Analysis of 4-aminobiphenyl-DNA adducts in human urinary bladder and lung by alkaline hydrolysis and negative ion gas chromatography-mass spectrometry

Analysis of carcinogen-DNA adducts has been regarded as a useful means of assessing human exposure to chemical carcinogens. We have established a method for quantitation of 4-aminobiphenyl (4-ABP)-DNA adducts by alkaline hydrolysis and gas chromatography with negative ion chemical ionization mass spectrometry (GC-NICI-MS). Aliquots of DNA (typically 100 μg/ml) were spiked with an internal standard,d₉-4-ABP, and were hydrolyzed in 0.05 N NaOH at 130°C overnight. The liberated 4-ABP was extracted with hexane and derivatized using pentafluoropropionic anhydride in trimethylamine for 30 min at room temperature prior to GC-NICI-MS. With in vitro [³H]Ahydroxy-4-ABP modified DNA standards, we observed 59 ± 7% (n = 9) recovery of the 4-ABP and a linear correlation between hydrolyzed 4-ABP and the adduct levels ranging from about 1 in 10⁸ to 1 in 10⁴ nucleotides (r = 0.999, n = 9). The method was further validated by comparison of the results with that obtained by the ³²P-postlabeling method. There was excellent agreement (r = 0.994, p<0.001) between the two methods for quantitation of the adduct in eight samples of Salmonella typhimurium DNA treated with 4-ABP and rat liver S9, although the ³²P-postlabeling method gave slightly higher values. The DNA adducts in 11 human lung and 8 urinary bladder mucosa specimens were then determined by our GC-NICI-MS method. The adduct levels were found to be <0.32 to 49.5 adducts per 10⁸ nucleotides in the lungs and <0.32 to 3.94 adducts per 10⁸ nucleotides in the bladder samples. Our results indicate that the alkaline hydrolysis/GC-NICI-MS method is sensitive, structure-selective, and accurate, and will be useful for molecular dosimetry of human exposure to this carcinogen.