MRP2及MRP2-GSH共转运体系对肝脏砷代谢影响的实验研究
摘要
目的 1．观察不同剂量砷染毒大鼠肝细胞膜转运蛋白(MRP2)表达水平的变化与砷代谢的关系
2．观察用BSO抑制大鼠谷胱甘肽(GSH)合成酶，对肝脏砷代谢的影响
方法 30只健康Wistar大鼠随机分为5组，每组6只，雌雄各半，灌胃染毒，受试物为亚砷酸钠(分析纯)，以生理盐水溶解。第1组为对照组，给予生理盐水；第2～4组为染砷组，分别给予4，10和20mg／kg体重的亚砷酸钠溶液，隔天染毒一次，2周后将动物处死；第5组为BSO干预组，在实验结束的前3天，每天大鼠预先腹腔注射2mmol／kg体重BSO溶液，4小时后再经口给予20mg／kg体重的亚砷酸钠溶液，其他处理同上。原子吸收分光光度法测定肝组织、胆汁和全血中的总砷含量。蛋白印迹法测定肝细胞膜上MRP2的含量改变。在电镜和光镜下观察形态学改变，同时测定血清谷丙转氨酶(ALT)，总胆红素，谷胱甘肽(GSH)，谷胱甘肽过氧化物酶(GSH-PX)，丙二醛(MDA)等指标，观察肝脏受损情况。
结果 随着染砷剂量的增加，胆汁和肝脏中砷含量逐渐增加，经方差检验差异均有统计学意义(P＜0.05)。两两比较发现，3个染毒组的血砷与对照组之间差异有统计学意义，而3个染毒组间差异无显著统计学意义；随染砷剂量增加，MRP2表达明显增加，且MRP2表达量与胆汁砷含量呈正相关关系；随着染砷剂量增加，血清ALT活性也随之增加，高剂量组胆红素
含量明显高于对照组。与对照组相比，高剂量组肝GSH含量及GSH一PX活力
显著降低，中、高剂量组MDA水平则显著升高;大鼠经BSO预处理后再给
予20 mg/kg体重的亚砷酸钠溶液，发现胆汁砷含量明显高于对照组，而低于
高剂量组;肝脏砷含量明显高于对照组和高剂量组;MPRZ表达量比高剂量
组略有下降;与高剂量组和对照组相比，血清ALT活性明显增高，胆红素含
量明显升高，GSH含量及GSH一PX活力显著降低，MDA水平则显著升高。光
镜下观察染毒组肝组织可见少量散在的脂肪性变，肝细胞坏死以及小叶间胆
管上皮细胞增生的表现。透射电镜观察可见，随染毒剂量的增加肝组织超微
结构病变有所加重，具体表现为核形不规则，线粒体显著肿胀，山脊减少，部
分靖断裂、消失，还可见胆小管微绒毛变得稀疏，微肿胀。
结论1.亚砷酸钠致使肝脏功能受损，并且随着染砷剂量增加，肝脏损害
不断加重。亚砷酸钠可以诱导MRPZ的表达，随染砷剂量增加，MR陀表达量
增加。MRPZ在砷及其代谢产物的胆汁排泄过程中发挥了重要作用。
2.MRPZ转运砷及其代谢产物的作用与细胞内GSH水平相关，BsO通
过抑制GSH合成，导致MRPZ一GSH共转运功能减弱，从而增加砷对肝脏的毒性
作用.
Objective To determine whether expression of MRP2 increased with increasing arsenic concentration and to observe effects of BSO, an inhibitor of GSH synthesis on hepatic arsenic metabolism in rats.
Methods Thirty healthy Wistar rats were divided randomly into five groups and challenged with different concentrations of sodium arsenite : 0, 4, 10 and 20 mgAs/kgBW. High dose animals were also pretreated with BSO before arsenic administration. All animals were sacrificed 2 weeks after this treatment. Arsenic in liven blood and bile were detected by atomic absorption spectroscopy (AAS), and expression of MRP2 in the membrane of hepatocyte was determined by Western-blot analysis. By light microscopy and eletric microscopy, the morphological changes of liver were observed. Five biochemical indexes: Alanine aminotransferase (ALT) , total bilirubin in serum, glutathione (GSH), glutathione peroxidase (GSH-PX),and malonicacid (MDA) were selected to show the toxic effects on rats.
