ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 334 (2004) 390–400 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.08.006 IdentiWcation and quantitation of benzo[a]pyrene-derived DNA adducts formed at low adduction level in mice lung tissue  Marzena Banasiewicz a , Garret Nelson b , Adam Swank b , Nenad Grubor a , JeVrey Ross b , Stephen Nesnow b , Harald Köfeler c , Gerald J. Small a , Ryszard Jankowiak a,¤ a Ames Laboratory–USDOE and Department of Chemistry, Iowa State University, Ames, IA 50011, USA b Environmental Carcinogenesis Division, US Environmental Protection Agency, Research Triangle Park, NC 27711, USA c Department of Chemistry, Washington University, St. Louis, MO 63130, USA Received 23 June 2004 Available online 17 September 2004 Abstract The two major metabolic pathways of benzo[a]pyrene (BP) that lead to DNA lesions are monooxygenation that results in diolep- oxides (BPDE) and one-electron oxidation that yields a BP radical cation. These pathways result in formation of stable and depuri- nating DNA adducts, respectively. Most in vivo animal studies with BP, however, have employed dosage/DNA adduct levels several orders of magnitude higher than the DNA damage level expected from environmentally relevant exposures. Presented are results of experiments in which A/J strain mice were intraperitoneally exposed to 50-g/g doses of BP. It is shown that non-line-narrowed Xuo- rescence and Xuorescence line-narrowing spectroscopies possess the selectivity and sensitivity to distinguish between helix-external, base-stacked, and intercalated conformations of DNA–BPDE adducts formed in lung tissue. Concentrations measured by 32 P post- labeling 2 and 3 days after intraperitoneal injection were 420–430 and 600–830 amol BPDE-type adducts per g DNA. The external and base-stacked conformations are attributed mainly to (+)-trans-anti-BPDE-N 2 dG and the intercalated conformations to (+)-cis- anti adducts. A stable adduct derived from 9-OH-BP-4,5-epoxide was also detected at a concentration about a factor of 10 lower than the above concentrations. The DNA supernatants were analyzed for the presence of depurinating BP-derived adducts by capil- lary electrophoresis laser-induced Xuorescence and high-performance liquid chromatography mass spectrometry.  2004 Elsevier Inc. All rights reserved. Keywords: Benzo[a]pyrene; Benzo[a]pyrene diolepoxide; Fluorescence line-narrowing spectroscopy; DNA adducts It is generally held that an essential, primary event in chemical carcinogenesis is covalent binding of the car- cinogen or its metabolites to DNA [1,2]. In this regard, benzo[a]pyrene (BP), 1 a ubiquitous and potent environ- mental carcinogen, has long been the subject of intensive studies [3–5]. DNA adducts derived from polycyclic  The research described in this article has been reviewed by the National Health and Environmental EVects Research Laboratory, US Environ- mental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reXect the views and the policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. * Corresponding author. Fax: +1 515 294 1699. E-mail address: jankowiak@ameslab.gov (R. Jankowiak). 1 Abbreviations used: AP, apurinic; BP, benzo[a]pyrene; BPDE, benzo[a]pyrene diolepoxide; dG, deoxyguanosine, BP-6-N7Gua, (benzo[a]pyren-6- yl)N7-guanine; BP-6-N7Ade, (benzo[a]pyren-6-yl)N7-adenine; BP-6-C8Gua, (benzo[a]pyren-6-yl)C8-guanine; BP-6-N3Ade, (benzo[a]pyren-6-yl)N3- adenine; anti-trans-BPDE-dG, 7R,8S,9S-trihydroxy-10S-(N 2 deoxyguanosyl-3'-phosphate)-7,8,9,10-tetrahydrobenzo[a]pyrene; FLNS, Xuorescence line-narrowing spectroscopy; i.p., intraperitoneal; LIF, laser-induced Xuorescence; MS, mass spectrometry; NLN, non-line-narrowed; PAH, polycy- clic aromatic hydrocarbon; SPE, solid-phase extraction; DOSS, dioctyl sulfosuccinate; DMSO, dimethyl sufoxide; ACN, acetonitrile; DCM, dichlo- romethane; CE, capillary electrophoresis; ZPL, zero-phonon line.