Study On Biomarkers Of Benzo[a]pyrene In Liver Tissue Of Rats

Objective: To detect the concentration of3-hydroxy-benzo[a]pyrene,(+)-anti-benzo[a]pyrene-diolepoxide-DNA adducts, superoxide dismutase, glutathionereductase, malonyl dialdehyde and8-hydroxy-2’-deoxyguanosine in liver tissue of ratsand study them as the exposure biomarker and the effect biomarker of benzo[a]pyrenein liver tissue.Methods:224rats were randomly divided into4groups (A, B, C, D) of56animals.Rats were dosed respectively the100mg/kg,50mg/kg,5mg/kg,0mg/kgbenzo[a]pyrene of body weight which was dissolved in corn oil. The liver tissuesamples, which were collected at0.5h,1h,2h,4h,8h,12h,24h following injection,were kept frozen at-20℃until the analysis.The rat tissue samples, collected from liver, were placed in a test tube. And fourtimes in Physiological saline were added in order to make into tissue homogenate. A100μL of tissue homogenate was placed in a test tube. And200μL of acetonitrile wereadded. After vortexing for1min and centrifuging10min, the organic phase wastransferred into test tube.The content of3-OHBaP in liver tissue of rats was measured by high-performanceliquid chromatography with fluorescence detection through external standard method.The chromatographic separation was achieved in CenturySIL BDS C18column(150×4.6mm,5μm) by using a binary mixture of Methanol-Water(97:3, v/v) as mobilephase, the flow rate:0.5mL/min, λex/λem=365/450nm with a injection volume of20μL.The liver tissues DNA were extracted by reagent kit. The DNA adducts werehydrolyzed in0.1mol/L HCL at90℃for4hours. The acid-hydrolysis a product(BaP-tetrol) of DNA adducts was extracted by ethyl acetate. The ethyl acetate phase wasevaporated in vacuum rotary evaporator and dried finally with a nitrogen stream, and200μL of dimethyl sulfoxid was added to redissolve. The content of BaP-tetrol wasmeasured by high-performance liquid chromatography with fluorescence detection through external standard method. The chromatographic separation was achieved inCenturySIL BDS C18column (150×4.6mm,5μm) by using a binary mixture ofMethanol-Water(55:45, v/v) as mobile phase, the flow rate:1.0mL/min,λex/λem=245/395nm with a injection volume of20μL. BaP-tetrol was reacted indirectlythe content of (+)-anti-BPDE-DNA adducts in liver tissue of rats.The concentration of SOD, GSH, MDA,8-OHDG were detected by kit,respectively.The results according with normal distribution, expressed as mean±standarddeviation, analyzed by SPSS17.0. The correlation between the exposure dose and itstwo metabolites was analysis with the Pearson method.Results: The samples, which in the High, medium and low exposure group (100,50,5mg/kg), were detected3-OHBaP and (+)-anti-BPDE-DNA adducts except(+)-anti-BPDE-DNA adducts in low exposure group. A positive correlation was foundbetween the concentration of intragastrical BaP and the concentration of3-OHBaP or(+)-anti-BPDE-DNA adducts. A positive correlation was found between theconcentration of intragastrical BaP and the concentration of MDA or8-OHDG. In allexposure concentration, GSH in liver reached the peak at5mg/kg and SOD in liverreached the peak at50mg/kg.Conclusion:3-OHBaP and (+)-anti-BPDE-DNA adducts can be considered as anexposure biomarker for assessing carcinogenic risks as it is a metabolite of BaP in livertissue. SOD、GSH、MDA、8-OHDG can be considered as an effect biomarker for BaPin liver tissue.