Role of phenobarbital-inducible cytochrome P450s as a source of active oxygen species in DNA-oxidation

Imaoka, Susumu; Osada, Mayuko; Minamiyama, Yukiko; Yukimura, Tokihito; Toyokuni, Shinya; Takemura, Shigekazu; Hiroi, Toyoko; Funae, Yoshihiko

doi:10.1016/j.canlet.2003.09.009

« Previous Next »Cancer Letters
Volume 203, Issue 2 , Pages 117-125, January 2004

Role of phenobarbital-inducible cytochrome P450s as a source of active oxygen species in DNA-oxidation

Susumu Imaoka
- Department of Chemical Biology, Osaka City University Medical School, Abeno-ku, Osaka, Japan
- Corresponding author. Address: Nanobiotechnology Research center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337, Japan. Fax: +81-79-565-7673
Mayuko Osada
- Department of Chemical Biology, Osaka City University Medical School, Abeno-ku, Osaka, Japan
Yukiko Minamiyama
- Department of Biochemistry, Osaka City University Medical School, Abeno-ku, Osaka, Japan
Tokihito Yukimura
- Department of Pharmacology, Osaka City University Medical School, Abeno-ku, Osaka, Japan
Shinya Toyokuni
- Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan
Shigekazu Takemura
- Department of Surgery, Osaka City University Medical School, Abeno-ku, Osaka, Japan
Toyoko Hiroi
- Department of Chemical Biology, Osaka City University Medical School, Abeno-ku, Osaka, Japan
Yoshihiko Funae
- Department of Chemical Biology, Osaka City University Medical School, Abeno-ku, Osaka, Japan

Received 10 June 2003; received in revised form 22 August 2003; accepted 1 September 2003.

Abstract

We investigated the biological effects of the active oxygen produced by P450s. First, we identified which isoforms of P450 efficiently produced active oxygen using electron spin resonance. Eight forms of P450 purified from rat liver were used. Of these, CYP1A2, 2B1, 2C11 and 3A2 produced hydroxyl radicals efficiently. Phenobarbital (PB) which is a typical inducer of CYP2B1 and 3A2 induced production of hydroxyl radicals by rat liver and ketoconazole, an inhibitor of P450, inhibited production of hydroxyl radicals in vitro. PB is a tumor promoter as well as the P450-inducer. We investigated oxidation of the genomic DNA by the hydroxyl radicals produced by PB-inducible P450 in vitro and in vivo. 8-Hydroxy-2′-deoxyguanosine (8-OHdG), a biomarker of DNA oxidation in vivo was assayed by HPLC. PB strongly induced the production of 8-OHdG in the rat liver. While ketoconazole inhibited the production of 8-OHdG in vivo. These results suggest that active oxygen produced by P450 oxidized genomic DNA and induction of P450 increased oxidative stress that may contribute to tumor initiation and promotion.

Keywords: Cytochrome P450, Superoxide, Hydroxyl radical, 8-Hydroxy-2′-deoxyguanosine, Electron spin resonance

Abbreviations: P450 or CYP, cytochrome P450, PB, phenobarbital, 3-MC, 3-methylcholanthrene, ESR, Electron spin resonance, DMPO, 5,5-dimethyl-1-pyrroline-N-oxide, DPIC, diphenyleneiodonium chloride, SOD, superoxide dismutase, 8-OHdG, 8-hydroxy-2′-deoxyguanosine, 4-HAE, 4-hydroxyalkenal, DLPC, dilauroylphosphatidylcholine