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: The formation of DNA adducts is considered essential for tumor initiation. Quantification of DNA adducts may be achieved by various techniques of which LC-MS/MS-based multiple reaction monitoring has become the most prominent in the past decade. Adducts of single nucleosides are analyzed following enzymatic break-down of a DNA sample following adduct enrichment usually by solid-phase extraction. LC-MS/MS quantification is carried out using stable isotope-labeled internal reference substances. An upcoming challenge is the use of DNA adducts as biomarkers either for internal exposure to electrophilic genotoxins or for the approximation of cancer risk. Here we review recent studies in which DNA adducts were quantified by LC-MS/MS in DNA samples from human matrices. We outline a possible way for future research to aim at the development of an "adductome" approach for the characterization of DNA adduct spectra in human tissues. The DNA adduct spectrum reflects the inner exposure of an individual's tissue to electrophilic metabolites and, therefore, should replace the conventional and inaccurate external exposure in epidemiological studies in the future.Advances in Experimental Medicine and Biology 01/2014; 806:383-397. · 2.01 Impact Factor
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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: 1-Methylpyrene, a carcinogenic polycyclic aromatic hydrocarbon, forms benzylic DNA adducts, in particular N(2)-(1-methylpyrenyl)-2´-deoxyguanosine, in mice and rats. It is bioactivated via 1-hydroxymethylpyrene (1-HMP) to electrophilic 1-sulfooxymethylpyrene (1-SMP). In this study we explored the role of individual mouse sulfotransferase (SULT) forms in this activation. First, we showed that all nine mouse SULTs tested were able to activate 1-HMP to a mutagen in the his(-) Salmonella typhimurium reversion test. Some activation was even observed with Sult2a3 and Sult5a1, orphan forms for which no substrates were identified hitherto. Subsequently we used cytosolic preparations from tissues of four mouse lines (wild-type, Sult1a1(-), Sult1d1(-), transgenic for human SULT1A1/2) for the activation of 1-HMP in the mutagenicity assay. The most prominent impacts of the genetic SULT status were: 96% decrease in hepatic activation by Sult1a1 knockout, 99% decrease in renal activation by Sult1d1 knockout, and 100-fold increase in pulmonary activation by transgenic human SULT1A1/2. Finally, we treated the various mice with 1-HMP (19.3 mg/kg, intraperitoneally), and then determined 1-SMP levels in plasma and DNA adducts in tissues. Transgenic human SULT1A1/2 strongly enhanced 1-SMP plasma levels and DNA adduct formation in liver, lung, heart and kidney, but not in colon. Sult1a1 and Sult1d1 knockout reduced plasma 1-SMP levels as well as DNA adduct formation in some tissues (strongest effects: 97% decrease in 1-SMP and 89% decrease in hepatic adducts in Sult1a1(-) mice). The adducts levels detected in various tissues did not accurately reflect the activation capacity of these tissues determined in vitro, probably due to the distribution of the reactive metabolite 1-SMP via the circulation. In conclusion, we demonstrated that many mouse SULT forms are able to activate 1-HMP. In vivo, we verified a prominent role of Sult1a1 in the hepatic and renal adduct formation and a smaller but unambiguous role of Sult1d1, and demonstrated a strong impact of transgenic human SULT1A1/2.Chemical Research in Toxicology 05/2014; · 4.19 Impact Factor