Acetaldehyde is a mutagen and carcinogen which occurs widely in the human environment, sometimes in considerable amounts, but little is known about its reactions with DNA. In this study, we identified three new types of stable acetaldehyde DNA adducts, including an interstrand cross-link. These were formed in addition to the previously characterized N(2)-ethylidenedeoxyguanosine. Acetaldehyde was allowed to react with calf thymus DNA or deoxyguanosine. The DNA was isolated and hydrolyzed enzymatically; in some cases, the DNA was first treated with NaBH(3)CN. Reaction mixtures were analyzed by HPLC, and adducts were isolated and characterized by UV, (1)H NMR, and MS. The major adduct was N(2)-ethylidenedeoxyguanosine (1), which was identified as N(2)-ethyldeoxyguanosine (7) after treatment of the DNA with NaBH(3)CN. The new acetaldehyde adducts were 3-(2-deoxyribos-1-yl)-5,6,7, 8-tetrahydro-8-hydroxy-6-methylpyrimido[1,2-a]purine-10(3H)one (9), 3-(2-deoxyribos-1-yl)-5,6,7,8-tetrahydro-8-(N(2)-deoxyguanosyl+ ++)- 6-methylpyrimido[1,2-a]purine-10(3H)one (12), and N(2)-(2, 6-dimethyl-1,3-dioxan-4-yl)deoxyguanosine (11). Adduct 9 has been previously identified in reactions of crotonaldehyde with DNA. However, the distribution of diastereomers was different in the acetaldehyde and crotonaldehyde reactions, indicating that the formation of 9 from acetaldehyde does not proceed through crotonaldehyde. Adduct 12 is an interstrand cross-link. Although previous evidence indicates the formation of cross-links in DNA reacted with acetaldehyde, this is the first reported structural characterization of such an adduct. This adduct is also found in crotonaldehyde-deoxyguanosine reactions, but in a diastereomeric ratio different than that observed here. A common intermediate, N(2)-(4-oxobut-2-yl)deoxyguanosine (6), is proposed to be involved in formation of adducts 9 and 12. Adduct 11 is produced ultimately from 3-hydroxybutanal, the major aldol condensation product of acetaldehyde. Levels of adducts 9, 11, and 12 were less than 10% of those of N(2)-ethylidenedeoxyguanosine (1) in reactions of acetaldehyde with DNA. As nucleosides, adducts 9, 11, and 12 were stable, whereas N(2)-ethylidenedeoxyguanosine (1) had a half-life of 5 min. These new stable adducts of acetaldehyde may be involved in determination of its mutagenic and carcinogenic properties.

Background and Purpose Diffusion tensor imaging tractography reconstruction of white matter pathways can help guide brain tumor resection. However, DTI tracts are complex mathematical objects and the validity of tractography-derived information in clinical settings has yet to be fully established. To address this issue, we initiated the DTI Challenge, an international working group of clinicians and scientists whose goal was to provide standardized evaluation of tractography methods for neurosurgery. The purpose of this empirical study was to evaluate different tractography techniques in the first DTI Challenge workshop. Methods Eight international teams from leading institutions reconstructed the pyramidal tract in four neurosurgical cases presenting with a glioma near the motor cortex. Tractography methods included deterministic, probabilistic, filtered, and global approaches. Standardized evaluation of the tracts consisted in the qualitative review of the pyramidal pathways by a panel of neurosurgeons and DTI experts and the quantitative evaluation of the degree of agreement among methods. Results The evaluation of tractography reconstructions showed a great inter-algorithm variability. Although most methods found projections of the pyramidal tract from the medial portion of the motor strip, only a few algorithms could trace the lateral projections from the hand, face, and tongue area. In addition, the structure of disagreement among methods was similar across hemispheres despite the anatomical distortions caused by pathological tissues. Conclusions The DTI Challenge provides a benchmark for the standardized evaluation of tractography methods on neurosurgical data. This study suggests that there are still limitations to the clinical use of tractography for neurosurgical decision-making.

