Assessment of N-Nitrosodimethylamine Dna Adducts in Rat Hepatocytes by High Performance Liquid Chromatography and Immunosorbent Assay

A rapid and efficient enzyme-linked immunosorbent assay (ELISA) for quantification of DNA adducts was developed using affinity-purified, polyclonal antibodies directed against O 6 -methylguanosine (O 6 -meGuo) coupled to bovine serum albumin (BSA). The specificity of the antibodies was characterized by competitive inhibition assay using a number of nucleosides, nucleobases and their analogues. The O 6 -methylguanine (O 6 -meG) adduct was quantified in rat hepatocytes pretreated in vitro with N-nitrosodimethylamine (NDMA) by high performance liquid chromatography (HPLC) and compared to the data obtained by ELISA, using amplification by the avidin-biotin (AB) system. The low, 5 mM NDMA, dose induced a low cell cytotoxicity and the highest formation of the O 6 -meG-DNA adduct. Thus, an inverse dose-response correlation was obtained by both methods for the cell viability determined as a function of NDMA concentration and subsequent formation of the O 6 -meG-DNA adducts, reflecting possibly the involvement of active cell metabolism in enzymatic activation of NDMA. Quantitation of the adduct formation vs concentration of NDMA used for the incubation of cells, expressed in pg O 6 -meG/μg DNA, showed a good correlation (r=0.992) for both analytical methods.