Identification of 4-(3-Pyridyl)-4-oxobutyl-2'-deoxycytidine Adducts Formed in the Reaction of DNA with 4-(Acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone: A Chemically Activated Form of Tobacco-Specific Carcinogens

Abstract

Metabolic activation of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1) and N'-nitrosonornicotine (NNN, 2) results in the formation of 4-(3-pyridyl)-4-oxobutyl (POB)-DNA adducts, several of which have been previously identified both in vitro and in tissues of laboratory animals treated with NNK or NNN. However, 2'-deoxycytidine adducts formed in this process have been incompletely examined in previous studies. Therefore, in this study we prepared characterized standards for the identification of previously unknown 2'-deoxycytidine and 2'-deoxyuridine adducts that could be produced in these reactions. The formation of these products in reactions of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc, 3), a model 4-(3-pyridyl)-4-oxobutylating agent, with DNA was investigated. The major 2'-deoxycytidine adduct, identified as its stable cytosine analogue O²-[4-(3-pyridyl)-4-oxobut-1-yl]-cytosine (12), was O²-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxycytidine (13), whereas lesser amounts of 3-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxycytidine (14) and N⁴-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxycytidine (15) were also observed. The potential conversion of relatively unstable 2'-deoxycytidine adducts to stable 2'-deoxyuridine adducts by treatment of the adducted DNA with bisulfite was also investigated, but the harsh conditions associated with this approach prevented quantitation. The results of this study provide new validated standards for the study of 4-(3-pyridyl)-4-oxobutylation of DNA, a critical reaction in the carcinogenesis by 1 and 2, and demonstrate the presence of previously unidentified 2'-deoxycytidine adducts in this DNA.

Scheme 1. Metabolic Activation of NNK and NNN by P450s and Hydrolysis of NNKOAc by Esterase Yield Reactive Intermediates That Form POB-DNA Adducts

POB-DNA adducts can release HPB upon acid hydrolysis.

Chart 1. Structures of DNA Adducts Discussed in the Text

Scheme 2. Synthesis of O²-POB-Cyt (12)

Scheme 3. Preparation of 3-POB-dCyd (14)

TBSCl, t-butyldimethylsilyl chloride; DMAP, 4-dimethylaminopyridine.

Scheme 4. Preparation of N⁴-POB-dCyd (15)

NCS, N-chlorosuccinimide.

Scheme 5. Preparation of O²-POB-dUrd (17)

NCS, N-chlorosuccinimide.

Scheme 6. Preparation of 3-POB-dUrd (16)

TBSCl, t-butyldimethylsilyl chloride; TBAF, tetrabutylammonium fluoride.

Figure 1

LC–MS/MS analysis of POB-DNA adducts for m/z 259.1 → 148.1. (A) Calf thymus DNA exposed to NNKOAc and subjected to enzymatic hydrolysis; (B) synthetic N⁴-POB-dCyd (15); (C) synthetic 3-POB-dCyd (14); (D) synthetic O²-POB-Cyt (12). Approximately 50% of the signal from adducts 14 and 15 exhibit in-source fragmentation and loss of the 2′-deoxyribose moiety.

Figure 2

LC–MS/MS analysis of bisulfite-treated POB-DNA. (A) DNA exposed to NNKOAc and subjected to base denaturation, bisulfite treatment, and enzymatic hydrolysis; (B) synthetic 3-POB-dUrd (16); (C) synthetic O²-POB-dUrd (17). N⁴-POB-dCyd (15) was also observed but is not shown.