铜离子急性胁迫对虎纹蛙肝脏中三羧酸循环及自由基代谢的影响
THE EFFECTS OF ACUTE COPPER STRESS ON TCA CYCLE AND FREE RADICAL METABOLISM IN THE LIVER OF HOPLOBATRACHUS CHINENSIS
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摘要: 为探明铜离子(Cu2+)对两栖动物肝脏线粒体中三羧酸(Tricarboxyl acid, TCA)循环及自由基代谢的毒理作用,采用静水暴露实验,研究了Cu2+不同浓度和不同暴露时间对虎纹蛙(Hoplobatrachus chinensis)肝脏线粒体中异柠檬酸脱氢酶(ICDHm)活性、-酮戊二酸脱氢酶(-KGDH)活性、抗超氧阴离子(anti-O2)活性、过氧化氢(H2O2)含量、抑制羟自由基(inhabit-OH)活性、一氧化氮(NO)含量以及一氧化氮合成酶(NOS)活性的影响。暴露实验共设置6个Cu2+浓度组(0.0、2.0、4.0、6.0、8.0和10.0 mg/L),分5个暴露时间(0、24h、48h、72h和96h)取材,对每个浓度的不同暴露时间分别取6个样本,测定TCA循环及自由基代谢的相关指标。结果显示,在TCA循环中随着Cu2+浓度的增加和暴露时间的延长,时间和浓度因素对ICDHm活性影响无显著性交互作用(P0.05),暴露时间的延长对ICDHm活性无显著性影响(P0.05),但随着Cu2+浓度的增加ICDHm活性逐渐减小;而时间和浓度因素对-KGDH活性影响有显著交互作用(P0.05),暴露处理后-KGDH活性下降,分别在24h和96h的4.0、6.0 mg/L时活性最低。在自由基代谢中,时间和浓度因素对抗O2活性、H2O2含量影响有显著交互作用(P0.05),而对抑制OH活性、NO含量、NOS活性的影响无显著性交互作用(P0.05)。不同时间随着Cu2+浓度的增加,抗O2活性均呈现出逐渐下降的趋势;实验处理后H2O2含量升高,在24h的6.0 mg/L时含量最大;随着暴露时间的延长和Cu2+浓度的增加抑制OH活性均逐渐降低;而NO含量和NOS活性的变化趋势基本相同,即随着Cu2+浓度的增加先增加后减少并趋近0浓度组,且都在6.0 mg/L时达到最大。研究结果表明急性Cu2+暴露对虎纹蛙肝脏线粒体中TCA循环及自由基代谢有显著的毒性作用。Abstract: In this study, we applied acute toxicity test to investigate the toxic effects of copper ion(Cu2+) on tricarbo-xylic acid cycle(TCA) and free radical metabolism in the mitochondria of the liver of Hoplobatrachus chinensis. We treated the animals with Cu2+ at different concentrations and for different exposure time, and tested the activities of TCA-related enzymes and free radicals. We found that although Cu2+ at high concentrations could reduce the activity of ICDHm, prolonged exposure time had no significant effect on ICDHm(P 0.05). We did not observe cross effects between the concentration of Cu2+ and the exposure time on the activity of ICDHm(P 0.05). However there were significant cross effects between the exposure time and the concentration of Cu2+ on the activity of -KGDH(P 0.05). The activity of -KGDH decreased when exposed to Cu2+, and the lowest activities were detected when the exposure times were 24h and 96h and concentrations were 4.0 and 6.0 mg/L. In terms of free radical metabolism, the exposure time and copper concentration had strong cross effects on the activities of anti-O2 and H2O2 (P 0.05), but not on the activity of inhibit-OH, the content of NO, and the activity of NOS. The activities of anti-O2 decreased along with the increase in the concentration of Cu2+. The content of H2O2 increased after the treatments and perked at 24h and 6.0 mg/L. There was a negative correlation between the activity of inhibit-OH and the exposure time and the concen-tration of Cu2+. Along with the increase in the concentration of Cu2+, the content of NO and the activity of NOS first increased and then decreased to the values of the control group, and the maximum values appeared in the 6.0 mg/L group. Our study demonstrated that the acute exposure of Cu2+ could have significant toxic effects on TCA cycle and free radical metabolism in the mitochondria of the liver of H. chinensis.
