Pyridylhydroxybutyl and pyridyloxobutyl DNA phosphate adduct formation in rats treated chronically with enantiomers of the tobacco-specific nitrosamine metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol

Abstract

The tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is metabolically converted to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in a reaction which is both stereoselective and reversible. NNAL is also a lung carcinogen, with both (R)-NNAL and (S)-NNAL inducing a high incidence of lung tumours in rats. Both NNAL and NNK undergo metabolic activation to intermediates which react with DNA to form pyridylhydroxybutyl and pyridyloxobutyl DNA adducts, respectively. DNA adduct formation by NNAL and NNK is an important step in their mechanisms of carcinogenesis. In this study, we quantified both pyridylhydroxybutyl and pyridyloxobutyl DNA phosphate adducts in the lung of rats treated with 5 ppm of (R)-NNAL or (S)-NNAL in drinking water for 10, 30, 50 and 70 weeks. In (R)-NNAL-treated rats, the pyridylhydroxybutyl and pyridyloxobutyl phosphate adducts were 4530-6920 fmol/mg DNA and 46-175 fmol/mg DNA, accounting for 45-51% and 0.3-1% of the total measured DNA phosphate and base adducts, respectively. In (S)-NNAL-treated rats, the two types of phosphate adducts were 3480-4180 fmol/mg DNA and 1180-4650 fmol/mg DNA, accounting for 30-36% and 11-38% of the total adducts, respectively. Distinct patterns of adduct formation were observed, with higher levels of NNAL-derived pyridylhydroxybutyl phosphate adducts and lower levels of NNK-derived pyridyloxobutyl phosphate adducts in the (R)-NNAL treatment group than the (S)-NNAL group. The persistence and increase over time of certain pyridylhydroxybutyl phosphate adducts over the course of the study suggest that these adducts could be useful biomarkers of chronic exposure to NNAL and NNK. The results of this study provide important new information regarding DNA damage by NNAL and NNK, and contribute to understanding mechanisms of tobacco-related carcinogenesis.

Methyl DNA Phosphate Adduct Formation in Rats Treated Chronically with 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and Enantiomers of Its Metabolite 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol

“…7 In another study, male Wistar rats were intraperitoneally injected with a single dose of NDMA at 10 mg/kg. TpMeT was detected and quantified in the liver with a half-life of 7 days, significantly longer than the corresponding O 6 -methylguanine with a half-life of 25 h. 24 The persistence of DNA phosphate adducts was also observed in our studies of pyridyloxobutyl 10 and pyridylhydroxybutyl 12,13 phosphate adducts in the NNK- and NNAL-treated rats. The persistence of these DNA phosphate adducts could be partially due to their chemical stability.…”

“…1,4,8,10,25 To determine the relative proportion of methyl phosphate adducts to the total measured DNA adducts formed in rats treated with NNK or NNAL, the levels of the other five types of adducts were obtained from our previous studies (Table 2). 4,10,12,13 The levels of total B 1 p(POB)B 2 phosphate adducts in the lung of NNK-treated rats were reported previously. 10 The levels of total B 1 p(POB)B 2 and B 1 p(PHB)B 2 phosphate adducts in the lung of ( R )- and ( S )-NNAL-treated rats and total B 1 p(PHB)B 2 phosphate adducts in the lung of NNK-treated rats were obtained from other studies.…”

“…10 The levels of total B 1 p(POB)B 2 and B 1 p(PHB)B 2 phosphate adducts in the lung of ( R )- and ( S )-NNAL-treated rats and total B 1 p(PHB)B 2 phosphate adducts in the lung of NNK-treated rats were obtained from other studies. 12,13 The levels of one methyl base adduct, O 6 -methylguanine, three major POB base adducts, O 2 -[4-(3-pyridyl)-4-oxobut-1-yl]thymidine, 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine, and O 6 -[4-(3-pyridyl)-4-oxobut-1-yl]deoxyguanosine, and two major PHB base adducts, O 2 -[4-(3-pyridyl)-4-hydroxybut-1-yl]thymidine and 7-[4-(3-pyridyl)-4-hydroxybut-1-yl]guanine, were reported previously. 4 Other NNK-derived methyl and POB base adducts have been previously identified from in vitro and in vivo studies, 1,9,26 but only the base adducts mentioned above were measured in the treated rats and included in this study.…”

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

“…In this vein, solid-phase extraction followed by LC-MS/MS analysis was previously used for quantifying several pyridyloxobutyl and pyridylhydroxybutyl DNA lesions. 17,21,23,26,28 Herein, we report the application of a highly sensitive nanoflow liquid chromatographynanoelectrospray ionization tandem mass spectrometry (nLC-nESI-MS/MS) coupled with the stable isotope-dilution technique for the simultaneous quantifications of O 4 -PHBdT, O 2 -PHBdT, and O 6 -PHBdG. This is the first reported measurement of O 4 -PHBdT in mammalian cells (Scheme 1).…”

Formalin-Fixed Paraffin-Embedded Tissues—An Untapped Biospecimen for Biomonitoring DNA Adducts by Mass Spectrometry

“…The DNA is typically digested with a cocktail of nucleases prior to adduct measurements by LC-MS. The digestion products contain adducts formed at the DNA bases of the 2′-deoxyribonucleosides, or in rarer cases, adducts are formed at the phosphate backbones [ 59 , 60 ]. A common feature for many DNA adducts is their tendency to lose the deoxyribose moiety (dR, 116 or 116.0473 Da in HRAMS), when subjected to collision-induced dissociation (CID) [ 61 ].…”