Role of phenobarbital-inducible cytochrome P450s as a source of active oxygen species in DNA-oxidation.
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.
Imaoka
Susumu
S
Department of Chemical Biology, Osaka City University Medical School, Abeno-ku, Osaka, Japan. imaoka@ksc.kwansei.ac.jp
Osada
Mayuko
M
Minamiyama
Yukiko
Y
Yukimura
Tokihito
T
Toyokuni
Shinya
S
Takemura
Shigekazu
S
Hiroi
Toyoko
T
Funae
Yoshihiko
Y
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism; D019362:Cytochrome P-450 CYP2B1 / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D004247:DNA / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D017665:Hydroxyl Radical; D007120:Immunochemistry; D007150:Immunohistochemistry; D007654:Ketoconazole / Q000494:pharmacology; D015227:Lipid Peroxidation; D008099:Liver / Q000201:enzymology* / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D010634:Phenobarbital / Q000494:pharmacology*; D020033:Protein Isoforms; D016755:Proto-Oncogene Proteins c-jun / Q000096:biosynthesis; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D039181:Steroid 16-alpha-Hydroxylase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism
D018691:Excitatory Amino Acid Antagonists; D008565:Membrane Proteins; D020033:Protein Isoforms; D016755:Proto-Oncogene Proteins c-jun; D016271:Proto-Oncogene Proteins c-myc; D013481:Superoxides; D017665:Hydroxyl Radical; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C450279:CYP2C11 protein, rat; C465260:Cyp3a2 protein, rat; D019388:Cytochrome P-450 CYP1A2; D019362:Cytochrome P-450 CYP2B1; D051544:Cytochrome P-450 CYP3A; D000072467:Cytochrome P450 Family 2; D039181:Steroid 16-alpha-Hydroxylase; D007654:Ketoconazole; D010100:Oxygen; D010634:Phenobarbital