Yusuke Hiraku1, Hiroyuki Goto2, Masaki Kohno2, Shosuke Kawanishi3, Mariko Murata2. 1. 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. 2. Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan. 3. Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Mie 513-8670, Japan.
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. GENERAL SIGNIFICANCE: This study would provide an insight into the mechanism of MB-induced carcinogenesis and its safety assurance for clinical use.
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 humantumor-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. GENERAL SIGNIFICANCE: This study would provide an insight into the mechanism of MB-induced carcinogenesis and its safety assurance for clinical use.