Quantification of DNA Lesions Induced by 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol in Mammalian Cells

Abstract

Quantitative measurement of DNA adducts in carcinogen-exposed cells provides the information about the frequency of formation and the rate of removal of DNA lesions in vivo, which yields insights into the initial events of mutagenesis. Metabolic activation of tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its reduction product 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), leads to pyridyloxobutylation and pyridylhydroxybutylation of DNA. In this study, we employed a highly robust nanoflow liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry (nLC-nESI-MS/MS) coupled with the isotope-dilution method for simultaneous quantification of O 6-[4-(3-pyridyl)-4hydroxylbut-1-yl]-2′-deoxyguanosine (O 6-PHBdG) and O 2-and O 4-[4-(3-pyridyl)-4hydroxylbut-1-yl]-thymidine (O 2-PHBdT and O 4-PHBdT). Cultured mammalian cells were exposed to a model pyridylhydroxybutylating agent, 4-(acetoxymethylnitrosamino)-1-(3pyridyl)-1-butanol (NNALOAc), followed by DNA extraction, enzymatic digestion, and sample enrichment prior to nLC-nESI-MS/MS quantification. Our results demonstrate, for the first time, that O 4-PHBdT is quantifiable in cellular DNA and naked DNA upon NNALOAc exposure. We also show that nucleotide excision repair (NER) machinery may counteract the formation of O 2-PHBdT and O 4-PHBdT, and O 6-alkylguanine DNA alkyltransferase (AGT) may be responsible for the repair of O 6-PHBdG and O 4-PHBdT in mammalian cells. Together, our study provides new knowledge about the occurrence and repair of NNAL-induced DNA lesions in mammalian cells.

Quantitation of DNA Adducts of Aristolochic Acids in Repair-Deficient Cells: A Mechanistic Study of the DNA Repair Mechanism

“…The elevated occurrence of the two adducts in the NER-deficient cells compared to their corresponding wild-type strain provided the first direct evidence that NER may be involved in the repair of these two lesions and that ALI-dA and ALI-dG are good substrates for NER. A similar dependence on NER machinery was also observed with repair of other bulky DNA lesions, including those formed by tobacco-specific nitrosamines. , …”

“…A similar dependence on NER machinery was also observed with repair of other bulky DNA lesions, including those formed by tobacco-specific nitrosamines. 20,21 Among the tested NER-deficient strains, the highest adduct levels were observed in JW4019, JW0762, and JW1898, which were deficient in the DNA repair excinuclease ABC subunit A (UvrA), B (UvrB), and C (UvrC), respectively. These proteins are known to act in concert, playing important roles in damage recognition, verification, and eventually adduct removal in the E. coli NER pathway.…”

Investigation of 2′-Deoxyadenosine-Derived Adducts Specifically Formed in Rat Liver and Lung DNA by N′-Nitrosonornicotine Metabolism

“…Similarly, α-methyl and α-methylene hydroxylation metabolically activate NNK to form pyridyloxobutyl (POB) diazonium ion 10 and methyl diazonium ion 11 , respectively. They are both strongly electrophilic and form the corresponding POB and methyl DNA adducts. − NNK is also enzymatically reduced to its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which also exerts strong carcinogenicity through a similar metabolic mechanism as described for NNK …”

Structural Rationalization for the Nonmutagenic and Mutagenic Bypass of the Tobacco-Derived O4-4-(3-Pyridyl)-4-oxobut-1-yl-thymine Lesion by Human Polymerase η: A Multiscale Computational Study

“…The DNA sequence was modified to ensure the sequence context surrounding the template base corresponds to the experimental work investigating O4-POB-T replication by pol η. 15,42 DNA duplex models based on the 5′-GCATGGCGXGCTATGC-3′ sequence (X = O4-POB-T) were built using Nucleic Acid Builder (NAB) and the lesion added using GaussView 6. 36 The sequence mirrors that used in experimental work on O4-POB-T bypass by pol η, 15 with two G:C pairs added to each strand end for stabilization.…”

“…When multiple orientations of an amino acid are viable based on experimental data, the residue was visually inspected and aligned to afford the most favorable interactions with neighboring residues or the highest occupied orientation was selected in the absence of potential interactions with surrounding residues. The DNA sequence was modified to ensure the sequence context surrounding the template base corresponds to the experimental work investigating O4-POB-T replication by pol η. , DNA duplex models based on the 5′-GCATGGCGXGCTATGC-3′ sequence (X = O4-POB-T) were built using Nucleic Acid Builder (NAB) and the lesion added using GaussView 6 . The sequence mirrors that used in experimental work on O4-POB-T bypass by pol η, with two G:C pairs added to each strand end for stabilization.…”