| Objective: Explore the absorption mechanism of the cinnabar,mercuric chloride and methyl mercury.Methods: 1 The establishment of UPLC-ICP/MS analysis method for the detection of inorganic mercury and methyl mercury in the sample.2 A complete Caco-2 cell model was prepared,after the model is successful,mercuric chloride,methyl mercury,mercuric sulfide and cinnabar were incubated with cells at soluble mercury molar concentration(0.1 ?mol/L),cell viability was measured by MTT assay and mercury content in AP side,BL side and cell sample was detected by UPLC-ICP / MS.3 The effect of hydrogen ion concentration on the absorption of mercuric chloride,methyl mercury,mercuric sulfide and cinnabar were studied.The experiment was divided into blank group,control group and hydrogen ions high concentration group.In AP side,the blank group was given 100 ?L of medium containing 10% fetal bovine serum;The control group was given 100 ?L of soluble mercury equimolar concentration(0.1 ?mol/L)of Mercury chloride,methyl mercury,mercuric sulfide and cinnabar prepared by DMEM(containing 10% FBS),hydrogen ion high concentration group was given 100 ?L of soluble mercury equimolar concentration(0.1 ?mol/L)of Mercury chloride,methyl mercury,mercuric sulfide and cinnabar prepared by MES-DMEM(containing 10% FBS).Given DMEM(containing 10% FBS)600 ?L in BL side,incubated with cells for 24 h,the AP side,BL side and cell samples were collected,using UPLC-ICP/MS to detect the mercury content.4 The effect of sodium ions on the absorption of mercury chloride,methyl mercury,mercuric sulfide and cinnabar,the experiment was divided into blank group,control group and no sodium group.In AP side,the blank group was given 100 ?L of medium containing 10% fetal bovine serum;The control group was given 100 ?L of soluble mercury equimolar concentration(0.1 ?mol/L)of Mercury chloride,methyl mercury,mercuric sulfide and cinnabar prepared by DMEM(containing 10% FBS),sodium free group was given 100 ?L of soluble mercury equimolar concentration(0.1 ?mol/L)of Mercury chloride,methyl mercury,mercuric sulfide and cinnabar prepared by no sodium ion reagent.Given DMEM(containing 10% FBS)600 ?L in BL side,incubated with cells for 24 h,the AP side,BL side and cell samples were collected,using UPLC-ICP/MS to detect the mercury content.5 The effect of ATP on the absorption of mercuric chloride,methyl mercury,mercuric sulfide and cinnabar.The experiment was divided into blank group,control group and ATP inhibitor group.In AP side,the blank group was given 100 ?L of medium containing 10% fetal bovine serum;The control group was given 100 ?L of soluble mercury equimolar concentration(0.1 ?mol/L)of mercury chloride,methyl mercury,mercuric sulfide and cinnabar prepared by DMEM(containing 10% FBS),ATP inhibitor group group was given 100 ?L of soluble mercury equimolar concentration(0.1 ?mol/L)of mercury chloride,methyl mercury,mercuric sulfide and cinnabar prepared by ATP inhibitors.Given DMEM(containing 10% FBS)600 ?L in BL side,incubated with cells for 24 h,the AP side,BL side and cell samples were collected,using UPLC-ICP/MS to detect the mercury content.6 Mercuric chloride,methyl mercury,mercuric sulfide and cinnabar were incubated with cells at soluble mercury molar concentration(0.1 ?mol/L),The expression of CTL1,CTL2,PEPT1,PEPT2,MCT1,LAT1,LAT2,CNT1,CNT2,SGLT6,P-gp,BCRP were detected by Real-Time PCR method.Results: 1 mercury content of saturated solution of 0.01 g mercury sulfide is 3.6 × 10-10 g,accounting for the amount of the amount of four millionths.mercury content of saturated solution of 0.01 g cinnabar is 3.45 × 10-10 g,accounting for the amount of the amount of three thousandths,mercury sulfide and cinnabar saturated solution contain inorganic mercury and methyl mercury,methyl mercury of mercuric sulfide is 13% of total mercury content in mercury sulfide saturated solution,methyl mercury of cinnabar is 21.5% of total mercury content in cinnabar saturated solution.2 The cell viability of mercury chloride,methyl mercury,mercuric sulfide and cinnabar was higher than 90% at the equimolar concentration of soluble mercury by MTT experiment.3 Using UPLC-ICP/MS detection of BL samples in the control group of mercury content in the BL side,mercuric chloride,mercury sulfide and cinnabar were respectively 14.08%,0.94% and 0.524%.The proportions of methyl mercury in methyl mercury,mercuric sulfide