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Keywords:
- Hoplobatrachus chinensis /
- Copper ion /
- TCA cycle /
- Free radical metabolism /
- Toxic effect
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[1] De Boeck G, Meeus W, Coen W D, et al. Tissue-specific Cu bioaccumulation patterns and differences in sensitivity to waterborne Cu in three freshwater fish:rainbow trout(Oncorhynchus mykiss), common carp(Cyprinus carpio), and gibel carp(Carassius auratus gibelio)[J]. Aquatic Toxicology, 2004, 70(3):179-188
[2] Wu F C, Feng C L, Cao Y J, et al. Aquatic life ambient freshwater quality criteria for copper in China[J]. Asian Journal of Ecotoxicology, 2011, 6(6):617-628[吴丰昌,冯承莲,曹宇静,等.我国铜的淡水生物水质基准研究.生态毒理学报, 2011, 6(6):617-628]
[3] Wang H D, Fang F M, Xie H F. Research situation and outlook on heavy metal pollution in water environment of China[J]. Guangdong Trace Elements Science, 2010, 17(1):14-18[王海东,方凤满,谢宏芳.中国水体重金属污染研究现状与展望.广东微量元素科学, 2010, 17(1):14-18]
[4] Zhu Y F, Hong W S, Lin J Z. Toxicity of Cu2+ to juvenile perch Lateolabrax maculatus[J]. Asian Journal of Ecotoxicology, 2011, 6(3):331-336[朱友芳,洪万树,林金忠.铜离子对中国花鲈幼鱼的毒性研究.生态毒理学报, 2011, 6(3):331-336]
[5] Zhang F, Liu H W, Song Z D. Effects of some heavy metals on superoxide anion(O2) production by coelomocytes of Stichopus japonicus[J]. Journal of Agro-Environment Science, 2006, 25(suppl):100-103[张峰,刘洪伟,宋志东.几种重金属对刺参体腔细胞超氧阴离子(O2)产生的影响.农业环境科学学报, 2006, 25(增刊):100-103]
[6] Gascon C, Collins J P, Moore R D, et al. Amphibian Conservation Action Plan. IUCN/SSC Amphibian Specialist Group[M]. Gland, Switzerland and Cambridge, UK. 2007, 64
[7] Lin Z H, Ji X. Sexual dimorphism in morphological traits and food habits in tiger frogs, Hoplobatrachus rugulosus in Lishui, Zhejiang[J]. Zoological Research, 2005, 26(3):255-262[林植华,计翔.浙江丽水虎纹蛙形态特征的两性异形和食性.动物学研究, 2005, 26(3):255-262]
[8] Lin X, Wang S K, Chen M F, et al. Study on tryptase in the mast cell in the digestive tract of indian bullfrog(Rana tigrina rugulosa) by an immunohistochemical method[J]. Acta Hydrobiologica Sinica, 2010, 34(1):29-34[林旋,王寿昆,陈梅芳,等.虎纹蛙消化道肥大细胞类胰蛋白酶免疫组化研究.水生生物学报, 2010, 34(1):29-34]
[9] Shao C, Wang Y, Qiao N. Isolation and characterization of microsatellite loci in tiger frog(Hoplobatrachus rugulosus)[J]. Conservation Genetics, 2009, 10(5):1601-1603
[10] Wang N, Shao C, Xie Z G, et al. Viability and changes of physiological functions in the tiger frog(Hoplobatrachus rugulosus) exposed to cold stress[J]. Acta Ecologica Sinica, 2012, 32(11):3538-3545[王娜,邵晨,颉志刚,等.低温胁迫下虎纹蛙的生存力及免疫和抗氧化能力.生态学报, 2012, 32(11):3538-3545]
[11] Gunter T E, Yule D I, Gunter K K, et al. Calcium and mitochondria[J]. Febs Letters, 2004, 567(1):96-102
[12] Wallace D C. A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer:a dawn for evolutionary medicine[J]. Annual Review of Genetics, 2005, 39:359-407
[13] Navarro A, Boveris A. The mitochondrial energy transduction system and the aging process[J]. American Journal of Physiology-Cell Physiology, 2007, 292(2):C670-C686
[14] Xu Z K, Guan G Q, Yin H, et al. Mitochondrial research of animal piroplasms[J]. Chinese Journal of Animal Infectious Diseases, 2011, 18(6):67-73[徐宗可,关贵全,殷宏,等.动物梨形虫线粒体研究进展.中国动物传染病学报, 2010, 18(6):67-73]
[15] Hao Z F, Yuan J C, Liu Y H. Role of isocitrate dehydrogenase on oxidative stress in plants[J]. Biotechnology Bulletin, 2012,(6):32-35[郝兆丰,袁进成,刘颖慧.异柠檬酸脱氢酶在植物抗氧化胁迫中的作用.生物技术通报, 2012,(6):32-35]
[16] Li K, Guo X, Yang Y, et al. Effects of high humidity environment on isocitrate dehydrogenase and alpha ketone glutaric acid dehydrogenase in liver of rat[J]. Journal of Third Military Medical University, 2013, 35(23):2595-2596[李昆,郭鑫,杨芸,等.高湿环境对大鼠肝脏异柠檬酸脱氢酶及-酮戊二酸脱氢酶的影响.第三军医大学学报, 2013, 35(23):2595-2596]
[17] Jo S H, Lee S H, Chun H S, et al. Cellular defence against UVB-induced phototoxicity by cytosolic NADP+-dependent isocitrate dehydrogenase[J]. Biochemical and Biophysical Research Communications, 2002, 292(2):542-549
