Haruo Kiyosawa

Cigarette smoking induces formation of 8-hydroxydeoxyguanosine, one of the oxidative DNA damages in human peripheral leukocytes

Active oxygen species (AOS) such as O2- and H2O2 have been shown to be generated from both gas and tar phases of cigarette smoke and it has been suggested that they are involved in carcinogenesis due to cigarette smoking. Therefore, we investigated the effect of cigarette smoking on oxidative DNA damages in human peripheral blood cells using 8-hydroxydeoxy-guanosine (8-OH-dG) as a marker. From ten healthy male volunteers aged 20-22 years, 5 ml of blood was taken before and 10 minutes after smoking 2 cigarettes in 10 minutes. After lysis of blood cell membranes leukocyte DNA was isolated using a DNA extractor and 8-OH-dG levels were determined using high performance liquid chromatography (HPLC) with electrochemical detection. The mean levels of 8-OH-dG increased significantly (P less than 0.05) from 3.3 +/- 0.8/10(6) dG (mean +/- SD) to 5.1 +/- 2.5 after smoking. These results indicate that cigarette smoking induces oxidative DNA damage in peripheral blood cells in a relatively short time.

DNA strand cleavage at 8-hydroxyguanine residues by hot piperidine treatment

The two findings indicate that 8-hydroxyguanine(8-OH-Gua) is a hot piperidine-sensitive lesion in DNA. These are cleavages of DNA containing 8-OH-Gua at the site of this residue and decomposition of 8-hydroxydeoxyguanosine when the DNA and nucleoside were treated in 1 M piperidine for 30 min at 90 degrees C. However, no cleavage was observed in DNA containing 8-hydroxyadenine or O6-methylguanine. 8-OH-Gua was found to be different from apurinic sites that are also alkali-labile lesions since the former was more resistant to alkali treatment. This property of 8-OH-Gua can be used as a check for the incorporation of this base into DNA after the synthesis of DNA containing 8-OH-Gua at a specific position or possibly can be one of the markers for the identification of 8-OH-Gua formed in DNA exposed to reactive oxygen species.

Formation of 8-Hydroxydeoxyguanosine in DNA and Carcinogenesis

Oxygen radicals are produced by ionizing radiation and many other environmental carcinogens. They are also produced endogenously in cells by oxygen metabolism. It seems important therefore to study DNA damage by oxygen radicals and its relation to carcinogenesis.