Development of liquid chromatography electrospray ionization tandem mass spectrometry methods for analysis of DNA adducts of formaldehyde and their application to rats treated with N-nitrosodimethylamine or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.
Reaction of formaldehyde with DNA in vitro produces a variety of adducts among which are observed the cross-link di-(N(6)-deoxyadenosyl)methane (dAdo-CH 2-dAdo, 1) and the hydroxymethyl adduct N(6)-hydroxymethyl-dAdo (N(6)-HOCH 2-dAdo, 2). While the structures of these adducts have been known for decades, there have been no reports of their formation in vivo. Formaldehyde is released during intracellular metabolism of carcinogenic N-nitrosomethyl compounds such as N-nitrosodimethylamine (NDMA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), but DNA adducts formed by this pathway have not been previously characterized. In this study, we addressed these questions by developing highly sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring methods for quantitation of adducts 1 and 2, the latter as N(6)-methyl-dAdo ( 3). Considerable effort was devoted to the problem of artifactual formation of 1, which can occur during storage of DNA samples by reaction of dAdo with 2. This problem was solved by the addition of adenosine deaminase during the DNA hydrolysis step, thus removing dAdo as a reactant. The instability of adduct 2 was another potential block to analysis, and this was solved by converting it to 3 with NaBH 3CN. Separate aliquots of DNA were analyzed for adducts 1 and 2, using the [(15)N]-labeled adducts as internal standards. The application of these methods to rat hepatic DNA to which adducts 1 and 3 were added demonstrated accuracy and precision. The detection limits for adducts 1 and 3 were 1-4 adducts per 10 (9) nucleotides using 100-150 microg of DNA. The method was applied to analyze hepatic and pulmonary DNA from rats treated with NDMA and NNK. The results clearly demonstrated the dose-dependent presence of N(6)-HOCH 2-dAdo ( 2) in all DNA samples. The cross-link adduct dAdo-CH 2-dAdo ( 1) was observed in hepatic DNA of NNK-treated rats, with lower amounts in pulmonary DNA. Levels of these adducts were generally less than those of DNA adducts formed by the classical diazohydroxide pathway of nitrosamine metabolism. The results of this study demonstrate for the first time the presence of formaldehyde DNA adducts in tissues of rats treated with carcinogenic nitrosamines and suggest that these adducts may play a role in cancer induction by these compounds.
University of Minnesota Cancer Center, MMC 806, 420 Delaware Street Southeast, Minneapolis, MN 55455, USA.
Mingyao
M
Wang
Guang
G
Cheng
Peter W
PW
Villalta
Stephen S
SS
Hecht
D000243:Adenosine Deaminase / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D018736:DNA Adducts / Q000032:analysis* / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D004128:Dimethylnitrosamine / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005557:Formaldehyde / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D008297:Male; D008956:Models, Chemical; D009602:Nitrosamines / Q000494:pharmacology*; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013997:Time Factors
D018736:DNA Adducts; D009602:Nitrosamines; D005557:Formaldehyde; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; D000243:Adenosine Deaminase; D004128:Dimethylnitrosamine