Study On The Metabolism And DNA Damage Of4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone In Vitro

Tobacco-specific nitrosamines are a group of carcinogens found only in tobaccosmoke and tobacco products. Eight tobacco-specific nitrosamines have been identifiedat percent, and4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) generallyoccurs in quantity greater than the others. Several evidences indicate that NNKrequires metabolic activation to express its carcinogenicity. Although five metabolicpathways contribute to NNK transfer, the key ingredient,-hydroxylation plays amajor role in carcinogenesis. In this work, studies were performed to investigate themetabolism of NNK and the formation of DNA adducts induced by NNK metabolitesin vitro.4-Hydroxy-1-(3-pyridyl)-1-butanone (HPB), a metabolite of NNK, wasquantified using [3,3,4,4-D4]HPB (D4-HPB) as internal standard. A quantitativemethod for HPB was established by high-performance liquidchromatography-atmosphere pressure chemical ion-tandem mass spectrometry(HPLC-APCI-MS/MS). Quantitative analyses of NNK were established which wasmetabolized under the catalysis of cytchrome P450enzymes in differentconcentrations and reaction times. Qualitative analyses of the formation of DNAadducts from metabolism of NNK were employed using high-performance liquidchromatography-electrospray ion-tandem mass spectrometry (HPLC-ESI-MS/MS).The contents are mainly involved in the following four aspects.Firstly, the HPB from metabolism of NNK was synthesized using the materialγ-butyrolactone and ethyl nicotinate. Then the product purified by columnchromatography with silica gel. The structure of the purified target compound wascharacterized by1H NMR, IR, UV and MS.Secondly, a quatitative analysis for HPB was established. HPB was separated byan Agilent Zorbax SB C18reversed phase column, and then quantified by the mode ofSelecting Reaction Monitoring (SRM) positive mode with the ions transition of m/z170â†'106and m/z166â†'106using HPB and D4-HPB as the standard samples. Thequatitative analysis for HPB was established by HPLC-APCI-MS/MS. The linearstandard curve was plotted with the concentration ratio of HPB vs D4-HPB asx-coordinate and the peak area ratio as y-coordinate. The correlation coefficient (R2)was0.9999with concentration ranged from0.2to400nmol/L. The intra-day relativestandard deviation and accuracy, and inter-day relative standard deviation andaccuracy, and recovery study showed that the method was with good stability and repeatability. The LOD and LOQ for HPB were0.025nmol/L and0.05nmol/L,respectively. The results indicated that the method was suitable for the quantitativeanalysis of HPB and could be used for the determination of this product of themetabolism from NNK.Thirdly, quantitative analyses of NNK were established which was metabolizedunder the action of cytchrome P450enzymes in different concentrations and reactiontimes. NNK was metabolic with the concentrations of0.01,0.1,1and10mmol/Lunder the action of cytchrome P450enzymes. After the remove of proteins, theproduct of HPB was quantified by HPLC-APCI-MS/MS. The results showed that ratliver microsomes solution was rich in NNK metabolic enzymes subtype. This systemcould be successfully metabolized NNK in vitro experiment. Concentration of theproduct HPB was increased with the NNK concentration and the reaction time.Finally, qualitative analyses of the formation of DNA adducts from metabolism ofNNK were employed using HPLC-ESI-MS/MS SRM mode. Calf thymus DNA wastreated with NNK with the concentration of10mmol/L and catalyzed by cytchromeP450enzymes. The samples were removed of protein and were hydrolyzed to freenucleosides by the enzymatic digestion. Then the pyridyloxobutylation DNA adducts(POB-DNA adducts) were quantified by HPLC-ESI-MS/MS in SRM mode. Theresults showed that, under the action of cytchrome P450enzymes, O6-POB-dGincreased obviously along with the reaction lasted3,6,9and12hours, respectively.The study included quantitative analysis NNK metabolite HPB and qualitativeanalysis POB-DNA adducts formation from metabolism of NNK. The study providednot only experimental evidences of NNK metabolic mechanism and carcinogenesis,but also a new research approach of neotype, simple and high accuracy method forinvestigation of tobacco-specific nitrosamines carcinogenic biomarkers.