Results The level of total arsenic in blood, bile and liver at all three different dose groups were higher than those in control groups. Significant difference can seen between two dose group in arsenic level of bile and liver, but there is no difference between two dose group in arsenic level of blood. Expression of MRP2 increased with increasing arsenic concentration. A clear tendency for a
positive correlation between biliary arsenic concentration and MRP2 levels was found in liver. The activity of ALT increased as the dose increasing and showed a dose-effect manner. Compared with controls, total bilirubin in serum was increased in high-dose group. The contents of GSH and the activity of GSH-PX in high-dose group were higher than those in controls. The level of MDA in middle and high-dose groups increased markedly. Pretreatment with BSO decreased GSH level and increased lipid peroxidation. Biliary arsenic of rats pretreated with BSO was lower than that in rats of high-dose group and pretreatment with BSO increased arsenic content in tissues of rats. Expression of MRP2 pretreated with BSO was slightly decreasing. There was a little proliferation and necrosis in hepatocytes by LM. In bile canaliculi microvilli became swelling and sparse and the shape of nuclear showed irregular and some of mitochondrial cristae missed or broken by EM compared with control groups. Conclusion Sodium arsenite can i
nduced expression of MRP2. The up-regulation of MRP2 proteins play an important role in the bile secretion of arsenite and its metabolies. Depletion of GSH reduced the function of MRP2-GSH cotransport system.
2．观察用BSO抑制大鼠谷胱甘肽(GSH)合成酶，对肝脏砷代谢的影响
方法 30只健康Wistar大鼠随机分为5组，每组6只，雌雄各半，灌胃染毒，受试物为亚砷酸钠(分析纯)，以生理盐水溶解。第1组为对照组，给予生理盐水；第2～4组为染砷组，分别给予4，10和20mg／kg体重的亚砷酸钠溶液，隔天染毒一次，2周后将动物处死；第5组为BSO干预组，在实验结束的前3天，每天大鼠预先腹腔注射2mmol／kg体重BSO溶液，4小时后再经口给予20mg／kg体重的亚砷酸钠溶液，其他处理同上。原子吸收分光光度法测定肝组织、胆汁和全血中的总砷含量。蛋白印迹法测定肝细胞膜上MRP2的含量改变。在电镜和光镜下观察形态学改变，同时测定血清谷丙转氨酶(ALT)，总胆红素，谷胱甘肽(GSH)，谷胱甘肽过氧化物酶(GSH-PX)，丙二醛(MDA)等指标，观察肝脏受损情况。
结果 随着染砷剂量的增加，胆汁和肝脏中砷含量逐渐增加，经方差检验差异均有统计学意义(P＜0.05)。两两比较发现，3个染毒组的血砷与对照组之间差异有统计学意义，而3个染毒组间差异无显著统计学意义；随染砷剂量增加，MRP2表达明显增加，且MRP2表达量与胆汁砷含量呈正相关关系；随着染砷剂量增加，血清ALT活性也随之增加，高剂量组胆红素
含量明显高于对照组。与对照组相比，高剂量组肝GSH含量及GSH一PX活力
显著降低，中、高剂量组MDA水平则显著升高;大鼠经BSO预处理后再给
予20 mg/kg体重的亚砷酸钠溶液，发现胆汁砷含量明显高于对照组，而低于
高剂量组;肝脏砷含量明显高于对照组和高剂量组;MPRZ表达量比高剂量
组略有下降;与高剂量组和对照组相比，血清ALT活性明显增高，胆红素含
量明显升高，GSH含量及GSH一PX活力显著降低，MDA水平则显著升高。光
镜下观察染毒组肝组织可见少量散在的脂肪性变，肝细胞坏死以及小叶间胆
管上皮细胞增生的表现。透射电镜观察可见，随染毒剂量的增加肝组织超微
结构病变有所加重，具体表现为核形不规则，线粒体显著肿胀，山脊减少，部
分靖断裂、消失，还可见胆小管微绒毛变得稀疏，微肿胀。
结论1.亚砷酸钠致使肝脏功能受损，并且随着染砷剂量增加，肝脏损害
不断加重。亚砷酸钠可以诱导MRPZ的表达，随染砷剂量增加，MR陀表达量
增加。MRPZ在砷及其代谢产物的胆汁排泄过程中发挥了重要作用。
2.MRPZ转运砷及其代谢产物的作用与细胞内GSH水平相关，BsO通
过抑制GSH合成，导致MRPZ一GSH共转运功能减弱，从而增加砷对肝脏的毒性
作用.