[18] Jo S H, Son M K, Koh H J, et al. Control of mitochondrial redox balance and cellular defense against oxidative damage by mitochondrial NADP+-dependent isocitrate dehydrogenase[J]. Journal of Biological Chemistry, 2001, 276(19):16168-16176
[19] Li M D, Zhao R, Jiang X L, et al. Effects of adding intermediate material in tricarboxylic acid cycle on the activity of key enzymes of Saccharomyces cerevisiae[J]. Microbiology China, 2010, 37(3):331-335[李明达,赵睿,姜晓雷,等. TCA循环中间产物对酿酒酵母胞内代谢关键酶活性的影响.微生物学通报, 2010, 37(3):331-335]
[20] Liu L M, Li Y, Shi Z P, et al. Enhancement of pyruvate productivity in Torulopsis glabrata:Increase of NAD+ availability[J]. Journal of Biotechnology, 2006, 126(2):173-185
[21] Jin F F, Wang L. Effects of cadmium on hepatopancreas mitochondrial free radical metabolism in freshwater crab Sinopotamon henanense[J]. Acta Scientiae Circumstantiae, 2012, 32(2):457-464[金芬芬,王兰.镉对河南华溪蟹肝胰腺线粒体自由基代谢的影响.环境科学学报, 2012, 32(2):457-464]
[22] Starkov A A, Fiskum G, Chinopoulos C, et al. Mitochondrial -ketoglutarate dehydrogenase complex generates reactive oxygen species[J]. Journal of Neuroscience, 2004, 24(36):7779-7788
[23] Huang H M, Ou H C, Xu H, et al. Inhibition of -ketoglutarate dehydrogenase complex promotes cyto-chrome c release from mitochondria, caspase-3 activation, and necrotic cell death[J]. Journal of Neuroscience Research, 2003, 74(2):309-317
[24] Lu Z F, Jia F, Qiu Y M, et al. Effect of mild hypothermia on the activities of -ketoglutarate dehydrogenase of mitochondria following traumatic brain injury[J]. Chinese Journal of Neurosurgery, 2007, 22(11):659-662[陆兆丰,贾锋,邱永明,等.亚低温对创伤性脑损伤后线粒体-酮戊二酸脱氢酶活性的影响.中华神经外科杂志, 2007, 22(11):659-662]
[25] Sarkar B. Metal replacement in DNA-binding zinc figer protein and its relebance to mutagenicity and carcinogenicity through free radica feneration[J]. Nutrition, 1995, 11(5 Suppl):646-649
[26] Del-Rio D, Stewart A J, Pellegrini N. A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress[J]. Nutrition Metabolism and Cardiovascular Diseases, 2005, 15(4):316-328
[27] Gill S S, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants[J]. Plant Physiology and Biochemistry, 2010, 48(12):909-930
[28] Anwer T, Sharma M, Pillai K K, et al. Protective effect of bezafibrate on streptozocin-induced oxidative stress and toxicity in rats[J]. Toxicology, 2007, 229(1/2):165-172
[29] Wang C G, Yu Q, Yu A, et al. Effect of benzo(a)pyrene and pyrene exposure on hepatic superoxide dismutase in Mugil so-iuy[J]. Marine Environmental Science, 2002, 21(4):10-13[王重刚,余群,郁昂,等.苯并(a)芘和芘暴露对梭鱼肝脏超氧化物歧化酶活性的影响.海洋环境科学, 2002, 21(4):10-13]
[30] Hultberg B, Andersson A, Isaksson A. Alterations of thiol metabolism in human cell lines induced by low amounts of copper, mercury of cadmium ions[J]. Toxicology, 1998, 126(3):203-212
[31] Riob N A, Melani M, Sanjun N, et al. The modulation of mitochondrial nitric-oxide synthase activity in rat brain development[J]. Journal of Biological Chemistry, 2002, 277(45):42447-42455
[32] Knowles R G, Moncada S. Nitric oxide synthases in mammals[J]. Biochemical Journal, 1994, 298(Pt 2):249-258
[33] Solien J, Haynes V, Giulivi C. Differential requirements of calcium for oxoglutarate dehydrogenase and mitochondrial nitric-oxide synthase under hypoxia:impact on the regulation of mitochondrial oxygen consumption[J]. Comparative Biochemistry and Physiology Part A:Molecular Integrative Physiology, 2005, 142(2):111-117
[34] Ghafourifar P, Colton C A. Mitochondria and nitric oxide[J]. Antioxidants Redox Signaling, 2003, 5(3):249-250
[35] Brown G C, Borutaite V. Nitric oxide and mitochondrial respiration in the heart[J]. Cardiovascular Research, 2007, 75(2):283-290
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