【作者】 李木兰；

【导师】 王永生；

【作者基本信息】 南华大学 ， 卫生毒理学， 2005， 硕士

【摘要】 DNA加合物是化学毒物经生物转化后的亲电活性产物与DNA分子形成的共价结合物,它既是一种暴露标志物,又是一种效应标志物,能综合反映生物体对化学毒物（致癌物）的暴露、吸收、分布、代谢以及机体对DNA的修复能力。研究醛类污染物与DNA形成加合物的机制,建立DNA加合物检测新方法对DNA加合物的研究具有重要意义。 在DNA加合物的形成机制研究方面,作者用紫外分光光度法和共振光散射法研究了醛类污染物与DNA的作用,结果表明:（1）在体外试管测试体系中,甲醛、乙醛与ctDNA作用,DNA的256nm处的紫外吸收峰发生红移（+）,且存在明显的剂量-反应关系;首次发现甲醛或乙醛与小牛胸腺DNA作用后也能引起DNA 218nm处紫外吸收峰的紫移,最大位移为16nm（-）。由此初步推断甲醛或乙醛能够加合到小牛胸腺DNA的亲核位点,引起了具有紫外吸收官能团的改变,形成了醛类-DNA加合物;（2）首次用共振光散射法研究了甲醛与DNA的相互作用。实验表明,在ctDNA-SDBS-JG体系中加入甲醛后,600nm附近的共振光散射峰发生明显的位移,共振光散射强度与甲醛加入量在0～0.34mol/L范围内成良好的线性关系,线性回归方程为:I=2.99c+311.3,r=0.994。可用本法来定性估计DNA加合物的形成量,并可用该法测定环境中的甲醛污染物的含量。实验还表明,SDBS、JG对甲醛与DNA的相互结合反应可能存在协同作用,即甲醛与DNA碱基作用形成加合物可能存在活化过程,即体内的带电荷的离子或极性分子诱导甲醛羰基的极化,促使甲醛聚集到DNA分子表面上,然后,其中部分嵌入到DNA碱基对中与DNA作用形成加合物。本文建立的共振光散射法可用于DNA加合物形成机制的研究;（3）根据实验结果和文献资料综合分析,提出了典型醛类污染物与DNA形成加合物的可能机制:典型醛类污染物在体内带电离子或极性分子的作用下,羰基极化或羰基氧原子与体内代谢所产生的H⁺结合,形成亲电活性分子,然后进攻DNA嘌呤碱基上的伯氨基（-NH₂）或嘧啶碱基上的仲胺基（-NH-）形成Schiff’s碱,Schiff’s碱不稳定,在体内还原体系的作用下,更多还原

【Abstract】 DNA adduct is a covalent complex formed by DNA and electrophilic activity production of toxic chemicals transformed in body, and it is not only an exposed biomarker but also an effective biomarker. DNA adduct is an effective measure of carcinogens’ affect and is directly related with carcinogenesis. It is important to research mechanism of DNA adducts formed by aldehyde pollutants with DNA and to set up a new method to detect DNA adduct.In the respect of DNA adducts formed mechanism, writer studied the mechanis -m interacted between aldehyde pollutants and DNA by ultraviolet spectrophotometry and resonance light scattering. Results indicated: (1) In the test sytem in vitro, the binding of formaldehyde, acetaldehyde with ctDNA were conducted by ultraviolet spectrophotometry, the red shifts of the 256nm UV absorption peak of ctDNA contam -inated by 2 kinds of aldehyde pollutants were significant; It was detected for the first time that violet shifts of 218nm UV absorption peak was 16nm, when the binding of formaldehyde, acetaldehyde with ctDNA were conducted by ultraviolet spectrophotometry. (2) The binding of formaldehyde with ctDNA , for the first time , was conducted by resonance light scattering . It indicated that the shift of 600nm resonance light scattering peak was significant after formaldehyde added in ctDNA-SDBS-JG system. And the scattering intensity was linear to the concern -tration of formaldehyde in the range of 0~0.34mol/L, the equation of linear regression was 1=2.99 c+ 311.3, r=0.994. This assay may be used to estimate the amount of DNA adducts and to determine formaldehyde pollutant in environment. It also hinted that the effect of SDBS and JG was synergistic to the binding of formaldehyde with DNA. It meant that they activiated the process of formaldehyde with DNA bases forming adducts. That was charged ion or polar molecule induced polarization of formaldehyde carbonyl group and formaldehyde was aggregated on DNA molecular surface; And some inserted in the DNA basic groups then adductswere formed. The resonance light scattering assay we built could be used to study the mechanism of DNA adduct forming.(3) According to the experiment results and literature analysis, a possible mechanism of DNA adducts formed by aldehydes pollutants with DNA was proposed: tyipical aldehydes pollutants were polarized on carbonyl group by charged ions or polar molecule in vivo, then the polarized carbonyl group or carboxide oxygen atom bound with H+ generated in vivo metabolism to form electrophile bioactive molecule, then formed Schiff s base by attack -NH2 of DNA purine bases or -NH- of DNA pyrimidine bases. But Schiff s base was instability in vivo, it can form stable DNA adduct on the effect of deoxydize system. Adduct of formaldehyde with DNA may be N2-hydroxymethyl-dG adduct, N6 -hydroxy methyl -dA adduct; typical aldehydes pollutants also can cause DNA-DNA cross linking and DNA-protein cross linking and so on. The violet shifts of UV absorption of formaldehyde and acetaldehyde with ctDNA may contribute to the cross linking of DNA-DNA bases and the demolishment of conjugated system; There may be other mechanisms in vivo , which will be taken advance study.In the respect of DNA adducts detection, a method of DNA adduct detection was set up based on the HSA fluorescence quenching: (1) An ATP detection method was based on the fluorescence quenching effect of ATP to HSA in Tris-HCl buffer solution at pH =7.0. In the condition selected( A- ex=290nm, A em=334) , the quenching value of J^/im was found linear to the concentration of ATP in the range of 1.70-42.3 7ug/mL (r=0.9991)with the detection limit of 1.70ug/mL. The RSD is 0.70%~1.36% and the recovery is 95%~101%; (2) DNA was digested with special nuclease, and adducts were excised as dinucleotides(XpN). T4 polynucleotide kinase can catalyze the transfer the Y -phosphate from ATP to the 5’-hydroxyl terminius of Oligonucleotide. Therefore, the amount of adducts in sample was in proportion to the amount of dephosphorylated ATP. The amount of ATP was detected by HSA fluorescence quenching analysis, and the amount of adducts was determined indirectly. At the same time, a calibration curve was built based on the use of 6-Oligo nucleotides (GACTCA) as standard mass, a new assay was initially set up to detect DNA adducts. The assay was used to detect the adduct of formaldehyde with ctDNA and the detection limit更多还原