Joint International Research Laboratory of Ethnomedicine, Key Laboratory of Basic Pharmacology of Ministry of Education, Zunyi Medical College, Guizhou, China.
Joint International Research Laboratory of Ethnomedicine, Key Laboratory of Basic Pharmacology of Ministry of Education, Zunyi Medical College, Guizhou, China; Department of Environmental Health, School of Public Health, Indiana University, Bloomington, IN, USA.
Environ Toxicol Pharmacol. 2019 Feb;66:83-90. doi: 10.1016/j.etap.2018.12.020. Epub 2018 Dec 26.
Cinnabar has a long history of uses in Chinese traditional medicines as an ingredient in various remedies. However, the detailed mechanism of cinnabar in medication remains unclear, and the toxicity of cinnabar has been a debate due to its containing mercury sulfide. This study was designed to investigate the differential transport mechanism of cinnabar and other Hg-containing compounds HgCl, MeHg and HgS, and to determine if organic anion transporters OAT1 and OAT3 were involved in the differential transport mechanism.
The 293T cells were employed to investigate and compare the differential transport mechanism of cinnabar and HgCl, MeHg and HgS. Cells were incubated with a low dose (5 μM HgCl and MeHg, 200 μM HgS and cinnabar), medium dose (10 μM HgCl and MeHg, 400 μM HgS and cinnabar), and high dose (20 μM HgCl and MeHg, 800 μM HgS and cinnabar) of HgCl, MeHg, HgS and cinnabar for 24 h. Following treatment, the cells were collected and the cell viability was determined by MTT assay. The intracellular mercury content was measured at 1, 4, and 24 h after treatment with 10 μM of the tested agents by an atomic fluorescence spectrophotometer. The effect of these tested agents on mitochondrial respiration was determined in a high-resolution oxygraphyat 24 h following treatment. Furthermore, the effect of modulation of expression of transporters OAT1 and OAT3 on the transport and cytotoxicity of the tested agents was evaluated. The up and down regulation of OAT1 and OAT3 were achieved by overexpression and siRNA transfection, respectively.
Compared with HgCl and MeHg, the cytotoxicity of cinnabar and HgS was lower, with cell viability at the high dose cinnabar and HgS being about 65%, while MeHg and HgCl were 40% and 20%, respectively. The intracellular mercury accumulation was time-dependent. At 24 h the intracellular concentrations of HgCl and MeHg were about 7 and 5 times higher, respectively, than that of cinnabar. No significant difference was found in the intracellular mercury content in cells treated with cinnabar compared to HgS. The knockdown and overexpression of the transporter OAT1 resulted in significant reduction and increase, respectively, in mercury accumulation in HgCl -treated cells in relative to control cells, while no significant changes were observed in cells treated with cinnabar, MeHg, and HgS. In addition, the knockdown and overexpression of the transporter OAT3 caused significant reduction and increase, respectively, in mercury accumulation in both HgCl and MeHg-treated cells in relative to control cells, while no significant changes were observed in cells treated with cinnabar and HgS. Furthermore, it was found that cells transfected with siOAT1 caused significant resistance to the cytotoxicity induced by HgCl, while no noticeable changes in cell viability were observed in cells treated with other tested agents. Additionally, cells transfected with OAT3 did not change cell sensitivity to cytotoxicity induced by all of the four tested agents.
This study demonstrates that differential transport and accumulation of mercury in 293T cells exists among cinnabar and the three mercury-containing compounds HgCl, MeHg and HgS, leading to distinct sensitivity to mercury induced cytotoxicity. The kidney organic anion transporters OAT1 and OAT3 are partially involved in the regulation of the transport of HgCl and MeHg, but not in the regulation of the transport of cinnabar.
辰砂在中国传统医学中作为各种药物的成分已有很长的使用历史。然而，辰砂在药物中的详细作用机制尚不清楚，由于其含有硫化汞，辰砂的毒性一直存在争议。本研究旨在研究辰砂与其他含汞化合物 HgCl、MeHg 和 HgS 的差异转运机制，并确定有机阴离子转运蛋白 OAT1 和 OAT3 是否参与差异转运机制。
采用 293T 细胞研究并比较辰砂与 HgCl、MeHg 和 HgS 的差异转运机制。细胞分别用低剂量（5μM HgCl 和 MeHg，200μM HgS 和辰砂）、中剂量（10μM HgCl 和 MeHg，400μM HgS 和辰砂）和高剂量（20μM HgCl 和 MeHg，800μM HgS 和辰砂）的 HgCl、MeHg、HgS 和辰砂孵育 24 小时。处理后，收集细胞，用 MTT 法测定细胞活力。用原子荧光分光光度计测定处理后 1、4 和 24 小时 10μM 测试剂处理后细胞内汞含量。处理后 24 小时，在高分辨率测氧仪中测定这些测试剂对线粒体呼吸的影响。此外，还评估了转运蛋白 OAT1 和 OAT3 表达调节对测试剂转运和细胞毒性的影响。通过过表达和 siRNA 转染分别实现 OAT1 和 OAT3 的上调和下调。
与 HgCl 和 MeHg 相比，辰砂和 HgS 的细胞毒性较低，高剂量辰砂和 HgS 的细胞活力约为 65%，而 MeHg 和 HgCl 分别为 40%和 20%。细胞内汞积累是时间依赖性的。24 小时时，HgCl 和 MeHg 的细胞内浓度分别约为辰砂的 7 倍和 5 倍。与 HgS 相比，辰砂处理的细胞内汞含量无明显差异。转运蛋白 OAT1 的敲低和过表达导致 HgCl 处理的细胞中汞积累分别显著减少和增加，而在辰砂、MeHg 和 HgS 处理的细胞中未观察到明显变化。此外，转运蛋白 OAT3 的敲低和过表达导致 HgCl 和 MeHg 处理的细胞中汞积累分别显著减少和增加，而在辰砂和 HgS 处理的细胞中未观察到明显变化。此外，发现转染 siOAT1 的细胞对 HgCl 诱导的细胞毒性有明显的抗性，而其他测试剂处理的细胞活力没有明显变化。此外，转染 OAT3 的细胞对四种测试剂诱导的细胞毒性均无明显改变细胞敏感性。
本研究表明，293T 细胞中辰砂与三种含汞化合物 HgCl、MeHg 和 HgS 之间存在差异转运和积累，导致对汞诱导的细胞毒性的敏感性不同。肾脏有机阴离子转运蛋白 OAT1 和 OAT3 部分参与了 HgCl 和 MeHg 的转运调节，但不参与辰砂的转运调节。
