Study On Biomarker Of Benzopyrene In Rats

Objective: To establish a method for the determination of the three benzopyrene metabolites (1-OHBaP ,3-OHBaP, BPDE-DNA) in rats blood by the HPLC -FLD. And to study the three metabolites concentration in the blood changes with the dose variation. By contrast sensitivity, detectability, stability, operation to measure the three metabolites of benzopyrene as the application of biomarkers.Methods: Purity of 96% benzopyrene dissolved in corn oil, configured as a high dose of (5~15)mg/ kg, the dose of (0.2~2)/ kg, (0.02~0.2)mg/ kg of irrigation with low doses of gastric juice aside. 280 male SD rats, weigh 180 ~ 220g, were randomly divided into A, B, C, D four groups. A, B, C group were exposed to the three groups, each group 90 rats, D Section 10 rats, non-exposed control group. 6h before treatment fasting water. A group of high-dose group, 90rats were divided into 10 groups, irrigation with high doses of diluted liquid gastric gavage dose were 5mg/kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg. B group in the dose gavage group, followed by intragastric concentration of the solution were diluted by irrigation of gastric juice concentration, exposure dose were 0.2 mg / kg, 0.4 mg / kg, 0.6 mg / kg, 0.8 mg / kg, 1.0 mg / kg, 1.2 mg / kg, 1.4 mg / kg, 1.6 mg / kg, 1.8 mg / kg, 2.0 mg / kg. C group, the low dose group, followed by irrigation of gastric juice were diluted by low concentrations of gastric irrigation, exposure dose of 0.02 mg / kg, 0.04 mg / kg, 0.06 mg / kg, 0.08 mg / kg, 0.10 mg / kg, 0.12 mg / kg, 0.14 mg / kg, 0 .16 mg / kg, 0.18 mg / kg, 0.20 mg / kg.D group was the control group, fed corn oil blank. 6h after the treatment interval of time. Handled three times. Fasting rats with water during processing. After the last treatment 2 hours after .Take the rats blood store in -20Blood samples were divided into two parts, whole blood to extract DNA. Another part of the centrifuge the plasma. plasma samples add acetone, vortex 5min, centrifuge 10min, take the upper organic phase, placed in the vacuum rotary evaporator, dried by nitrogen. Then add 150 L acetone, 50 L ethyl iodide fluorescence derivatization agent, and 0.1g of anhydrous sodium sulfate, 90 water bath for 10min. Take 20 L, methanol / water (14/86), PH = 7.45; flow rate: 1.0mL/min, excitation wavelength:280nm; detection wavelength:330nm,injection chromatographic conditions, record the chromatogram.Take whole blood samples, whole blood DNA extraction kit processing, purification of plasma DNA in the 0.1 nmol / L HCl, 90 constant temperature water bath in the acid hydrolysis of 4 h, extracted with ethyl acetate acid hydrolysis products - tetraol - benzo (a) pyrene, ethyl acetate after extraction solution used in the vacuum evaporator and evaporated to dryness, re-dissolved in 200 L acetone, take 20 Lsample, methanol / water (17/83), PH = 7.45; flow rate: 1.0mL/min, excitation wavelength:265nm, detection wavelength:425nm,injection chromatographic conditions, record the chromatogram.Establish standard curve calculation of the various exposed groups of three metabolites of blood 1-OHBaP,3-OHBaP, BPDE-DNA. do scatter plots of blood concentration relative exposure levels with blood levels .Results: The experimental method can effectively detect three metabolites. The extraction precision stability was good.in the three dose groups 1-OHBaP, 3-OHBaP were effectively detected .in the low dose group 1-OHBaP, 3-OHBaP were significant positive dose-related relations .in the high dose group 1-OHBaP ,3-OHBaP exceeded the enzyme saturation, there is no significant positive correlation. BPDE-DNA in the low dose group have no effective detection, in the middle and high dose group have effectively detect, in the middle dose group showed significantly positive. at high dose group in the enzyme due to over saturation, found no significant positive correlation.In the three dose group in the ,3-OHBaP concentrations are much higher than 1-OHBaP and the concentration of BPDE with good detectability. And 3-OHBaP the sensitivity of the relative dose was higher than 1-OHBaP and BPDE-DNA.