title: Improved high-performance liquid chromatography analysis of 32P-postlabeled 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-DNA adducts using in-line precolumn purification.
abstract: An improved HPLC-based 32P-postlabeling assay has been developed for the analysis of DNA modified with the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Postlabeled samples are loaded onto a C18 precolumn and adducted bases are retained while excess radioactivity and unmodified DNA bases are eluted directly to waste through a switching valve. The use of this HPLC in-line precolumn purification (HIPP) technique allows entire postlabeled samples to be analyzed without prior removal of inorganic phosphate and unmodified DNA bases. The method has a sample to sample precision of 15% and accuracy of 20%, at adduct levels of 2 adducts/10(7) bases and shows a linear relationship between signal and adduction levels from 1 adduct per 10(4) to approximately 2 +/- 1 adducts per 10(9) bases. Individual postlabeled DNA samples can be analyzed by HPLC in less than 1 h, allowing high throughput. The use of calf-thymus DNA (CT-DNA), highly modified with PhIP, or DNA isolated from mice chronically fed a PhIP-modified diet shows two major PhIP-DNA adduct peaks and three additional minor adduct peaks when labeled under ATP-limiting conditions. Isolation of the HPLC purified peaks and analysis by thin layer chromatography (TLC) matches the five HPLC peaks to the spots typically seen by TLC, including N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (dG-C8-PhIP). Variations in digestion techniques indicate a potential resistance of the PhIP-DNA adducts to the standard enzymatic digestion methods. Attempts at adduct intensification by solid phase extraction, nuclease P1 enrichment or 1-butanol extraction decreased PhIP-DNA adduct peaks and introduced a large early eluting peak. Removal of the 3'-phosphate with nuclease P1 following the kinase labeling reaction simplifies the HPLC profile to one major peak (dG-C8-PhIP monophosphate) with several minor peaks. In addition to the high resolution provided by HPLC separation of the PhIP-DNA adducts, this method can be adjusted for analysis of other DNA adducts and is readily automated for high throughput.
authors.affiliation: Biology and Biotechnology Program, Lawrence Livermore National Laboratory, CA 94550, USA.
authors.forename: R J
authors.initials: RJ
authors.lastname: Mauthe
authors.forename: G A
authors.initials: GA
authors.lastname: Marsch
authors.forename: K W
authors.initials: KW
authors.lastname: Turteltaub
mesh_terms: D000818:Animals; D002273:Carcinogens / Q000737:chemistry*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation*; D002855:Chromatography, Thin Layer; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D018736:DNA Adducts / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D007093:Imidazoles / Q000737:chemistry*; D051379:Mice; D008836:Micrococcal Nuclease / Q000378:metabolism; D009153:Mutagens / Q000737:chemistry*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013950:Thymus Gland / Q000737:chemistry
chemical_list: D002273:Carcinogens; D018736:DNA Adducts; D007093:Imidazoles; D009153:Mutagens; D010761:Phosphorus Radioisotopes; D004247:DNA; C049584:2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine; D008836:Micrococcal Nuclease; D010727:Phosphoric Diester Hydrolases