Collect. Czech. Chem. Commun. 1999, 64, 1335-1347

32P-Postlabelling Analysis of DNA Adducts with 1-(Phenylazo)-2-naphthol (Sudan I, Solvent Yellow 14) Formed in vivo in Fisher 344 Rats

Marie Stiborov√°a,*, Heinz H. Schmeiserb, Andrea Breuerb and Eva Freib

a Department of Biochemistry, Charles University, 128 40 Prague 2, Czech Republic
b Department of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany


We report the analysis of DNA adducts with 1-(phenylazo)-2-naphthol in the liver and urinary bladder of Fisher 344 rats treated orally with this dye. DNA adducts were detected and quantitated using the nuclease P1-enhanced version of the 32P-postlabelling assay. Two variations of multidirectional chromatographic systems were used to resolve either bulky and/or smaller (polar) 32P-labelled adducts by TLC. In the present study, a double oral administration of the dye (500 mg/kg) for one day yielded negative results in 32P-postlabelling assay of liver DNA (24 h after dosing). However, three DNA adducts in the urinary bladder were detected under the same conditions of treatment. Chromatography experiments indicated that the two principal DNA adducts detected in the urinary bladder of Fisher 344 rats were the same as those detected in DNA modified by 1-(phenylazo)-2-naphthol and its metabolite 1-(phenylazo)naphthalene-2,6-diol after their activation with peroxidase in vitro. The results presented here strongly suggest that peroxidase itself or in a combination with cytochrome P450 participates in the initiation phase of 1-(phenylazo)-2-naphthol carcinogenesis in the urinary bladder.

Keywords: Carcinogens; Carcinogenesis; Azo dyes; Sudan I; DNA adducts; 32P-postlabelling; Nucleotides; CYP450; Peroxidases.