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

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• 作者：Bernhard H. Monien ; Carolin Mü ; ller ; Wolfram Engst ; Heinz Frank ; Albrecht Seidel ; Hansruedi Glatt
• 刊名：Chemical Research in Toxicology
• 出版年：2008
• 出版时间：October 20, 2008
• 年：2008
• 卷：21
• 期：10
• 页码：2017-2025
• 全文大小：264K
• 年卷期：v.21,no.10(October 20, 2008)
• ISSN：1520-5010

文摘

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 N²-(1-methylpyrenyl)-2′-deoxyguanosine (MP-dGuo) and N⁶-(1-methylpyrenyl)-2′-deoxyadenosine (MP-dAdo) formed in DNA in the presence of 1-SMP. DNA samples were spiked with stable isotope internal standards, [¹⁵N₅,¹³C₁₀]MP-dGuo and [¹⁵N₅]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 10⁸ 2′-deoxynucleosides using 100 μg 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 ∼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 ³²P-postlabeling. MRM allowed the more precise quantification of specific 1-MP adducts, in addition to a time reduction of the analysis when compared with ³²P-postlabeling.