改性超细炭黑诱发氧化应激的毒性效应与机理
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摘要
为研究改性超细炭黑(modified ultrafine carbon black, MCB)诱发氧化应激的毒性效应与机理,将小鼠肝细胞和过氧化氢酶(catalase, CAT)暴露于不同质量浓度的MCB溶液中。采用CCK-8、丙二醛(malondialdehyde, MDA)含量和CAT活力检测等方法评价MCB的细胞毒性;利用荧光光谱,紫外-可见吸收光谱,圆二色谱等方法探究MCB对CAT构象的影响。结果表明:细胞活力随MCB质量浓度的升高而降低,低质量浓度的MCB(<30 mg/L)刺激细胞提高CAT的活力来保护自身免受氧化损伤,高质量浓度的MCB(>30 mg/L)使MDA在细胞内累积并造成氧化还原失衡,造成肝脏的氧化损伤;光谱学研究发现,MCB会改变CAT的二、三级结构和氨基酸微环境,使肽链紧缩极性增强,骨架结构的变化降低了CAT的活性。阐明MCB造成氧化应激效应的毒性机理,同时为纳米材料的毒理研究提供了参考。
To study the toxic effects and mechanisms of oxidative stress induced by modified ultrafine carbon black(MCB), mouse hepatocytes and catalase(CAT) were exposed to MCB solutions. The cytotoxicity of MCB was assessed by CCK-8 kit, malondialdehyde(MDA) kit and CAT activity assay. The effects on CAT structure and function of MCB were investigated by utilizing fluorescence, UV—vis absorption and circular dichroism spectroscopy. The experiment results demonstrated that the hepatocyte viability decreased with the increase of MCB concentration. Low MCB doses(< 30 mg/L) increased CAT activity to protectcells from oxidative damage while high doses of MCB(> 30 mg/L) caused accumulation of MDA and redox imbalance in the cells, which induced oxidative damage in the liver. Spectroscopy studies found that MCB destroyed the secondary and tertiary structure of CAT and changed the microenvironment of amino acids, which made denaturation of the peptide chain. As a result, changes of skeleton structure reduced the activity of CAT. This study clarified the oxidative mechanism of MCB causing oxidative stress effects and provided a reference for the toxicity mechanism of nanomaterials.
To study the toxic effects and mechanisms of oxidative stress induced by modified ultrafine carbon black(MCB), mouse hepatocytes and catalase(CAT) were exposed to MCB solutions. The cytotoxicity of MCB was assessed by CCK-8 kit, malondialdehyde(MDA) kit and CAT activity assay. The effects on CAT structure and function of MCB were investigated by utilizing fluorescence, UV—vis absorption and circular dichroism spectroscopy. The experiment results demonstrated that the hepatocyte viability decreased with the increase of MCB concentration. Low MCB doses(< 30 mg/L) increased CAT activity to protectcells from oxidative damage while high doses of MCB(> 30 mg/L) caused accumulation of MDA and redox imbalance in the cells, which induced oxidative damage in the liver. Spectroscopy studies found that MCB destroyed the secondary and tertiary structure of CAT and changed the microenvironment of amino acids, which made denaturation of the peptide chain. As a result, changes of skeleton structure reduced the activity of CAT. This study clarified the oxidative mechanism of MCB causing oxidative stress effects and provided a reference for the toxicity mechanism of nanomaterials.
引文
[1] KHAN M B,PARVAZ M,KHAN Z H,et al.Graphene oxide:synthesis and characterization[M].Singapore:Springer Singapore,2017:1-28.
[2] LEE J H,WEE S B,KWON M S,et al.Strategic dispersion of carbon black and its application to ink-jet-printed lithium cobalt oxide electrodes for lithium ion batteries[J].Journal of Power Sources,2011,196(15):6449-6455.
[3] SENGUPTA R,BHATTACHARYA M,BANDYOPADHYAY S,et al.A review on the mechanical and electrical properties of graphite and modified graphite reinforced polymer composites[J].Progress in Polymer Science,2011,36(5):638-670.
[4] ZHAO X C,LU D W,HAO F,et al.Exploring the diameter and surface dependent conformational changes in carbon nanotube-protein corona and the related cytotoxicity[J].Journal of Hazardous Materials,2015,292:98-107.