The tobacco specific carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N‘-nitrosonornicotine (NNN) are metabolically activated to 4-oxo-4-(3-pyridyl)-1-butanediazohydroxide (7), which is known to pyridyloxobutylate DNA. A substantial proportion of the adducts in this DNA releases 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB, 11) under various hydrolysis conditions, including neutral thermal hydrolysis. These HPB-releasing DNA adducts have been detected in target tissues of animals treated with NNK and NNN as well as in lung tissue from smokers. Although their presence in pyridyloxobutylated DNA was conclusively demonstrated 15 years ago, their structures have not been previously determined. We investigated this question in the present study by determining the structures of products formed in reactions with dGuo and DNA of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKCH2OAc, 3), a stable precursor to 7. Reaction mixtures from NNKCH2OAc and dGuo were analyzed by liquid chromatography−electrospray ionization−mass spectrometry (LC-ESI-MS) with selected ion monitoring at m/z 415. A major peak was detected and identified as 7-[4-oxo-4-(3-pyridyl)but-1-yl]dGuo (37) by its ESI-MS fragmentation pattern and by neutral thermal hydrolysis, which converted it to 11 and 7-[4-oxo-4-(3-pyridyl)but-1-yl]Gua (26). The latter was identified by comparison to synthetic 26 using LC-ESI-MS with selected ion monitoring at m/z 299, M + 1 of 26. Further evidence was obtained by NaBH4 reduction of 26 to 7-[4-hydroxy-4-(3-pyridyl)but-1-yl]Gua, which was also matched with a standard. Adduct 37 was similarly identified in enzyme hydrolysates of DNA reacted with NNKCH2OAc, accounting for 30−35% of the HPB-releasing adducts in this DNA. Several other adducts resulting from pyridyloxobutylation of the N 2- and O 6-positions of Gua were also identified as products in the dGuo or DNA reactions by comparison to standards; their concentrations were considerably less than that of 37. These adducts were N 2-[4-oxo-4-(3-pyridyl)but-1-yl]dGuo (23), N 2-[4-oxo-4-(3-pyridyl)but-2-yl]dGuo (25), N 2-[2-(3-pyridyl)tetrahydrofuran-2-yl]dGuo (31a) (or its open chain tautomer 31b), and O 6-[4-oxo-4-(3-pyridyl)but-1-yl]dGuo (10). Adducts 23, 25, and 10 did not release HPB upon neutral thermal hydrolysis. The results of this study provide the first structural identification of an HPB-releasing DNA adduct of the tobacco specific nitrosamines NNK and NNN.

We investigated the reactions of formaldehyde plus acetaldehyde with dGuo and DNA in order to determine whether certain 1,N(2)-propano-dGuo adducts could be formed. These adducts-3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-(3H)-one (1) and 3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-(3H)-one (3a,b)-have been previously characterized as products of the reaction of acrolein with dGuo and DNA. Adduct 1 predominates in certain model lipid peroxidation systems [Pan, J., and Chung, F. L. (2002) Chem. Res. Toxicol. 15, 367-372]. We hypothesized that this could be due to stepwise reactions of formaldehyde and acetaldehyde with dGuo, rather than by reaction of acrolein with dGuo. The results demonstrated that adducts 1 and 3a,b were relatively minor products of the reaction of formaldehyde and acetaldehyde with dGuo and that there was no selectivity in their formation. These findings did not support our hypothesis. However, substantial amounts of previously unknown cyclic dGuo adducts were identified in this reaction. The new adducts were characterized by their MS, UV, and NMR spectra as diastereomers of 3-(2'-deoxyribosyl)-6-methyl-1,3,5-diazinan[4,5-a]purin-10(3H)-one (10a,b). Adducts 10a,b were apparently formed by addition of formaldehyde to N1 of N(2)-ethylidene-dGuo, followed by cyclization. An analogous set of four diastereomers of 3-(2'-deoxyribosyl)-6,8-dimethyl-1,3,5-diazinan[4,5-a]purin-10(3H)-one (12a-d) were formed in the reactions of acetaldehyde with dGuo. These products are the first examples of exocyclic dGuo adducts of the pyrimido[1,2-a]purine type in which an oxygen atom is incorporated into the exocyclic ring. Formaldehyde-derived adducts were the other major products of the reactions of formaldehyde plus acetaldehyde with dGuo. Prominent among these were N(2)-hydroxymethyl-dGuo (9) and the cross-link di-(N(2)-deoxyguaonosyl)methane (13). We did not detect adducts 1, 3a,b, or 10a,b in enzymatic hydrolysates of DNA that had been allowed to react with formaldehyde plus acetaldehyde. However, we did detect substantial amounts of the formaldehyde cross-links di-(N(6)-deoxyadenosyl)methane (17), with lesser quantities of (N(6)-deoxyadenosyl-N(2)-deoxyguanosyl)methane (18), di-(N(2)-deoxyguanosyl)methane (13), and N(6)-hydroxymethyl-dAdo (19). Schiff base adducts of formaldehyde and acetaldehyde were also detected in these reactions. These results demonstrate that the reactions of formaldehyde plus acetaldehyde with dGuo are dominated by newly identified cyclic adducts and formaldehyde-derived products whereas the reactions with DNA result in the formation of formaldehyde cross-link adducts. The carcinogens formaldehdye and acetaldehyde occur in considerable quantities in the human body and in the environment. Therefore, further research is required to determine whether the adducts described here are formed in animals or humans exposed to these agents.

Metabolic hydroxylation of the methyl group of the tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) results in the formation of intermediates that can alkylate DNA. Similarly, metabolic hydroxylation of the 2'-position of the tobacco specific carcinogen N'-nitrosonornicotine gives DNA alkylating intermediates. The resulting pyridyloxobutyl and pyridylhydroxybutyl adducts with dGuo have been characterized, but there are no reports of pyrimidine adducts. Therefore, in this study, we investigated the reactions of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKCH(2)OAc) and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol (NNALCH(2)OAc) with DNA, dCyd, and dThd. NNKCH(2)OAc and NNALCH(2)OAc are stable precursors to the products formed upon metabolic methyl hydroxylation of NNK and NNAL. Analysis by LC-ESI-SIM of enzyme hydrolysates of DNA that had been allowed to react with NNKCH(2)OAc and NNALCH(2)OAc demonstrated the presence of major adducts with dCyd and dThd. The dCyd adducts were thermally unstable, releasing 4-HPB (18) or 4-hydroxy-1-(3-pyridyl)-1-butanol (25) upon treatment at 100 degrees C, pH 7.0. The dThd adducts were stable under these conditions. The dCyd adduct of NNALCH(2)OAc was characterized by its MS and UV and by conversion upon neutral thermal hydrolysis to the corresponding Cyt adduct, which was identified by MS, UV, and NMR. The dCyd and Cyt adducts of NNKCH(2)OAc were similarly characterized. The dThd adduct of NNKCH(2)OAc was identified by MS, UV, and NMR. Treatment of this adduct with NaBH(4) gave material, which was identical to that produced upon reaction of NNALCH(2)OAc with DNA or dThd. These data demonstrate that the major pyrimidine adducts formed in the reactions of NNKCH(2)OAc with DNA are O(2)[4-(3-pyridyl)-4-oxobut-1-yl]dCyd (26) and O(2)[4-(3-pyridyl)-4-oxobut-1-yl]dThd (30) while those produced from NNALCH(2)OAc are O(2)[4-(3-pyridyl)-4-hydroxybut-1-yl]dCyd (28) andO(2)[4-(3-pyridyl)-4-hydroxybut-1-yl]dThd (31). Levels of these pyrimidine adducts of NNKCH(2)OAc in DNA were substantially greater than those of the dGuo adducts of NNKCH(2)OAc, based on MS peak area. Furthermore, 26 was identified as a major 4-HPB releasing adduct of NNKCH(2)OAc. These results suggest that pyrimidine adducts of tobacco specific nitrosamines may be important contributors to their mutagenic and carcinogenic activity.