A novel method for the isolation and identification of stable DNA adducts formed by dibenzo[a,l]pyrene and dibenzo[a,l]pyrene 11,12-dihydrodiol 13,14-epoxides in vitro

Our laboratory previously reported the identification and quantification of depurinating DNA adducts of dibenzo[a,l]pyrene (DB[a,l]P) in vitro, which comprise about 84% of all the DNA adducts that are formed [Li, K.-M., et al . (1995) Biochemistry 34, 8043-8049]. To determine a complete adduct profil e and identify both stable and depurinating DNA adducts, we have developed a relatively simple, nonradioactive method for the identification of stable DNA adducts by combining enzymatic digestion, HPLC, and fluorescence line- narrowing spectroscopy (FLNS) techniques. Calf thymus DNA, bound to either (+/-)-anti- or (+/-)-syn-DB[a,l]PDE or rat liver microsome-activated DB[a,l ]P, was first digested to 3'-mononucleotides with micrococcal nuclease and spleen phosphodiesterase. The adducts were then separated by HPLC with an i on-pair column and identified by FLNS by using the spectra of standards for comparison. In reactions with (+/-)-anti-DB[a,l]PDE, three adducts, an ant i-cis-DB[a,l]PDE-dGMP, an anti-trans-DB[a,l]PDE-dAMP, and an anti-cis-DB[a, l]PDE-dAMP, were identified by HPLC and FLNS. In reactions with (+/-)-syn-D B[a,l]PDE, a pair of syn-trans-DB[a,l]PDE-dGMP adducts as well as a syn-cis -DB[a,l]PDE-dGMP, a syn-cis-DB[a,l]PDE-dAMP, and a pair of syn-trans-DB[a,l ]PDE-dAMP adducts were identified. From the digest of microsome-activated D B[a,l]P-bound DNA, a syn-tl ans-DB[a,l]PDE-dGMP, an anti-cis-DB[a,l]PDE-dGM P, a syn-trans-DB[a,l]PDE-dAMP, and a syn-cis-DB[a,l]PDE-dAMP adduct were i dentified. An anti-cis-DB[a,l]PDE-dAMP adduct was identified only by P-32-p ostlabeling. A total of five of the stable adducts formed by DB[a,l]P and n ine of the stable adducts formed by DB[a,l]PDE in vitro have been identifie d. These adducts were also correlated to adduct spots in the P-32-postlabel ing method by cochromatography with standards. Approximately 93% of the sta ble adducts formed in reactions with (+/-)-anti-DB[a,l]PDE, 90% of adducts with (+/-)-syn-DB[a,l]PDE, and 85% of adducts formed with microsome-activat ed DB[a,l]P have been identified as Gua or Ade adducts. Equal amounts of st able Gua and Ade adducts were observed in the microsome-catalyzed binding o f DB[a,l]P to calf thymus DNA, while 1.4 times more Gua adducts than Ade ad ducts were obtained in reactions with (+/-)-anti- or (+/-)-syn-DB[a,l]PDE.