases Complex in DNA Double-Strand Break Response

“…Moreover, we are particularly interested in the response to DSBs, and bleomycin is more efficient in producing DSBs than the often used ionizing radiation because it binds to the DNA and can cleave the DNA at two opposing strands, resulting in a DSB (14). The ratio between double-and single-strand breaks is 1:9 for bleomycin, and 1:100 for ionizing radiation (15 samples were analyzed using two-dimensional difference in gel electrophoresis (DiGE), allowing the simultaneous analysis of many samples which could not be easily done using any of the alternative approaches in quantitative proteomics. The use of fluorescent dyes permitted the analysis of small changes in protein expression both for relatively low and high protein levels, and the use of an internal standard to normalize the gel-to-gel variation.…”