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, [ (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.
Monien
Bernhard H
BH
Department of Toxicology, German Institute of Human Nutrition (DIfE), 14558 Nuthetal, Germany. monien@dife.de
Müller
Carolin
C
Engst
Wolfram
W
Frank
Heinz
H
Seidel
Albrecht
A
Glatt
Hansruedi
H
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D018736:DNA Adducts / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D003839:Deoxyadenosines / Q000378:metabolism; D003849:Deoxyguanosine / Q000378:metabolism; D007201:Indicator Dilution Techniques; D008099:Liver / Q000737:chemistry* / Q000378:metabolism*; D008297:Male; D015394:Molecular Structure; D010761:Phosphorus Radioisotopes; D011721:Pyrenes / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013094:Spermatozoa / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors
D018736:DNA Adducts; D003839:Deoxyadenosines; D010761:Phosphorus Radioisotopes; D011721:Pyrenes; D003577:Cytochrome P-450 Enzyme System; C053035:1-methylpyrene; D003849:Deoxyguanosine