Mechanism of formation and 32P-postlabeling of DNA adducts derived from peroxidative activation of carcinogenic non-aminoazo dye 1-phenylazo-2-hydroxynaphthalene (Sudan I).
Horseradish peroxidase in the presence of hydrogen peroxide mediates the activation of carcinogenic 1-phenylazo-2-hydroxynaphthalene (Sudan I) to DNA-bound products in vitro. The peroxidase activating system is greater than 10 times more effective with respect to DNA modification by Sudan I than the microsomal enzymes containing cytochrome P450. The DNA-binding reaction of the Sudan I metabolite(s) formed by the peroxidase system is dependent on Sudan I and H2O2 concentration and pH. Reactive intermediate(s) or product(s) of the Sudan I oxidation by peroxidase with a short half-life are responsible for the DNA modification. DNA modified by peroxidase-activated Sudan I becomes colored and has an absorption maximum at approximately 480 nm. The modification of DNA by Sudan I metabolites(s) formed by the peroxidase system is inhibited by some compounds of physiological importance (ascorbate, glutathione, Mg2+ ions) and by radical trapping agents (nitrosobenzene, methyl viologen). 32P-Postlabeling assay of the DNA modified by Sudan I activated by the peroxidase system indicates that the covalent DNA adduct formation is the principal type of the DNA modification. Four major and several minor adducts of deoxyribonucleotide 3',5'-bisphosphate from DNA with Sudan I metabolite(s) were detected by the classical Randerath 32P-postlabelling assay as well as by the nuclease P1 version of the same method.
Department of Biochemistry, Faculty of Natural Sciences, Charles University Prague, Czechoslovakia.
M
M
Stiborová
E
E
Frei
H H
HH
Schmeiser
M
M
Wiessler
J
J
Hradec
D000818:Animals; D001345:Autoradiography; D001571:Benzoflavones / Q000494:pharmacology; D001711:Biotransformation; D002250:Carbon Radioisotopes; D002273:Carcinogens / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D009284:Naphthols / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D051381:Rats; D011919:Rats, Inbred Strains; D019324:beta-Naphthoflavone
D001571:Benzoflavones; D002250:Carbon Radioisotopes; D002273:Carcinogens; D009284:Naphthols; D010761:Phosphorus Radioisotopes; C024336:1-phenylazo-2-naphthol; D019324:beta-Naphthoflavone; D004247:DNA; D006735:Horseradish Peroxidase