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

High-Performance Liquid Chromatography for Analysis of 32P-Postlabeled DNA Adducts Magnus Zeisig Center for Nutrition and Toxicology, Department of Bioscience at Novum, Karolinska Institutet, Novum, S-141 57 Huddinge, SwedenThe formation of DNA adducts, i.e. the covalent binding of chemicals and chemical groups to DNA,isbelieved to be an important step in chemical carciwgenesis. DNA adducts are usually formed at very lowlevels which requires very sensitive methods to analyze them. One of the most sensitive ways to analyzeDNA 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. Chromatographicseparation is usually performed by multi-directional thin-layer chromatography (TLC), followed byautoradiography, 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 equipmentand offers high sensitivity and a rather high resolution of adducts. HPLC analysis usually offers a higherresolution and a better reproducibility but a lower sensitivity than TLC analysis. The HPLC equipment isalso rather expensive.To facilitate analysis of DNA adducts a new 32P-HPLC method for analysis of 32P-postlabeled DNAadducts was developed. It employed a reverse-phase column, a high salt (2 M ammonium formate) eluentwith a gradient of acetonitrile, and on-line radioactivity detection. The method required no extra samplepreparations 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 normalDNA compounds and lipophilic DNA adducts formed in vitro, in laboratory animals and in humans. Thesensitivity was approximately I adduct per 109 normal nucleotides both in vitro and in vivo. Polar andlipophilic compounds were resolved in the same analysis. The resolving power enabled the detection of 21DNA adducts or a total of 51 DNA components in one analysis. The resolution could be further enhancedto allow for the detection of 108 DNA components in one sample by repeated analyses, or for separationof DNA adduct stereo-isomers. 32P-HPLC was used for characterization of the major DNA adduct from theair pollutant 2-nitrofluorene in rat, and to study the effect of variations in workup procedunes on analysis.It was used for analysis, partial characterization and comparisons of DNA adducts from a wide variety ofsubstances in vitro and in vivo. Seasonal variations of diffenent DNA adducts or groups of DNA adducts inhumans were also studied by 32P-HPLC.Key Words: DNA adducts, 32P-postlabeling, HPLC, method development ISBN 91-628-2028-