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 qu antification of DNA adducts was developed using affinity-purified, pol yclonal antibodies directed against O6-methylguanosine (O6-meGuo) coup led to bovine serum albumin (BSA). The specificity of the antibodies w as characterized by competitive inhibition assay using a number of nuc leosides, nucleobases and their analogues. The O6-methylguanine (O6-me G) 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 amplificatio n by the avidin-biotin (AB) system. The low, 5 mM NDMA, dose induced a low cell cytotoxicity and the highest formation of the O6-meG-DNA add uct. Thus, an inverse dose-response correlation was obtained by both m ethods for the cell viability determined as a function of NDMA concent ration and subsequent formation of the O6-meG-DNA adducts, reflecting possibly the involvement of active cell metabolism in enzymatic activa tion of NDMA. Quantitation of the adduct formation vs concentration of NDMA used for the incubation of cells, expressed in pg O6-meG/mug DNA , showed a good correlation (r = 0.992) for both analytical methods.