Study The Intake And Efflux Differences Of Cinnabar And Other Mercury-containing Compounds In The Kidney

Objective:To study and compare the intake and efflux differences of cinnabar,HgS,HgCl₂ and MeHg in the kidney.Methods:1.UPLC-ICP/MS methods were developed and validated for the determination of inorganic mercury and organic mercury in intracellular and incubation medium.2.The HEK 293T cells were incubated in 6-well plates and divided into five groups:control group,HgCl₂ group?0.2?M?,MeHg group?0.2?M?,HgS group?0.2?M?,cinnabar group?0.2?M?)with 6 wells per group.After the pretreatment of each test sample,they were incubated with the cells for 48h.Then 20?L of MTT was added in cells under dark conditions.After 4h,the supernatant was carefully discarded and 150?L of DMSO was added to measure the absorbance value at a wavelength of 490 nm.3.The HEK 293T cells were incubated in 6-well plates and divided into five groups:control group,HgCl₂ group?0.2?M?,MeHg group?0.2?M?,HgS group?0.2?M?,cinnabar group?0.2?M?with 6 wells per group.After the pretreatment of each test sample,they were incubated with the cells for 24h,36h and 48h,respectively.Then washed cells once with PBS.Finally,the cells were collected in 2 mL tubes,centrifuged at 179?g for 3 min at room temperature,discarded the supernatant,and repeated the above operation twice.Then 2 mL 0.3%Triton X-100 stationary cells were added in cells for 10 min.Intracellular mercury content was measured by UPLC-ICP/MS.4.The HEK 293T cells were incubated in 6-well plates and divided into five groups:control group,HgCl₂ group?0.2?M?,MeHg group?0.2?M?,HgS group?0.2?M?,cinnabar group?0.2?M?with 6 wells per group.After the pretreatment of each test sample,they were incubated with cells for 48h.After the cells were washed three times with PBS,fresh 9%fetal bovine serum medium added in cells and cultured 24h,36h again,?co-incubation culture 72h,84h?respectively.And the last the extracellular fluid was collected.Extracellular mercury content was measured by UPLC-ICP/MS.5.The HEK 293T cells were incubated in 6-well plates and divided into five groups:control group,HgCl₂ group?0.2?M?,MeHg group?0.2?M?,HgS group?0.2?M?,cinnabar group?0.2?M?with 6 wells per group.After the pretreatment of each test sample,they were incubated with the cells 24h,36h,48h,respectively.After the cells were washed three times with PBS,1 mL of Trizol lysis cells was added to each well.Similarly,the HEK 293T cells were incubated in 6-well plates and divided into five groups:control group,HgCl₂ group?0.2?M?,MeHg group?0.2?M?,HgS group?0.2?M?,cinnabar group?0.2?M?with 6 wells per group.After the pretreatment of each test sample,they were incubated with the cells 48h.Next,the cells washed three times with PBS,fresh 9%fetal bovine serum medium were added in cells and cultured 24h,36h again,?co-incubation culture 72h,84h?respectively.After the cells were washed three times with PBS,1 mL of Trizol lysis cells was added to each well.Real-time PCR was used to detect the expression of the kidney transporters mRNA.Results:1.A simple,specific and reproducible UPLC-ICP/MS method has been developed and validated for determination of inorganic mercury and methyl mercury in intracellular and incubation medium,respectively.2.After treatment of 0.2?M of mercuric sulfide,cinnabar,mercuric chloride and methyl mercury for 48h,the cell viability was 90.97%,90.32%,91.88%and 91.45%,respectively.3.Intracellular mercury experiments showed that the content of inorganic mercury in the mercury chloride group within 24h was 0.48%?%of the added amount?,but inorganic mercury content were lower than 0.039?g/L in mercuric sulfide and cinnabar.?low limit of quantification was 0.039?g/L?.The organic mercury in the methyl mercury group was 8.43%?%of the added amount?,while organic mercury content were lower than 0.039?g/L in mercury sulfide and cinnabar groups?low limit of quantification was 0.039?g/L?.The content of inorganic mercury in the intracellular mercury chloride group,the mercury sulfide group,and the cinnabar group for 36 h was 2.41%,1.58%,and 0.36%,respectively.?%of the added amount?.The organic mercury contents of methyl mercury,mercury sulfide,and cinnabar were10.31%,0.77%,and 0.14%,respectively.?%of the added amount?.The content of inorganic mercury for 48h,mercury chloride group,the mercury sulfide group,and the cinnabar group was 3.00%,2.40%,and 2.20%,respectively.?%of the added amount?.48h intracellular methyl mercury group,mercury sulfide group,cinnabar group of organic content were 15.38%,0.79%,0.19%,respectively.?%of the added amount?.The results showed that the inorganic mercury of the cinnabar group and the mercury sulfide group increased continuously with the increase of time,and reached the peak at 48h.4.The extracellular mercury experiments showed that the extracellular mercury content of inorganic mercury in the chloride group,mercury sulfide group,and cinnabar group at 24h?co-incubation for 72h?was 68.28%,74.01%,and 81.35%?%of intracellular mercury content?.The extracellular mercury of organic mercury content in the methyl mercury group,mercury sulfide,and cinnabar group were63.55%,1.06%,and 1.14%?%of intracellular mercury content?.The extracellular mercury of inorganic mercury content in the chloride group,mercury sulfide group,and cinnabar group at 36h?co-incubation for 84h?was 71.80%,81.17%,89.02%,respectively.?%of intracellular mercury content?.The extracellular mercury of organic mercury content in the methyl mercury group,mercury sulfide,and cinnabar group were 66.96%,1.34%,and 1.40%,respectively?%of intracellular mercury content?.At 36h,the cinnabar group content of inorganic mercury outside cells was the highest.5.According to RT-PCR results,the mercury sulfide and cinnabar group significantly increased mRNA expression of Mdr1 at 72h.Then the cinnabar group and the mercury sulfide group increased mRNA levels of Mrp2 at 72h and 84h,respectively.The methyl mercury group and the mercuric chloride group had little effect on the mRNA of Mrp1,Mrp2,and Mrp4.Conclusion:1.The transportation of cinnabar and other mercury compounds were different,and its cell-intake speed is much lower than that of mercuric chloride and methyl mercury.The speed of cinnabar cell-efflux is faster than that of mercuric chloride and methyl mercury.2.The efflux of mercury sulfide and cinnabar may be related to transporters Mdr1and Mrp2.