Evidence for activation of carcinogenic o-anisidine by prostaglandin H synthase: 32P-postlabelling analysis of DNA adduct formation.
2-Methoxyaniline (o-anisidine) is a urinary bladder carcinogen in both mice and rats. Since the urinary bladder contains substantial peroxidase activity, we examined the ability of prostaglandin H synthase (PHS), a prominent enzyme in the urinary bladder, to activate this carcinogen to metabolites binding to macromolecules. Using [14C]-labeled o-anisidine, we observed substantial PHS-dependent binding of o-anisidine to protein, DNA and polydeoxyribonucleotides [poly(dX)]. This binding is inhibited by radical scavengers glutathione, ascorbate and NADH. The nuclease P1 and 1-butanol extraction enrichment procedure of the 32P-postlabeling analysis of DNA modified by activated o-anisidine provide evidence that covalent binding to DNA is the principal type of DNA modification. Deoxyguanosine is determined to be the major target for binding of o-anisidine in DNA. The possibility that o-anisidine is carcinogenic to the rodent urinary bladder via its activation by bladder PHS is suggested. The results presented here are the first report demonstrating a PHS-mediated activation of o-anisidine to reactive species forming covalent DNA adducts.
Stiborová
M
M
Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic. stiborov@prfdec.natur.cuni.cz
Schmeiser
H H
HH
Breuer
A
A
Frei
E
E
D000814:Aniline Compounds; D000818:Animals; D002273:Carcinogens; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism; D018736:DNA Adducts; D016166:Free Radical Scavengers; D006735:Horseradish Peroxidase / Q000378:metabolism; D008297:Male; D008861:Microsomes / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D051381:Rats; D012669:Seminal Vesicles; D001749:Urinary Bladder Neoplasms / Q000139:chemically induced*
D000814:Aniline Compounds; D002273:Carcinogens; D018736:DNA Adducts; D016166:Free Radical Scavengers; D004247:DNA; D006735:Horseradish Peroxidase; D011451:Prostaglandin-Endoperoxide Synthases; C003898:2-anisidine