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 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-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 (32)P-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 (32)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 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.
Devanesan
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P
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, Nebraska 68198-6805, USA.
Ariese
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F
Jankowiak
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Small
G J
GJ
Rogan
E G
EG
Cavalieri
E L
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D000818:Animals; D001345:Autoradiography; D001580:Benzopyrenes / Q000737:chemistry*; D002273:Carcinogens / Q000737:chemistry*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018736:DNA Adducts / Q000032:analysis / Q000302:isolation & purification*; D003838:Deoxyadenine Nucleotides / Q000032:analysis; D003848:Deoxyguanine Nucleotides / Q000032:analysis; D004852:Epoxy Compounds / Q000737:chemistry*; D005092:Exonucleases / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D051381:Rats; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism
D001580:Benzopyrenes; D002273:Carcinogens; D018736:DNA Adducts; D003838:Deoxyadenine Nucleotides; D003848:Deoxyguanine Nucleotides; D004852:Epoxy Compounds; D010761:Phosphorus Radioisotopes; C121930:dibenzo(a,l)pyrene diol epoxide; C012873:2'-deoxy-5'-adenosine monophosphate; C007257:2'-deoxyguanosine 5'-phosphate; D004247:DNA; D005092:Exonucleases; C022650:spleen exonuclease; D008836:Micrococcal Nuclease; C041517:dibenzo(a,l)pyrene