Study On Exposure Biomakers Of Benzo(a)pyrene

Objective: To establish a simple and highly sensitive method for the determinationof3-hydroxybenzo[a]pyrene(3-OHBaP) and benzo[a]pyrene-tetrol I-1(BaP-tetrol I-1)released after acid hydrolysis of (+)-anti-benzo(a)pyrene diol-epoxide(BPDE)-DNAadducts metabolited from BaP in rats blood and tissues by the high performance liquidchromatography with fluorescence detection （HPLC/FD）. And to study thetime-dose-effect relationship of3-OHBaP in rat blood, heart, spleen, lung, kidney,stomach, intestine, brain and muscles and the time-dose-effect relationship of(+)-anti-BPDE-DNA in rat blood, lung, kidney and brain and the correlation ofconcentrations of3-OHBaP and (+)-anti-BPDE-DNA in blood and that in tissues. Inorder to discuss the application of3-OHBaP and (+)-anti-BPDE-DNA in blood asexposure biomarkers of BaP.Methods: Purity of96%BaP was dissolved in corn oil, configured as the high doseof10mg/mL, the middle dose of5mg/mL and the low dose of0.5mg/mL of gastricjuice aside, respectively.224male SD rats, weigh180-220g, were randomLy dividedinto the high dose group (100mg/kg), middle dose group (50mg/kg), low dose group (5mg/kg) and control group (0mg/kg). Control animals were received the same volume ofcorn oil only. Each group contained56rats. Fasting for12h and free drinking waterbefore treatment, the volume of gastric was2mL.8rats were killed by decapitation at0.5h,1h,2h,4h,8h,12h,24h following gavage, respectively. The heart, liver, spleen,lung, kidney, stomach, intestine, brain and muscles were removed quickly and put theminto the physiological saline, washed and dryed surface with filter paper. At the sametime, blood and plasma samples were collected. All of samples were kept frozen at-20℃until the analysis.The plasma sample was for determination of3-OHBaP. A1mL of plasma samplewas placed into a test tube added2mL of acetone. After vortexing for5min andcentrifuging for10min, the organic phase was transferred into test tube, evaporated anddried finally. In the residue,150μL of acetone,50μL of iodoethane as derivatizing agent and0.1g of potassium carbonate were added at90℃for10min. After cooling,20μL ofthe residue was for detection.Average each tissue into two parts, one part was for determination of3-OHBaP andanother one was for determination of (+)-anti-BPDE-DNA adducts. The part fordetermination of3-OHBaP was placed in a test tube and four times in Physiologicalsaline were added in order to made into tissue homogenate. A100μL of tissuehomogenate was placed in a test tude. And200μL of Acetonitrile were added. Aftervortexing for5min and centrifuging10min, the organic phase was transferred into testtube. The part for determination of (+)-anti-BPDE-DNA was managed by tissue DNAextraction kit.The whole blood DNA and the tissue DNA were extracted by reagent kit. The(+)-anti-BPDE-DNA adducts were hydrolyzed in0.1mol/L HCl at90℃for4hours.The acid-hydrolysis products (BaP-tetrol I-1) of DNA adducts were extracted byethylacetate. After the ethylacetate was evaporated, the residue was redissolved in200μL of DMSO.Both of3-OHBaP and BaP-tetrol were measured by high-performance liquidchromatography with fluorescence detection. For3-OHBaP and BaP-tetrol I-1, thechromatographic separation were achieved in CenturySIL BDS C18column(150×4.6mm,5μm) by using a binary mixture of Methanol-Water(97:3, v/v) andMethanol-Water(pH7.0)(55:45, v/v)as mobile phase, respectively. The flow rate fordetermination of3-OHBaP and BaP-tetrol I-1were0.5mL/min and1mL/min,respectively. The3-OHBaP and BaP-tetrol I-1were performed under FD atλex/λem=365/450nm and λex/λem=245nm/395nm with a injection volume of20μL,respectively.The standard curve calculation of the various exposed groups of3-OHBaP and(+)-anti-BPDE-DNA adducts were established, respectively. Calculating theconcentrations of3-OHBaP and (+)-anti-BPDE-DNA adducts in rat blood and tissues,using the SPSS17.0to analysis and discussing both of the time-dose-effect relationshipof3-OHBaP and (+)-anti-BPDE-DNA adducts and the correlation of concentrations of3-OHBaP and (+)-anti-BPDE-DNA adducts in rat blood and that in tissues were did.Results: The methods for determination of3-OHBaP and (+)-anti-BPDE-DNAadducts were accurate and effective and the extraction recovery and the stability weregood. The dose-effect relationship of3-OHBaP in blood, heart, spleen, lung, kidney, stomach, intestine, brain and muscles and that of (+)-anti-BPDE-DNA adducts in blood,lung and brain were good. There was significant correlation of concentrations of3-OHBaP in heart, spleen, lung, kidney, brain, muscles and that in blood. Thecorrelation of concentrations of (+)-anti-BPDE-DNA in lung, brain and that in bloodwas good as well.Conclusion: A high-efficiency and stabilized high-performance liquid chromato-graphy with fluorescence method was improved for the determination of3-OHBaP and(+)-anti-BPDE-DNA adducts in blood and tissues. Both of concentrations of3-OHBaPand (+)-anti-BPDE-DNA adducts in blood were recommend to be BaP exposurebiomakers.