细胞色素P450酶催化烟草特异性亚硝胺N’-亚硝基新烟草碱和N’-亚硝基假木贼碱代谢活化的分子机制研究

Mechanistic Insight into Cytochrome P450-catalyzed Activation of Tobacco-specific N’-Nitrosoanatabine and N’-Nitrosoanabasine

烟草特异性亚硝胺N’-亚硝基新烟草碱(NAT)和N’-亚硝基假木贼碱(NAB)不仅在所有烟草制品和烟草烟雾中广泛存在,还存在于大气颗粒物中,表明这2类污染物存在不可避免的暴露风险。NAT和NAB被细胞色素P450酶代谢活化是发挥其致癌活性的重要前提,但到目前为止反应机理细节尚未被系统研究。因此,本文通过密度泛函理论(DFT)计算系统揭示了NAT和NAB代谢的α-羟基化致癌路径,明确了反应的机理细节及能量关系。计算表明,P450催化的NAT和NABα-羟基化路径主要包括:(1)氢夺取反应(能垒为12.7~21.6 kcal·mol^1),形成放热但不稳定的Cα自由基中间体;(2)无能垒的羟基转移反应,形成剧烈放热的α-羟基化中间体;(3)α-羟基化中间体的自发分解反应(能垒为14.1~20.1 kcal·mol^1),产生最终有致癌潜力的重氮氢氧化代谢物。进一步比较发现,NAT 2-羟基化和6-羟基化路径都是其代谢的潜在致癌路径,而NAB的2’-羟基化路径是其主要致癌路径。此外,尽管NAT和NAB在结构上具有很大的相似性,但是后者的致癌活性可能要略高于前者。上述研究结果有助于全面理解NAT和NAB的代谢活化机制,并有助于识别潜在的生物标志物用于合理评估这2种亚硝胺污染物暴露的健康风险。

Tobacco-specific nitrosamines N’-nitrosoanatabine(NAT) and N’-nitrosoanabasine(NAB) are abundantly present in all tobacco products,cigarette smoke,and even atmospheric particulate matters. The toxification routes of NAT and NAB metabolisms catalyzed by cytochrome P450 enzymes are important triggers for their carcinogenicity;however,the molecular details of respective toxification mechanisms are not well understood until now,which hampers a comprehensive risk assessment of NAT and NAB exposures. In the present investigation,the density functional theory(DFT) calculations were performed to unravel the mechanistic details of P450-catalyzed activations of these two contaminants. NAT and NAB α-hydroxylations occurring at two Cαsites adjacent to nitrosamine group include the following elementary steps:(1) initial Hαatom abstraction(reaction barrier 12.7 ~ 21.6 kcal·mol^-1) by the highly reactive FeIV= O unit to form exothermic and unstable Cαradical intermediate,(2) energy-free OH rebound from FeIV-OH to Cαradical to yield stable α-hydroxyNAT/α-hydroxyNAB intermediate,and(3)spontaneous intramolecular decomposition of α-hydroxynitrosamine(barrier 14.1 ~ 20.1 kcal·mol^-1),generating reactive diazohydroxides as potential genotoxic metabolites to alkylate DNA. NAT and NAB have similar structural features,and correspondingly show comparable α-hydroxylation routes. NAT 2-hydroxylation shows higher H atom abstraction barrier but lower decomposition barrier than 6-hydroxylation,suggesting that these two routes are kinetically feasible for NAT metabolism. In contrast,2’-hydroxylation is likely to dominate the metabolic fate of NAB.Moreover,the results further suggest that NAB may have slightly higher carcinogenic potential than NAT for the P450-catalyzed metabolism. Overall,the calculation results can provide a comprehensive understanding of metabolic activations of NAT and NAB,and further enable the identification of mechanistic-specific metabolites and biomarkers for assessing the health risk of both NAT and NAB exposures.