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Biochim Biophys Acta. 2014 Sep;1840(9):2776-82. doi: 10.1016/j.bbagen.2014.04.020. Epub 2014 May 2.

Metal-mediated oxidative DNA damage induced by methylene blue.

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Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan. Electronic address:
Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan.
Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Mie 513-8670, Japan.



Methylene blue (MB) is used for various clinical purposes, including chromoendoscopy and methemoglobinemia treatment. However, MB induces tumors of pancreatic islets and small intestine in experimental animals. This finding raises a possibility that MB induces carcinogenicity in these organs via light-independent mechanisms, although MB is known to cause light-dependent DNA damage.


We investigated the mechanism of MB-induced DNA damage using (32)P-5'-end-labeled DNA fragments of human tumor-relevant genes. We investigated the redox reaction of MB by UV-visible spectrometry.


MB induced DNA damage at the 5'-ACG-3' sequence, a hot spot of the p53 gene, in the presence of NADH and Cu(II). DNA damage was inhibited by catalase and bathocuproine, a Cu(I)-specific chelator. MB induced DNA damage at every nucleotide in the presence of NADH and Fe(III)-ethylenediaminetetraacetic acid, which was inhibited by OH scavengers and catalase. MB significantly increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine, an oxidative DNA lesion, in the presence of NADH and metal ions. UV-visible spectrometry revealed that the absorbance of oxidized form of MB at 668nm was decreased by NADH, and the addition of metal ions attenuated the spectral change.


MB undergoes NADH-dependent reduction followed by metal ion-mediated reoxidation. Reduced metal ions [Cu(I) and Fe(II)] interact with H2O2, generated during the redox reaction, to produce Cu(I)OOH and OH that cause DNA damage, respectively. These findings suggest that metal-mediated DNA damage contributes to MB-mediated carcinogenesis.


This study would provide an insight into the mechanism of MB-induced carcinogenesis and its safety assurance for clinical use.


Carcinogenesis; DNA damage; Metal ion; Methylene blue; NADH; Reactive oxygen species

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