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 profile 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 ion-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 anti- cis-DB[a,l]PDE-dGMP, an antitrans-DB[a,l]PDE-dAMP, and an anti-cis- DB[a,l]PDE-dAMP, were identified by HPLC and FLNS. In reactions with (±)- syn-DB[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 DB[a,l]P-bound DNA, a syn-trans-DB[a,l]PDE-dGMP, an anti-cis- DB[a,l]PDE-dGMP, a syn-trans-DB[a,l]PDE-dAMP, and a syn-cis-DB[a,l]PDE-dAMP adduct were identified. An anti-cis-DB[a,l]PDE-dAMP adduct was identified only by ³²P-postlabeling. A total of five of the stable adducts formed by DB[a,l]P and nine of the stable adducts formed by DB[a,l]PDE in vitro have been identified. These adducts were also correlated to adduct spots in the ³²P-postlabeling method by cochromatography with standards. Approximately 93% of the stable 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-activated DB[a,l]P have been identified as Gua or Ade adducts. Equal amounts of stable Gua and Ade adducts were observed in the microsome- catalyzed binding of DB[a,l]P to calf thymus DNA, while 1.4 times more Gua adducts than Ade adducts were obtained in reactions with (±)-anti- or (±)- syn-DB[a,l]PDE.