[5] THROWER P A.Toxicology of carbon nanomaterials[J].Carbon,2006,44(6):1027.
[6] CHANG C C,CHEN C Y,CHIU H F,et al.Elastases from inflammatory and dendritic cells mediate ultrafine carbon black induced acute lung destruction in mice[J].Inhalation Toxicology,2011,23(10):616-626.
[7] INOUE K,YANAGISAWA R,KOIKE E,et al.Effects of carbon black nanoparticles on elastase-induced emphysematous lung injury in mice[J].Basic & Clinical Pharmacology & Toxicology,2011,108(4):234-240.
[8] BUCHNER N,ALE-AGHA N,JAKOB S,et al.Unhealthy diet and ultrafine carbon black particles induce senescence and disease associated phenotypic changes[J].Experimental Gerontology,2013,48(1):8-16.
[9] INOUE H,SHIMADA A,KAEWAMATAWONG T,et al.Ultrastructural changes of the air-blood barrier in mice after intratracheal instillation of lipopolysaccharide and ultrafine carbon black particles[J].Experimental and Toxicologic Pathology,2009,61(1):51-58.
[10] TIMBLIN C R,SHUKLA A,BERLANGER I,et al.Ultrafine airborne particles cause increases in protooncogene expression and proliferation in alveolar epithelial cells[J].Toxicology and Applied Pharmacology,2002,179(2):98-104.
[11] WU Z C,ZHANG B,YAN B.Regulation of enzyme activity through interactions with nanoparticles[J].International Journal of Molecular Sciences,2009,10(10):4198-4209.
[12] ZHANG R,WU Q Q,LIU R T.Characterizing the binding interaction between ultrafine carbon black (UFCB) and catalase:electron microscopy and spectroscopic analysis[J].RSC Advances,2017,7(67):42549-42558.
[13] CASTILLO C,PEREIRA V,ABUELO A,et al.Preliminary results in the redox balance in healthy cats:influence of age and gender[J].Journal of Feline Medicine and Surgery,2013,15(4):328-332.
[14] HU X L,CUI S Y,LIU J Q.Fluorescence studies of interaction between flavonol p-coumaroylglucoside tiliroside and bovine serum albumin[J].Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy,2010,77(2):548-553.
[15] ZHANG Y Z,ZHOU B,LIU Y X,et al.Fluorescence study on the interaction of bovine serum albumin with p-aminoazobenzene[J].Journal of Fluorescence,2008,18(1):109-118.
[16] MONDAL S,DAS S,GHOSH S.Interaction of myoglobin with cationic gemini surfactants in phosphate buffer at pH 7.4[J].Journal of Surfactants and Detergents,2015,18(3):471-476.
[17] JING M,LIU Y,SONG W,et al.Oxidative damage induced by copper in mouse primary hepatocytes by single-cell analysis[J].Environmental Science & Pollution Research,2016,23(2):1335-1343.
[18] SRIKANTH K,PEREIRA E,DUARTE A C,et al.Assessment of cytotoxicity and oxidative stress induced by titanium oxide nanoparticles on chinook salmon cells[J].Environmental Science & Pollution Research,2015,22(20):15579-15586.
[19] MITRY R R,HUGHES R D,AW M M,et al.Human hepatocyte isolation and relationship of cell viability to early graft function[J].Cell Transplantation,2003,12(1):69-74.
[20] GUAN S,GE D,LIU T Q,et al.Protocatechuic acid promotes cell proliferation and reduces basal apoptosis in cultured neural stem cells[J].Toxicology in Vitro,2009,23(2):201-208.
[21] GU Q,KENNY J E.Improvement of inner filter effect correction based on determination of effective geometric parameters using a conventional fluorimeter[J].Analytical Chemistry,2009,81(1):420-426.
[22] WU H,CHEN M M,SHANG M T,et al.Insights into the binding behavior of bovine serum albumin to black carbon nanoparticles and induced cytotoxicity[J].Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy,2018,2005:1-7.
[23] WU Y K,GUO Y F,SONG H Y,et al.Oxygen content determines the bio-reactivity and toxicity profiles of carbon black particles[J].Ecotoxicology and Environmental Safety,2018,150:207-214.
[24] FOUCAUD L,GOULAOUIC S,BENNASROUNE A,et al.Oxidative stress induction by nanoparticles in THP-1 cells with 4-HNE production:stress biomarker or oxidative stress signalling molecule?[J].Toxicology in Vitro,2010,24(6):1512-1520.
