Yusuke Hiraku

Superoxide formation and DNA damage induced by a fragrant furanone in the presence of copper(II)

2,5-Dimethyl-4-hydroxy-3(2H)-furanone (2,5-DMHF), a caramel-like fragrant compound found in may processed foodstuff, has been reported to be mutagenic. 4,5-Dimethyl-3-hydroxy-2(5H)-furanone (4,5-DMHF), which is a similar characteristic fragrant compound, has no report concerning its mutagenicity. DNA damage by 2,5-DMHF and 4,5-DMHF was investigated by using DNA fragments obtained from the p53 tumor suppressor gene. 2,5-DMHF induced DNA damage extensively in the presence of Cu(II), but only slightly in the presence of Fe(III). 4,5-DMHF did not cause metal-dependent DNA damage. Bathocuproine, a Cu(I)-specific chelator, and catalase inhibited DNA damage induced by 2,5-DMHF plus Cu(II), whereas free hydroxyl radical scavengers did not. The order of DNA cleavage sites was thymine, cytosine > guanine residues. The site-specific DNA damage and effects of scavengers show that DNA-copper-oxygen complex rather than free .OH are involved in the DNA damage. Formation of 8-oxodeoxyguanosine (8-oxodG) by 2,5-DMHF increased with its concentration in the presence of Cu(II), whereas 8-oxodG formation increased only slightly in the presence of Fe(III). Degradation of 2,5-DMHF was efficiently accelerated by Cu(II), but only slightly accelerated by Fe(III). The degradation of 4,5-DMHF was little even in the presence of metal ions. Examination using cytochrome c suggest that superoxide was generated from 2,5-DMHF. Stoichiometric study of Cu(II) reduction revealed that autoxidation of 2,5-DMHF could offer 4-electron reduction. These results suggest that, at least in vitro and in an acellular system, 2,5-DMHF generates superoxide and subsequently hydrogen peroxide to induce metal-dependent DNA damage.

NADH-mediated DNA damage induced by a new coenzyme, pyrroloquinoline quinone, in the presence of copper(II) ion

Pyrroloquinoline quinone (PQQ) plays a role as a vitamin or growth factor. Low concentrations of PQQ induced DNA cleavage sites frequently at thymine and cytosine residues in the presence of NADH and Cu(II). Catalase and bathocuproine inhibited DNA damage, whereas free hydroxyl radical scavengers did not. Electron spin resonance and UV‐visible spectrometries showed generation of semiquinone radical and superoxide during the reaction of PQQ with NADH. These results suggest that NADH‐dependent PQQ redox cycle generated superoxide and hydrogen peroxide to mediate copper‐dependent DNA damage. The present study has proposed a requirement to investigate the potentiality of PQQ carcinogenicity.