Mechanistic Studies On The Metabolic Activations Of Tobacco-specific Nitrosamines Catalyzed By Cytochrome P450 Enzymes

Tobacco-specific nitrosamines(TSNAs)are a class of special nitrosamines carcinogens widely existed in all tobacco products.It is one of the main factors that lead to lung cancer and oral cancer.The structure of the TSNAs compound itself is relatively stable,but its metabolites show obvious carcinogenic activity after being activated by cytochrome P450 in vivo.TSNAs have different structures and also show great differences in oncogenic activities,and the position of the tumor is also specific.Research showed that the metabolic pathways of TSNAs were mainly catalyzed by P450 enzymes,at first,α-hydroxylation reaction produced the α-hydroxy nitrosamines,then after the product of the decomposition of α-hydroxy nitrosamines,which bond to DNA,can make the DNA alkylation and eventually lead to the production of the tumor.Experimental studies on the metabolism and carcinogenicity of TSNAs in P450 enzymes have been carried out experimentally,but different activation pathways have not been explored in depth.Besides,the chemical mechanism and general rules of toxic effects of metabolites are also unclear.Therefore,in this paper,the hydroxylation of nitrosamines was simulated by quantum chemical calculation method,which can provide a theoretical basis for the experimental studies on TSNAs carcinogenicity.1.By utilizing simplified Compound I(Cpd I)model to stimulate the active center of P450 enzyme and based on Density Functional Theory(DFT),the metabolic pathways for α-and β-hydroxyl of N’-nitrocarnitine(NNN)were provided with theoretical stimulation.Calculation results showed that NNN hydroxylation reaction can happen both on potential energy surfaces of high spin(HS)and low spin(LS)state.The reaction went through two characteristic stages.Firstly,it was NNN and Cpd I to form initial complex for high and low spin state,and α-hydrogen atom in NNN was extracted and transferred to oxygen atom connected to Fe in Cpd I to form PorFe-OH and α-radical intermediate.Then OH on PorFe-OH was transferred toα-radical intermediate to form α-hydroxylation NNN.Although the stability of α-hydroxylation NNN was relatively high,interaction between nitrosamine groups andα-hydroxylation can make α-hydroxylation NNN further decomposed into diazohydroxide with high carcinogenic activity.α-hydroxylation was the main metabolic pathway on LS potential energy surface.But on HS potential energy surface,NNN denitrification can compete with α-hydroxylation to form alexipharmic myosmine.Therefore,at the time of researching NNN metabolism,carcinogenic α-hydroxylation and alexipharmic denitrification shall be considered to fairly evaluate NNN carcinogenicity.The Paper further researched pyridine N-oxidation for NNN and forming way of nornicotine and epoxide,and improved researches on metabolic process inside body of NNN.Mechanism model for NNN metabolic process was established,and powerful theoretical support was provided for the subsequent related experiments.2.Based on DFT,4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone(NNK)catalyzed by Cpd I active center of P450 enzyme to happen α-methyl(1′)andα-methylene(3′)hydroxylation were calculated and researched.The pathway of reaction was consistent with NNN.In reaction,extraction of hydrogen atom determined speed rate of reaction.In the reaction of α-hydroxyl NNK,reaction energy barrier for 1′-hydroxyl NNK was 16.0/14.9 kcal/mol(HS/LS),and for 3′-hydroxyl NNK was 15.3/14.3 kcal/mol(HS/LS).The results showed that in the process of NNK α-hydroxylation,energy barrier that need to be crossed on LS potential energy surface was lower than HS at the time of forming α-hydroxyl NNK.Therefore,α-hydroxylation on LS potential energy surface was the main metabolic pathway.In addition,compareed with energy information of 1′-and 3′-hydroxylation NNK,it was find that 3′-hydroxylation pathway was the dominant carcinogenic pathway.3.Based on DFT calculation,reaction mechanism for metabolic activation of N’-nitrosoanabasine(NAB)and N’-nitrosoanatabine(NAT)catalyzed by Cpd I was further researched.Results showed that hydroxylation process of NAB and NAT were similar to NNN and NNK.Extraction process of hydrogen atom was the rate determining step of reaction,and there was no obvious spin selectivity in reaction,similar to process of alkane hydroxylation reaction,conforming to Two-State Reactivity(TSR).In the reaction,the energy barrier of 2′-hydroxyl NAB was20.5/18.8 kcal/mol(HS/LS),and for 6′-hydroxyl NAB was 11.5/13.6 kcal/mol(HS/LS);reaction energy barrier for 2′-hydroxyl NAT was 22.3/23.6 kcal/mol(HS/LS),and for 6′-hydroxyl NAT was 13.0/12.9 kcal/mol(HS/LS).Comprehensively compared,energy barrier that need to be crossed by NAB was lower than NAT,and metabolic activation reaction was easier to occur.Therefore,NAB was the dominant metabolic pathway in hydroxylation reaction.On the basis of studying and discussing the metabolic mechanism of four kinds of TSNAs in tobacco,the corresponding chemical reaction model was constructed.For different TSNAs,the metabolic process is different,the energy barrier height and the dominant pathway are different.All these provide effective theoretical support for the related research,and further improve the overall level of related research.