phosphorus-radioisotopes and 1-phenylazo-2-naphthol

A 32P-postlabeling assay was employed for detection and quantitation of DNA adducts formed with carcinogenic 1-phenylazo-2-hydroxynaphthalene (Sudan I, Solvent Yellow 14) activated by a peroxidase system. Enrichment of adducts by digestion with nuclease P1 or by extraction into n-butanol prior to 32P-labeling was used. Both enrichment procedures exhibited comparable results for recovery of individual DNA adduct spots. Co-chromatographic analyses of adduct spots obtained by reaction with DNA and homopolydeoxy-ribonucleotides showed that four out of the eight major Sudan I-DNA adducts were formed by reaction of activated Sudan I with deoxyadenosine or deoxyguanosine in DNA. The accuracy of quantitation of adducts by 32P-postlabeling procedure is discussed.

Topics: Autoradiography; Carcinogens; Chromatography, Thin Layer; DNA; Naphthols; Phosphorus Radioisotopes; Polydeoxyribonucleotides; Radioisotope Dilution Technique; Single-Strand Specific DNA and RNA Endonucleases

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.

Topics: Animals; Autoradiography; Benzoflavones; beta-Naphthoflavone; Biotransformation; Carbon Radioisotopes; Carcinogens; DNA; Horseradish Peroxidase; Kinetics; Male; Microsomes, Liver; Naphthols; Phosphorus Radioisotopes; Rats; Rats, Inbred Strains