High-performance liquid chromatography for analysis of 32P-postlabeled DNA adducts

Abstract: The formation of DNA adducts, i.e. the covalent binding of chemicals and chemical groups to DNA, is believed to be an important step in chemical carciogenesis. DNA adducts are usually formed at very low levels which requires very sensitive methods to analyze them. One of the most sensitive ways to analyze DNA adducts is the 32P-postlabeling assay, i.e. isolation and digestion of DNA followed by enrichment, 32P-labeling and chromatographic separation of the resulting labeled nucleotide adducts. Chromatographic separation is usually performed by multi-directional thin-layer chromatography (TLC), followed by autoradiography, but high-performance liquid chromatography (HPLC) with radioactive detection on-lineor in collected fractions of the HPLC eluate is also used. TLC analysis requires little expensive equipment and offers high sensitivity and a rather high resolution of adducts. HPLC analysis usually offers a higher resolution and a better reproducibility but a lower sensitivity than TLC analysis. The HPLC equipment is also rather expensive. To facilitate analysis of DNA adducts a new 32P-HPLC method for analysis of 32P-postlabeled DNA adducts was developed. It employed a reverse-phase column, a high salt (2 M ammonium formate) eluent with a gradient of acetonitrile, and on-line radioactivity detection. The method required no extra sample preparations compared to TLC analysis and could be coupled with an autosampler, thereby requiring aminimum of handling of the 32P-postlabeled samples. The 32P-HPLC method enabled analyses of normal DNA compounds and lipophilic DNA adducts formed in vitro, in laboratory animals and in humans. The sensitivity was approximately I adduct per 109 normal nucleotides both in vitro and in vivo. Polar and lipophilic compounds were resolved in the same analysis. The resolving power enabled the detection of 21 DNA adducts or a total of 51 DNA components in one analysis. The resolution could be further enhanced to allow for the detection of 108 DNA components in one sample by repeated analyses, or for separation of DNA adduct stereo-isomers. 32P-HPLC was used for characterization of the major DNA adduct from the air pollutant 2-nitrofluorene in rat, and to study the effect of variations in workup procedures on analysis. It was used for analysis, partial characterization and comparisons of DNA adducts from a wide variety of substances in vitro and in vivo. Seasonal variations of different DNA adducts or groups of DNA adducts in humans were also studied by 32P-HPLC.