Objective To determine whether expression of MRP2 increased with increasing arsenic concentration and to observe effects of BSO, an inhibitor of GSH synthesis on hepatic arsenic metabolism in rats.
Methods Thirty healthy Wistar rats were divided randomly into five groups and challenged with different concentrations of sodium arsenite : 0, 4, 10 and 20 mgAs/kgBW. High dose animals were also pretreated with BSO before arsenic administration. All animals were sacrificed 2 weeks after this treatment. Arsenic in liven blood and bile were detected by atomic absorption spectroscopy (AAS), and expression of MRP2 in the membrane of hepatocyte was determined by Western-blot analysis. By light microscopy and eletric microscopy, the morphological changes of liver were observed. Five biochemical indexes: Alanine aminotransferase (ALT) , total bilirubin in serum, glutathione (GSH), glutathione peroxidase (GSH-PX),and malonicacid (MDA) were selected to show the toxic effects on rats.
Results The level of total arsenic in blood, bile and liver at all three different dose groups were higher than those in control groups. Significant difference can seen between two dose group in arsenic level of bile and liver, but there is no difference between two dose group in arsenic level of blood. Expression of MRP2 increased with increasing arsenic concentration. A clear tendency for a
positive correlation between biliary arsenic concentration and MRP2 levels was found in liver. The activity of ALT increased as the dose increasing and showed a dose-effect manner. Compared with controls, total bilirubin in serum was increased in high-dose group. The contents of GSH and the activity of GSH-PX in high-dose group were higher than those in controls. The level of MDA in middle and high-dose groups increased markedly. Pretreatment with BSO decreased GSH level and increased lipid peroxidation. Biliary arsenic of rats pretreated with BSO was lower than that in rats of high-dose group and pretreatment with BSO increased arsenic content in tissues of rats. Expression of MRP2 pretreated with BSO was slightly decreasing. There was a little proliferation and necrosis in hepatocytes by LM. In bile canaliculi microvilli became swelling and sparse and the shape of nuclear showed irregular and some of mitochondrial cristae missed or broken by EM compared with control groups. Conclusion Sodium arsenite can i
nduced expression of MRP2. The up-regulation of MRP2 proteins play an important role in the bile secretion of arsenite and its metabolies. Depletion of GSH reduced the function of MRP2-GSH cotransport system.
引文
[1] JA Offergelt, H Roels, JP Buchet, M Boeckx, R Lauwerys. Relation between airborne arsenic trioxide and urinary excretion of inorganic arsenic and its methylated metabolited. Br J Ind Med 1992, 49:387-393
[2] E Hakala, L Pyy. Assessment of exposure to inorganic arsenic by determining the arsenic species excreted in urine. Toxicol Lett 1995, 77:249-258
[3] D. Kirk Nordstrom PUBLIC HEALTH: Enhanced: Worldwide Occurrences of Arsenic in Ground Water Science magazine Volume 296, Number 5576, Issue of 21 2002 Jun: 2143-2145
[4] 王福旭，董作仁，姚尔同．肿瘤及白血病细胞多釣耐药相关蛋白基因的研究进展．国外医学输血及血液学分册1996，19：135-138．
[5] Chen GQ, Shi XG, Tang W, Xiong SW, Zhu J, et al..Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL). Blood 89:3345-3353
[6] 王树叶 胡龙虎 三氧化二砷注射液治疗72例急性早幼粒细胞白血病张鹏，中华血液学杂志1996．2．14，17(2)：58—60，
[7] Kirk T. Kitchin Recent advances in arsenic carcinogenesis: modes of action, animal model systems, and methylated arsenic metabolites. Toxicol. Appl. Pharmacol. 172, 249-261. 2001
[8] Cole SPC, Bhardwaj G, Gerlach JH, et al. Overexpression of a transporter gene in a multidrug-resistance human lung cancer cell line. Science, 1992, 258:1650-1654.
