title: Metal-mediated oxidative DNA damage induced by methylene blue.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
  
  
  CONCLUSIONS
  This study would provide an insight into the mechanism of MB-induced carcinogenesis and its safety assurance for clinical use.
authors.affiliation: Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan. Electronic address: y-hiraku@doc.medic.mie-u.ac.jp.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Hiraku
authors.affiliation: Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Goto
authors.affiliation: Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Kohno
authors.affiliation: Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Mie 513-8670, Japan.
authors.forename: Shosuke
authors.initials: S
authors.lastname: Kawanishi
authors.affiliation: Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan.
authors.forename: Mariko
authors.initials: M
authors.lastname: Murata
mesh_terms: D003300:Copper / Q000737:chemistry*; D004249:DNA Damage; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry*; D007501:Iron / Q000737:chemistry*; D008751:Methylene Blue / Q000737:chemistry*; D010084:Oxidation-Reduction; D010618:Phenanthrolines / Q000737:chemistry*
chemical_list: D010618:Phenanthrolines; D003300:Copper; C002478:bathocuproine; D006861:Hydrogen Peroxide; D007501:Iron; D008751:Methylene Blue
keywords: Carcinogenesis; DNA damage; Metal ion; Methylene blue; NADH; Reactive oxygen species