Investigation On The Interaction Between Three Phthalic Acid Esters Plasticizers And DNA

In recent years,with the increase efforts in environmental protection,some common environmental pollutants are familiar to the public.However,since phthalate plasticizers are classified as environmental pollutants,their toxicological properties are not well understood at present.It has been reported that plasticizers have potential carcinogenic,teratogenic and mutagenic effects,at the same time,as a kind of endocrine disruptors,they also have certain reproductive toxicity,such as reducing male fertility as well as promoting precocious puberty in female children.Due to the extensive use of plasticizers in the industrial production process,may cause an enrichment in the product and lead to security risks,such as some commercially available drinks have been detected the plasticizer in 2011 in Taiwan,in 2012 a mainland liquor also has been detencted the excessive plasticizer.So that,a special attention should be paid to the harm and potential toxicity of plasticizer.DNA is an important genetic material and a major target for exogenous small molecule compounds.Therefore,the study of the interaction mechanism between phthalate plasticizers and DNA will help to assess the toxicological properties of plasticizers at molecular level.In this thesis,the interaction mechanisms between three kinds of phthalate plasticizers（DMP,DBP,DEHP）and DNA were studied by using multispectroscopy method,chemometrics,molecular simulation and gel electrophoresis technique in a simulated physiological acidity（pH 7.4）.Meanwhile,the preparation of the inclusion compound of DMP-hydroxypropyl-β-cyclodextrin as well as probing its interaction with ctDNA.In addition,the effects of Ca²⁺,Cu²⁺and Mg²⁺on the interaction between DBP and ctDNA also been studied.The main contents and results are as following:1.The binding properties between three kinds of phthalate plasticizers（DMP,DBP and DEHP）and ctDNA were studied by fluorescence spectroscopy,UV–vis absorption and chemometrics（multivariate curve resolution-alternating least squares method,MCR-ALS）.MCR-ALS was used to analyze the expanded UV-vis datamatrix,indicated that the three plasticizers formed corresponding complexes with ctDNA,the pure spectra and concentration trend of each components were obtained from the highly overlapping spectra.Fluorescence quenching experiments showed that the endogenous fluorescence of the three plasticizers could be quenched by ctDNA,and all the quenching process were static quenching.Based on the calculated thermodynamic parameters,the main driving forces were hydrophobic interaction and hydrogen,for the binding interaction of three kinds of plasticizers with ct DNA.2.The binding modes of DMP,DBP and DEHP with ctDNA were studied by viscosity measurement,melting experiment and salt effect.All experimental results indicated that the three plasticizers interacted with ctDNA through groove binding mode.Infrared and circular dichroism spectroscopy showed that the main binding sites of DMP,DBP and DEHP with ctDNA were A-T rich region,and the conformation of ctDNA was slightly disturbed,but did not change the original type B conformation of ctDNA.The results of molecular simulation showed that DMP,DBP and DEHP interacted with DNA（ID: 453D）at A-T rich region of minor groove,which further supported the experimental results of FT-IR spectroscopy.DNA damage experiments were performed on agarose gel electrophoresis,the results showed that DMP,DBP and DEHP did not produce significant cleavages on plasmid DNA.3.The inclusion compound of DMP-Hp-β-CD was obtained by ultrasonic method,the inclusion ratio was determined to be 1:1.The preparation processes were followed by filtration,concentration and drying.Characterization of DMP-Hp-β-CD inclusion compound was proved by infrared spectroscopy,circular dichroism and scanning electron microscopy.In addition,the interaction between DMP-Hp-β-CD inclusion compound and ctDNA was further studied.The results showed that the binding constant between DMP and ctDNA was decreased while DMP included by Hp-β-CD,but the binding mode did not change,and the binding region was transferred from the minor groove to the major groove.4.The interaction between DBP and ctDNA in the presence of Mg²⁺,Ca²⁺ and Cu²⁺ in a simulated physiological acidity environment were investigated through multi-spectroscopy methods combined with DNA melting and viscositymeasurements.The results showed that Mg²⁺,Ca²⁺and Cu²⁺did not change the groove binding mode of DBP with ctDNA.However,the different binding constants were concentration-dependent of Mg²⁺,Ca²⁺ and Cu²⁺.The differences were mainly due to the binding ability of metal ions with DBP and DNA（mainly bases and phosphate groups）.In addition,the presence of Mg²⁺,Ca²⁺ and Cu²⁺ did not disturb the ctDNA conformation significantly,but interfered binding sites of DBP with ctDNA.