The Study Of BPDE-DNA Adducts In Vitro And Vivo

Benzo(a)pyrene (BaP) is generally recognized as one of human carcinogens with extraordinary strong teratogenecity, mutagenesis and carcinogenesis. BaP presents everywhere in living environment of human beings, and its detection is one of monitoring items in environmental pollution either in home or abroad. When BaP was absorbed by human or animals, after metabolic activation, it could covalently bind to DNA and form DNA adducts. DNA adduct, one most important and generally chemical lesions of DNA, is an important index in biological monitoring. If these adducts escaped from auto-restoration system of cells, they could play a key role in the early stages of mutagenesis and carcinogenesis. In this experiment, we studied the formation conditions of BPDE-DNA [BPDE is the abbraviation for (7R, 8S) - dihydroxy - (9S, 10R)-epoxy-7, 8, 9, 10 - tetrahydro benzo[a]pyrene] adducts in vitro and vivo and established a method of measurement of BPDE-DNA adducts by high performance liquid chromatography.The research in vitro: After incubation of the mixtures of BaP, DNA, S9 at a certain proportion (BaP:DNA:S9=1: 12:35) at 37℃for 21h, the ultraviolet spectrum of the mixtures was scanned with a UV spectrophotometer, to inspect whether the DNA structure had changed or not, and the BaP, BaP metabolites (the most important one was BPDE) and acid-hydrolysis products of DNA adducts (BP-tetrol) were detected by HPLC and confirmed by HPLC-MS.Animal studies: The SD rats were treated with single i.p. dose of BaP-DMSO (cosolvent:1% sodium carboxymethyl cellulose), 150mg/kg rat weight. The blood of femoral vein was withdrawn 5h later, and anticoagulated using 4% EDTA. The blood DNA was extracted with kit and confirmed the extraction by agarose gel electrophoresis. After extraction, the DNA was hydrolyzed in 0.1nmol/L HC1 at 90℃water bath for 4h. The acid-hydrolysis products (BP-tetrol) of DNA adducts were extracted by ethyl acetate and measured by HPLC. Finally it was confirmed by HPLC-MS.The measurement parameters: analytical column: Phenomenex (USA), C₁₈ (ODS), 5um, 250×4.6mm; column temperature: room temperature; mobile phase A: methanol, mobile phase B: HAC-NH₄AC (pH4.5±0.2); gradient elution with flow rate of 1.0ml/min; fluorescence detector (BPDE:λ_ex=338nm,λ_em=425nm; BP-tetrol:λ_ex=344nm,λ_em=398nm). The results of HPLC indicated that there were BPDE-DNA adducts and the formation of BPDE and BP-tetrol was confirmed by HPLC-MS.In vitro research, the UV wavelength of the maximum absorption of DNA after incubation with BaP and S9 has been red shift compared with that of DNA standard which incubated in the same way but without BaP and S9 (DNA standard: 259.1nm; DNA adducts: 305.3nm), the change of spectrum indicated that structure of DNA had been changed; and there appeared new peaks in the incubation mixture with BaP and S9 in the HPLC chromatogram as well as in the acid-hydrolysis solution. The new peaks were confirmed by HPLC-MS to be BPDE and BP-tetrol. In animal studies, there were new peaks in the rats treated with BaP compared with the control ones in chromatogram. One of the new peaks was confirmed by HPLC-MS to be BP-tetrol.According to the results of UV spectrum, HPLC and LC-MS, we could make a conclusion that under the experimental conditions, BaP could be transformed to BPDE, and to form BPDE-DNA adducts both in vitro and vivo. The HPLC method established in this study was applied to measure BPDE-DNA adducts, and found it was simple, time-saving and low-cost.