[25] HUSSAIN S,THOMASSEN L C J,FERECATU I,et al.Carbon black and titanium dioxide nanoparticles elicit distinct apoptotic pathways in bronchial epithelial cells[J].Particle and Fibre Toxicology,2010,7:17.
[26] GAWEL S,WARDAS M,NIEDWOROK E,et al.Malondialdehyde (MDA) as a lipid peroxidation marker[J].Wiadomosci Lekarskie,2004,57(9-10):453-455.
[27] AYALA A,MUNOZ M F,ARGUELLES S.Lipid peroxidation:production,metabolism,and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal[J].Oxidative Medicine and Cellular Longevity,2014,2014:360438.
[28] YU H,ZHANG J,TONG Q,et al.Icariin attenuates carbon black-induced pulmonary inflammatory response and oxidative stress in mice[J].International Journal of Clinical and Experimental Pathology,2016,9(1):81-88.
[29] LIU S,GUO X C,ZHANG X X,et al.Impact of iron precipitant on toxicity of arsenic in water:a combined in vivo and in vitro study[J].Environmental Science & Technology,2013,47(7):3432-3438.
[30] TENG Y,ZHANG H,LIU R T.Molecular interaction between 4-aminoantipyrine and catalase reveals a potentially toxic mechanism of the drug[J].Molecular Biosystems,2011,7(11):3157-3163.
[31] BANERJEE M,BALLAL A,APTE K.Mn-catalase (Alr0998) protects the photosynthetic,nitrogen-fixing cyanobacterium anabaena PCC7120 from oxidative stress[J].Environmental Microbiology,2012,14(11):2891-2900.
[32] LIU W,ROSE J,PLANTEVIN S,et al.Protein corona formation for nanomaterials and proteins of a similar size:hard or soft corona?[J].Nanoscale,2013,5(4):1658-1668.
[33] KOKKINOPOULOU M,SIMON J,LANDFESTER K,et al.Visualization of the protein corona:towards a biomolecular understanding of nanoparticlecell-interactions[J].Nanoscale,2017,9(25):8858-8870.
[34] XU M H,SHENG Z H,LU W Y,et al.Probing the interaction of mutiwalled carbon nanotubes and catalase:mutispectroscopic approach[J].Journal of Biochemical and Molecular Toxicology,2012,26(12):493-498.
[35] PAUL B K,BHATTACHARJEE K,BOSE S,et al.A spectroscopic investigation on the interaction of a magnetic ferrofluid with a model plasma protein:effect on the conformation and activity of the protein[J].Physical Chemistry Chemical Physics,2012,14(44):15482-15493.
[36] ALBANI J R.Principles and applications of fluorescence spectroscopy[M].Oxford,UK:Blackwell Science,2007:101-138.
[37] MALLICK A,HALDAR B,CHATTOPADHYAY N.Spectroscopic investigation on the interaction of ICT probe 3-acetyl-4-oxo-6,7-dihydro-12H indolo-2,3-a quinolizine with serum albumins[J].Journal of Physical Chemistry B,2005,109(30):14683-14690.
[38] WANG Y Q,ZHANG H M,CAO J,et al.Interaction of methotrexate with trypsin analyzed by spectroscopic and molecular modeling methods[J].Journal of Molecular Structure,2013,1051:78-85.
[39] ZHANG H M,TANG B P,WANG Y Q.The interaction of lysozyme with caffeine,theophylline and theobromine in solution[J].Molecular Biology Reports,2010,37(7):3127-3136.
[40] MU Y,LIN J,LIU R T.Interaction of sodium benzoate with trypsin by spectroscopic techniques[J].Spectrochimica Acta Part a-Molecular and Biomolecular Spectroscopy,2011,83(1):130-135.
[41] WU T Q,WU Q,GUAN S Y,et al.Binding of the environmental pollutant naphthol to bovine serum albumin[J].Biomacromolecules,2007,8(6):1899-1906.
[42] VARLAN A,HILLEBRAND M.Study on the interaction of 2-carboxyphenoxathiin with bovine serum albumin and human serum albumin by fluorescence spectroscopy and circular dichroism[J].Revue Roumaine De Chimie,2010,55(1):69-77.
