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. Calfthymus DNA, bound to either (+_)-anti- or (+_)-syn-DB[a,l]PDE or rat liver microsome-activated DB[a,l]P, was first digested to 3ʹ-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]PDʹE-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-transDB[a,l]PDE-dAMP adducts were identified. From the digest of microsome-activated DB[a,Z]Pbound DNA, a syn-trans-DB[a,l]PDE-dGMP, an anti-cis-DB[a,l]PDE-dGMP, a syn-transDB[a,l]PDE-dAMP, and a syn-cis-DB[a,l]PDE-dAMP adduct were identified. An anti-cisDB[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 ^P-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 calfthymus DNA, while 1.4 times more Gua adducts than Ade adducts were obtained in reactions with (±)-anti- or (+_)-syn-DB[a,l]PDE.