Time Course of Hepatic 1-Methylpyrene DNA Adducts in Rats Determined by Isotope Dilution LC-MS/MS and 32P-Postlabeling

The alkylated polycyclic aromatic hydrocarbon 1-methylpyrene is a carcinogen in rodents and has been detected in various environmental matrices and foodstuffs. It is activated metabolically by benzylic hydroxylation to 1-hydroxymethylpyrene followed by sulfoconjugation to yield electrophilic 1-sulfooxymethylpyrene (1-SMP) that is prone to form DNA adducts. An LC-MS/MS method using multiple reaction monitoring (MRM) of fragment ions has been developed for specific detection and quantification of N-2-(1-methylpyrenyl)-2'-deoxyguanosine(MP-dGuo) and N-6-(1-methylpyrenyl)-2'-deoxyadenosine (MP-dAdo) formed in DNA in the presence of 1-SMP. DNA samples were spiked with stable isotope internal standards, [N-15(5), C-13(10)]MP-dGuo and [N-15(5)]MP-dAdo, followed by enzymatic digestion to 2'-deoxynucleosides and solid-phase extraction to remove unmodified 2'-deoxynucleosides prior to analysis by LC-MS/MS. The limits of detection were 10 fmol of MP-dGuo and 2 fmol of MP-dAdo or three molecules of MP-dGuo and 0.6 molecules of MP-dAdo per 10(8) 2'-deoxynucleosides using 100 mu g of herring sperm DNA as the sample matrix. The method was validated with herring sperm DNA reacted with 1-SMP in vitro. Hepatic DNA was analyzed from rats that were dosed intraperitoneally with 9.3 mg 1-SMP per kg body weight and killed after various time periods. Levels of MP-dGuo and MP-dAdo in rat liver were found to increase, reaching their maxima at similar to 3 h, and then decrease over time. A good correlation was observed between the results obtained using LC-MS/MS and MRM and those from P-32-postlabeling. MRM allowed the more precise quantification of specific 1-MP adducts, in addition to a time reduction of the analysis when compared with P-32-postlabeling.