不同价态染砷大鼠肝脏MRP2表达水平与砷甲基化产物关联性

Association of MRP2 expression level and arsenic methylation in rats＇ liver treated with sodium arsenite and sodium arsenate

目的分析亚砷酸钠（iAs3＋）和砷酸氢钠（iAs5＋）染毒后，对大鼠多药耐药相关蛋白2（MRP2）表达的影响，及其与肝脏砷及甲基化产物的关联性，寻找不同价态砷体内代谢转运间的差异，为进一步阐明砷毒作用机制提供依据。方法70只Wistar大鼠分成7组，第l组为正常对照组，给予饮用去离子水；第2—4组为染毒iAs3＋高、中、低剂量组，分别给予20．0、6．7、2．2mg／kg体质量亚砷酸钠溶液；第5—7组为染毒iAs5＋高、中、低剂量组，分别给予20．0、6．7、2．2mg／kg体质量砷酸氢钠溶液。各组大鼠于染毒90d后处死，取肝脏组织，用蛋白印迹法（Western—blotting）测定MRP2表达的变化。采用高效液相一氢化物发生原子荧光光谱分析法（HPLC—HGAFS）分析各组肝脏中砷及甲基化产物含量，并运用Pearson相关法分析MRP2表达与砷及甲基化产物的相关性。结果对照组、iAs3＋和iAs5＋高、中、低剂量组MRP2蛋白表达分别为1．21±0．13、1．85±0．09、1．65±0．19、1．61±0．18、1．69±0．04、1．42±0．19、1．27±0．10，iAs3＋的高、中、低剂量组和iAs0‘高、中剂量组MRP2蛋白表达均高于对照组（P均〈0．05）；相同受试物比较，iAs3＋和iAs5＋的高剂量组MRP2蛋白表达均高于低剂量组（P均〈0．05）；不同受试物同一剂量组比较，iAs“高、中、低剂量组的MRP2蛋白表达均强于iAs5＋高、中、低剂量组（P均〈0．05）。同时，iAs3＋染毒组肝脏中MRP2蛋白表达量与iAs3＋和砷甲基化代谢产物MMA和DMA的含量均呈正相关关系（r值分别为0．575、0．678、0．395，P均〈0．05）；iAs5＋染毒组肝脏中MRP2表达量与MMA和DMA的含量均呈正相关关系（r值分别为0．593、0．643，P均〈0．05），与iAs3＋的含量无相关关系（P〉0．05）。结论随着砷染毒剂量的增加，MRP2表达逐渐上调，从而致使肝细胞膜上MRP2表达代偿性改变。MRP2外排通路可能在iAs3＋代谢径路中发挥重要作用。肝脏MRP2与不同价态砷甲基化产物的负荷或水平密切相关。

Objective To explore the effect of sodium arsenite （iAs3＋） and sodium hydrogen arsenate （iAs5＋） exposure on rat multidrug resistance protein 2 （MRP2） expression, and the correlation of liver arsenic and its methylation products, and to provide a basis for further elucidating the mechanism of arsenic toxicity. Methods Seventy wistar rats were randomly divided into seven groups, 10 rats in each group. Control group was administrated with deionized water. Sodium arsenite high-dose group, middle-dose group, low-dose group were administrated with different concentrations of sodium arsenite： 20.0, 6.7 and 2.2 mg/kg BW every day, respectively. Animals were sacrificed 90 days later by cervical dislocation to collect liver, the expression of MRP2 in the membrane of hepatocyte was determined by Western blotting. HPLC-HGAFS was employed to determine the content or level of arsenic and its methylation products. Correlation between expression of MRP2 and arsenic methylation products was analyzed using Pearson＇s linear correlation method. Results The MRP2 protein levels of control group, iAs3＋ and iAs5＋ high-, middle-, low-dose groups were 1.21 ± 0.13, 1.85 ± 0.09, 1.65 ± 0.19, 1.61 ± 0.18, 1.69 ± 0.04, 1.42 ± 0.19, 1.27 ± 0.10. Compared to control group, MRP2 protein levels of iAs3± high-, middle-, low-dose group and iAs5＋ high-dose group were increased （P 〈 0.05） ; Compared with the low-dose group, MRP2 protein levels of iAs3＋ and iAs5＋ high dose groups were increased （P 〈 0.05） ; Comparing the matched doses group of iAs5± and iAs3±, MRP2 protein levels of iAs3＋ high-, middle-, low-dose group were higher than iAs5＋（P 〈 0.05）. Meanwhile, there was a significant positive relationship between the expression of MRP2 and the content of iAs3＋ , MMA and DMA in iAs3＋exposed group（r = 0.575, 0.678, 0.395, all P 〈 0.05）. There was also a significant correlation between the expression of MRP2 and MMA and DMA in iAs5＋ exposed group（r = 0.593, 0.643, all P 〈 0.05）. There was no significant relationship between the expression of MRP2 and the content of lAs5＋ in iAs5＋ exposed group. Conclusions The expression of MRP2 is increased with increasing dose of arsenic exposure, thus resulting in compensatory changes on MRP2 expression in the liver cell membrane. MRP2 efflux may play an important role in the metabolic pathways of iAs3. The level or load of arsenic methylation in liver is closely related with MRP2 expression.