[9] Hipfner DR, Deeley RG, Cole SPC. Structual mechanistic and clinical aspects of MRP1. Biochem Biophys Acta, 1999, 1461:276-359
[10] K(?)nig J, Nies AT, Cui Y, Leier I and Keppler D Conjugate export pumps of the multidrug resistance protein (MRP) family: localisation, substrate specificity, and MRP2-mediated drug resistance. Biochim Biophys Acta 1999 1461:377-394
[11] Subbarao V.Kala, Matthew W. Neely, Geeta Kala, Christopher I.Prater, Donna W.Atwood, Jeffrey S. Rice, and Michael W.Lieberman The mrp2/cMOAT Transporter and Arsenic-Glutathione Complex Formation Are Required for Biliary Excretion of Arsenic J. Biol. Chem., 2000,Vol. 275, Issue 43, 33404-33408, October 27,
[12] MICHAEL TRAUNER, MARCO ARRESE, CAROL J, et al. The rat canalicular conjugate export pump(Mrp2) isdown-regulated in intrahepatic and obstructive cholestasis: Gastroenterology 1997:113:255-264
[13] Kool M, de Haas M, Scheffer GL, Scheper RJ, et al. Analysis of expression of cMOAT(MRP2), MRP3, MRP4, and MRP5, homologues of themultidrug resistance-associated protein gene(MRP1), in human cancer cell lines. Cancer Res 1997: 57:3537-47
[14] Zaman GJ, Lankelma J, van Tellingen O, et al. Role of glutathione in the export of compounds from cells by the multidrug-resistance associated protein. Proc Natl Acad Sci USA, 1995,92:7690-7694.
[15] 卢圣栋《现代分子生物学实验技术》 中国协和医科大学出版社1999．12 382-386
[16] 陈志，杨凤山，张丽玲等我国地方性砷中毒及防治研究。中国地方病防治杂志 1998，13(6)：342-343
[17] Paul PC, Misbahuddin M, Ahmed AN, Dewan ZF, Mannan MA. Accumulation of arsenic in tissues of iron-deficient rats. Toxicol Lett 2002 Oct 5;135(3): 193-7
[18] Cohen SM, Arnold LL, Uzvolgyi E, Cano M, St John M, Yamamoto S, Lu X, Le XC Possible role of dimethylarsinous acid in dimethylarsinic acid-induced urothelial toxicity and regeneration in the rat Chem Res Toxicol 2002 Sep;15(9):1150-7
[19] Sheabar F, et al..In vitro effects of cadmium and arsenite on glare peroxidase, aspercate and lanine amiantransferases; choinestrerase and gloose 6-phosphate dehydro-genase activity in blood. Vel Hum Toxicol,1989 (6):518-531
[20] 刘继文，张晨等 氟砷染毒对大鼠子代肝脏氧化性损伤的研究。环境与健康杂志2000 17 (3)：139—142
[21] Huesker M, Folmer Y, Schneider M, Fulda C, Blum HE, Hafkemeyer R Reversal of drug resistance of hepatocellular carcinoma cells by adenoviral delivery of anti-MDR 1 ribozymes. Hepatology 2002 Oct;36(4 Pt 1):874-84
[22] Wijnholds J, Scheffer GL, van der Valk M, van der Valk P, Beijnen JH, Scheper R J. et al. Multidrug resistance protein 1 protects the oropharyngeal mucosal layer and the testicular tubules against drug-induced damage. J Exp Med 1998;188:797-808.
