Reversed-phase high-performance liquid chromatographic separation of P-32-labeled nucleotide adducts formed by food-derived carcinogenic aminoimidazoazarenes

Carcinogenic heterocyclic amines are formed on the surface of grilled or fr ied meat and fish. These heterocyclic amines have been shown to form the co valent DNA adducts considered to be the first stage of the process of carci nogenesis. In this paper we describe the separation of DNA adducts formed b y six food-derived mutagenic aminoimidazoazarenes. P-32-Postlabeling is the most sensitive technique for analysis of DNA adducts formed by covalent bo nding of chemical carcinogens to DNA. An improved method developed by combi ning ion-exchange thin-layer chromatography with reversed-phase high-perfor mance liquid chromatography enabled the resolution of the P-32-labeled mono phosphate nucleotides modified with reactive derivatives of these food muta gens. Optimum conditions for the separation were obtained by use of a Zorba x phenyl-modified silica gel column and a gradient system consisting of ace tonitrile and a phosphate buffer of high ionic strength (0.5 M) at pH 2.0. Sub-femtomole quantities of labeled adducts were detected with a radioactiv ity flow detector coupled to the HPLC equipment. This method can be used fo r the characterization of DNA adducts detected in human tissues and for ana lysis of mixtures of DNA adducts formed in experiments on combination genot oxic effects of mixtures of food-derived heterocyclic amines.