[43] KOSHLAND D E.Correlation of structure and function in enzyme action[J].Science,1963,142(359):1533-1541.
[2] LEE J H,WEE S B,KWON M S,et al.Strategic dispersion of carbon black and its application to ink-jet-printed lithium cobalt oxide electrodes for lithium ion batteries[J].Journal of Power Sources,2011,196(15):6449-6455.
[3] SENGUPTA R,BHATTACHARYA M,BANDYOPADHYAY S,et al.A review on the mechanical and electrical properties of graphite and modified graphite reinforced polymer composites[J].Progress in Polymer Science,2011,36(5):638-670.
[4] ZHAO X C,LU D W,HAO F,et al.Exploring the diameter and surface dependent conformational changes in carbon nanotube-protein corona and the related cytotoxicity[J].Journal of Hazardous Materials,2015,292:98-107.
[5] THROWER P A.Toxicology of carbon nanomaterials[J].Carbon,2006,44(6):1027.
[6] CHANG C C,CHEN C Y,CHIU H F,et al.Elastases from inflammatory and dendritic cells mediate ultrafine carbon black induced acute lung destruction in mice[J].Inhalation Toxicology,2011,23(10):616-626.
[7] INOUE K,YANAGISAWA R,KOIKE E,et al.Effects of carbon black nanoparticles on elastase-induced emphysematous lung injury in mice[J].Basic & Clinical Pharmacology & Toxicology,2011,108(4):234-240.
[8] BUCHNER N,ALE-AGHA N,JAKOB S,et al.Unhealthy diet and ultrafine carbon black particles induce senescence and disease associated phenotypic changes[J].Experimental Gerontology,2013,48(1):8-16.
[9] INOUE H,SHIMADA A,KAEWAMATAWONG T,et al.Ultrastructural changes of the air-blood barrier in mice after intratracheal instillation of lipopolysaccharide and ultrafine carbon black particles[J].Experimental and Toxicologic Pathology,2009,61(1):51-58.
[10] TIMBLIN C R,SHUKLA A,BERLANGER I,et al.Ultrafine airborne particles cause increases in protooncogene expression and proliferation in alveolar epithelial cells[J].Toxicology and Applied Pharmacology,2002,179(2):98-104.
[11] WU Z C,ZHANG B,YAN B.Regulation of enzyme activity through interactions with nanoparticles[J].International Journal of Molecular Sciences,2009,10(10):4198-4209.
[12] ZHANG R,WU Q Q,LIU R T.Characterizing the binding interaction between ultrafine carbon black (UFCB) and catalase:electron microscopy and spectroscopic analysis[J].RSC Advances,2017,7(67):42549-42558.
[13] CASTILLO C,PEREIRA V,ABUELO A,et al.Preliminary results in the redox balance in healthy cats:influence of age and gender[J].Journal of Feline Medicine and Surgery,2013,15(4):328-332.
[14] HU X L,CUI S Y,LIU J Q.Fluorescence studies of interaction between flavonol p-coumaroylglucoside tiliroside and bovine serum albumin[J].Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy,2010,77(2):548-553.
[15] ZHANG Y Z,ZHOU B,LIU Y X,et al.Fluorescence study on the interaction of bovine serum albumin with p-aminoazobenzene[J].Journal of Fluorescence,2008,18(1):109-118.
[16] MONDAL S,DAS S,GHOSH S.Interaction of myoglobin with cationic gemini surfactants in phosphate buffer at pH 7.4[J].Journal of Surfactants and Detergents,2015,18(3):471-476.
[17] JING M,LIU Y,SONG W,et al.Oxidative damage induced by copper in mouse primary hepatocytes by single-cell analysis[J].Environmental Science & Pollution Research,2016,23(2):1335-1343.
[18] SRIKANTH K,PEREIRA E,DUARTE A C,et al.Assessment of cytotoxicity and oxidative stress induced by titanium oxide nanoparticles on chinook salmon cells[J].Environmental Science & Pollution Research,2015,22(20):15579-15586.
[19] MITRY R R,HUGHES R D,AW M M,et al.Human hepatocyte isolation and relationship of cell viability to early graft function[J].Cell Transplantation,2003,12(1):69-74.
[20] GUAN S,GE D,LIU T Q,et al.Protocatechuic acid promotes cell proliferation and reduces basal apoptosis in cultured neural stem cells[J].Toxicology in Vitro,2009,23(2):201-208.
