Journal of Chromatography B, 802 (2004) 155–166 Review Formation and analysis of heterocyclic aromatic amine–DNA adducts in vitro and in vivo Robert J. Turesky a,∗ , Paul Vouros b a Division of Chemistry, National Center for Toxicological Research, 3900 NCTR Road, Jefferson, AR 72079, USA b Department of Chemistry and Barnett Institute, Northeastern University, Boston, MA 02115, USA Abstract The detection and quantification of heterocyclic aromatic amine (HAA)–DNA adducts, critical biomarkers in interspecies extrapolation of toxicity data for human risk assessment, remains a challenging analytical problem. The two main analytical methods currently in use to screen for HAA–DNA adducts are the 32 P-postlabeling assay and mass spectrometry, using either accelerated mass spectrometry (AMS) or liquid chromatography and electrospray ionization mass spectrometry (LC–ESI–MS). In this review, the principal methods to synthesize and characterize DNA adducts, and the methods applied to measure HAA–DNA adduct in vitro and vivo are discussed. © 2003 Elsevier B.V. All rights reserved. Keywords: Reviews; Heterocyclic aromatic amine–DNA adducts; DNA Contents 1. Introduction ................................................................................................... 155 2. Genotoxic heterocyclic aromatic amine metabolites and DNA adduct formation ..................................... 157 3. HAA–DNA adducts in experimental animal studies assayed by 32 P-postlabeling .................................... 158 4. HAA–DNA adducts analyses in vitro and in experimental animal studies using mass spectrometry ................... 161 5. HAA–DNA adduct formation in human tissues ................................................................... 162 6. Relevance of HAA–DNA adduct measurements and future directions .............................................. 164 Acknowledgements ................................................................................................. 165 References ......................................................................................................... 165 1. Introduction Heterocyclic aromatic amines (HAAs) induce cancer at multiple sites in experimental animals during long-term feeding studies [1]. Because of their wide spread occurrence in cooked meat products [2], HAAs may contribute to com- mon forms of human cancers including colorectal, prostate, and breast that are associated with frequent consumption of diets high in meats and fat [3–5]. The adduction of genotoxic carcinogens such as HAAs to DNA is believed to be the first step in chemically induced ∗ Corresponding author. Tel.: +1-870-543-7301; fax: +1-870-543-7686. E-mail address: rturesky@nctr.fda.gov (R.J. Turesky). carcinogenesis [6,7], and the identification of biomarkers representing genotoxic damage, such as DNA adducts, may aid in assessing human health risks [7,8]. Over the past sev- eral decades, different analytical methods have been estab- lished to detect and measure DNA adduct formation in exper- imental animals and humans. Early studies in animals used tritium- or radiocarbon-labeled aromatic amine carcinogens to assess DNA binding. Adduct identification was deter- mined by high performance liquid chromatography (HPLC) with co-migration of non-radiolabeled adducts serving as UV markers [9]. A major limitation in these studies was the use of large amounts of radioactivity that precluded investi- gations in humans. More recently Randerath et al. have developed the 32 P-postlabeling assay [10], which uses polynucleotide 1570-0232/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2003.10.053