and cinnabar were respectively 38.5%,2.36% and 4.4%.The mercury content in cinnabar group was 34.97% of the mercuric chloride group,was 12.79% of the methyl mercury group.The mercury content of mercury sulfide and cinnabar into the BL side is lower than mercuric chloride and methyl mercury.4 Compared with the control group,the content of inorganic mercury in the mercuric chloride group decreased by 8%,and the contents of inorganic mercury in mercuric sulfide and cinnabar were not significantly changed.methyl mercury,mercuric sulphide and cinnabar were decreased by 39.7%,40.28% and 30.8%,respectively.In cells,the content of inorganic mercury in the mercury chloride group increased by 26.8%,and the contents of inorganic mercury in mercuric sulfide and cinnabar were the content of methyl mercury in mercury and mercuric soda increased by 126% and 88.7%,respectively.In the BL side,mercuric chloride,mercuric sulfide and cinnabar were in the range of inorganic mercury the content of methyl mercury in methyl mercury,mercuric sulfide and cinnabar was increased by 35.5%,61.14% and 104%,respectively.That is: to increase the concentration of hydrogen ions on the side of the administration,and promote the absorption of methyl mercury in mercuric chloride,methyl mercury and mercuric sulfide and cinnabar.5 Compared with the control group,the contents of inorganic mercury in the mercury chloride group decreased by 18.8%,and the contents of inorganic mercury in mercuric sulfide and cinnabar were not changed,mercuric sulfide and cinnabar were increased by 28.8% and 51.5%,respectively.In cells,the content of inorganic mercury in the mercury chloride group increased by 32.8%,and the contents of inorganic mercury in mercuric sulfide and cinnabar were increased 63.2% and 66% respectively.There was no significant difference in the content of methyl mercury in methyl mercury,mercuric sulfide and cinnabar.There was no significant difference in the content of inorganic mercury in the BL side,mercuric chloride,mercuric chloride and cinnabar,and methyl mercury in cinnabar decreased by 16.4%,35.3% and 40.3%,respectively.To reduce the concentration of sodium ions on the side of the administration,the absorption of mercuric chloride and mercuric sulfide and inorganic mercury in cinnabar is promoted,and the absorption of methyl mercury in methyl mercury,mercuric sulfide and cinnabar is inhibited.6 Compared with the control group,the content of inorganic mercury in the mercury chloride group was 14.29% higher than that in the control group.The contents of inorganic mercury in mercuric sulfide and cinnabar were significantly different,and the content of methyl mercury in the methyl mercury group increased 50.7%,mercury sulfide and cinnabar were increased by 40.7% and 39.7%,respectively.There was no significant difference in mercury,methyl mercury,mercuric sulfide and cinnabar content in cells.On BL side,mercuric sulfide and cinnabar were not significantly different,and the contents of methyl mercury in methyl mercury,mercuric sulfide and cinnabar decreased by 71%,57.4% and 71%,respectively.That is: when the intracellular ATP is inhibited in the case of inhibition of mercury chloride,methyl mercury and mercuric sulfide and cinnabar methyl mercury absorption.7 Compared with the blank group,the expression of PEPT1 m RNA in mercuric sulfide group and cinnabar group was significantly decreased(P <0.05).The expression of LAT1 m RNA of the sodium-dependent transporter in mercuric chloride group and methyl mercury group was significantly increased(P <0.05).The expression of ATP-dependent transporter MRP3 m RNA was significantly increased in mercuric chloride group and methyl mercury group(P <0.05).Conclusion: Hydrogen ion concentration,sodium ion concentration and ATP affect the absorption of methyl mercury of methyl mercury,mercuric sulfide and cinnabar,sodium ion concentration has a significant effect on the absorption of inorganic mercury in mercuric chloride,mercuric sulfide and cinnabar;Based on the Caco-2 cell model,the absorption of mercuric sulfide and cinnabar on the BL side is much lower than that of mercuric chloride and methyl mercury,which mercuric sulfide and cinnabar may decrease the expression of PEPT1 m RNA,mercuric chloride and methyl mercury may increase the expression of LAT1 m RNA and MRP3 m RNA. |
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