[23] Qiu-Ling Pei, Yoshinao Kobayashi, Yuji Tanaka, et al. Increased expression of multidrug resistance-associated protein 1 (mrpl) in hepatocyte basolateral membrane and renal tubular epithelia after bile duct ligation in rats. Hepatology Research, 2002, 22:58-64
[24] Cole SPC, Spark KE, Fraser K, et al. Pharmacological characterization of multidrug resistant MRP-transfected human tumor cells. Cancer Res,1994, 54:5902-5910.
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[27] Huesker M, Folmer Y, Schneider M, Fulda C, Blum HE, Hafkemeyer R Reversal of drug resistance of hepatocellular carcinoma cells by adenoviral delivery of anti-MDR 1 ribozymes. Hepatology 2002 Oct;36(4 Pt 1):874-84
[28] Lorico A, Rappa G, Finch RA, Yang D, Flavell RA, Sartorelli AC. Disruption of the murine MRP (multidrug resistance protein) gene leads to increased sensitivity to etoposide (VP-16) and increased levels of glutathione. Cancer Res 1997;57:5238-42.
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[33] I Leier, G Jedlitschky, U Buchholz, SP Cole, RG Deeley and D Keppler The MRP gene encodes an ATP-dependent export pump for leukotriene C4 and structurally related conjugates J. Biol. Chem., Vol. 269, Issue 45, 27807-27810, Nov, 1994
[34] Loe DW, Almquist KC, Deeley RG, Cole SP. Multidrug resistance protein (MRP)-mediated transport of leukotriene C4 and chemotherapeutic agents in membrane vesicles. J Biol Chem 1996;271:9675-82.
[35] Keppler D, Leier I, Jedlitschky G, Konig J. ATP-dependent transport of glutathione S-conjugates by the multidrug resistance protein MRP1 and its apical isoform MRP2. Chem Biol Interact 1998; 111-112:153-61.
[36] Habig WH ,Keppler D, Leier I, Jedlitschky G, Konig J. ATP-dependent transport of glutathione S-conjugates by the multidrugresistance protein MRP1 and its apical isoform MRP2. Chem Biol Interact 1998;111-112:153-61.
[37] I Leier, G Jedlitschky, U Buchholz, SP Cole, RG Deeley and D Keppler The MRP gene encodes an ATP-dependent export pump for leukotriene C4 and structurally related conjugates J. Biol. Chem., Vol. 269, Issue 45, 27807-27810, Nov, 1994
[38] Jie Liu,Hua Chen, Leier I, Jedlitschky G, Konig J. Over expression of glutathione S-transferase and multidrug resistance transport proteins is associated with acquired tolerance to inorganic arsenic. Mol Pharm 2001,60:302-309
[39] Vanhoefer U,Cao S, S. Ryu, T. Kawabe, S. Nada, A, Yamaguchi, Identification of basic residues involved in drug export function of human multidrug resistrance-assiciated protein 2, J. Biol. Chem. 275N(2000) 39617-39624
[2] E Hakala, L Pyy. Assessment of exposure to inorganic arsenic by determining the arsenic species excreted in urine. Toxicol Lett 1995, 77:249-258
[3] D. Kirk Nordstrom PUBLIC HEALTH: Enhanced: Worldwide Occurrences of Arsenic in Ground Water Science magazine Volume 296, Number 5576, Issue of 21 2002 Jun: 2143-2145
[4] 王福旭，董作仁，姚尔同．肿瘤及白血病细胞多釣耐药相关蛋白基因的研究进展．国外医学输血及血液学分册1996，19：135-138．
[5] Chen GQ, Shi XG, Tang W, Xiong SW, Zhu J, et al..Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL). Blood 89:3345-3353
[6] 王树叶 胡龙虎 三氧化二砷注射液治疗72例急性早幼粒细胞白血病张鹏，中华血液学杂志1996．2．14，17(2)：58—60，
[7] Kirk T. Kitchin Recent advances in arsenic carcinogenesis: modes of action, animal model systems, and methylated arsenic metabolites. Toxicol. Appl. Pharmacol. 172, 249-261. 2001
[8] Cole SPC, Bhardwaj G, Gerlach JH, et al. Overexpression of a transporter gene in a multidrug-resistance human lung cancer cell line. Science, 1992, 258:1650-1654.
