title: Detection and quantitation of benzo[a]pyrene-derived DNA adducts in mouse liver by liquid chromatography-tandem mass spectrometry: comparison with 32P-postlabeling.
abstract: The polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P) is a proven animal carcinogen that is potentially carcinogenic to humans. B[a]P is an ubiquitous environmental pollutant and is also present in tobacco smoke, coal tar, automobile exhaust emissions, and charred food. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using electrospray ionization and selected reaction monitoring (SRM) has been developed for the detection of 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE-N(2)dG) adducts formed in DNA following the metabolic activation of B[a]P to benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (B[a]PDE). The method involves enzymatic digestion of the DNA sample to 2'-deoxynucleosides following the addition of a stable isotope internal standard, [(15)N(5)]B[a]PDE-N(2)dG, and then solid phase extraction to remove unmodified 2'-deoxynucleosides prior to analysis by LC-MS/MS SRM. The limit of detection of the method was 10 fmol (approximately 3 B[a]PDE-N(2)dG adducts per 10(8) 2'-deoxynucleosides) using 100 microg of calf thymus DNA as the matrix. Calf thymus DNA reacted with B[a]PDE in vitro and mouse liver DNA samples at different time points following dosing intraperitoneally with 50, 100, and 200 mg/kg B[a]P was analyzed. Three stereoisomers of the B[a]PDE-N(2)dG adduct were detected following the reaction of calf thymus DNA with B[a]PDE in vitro. The levels of B[a]PDE-N(2)dG DNA adducts in the mice livers were found to increase in a dose-dependent manner with adducts reaching maximal levels at 1-3 days and then gradually decreasing over time but still detectable after 28 days. A very good correlation (r = 0.962, p < 0.001) was observed between the results obtained for the mouse liver DNA samples using LC-MS/MS SRM as compared to those obtained using a (32)P-postlabeling method. However, the levels of adducts observed following (32)P-postlabeling using butanol enrichment were approximately 3.7-fold lower. The LC-MS/MS method allowed the more precise quantitation of DNA adduct levels that were structurally characterized, in addition to a reduction in the time taken to perform the analysis when compared with the (32)P-postlabeling method.
authors.affiliation: Cancer Biomarkers and Prevention Group, Biocenter, University of Leicester, UK. rs25@le.ac.uk
authors.forename: Rajinder
authors.initials: R
authors.lastname: Singh
authors.forename: Margaret
authors.initials: M
authors.lastname: Gaskell
authors.forename: Rachel C
authors.initials: RC
authors.lastname: Le Pla
authors.forename: Balvinder
authors.initials: B
authors.lastname: Kaur
authors.forename: Ali
authors.initials: A
authors.lastname: Azim-Araghi
authors.forename: Jonathan
authors.initials: J
authors.lastname: Roach
authors.forename: George
authors.initials: G
authors.lastname: Koukouves
authors.forename: Vassilis L
authors.initials: VL
authors.lastname: Souliotis
authors.forename: Soterios A
authors.initials: SA
authors.lastname: Kyrtopoulos
authors.forename: Peter B
authors.initials: PB
authors.lastname: Farmer
mesh_terms: D000818:Animals; D001564:Benzo(a)pyrene / Q000737:chemistry*; D002417:Cattle; D004247:DNA / Q000737:chemistry; D018736:DNA Adducts / Q000737:chemistry* / Q000235:genetics*; D003849:Deoxyguanosine / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D008099:Liver / Q000737:chemistry* / Q000378:metabolism*; D008297:Male; D051379:Mice; D015394:Molecular Structure; D010761:Phosphorus Radioisotopes
chemical_list: D018736:DNA Adducts; D010761:Phosphorus Radioisotopes; D001564:Benzo(a)pyrene; D004247:DNA; C427203:calf thymus DNA; D003849:Deoxyguanosine