Reversed-phase high-performance liquid chromatographic separation of32P-labeled nucleotide adducts formed by food-derived carcinogenic aminoimidazoazarenes

Abstract

Carcinogenic heterocyclic amines are formed on the surface of grilled or fried meat and fish. These heterocyclic amines have been shown to form the covalent DNA adducts considered to be the first stage of the process of carcinogenesis. In this paper we describe the separation of DNA adducts formed by six food-derived mutagenic aminoimidazoazarenes.32P-Postlabeling is the most sensitive technique for analysis of DNA adducts formed by covalent bonding of chemical carcinogens to DNA. An improved method developed by combining ion-exchange thin-layer chromatography with reversed-phase high-performance liquid chromatography enabled the resolution of the32P-labeled monophosphate nucleotides modified with reactive derivatives of these food mutagens. Optimum conditions for the separation were obtained by use of a Zorbax phenyl-modified silica gel column and a gradient system consisting of acetonitrile and a phosphate buffer of high ionic strength (0.5m) at pH 2.0. Sub-femtomole quantities of labeled adducts were detected with a radioactivity flow detector coupled to the HPLC equipment. This method can be used for the characterization of DNA adducts detected in human tissues and for analysis of mixtures of DNA adducts formed in experiments on combination genotoxic effects of mixtures of food-derived heterocyclic amines.