Time course of hepatic 1-methylpyrene DNA adducts in rats determined by isotope dilution LC-MS/MS and 32P-postlabeling.
ABSTRACT 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, [ (15)N 5, (13)C 10]MP-dGuo and [ (15)N 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 mug 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 approximately 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 (32)P-postlabeling. MRM allowed the more precise quantification of specific 1-MP adducts, in addition to a time reduction of the analysis when compared with (32)P-postlabeling.
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ABSTRACT: DNA adducts represent an important category of biomarkers for detection and exposure surveillance of potential carcinogenic and genotoxic chemicals in the environment. Sensitive and specific analytical methods are required to detect and differentiate low levels of adducts from native DNA from in vivo exposure. In addition to biomonitoring of environmental pollutants, analytical methods have been developed for structural identification of adducts which provides fundamental information for determining the toxic pathway of hazardous chemicals. In order to achieve the required sensitivity, mass spectrometry has been increasingly utilized to quantify adducts at low levels as well as to obtain structural information. Furthermore, separation techniques such as chromatography and capillary electrophoresis can be coupled to mass spectrometry to increase the selectivity. This review will provide an overview of advances in detection of adducted and modified DNA by mass spectrometry with a focus on the analysis of nucleosides since 2007. Instrument advances, sample and instrument considerations, and recent applications will be summarized in the context of hazard assessment. Finally, advances in biomonitoring applying mass spectrometry will be highlighted. Most importantly, the usefulness of DNA adducts measurement and detection will be comprehensively discussed as a tool for assessment of in vitro and in vivo exposure to environmental pollutants.Talanta 12/2014; 130C:475-494. · 3.50 Impact Factor
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ABSTRACT: The common polycyclic aromatic hydrocarbon 1-methylpyrene is hepatocarcinogenic in the newborn mouse assay. In vitro studies showed that it is metabolically activated via benzylic hydroxylation and sulphation to a reactive ester, which forms benzylic DNA adducts, N (2)-(1-methylpyrenyl)-2'-deoxyguanosine (MPdG) and N (6)-(1-methylpyrenyl)-2'-deoxyadenosine (MPdA). Formation of these adducts was also observed in animals treated with the metabolites, 1-hydroxymethylpyrene and 1-sulphooxymethylpyrene (1-SMP), whereas corresponding data are missing for 1-methylpyrene. In the present study, we treated mice with 1-methylpyrene and subsequently analysed blood serum for the presence of the reactive metabolite 1-SMP and tissue DNA for the presence of MPdG and MPdA adducts. We used wild-type mice and a mouse line transgenic for human sulphotransferases (SULT) 1A1 and 1A2, males and females. All analyses were conducted using ultra-performance liquid chromatography coupled with tandem mass spectrometry, for the adducts with isotope-labelled internal standards. 1-SMP was detected in all treated animals. Its serum level was higher in transgenic mice than in the wild-type (p < 0.001). Likewise, both adducts were detected in liver, kidney and lung DNA of all exposed animals. The transgene significantly enhanced the level of each adduct in each tissue of both sexes (p < 0.01-0.001). Adduct levels were highest in the liver, the target tissue of carcinogenesis, in each animal model used. MPdG and MPdA adducts were also observed in rats treated with 1-methylpyrene. Our findings corroborate the hypothesis that 1-SMP is indeed the ultimate carcinogen of 1-methylpyrene and that human SULT are able to mediate the terminal activation in vivo.Archives of Toxicology 12/2013; · 5.22 Impact Factor
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ABSTRACT: Japanese medaka (Oryzias latipes) embryos were exposed to sediments spiked with environmental concentrations (300 and 3,000 ng/g dry weight) of pyrene (Pyr) and methylpyrene (MePyr) throughout their development. Embryotoxicity, teratogenicity, and transcriptional responses (qRT-PCR) were analyzed in embryos and newly hatched larvae. The genotoxicity of the two polycyclic aromatic hydrocarbons (PAHs) was also tested in prolarvae using the comet assay. Exposure to each compound had a clear impact on embryonic development and resulted in several teratogenic effects, including cardiovascular injuries, reduced absorption of yolk sac reserves, and jaw and spinal deformities. Interestingly, the overall toxic effects of Pyr and MePyr considerably overlapped those induced following dioxin exposure. qRT-PCR analysis revealed the transcriptional induction of genes involved in mitochondrial energetic metabolism (coxI), xenobiotic biotransformation (cyp1a), and cell cycle regulation (wnt1) by the two PAHs. MePyr also activated cell cycle arrest (p53), oxidative DNA damage repair (ogg1), and retinoid-mediated (raldh2 and rarα1) gene transcription. DNA damage was not found to be significantly increased following Pyr and MePyr exposure. The lack of significant genotoxic effect in comparison to the control might be the consequence of the efficient onset of DNA damage repair mechanisms as suggested by ogg1 gene transcription upregulation. Results reported in the present study have brought new insights into the modes of action of Pyr, and the effects of MePyr exposure have been investigated in fish ELS for the first time.Environmental Science and Pollution Research 04/2014; · 2.76 Impact Factor