Mechanism And Determination Of DNA Adducts Formed By Typical Aldehyde Pollutants With DNA

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...