[9] Hipfner DR, Deeley RG, Cole SPC. Structual mechanistic and clinical aspects of MRP1. Biochem Biophys Acta, 1999, 1461:276-359
[10] K(?)nig J, Nies AT, Cui Y, Leier I and Keppler D Conjugate export pumps of the multidrug resistance protein (MRP) family: localisation, substrate specificity, and MRP2-mediated drug resistance. Biochim Biophys Acta 1999 1461:377-394
[11] Subbarao V.Kala, Matthew W. Neely, Geeta Kala, Christopher I.Prater, Donna W.Atwood, Jeffrey S. Rice, and Michael W.Lieberman The mrp2/cMOAT Transporter and Arsenic-Glutathione Complex Formation Are Required for Biliary Excretion of Arsenic J. Biol. Chem., 2000,Vol. 275, Issue 43, 33404-33408, October 27,
[12] MICHAEL TRAUNER, MARCO ARRESE, CAROL J, et al. The rat canalicular conjugate export pump(Mrp2) isdown-regulated in intrahepatic and obstructive cholestasis: Gastroenterology 1997:113:255-264
[13] Kool M, de Haas M, Scheffer GL, Scheper RJ, et al. Analysis of expression of cMOAT(MRP2), MRP3, MRP4, and MRP5, homologues of themultidrug resistance-associated protein gene(MRP1), in human cancer cell lines. Cancer Res 1997: 57:3537-47
[14] Zaman GJ, Lankelma J, van Tellingen O, et al. Role of glutathione in the export of compounds from cells by the multidrug-resistance associated protein. Proc Natl Acad Sci USA, 1995,92:7690-7694.
[15] 卢圣栋《现代分子生物学实验技术》 中国协和医科大学出版社1999．12 382-386
[16] 陈志，杨凤山，张丽玲等我国地方性砷中毒及防治研究。中国地方病防治杂志 1998，13(6)：342-343
[17] Paul PC, Misbahuddin M, Ahmed AN, Dewan ZF, Mannan MA. Accumulation of arsenic in tissues of iron-deficient rats. Toxicol Lett 2002 Oct 5;135(3): 193-7
[18] Cohen SM, Arnold LL, Uzvolgyi E, Cano M, St John M, Yamamoto S, Lu X, Le XC Possible role of dimethylarsinous acid in dimethylarsinic acid-induced urothelial toxicity and regeneration in the rat Chem Res Toxicol 2002 Sep;15(9):1150-7
[19] Sheabar F, et al..In vitro effects of cadmium and arsenite on glare peroxidase, aspercate and lanine amiantransferases; choinestrerase and gloose 6-phosphate dehydro-genase activity in blood. Vel Hum Toxicol,1989 (6):518-531
[20] 刘继文，张晨等 氟砷染毒对大鼠子代肝脏氧化性损伤的研究。环境与健康杂志2000 17 (3)：139—142
[21] Huesker M, Folmer Y, Schneider M, Fulda C, Blum HE, Hafkemeyer R Reversal of drug resistance of hepatocellular carcinoma cells by adenoviral delivery of anti-MDR 1 ribozymes. Hepatology 2002 Oct;36(4 Pt 1):874-84
[22] Wijnholds J, Scheffer GL, van der Valk M, van der Valk P, Beijnen JH, Scheper R J. et al. Multidrug resistance protein 1 protects the oropharyngeal mucosal layer and the testicular tubules against drug-induced damage. J Exp Med 1998;188:797-808.
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