Metabolism of carcinogenic urethane to nitric oxide is involved in oxidative DNA damage.
Carcinogenic urethane (ethyl carbamate) forms DNA adduct via epoxide, whereas carcinogenic methyl carbamate can not. To clarify a mechanism independent of DNA adduct formation, we examined DNA damage induced by N-hydroxyurethane, a urethane metabolite, using 32P-5'-end-labeled DNA fragments. N-hydroxyurethane induced Cu(II)-mediated DNA damage especially at thymine and cytosine residues. DNA damage was inhibited by both catalase and bathocuproine, suggesting a role for H(2)O(2) and Cu(I) in DNA damage. Free (*) OH scavengers did not inhibit the DNA damage, although methional did inhibit it. These results suggest that reactive species, such as the Cu(I)-hydroperoxo complex, cause DNA damage. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) was increased by N-hydroxyurethane in the presence of Cu(II). When treated with esterase, N-hydroxyurethane induced 8-oxodG formation to a similar extent as that induced by hydroxylamine. Enhancement of DNA cleavages by endonuclease IV suggests that hydroxylamine induced depurination. Furthermore, hydroxylamine induced a significant increase in 8-oxodG formation in HL-60 cells but not in its H(2)O(2)-resistant clone HP 100 cells. o-Phenanthroline significantly inhibited the 8-oxodG formation in HL-60 cells, confirming the involvement of metal ions in the 8-oxodG formation by hydroxylamine. Electron spin resonance spectroscopy, utilizing Fe[N-(dithiocarboxy)sarcosine](3), demonstrated that nitric oxide (NO) was generated from hydroxylamine and esterase-treated N-hydroxyurethane. It is concluded that urethane may induce carcinogenesis through oxidation and, to a lesser extent, depurination of DNA by its metabolites.
Department of Hygiene, Mie University School of Medicine, Mie, Japan.
Katsuhisa
K
Sakano
Shinji
S
Oikawa
Yusuke
Y
Hiraku
Shosuke
S
Kawanishi
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D002273:Carcinogens; D002417:Cattle; D002460:Cell Line; D004247:DNA / Q000187:drug effects; D004249:DNA Damage; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004950:Esterases / Q000378:metabolism; D005609:Free Radicals; D006801:Humans; D008954:Models, Biological; D009569:Nitric Oxide / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D013950:Thymus Gland / Q000378:metabolism; D014520:Urethane / Q000031:analogs & derivatives* / Q000378:metabolism*
D002273:Carcinogens; D005609:Free Radicals; D009569:Nitric Oxide; D014520:Urethane; C011286:N-hydroxyurethane; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D004950:Esterases; D003849:Deoxyguanosine; D010100:Oxygen