多环芳烃与胞外DNA非共价结合及机制

Non-covalent binding interaction and mechanism between polycyclic aromatic hydrocarbons and extracellular DNA

多环芳烃(PAHs)作为持久性有机污染物,因具有"三致"效应,其环境污染与人体健康问题一直是环境领域的研究热点.PAHs与生命遗传物质DNA结合形成PAHs-DNA加合物进而阻碍细胞正常复制是其诱发癌症的主要机理.然而,该过程尚未引起足够重视.本文采用荧光猝灭法,分别研究了低、中、高环PAHs与水中胞外DNA之间的结合强度及机制.结果表明,静态猝灭与动态猝灭过程共存,且PAHs可与DNA结合形成稳定的PAHs-DNA复合物,二者之间的结合常数K_(A)分别为2.05×10^(10)L/mol(苊烯)、4.42×10^(7)L/mol(苊)、1.71×10^(4)L/mol(荧蒽)、1.20×10^(4)L/mol(芘)、1.80×10^(2)L/mol(苯并[a]蒽)、4.96×10^(4)L/mol(屈)、1.11×10^(2)L/mol(苯并[k]荧蒽)和4.86×10^(2)L/mol(苯并[a]芘).红外光谱结果证实胸腺嘧啶为PAHs与DNA结合的主要作用位点.分子对接和量子化学计算明确了PAHs可通过范德华力、π-π堆积与DNA结合并插入其双螺旋结构的小沟中.紫外可见光谱分析结果表明,PAHs与DNA结合可导致DNA分子的紫外吸收光谱产生增色效应,造成DNA分子局部损伤、扭曲或降解.相关性分析结果发现,PAHs理化性质(分子量、熔点、沸点、水溶性、logK_(ow)、logK_(oc)、logK_(oa))与猝灭常数显著相关,但与结合常数无相关关系,表明PAHs本身理化性质差异可影响PAHs与DNA结合的动态猝灭过程,但未影响二者间静态猝灭过程.本研究对认识水环境中PAHs污染的分子生态效应具有重要的科学意义.

Polycyclic aromatic hydrocarbons(PAHs),as persistent organic pollutants,can induce teratogenic,carcinogenic and mutagenic effects.Thus,their environmental pollution and human health issues have always been hotspots in the environmental field.PAHs binding with DNA to form PAHs-DNA adducts is the main carcinogenic mechanism.However,this important process has not attracted enough attention.Few previous studies reported the binding interactions between hydrophilic organic pollutants(such as pesticides,antibiotics,etc.)and DNA,including binding affinities,binding modes,binding mechanisms,and influencing factors.However,reports on the in vitro binding of hydrophobic organic pollutants(e.g.,PAHs)to DNA are scarce.In this study,fluorescence quenching tests were performed to study the binding interactions between PAHs with low,medium,high rings and extracellular DNA in water.During the binding interaction,both static quenching and dynamic quenching processes occurred simultaneously.PAHs bound to DNA to form stable PAHs-DNA complexes.The binding constants(K_(A))for PAHs-DNA were 2.05×10^(10)L/mol(acenaphthylene),4.42×10^(7)L/mol(acenaphthene),1.71×10^(4)L/mol(fluoranthene),1.20×10^(4)L/mol(pyrene),1.80×10^(2)L/mol(benzo[a]anthracene),4.96×10^(4)L/mol(flexion),1.11×10^(2)L/mol(benzo[k]fluoranthene),and 4.86×10^(2)L/mol(benzo[a]pyrene),respectively.In addition,PAHs-DNA binding sites can be characterized by the attenuated total reflection-Fourier transform infrared spectroscopy(ATR-FTIR)to detect the vibration changes of specific functional groups in DNA molecules.The infrared vibration peaks of thymine were shifted from 1653 to 1647 cm-1after binding with PAHs,indicating that thymine was the main binding site for PAHs-DNA binding interaction.After that,molecular docking and quantum chemical calculations were performed to identify the binding modes and driving forces of PAHs-DNA binding interaction,respectively.On one hand,molecular docking results of PAHs-DNA binding clarified that PAHs could insert into the minor grooves of DNA double helix structure.On the other hand,the results of quantum chemical calculations confirmed that van der Waals force andπ-πstacking interaction were the main driving forces for the binding interactions between PAHs and thymine.Furthermore,UV-vis spectroscopy was used to analyze the influence of PAHs on DNA molecular structure after binding with DNA.The results showed that the characteristic peak intensities of DNA at 260 nm were gradually increased with the concentrations of PAHs increasing.This result indicated that the binding of PAHs and DNA could cause hyperchromic effect in the absorption spectra of DNA molecules,and lead to local damage,distortion or degradation of the DNA molecular.In order to clarify the relationship between the PAHs-DNA binding strength and the physicochemical properties of PAHs,we used Pearson correlation analysis in SPSS software to analyze the correlation between physicochemical properties of PAHs and the quenching constants and binding constants of the fluorescence quenching process.The results suggested that the physicochemical properties of PAHs(molecular weight,melting point,boiling point,water solubility,logK_(ow),logK_(oc),and logK_(oa))were significantly correlated with quenching constants;but there was no correlation with the binding constants,indicating that the physical-chemical properties of PAHs were closely related to dynamic quenching process but had nothing to do with static quenching process.In all,this work investigated the binding strength and mechanism of eight PAHs with extracellular DNA.The research results probably enhance people’s knowledge and understanding of the relationship between PAHs and genetic materials such as DNA,and can provide a theoretical basis for further exploring the molecular toxicology of PAHs and their derivatives in the actual environmental system of human cells in the future.