[21] GU Q,KENNY J E.Improvement of inner filter effect correction based on determination of effective geometric parameters using a conventional fluorimeter[J].Analytical Chemistry,2009,81(1):420-426.
[22] WU H,CHEN M M,SHANG M T,et al.Insights into the binding behavior of bovine serum albumin to black carbon nanoparticles and induced cytotoxicity[J].Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy,2018,2005:1-7.
[23] WU Y K,GUO Y F,SONG H Y,et al.Oxygen content determines the bio-reactivity and toxicity profiles of carbon black particles[J].Ecotoxicology and Environmental Safety,2018,150:207-214.
[24] FOUCAUD L,GOULAOUIC S,BENNASROUNE A,et al.Oxidative stress induction by nanoparticles in THP-1 cells with 4-HNE production:stress biomarker or oxidative stress signalling molecule?[J].Toxicology in Vitro,2010,24(6):1512-1520.
[25] HUSSAIN S,THOMASSEN L C J,FERECATU I,et al.Carbon black and titanium dioxide nanoparticles elicit distinct apoptotic pathways in bronchial epithelial cells[J].Particle and Fibre Toxicology,2010,7:17.
[26] GAWEL S,WARDAS M,NIEDWOROK E,et al.Malondialdehyde (MDA) as a lipid peroxidation marker[J].Wiadomosci Lekarskie,2004,57(9-10):453-455.
[27] AYALA A,MUNOZ M F,ARGUELLES S.Lipid peroxidation:production,metabolism,and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal[J].Oxidative Medicine and Cellular Longevity,2014,2014:360438.
[28] YU H,ZHANG J,TONG Q,et al.Icariin attenuates carbon black-induced pulmonary inflammatory response and oxidative stress in mice[J].International Journal of Clinical and Experimental Pathology,2016,9(1):81-88.
[29] LIU S,GUO X C,ZHANG X X,et al.Impact of iron precipitant on toxicity of arsenic in water:a combined in vivo and in vitro study[J].Environmental Science & Technology,2013,47(7):3432-3438.
[30] TENG Y,ZHANG H,LIU R T.Molecular interaction between 4-aminoantipyrine and catalase reveals a potentially toxic mechanism of the drug[J].Molecular Biosystems,2011,7(11):3157-3163.
[31] BANERJEE M,BALLAL A,APTE K.Mn-catalase (Alr0998) protects the photosynthetic,nitrogen-fixing cyanobacterium anabaena PCC7120 from oxidative stress[J].Environmental Microbiology,2012,14(11):2891-2900.
[32] LIU W,ROSE J,PLANTEVIN S,et al.Protein corona formation for nanomaterials and proteins of a similar size:hard or soft corona?[J].Nanoscale,2013,5(4):1658-1668.
[33] KOKKINOPOULOU M,SIMON J,LANDFESTER K,et al.Visualization of the protein corona:towards a biomolecular understanding of nanoparticlecell-interactions[J].Nanoscale,2017,9(25):8858-8870.
[34] XU M H,SHENG Z H,LU W Y,et al.Probing the interaction of mutiwalled carbon nanotubes and catalase:mutispectroscopic approach[J].Journal of Biochemical and Molecular Toxicology,2012,26(12):493-498.
[35] PAUL B K,BHATTACHARJEE K,BOSE S,et al.A spectroscopic investigation on the interaction of a magnetic ferrofluid with a model plasma protein:effect on the conformation and activity of the protein[J].Physical Chemistry Chemical Physics,2012,14(44):15482-15493.
[36] ALBANI J R.Principles and applications of fluorescence spectroscopy[M].Oxford,UK:Blackwell Science,2007:101-138.
[37] MALLICK A,HALDAR B,CHATTOPADHYAY N.Spectroscopic investigation on the interaction of ICT probe 3-acetyl-4-oxo-6,7-dihydro-12H indolo-2,3-a quinolizine with serum albumins[J].Journal of Physical Chemistry B,2005,109(30):14683-14690.
[38] WANG Y Q,ZHANG H M,CAO J,et al.Interaction of methotrexate with trypsin analyzed by spectroscopic and molecular modeling methods[J].Journal of Molecular Structure,2013,1051:78-85.
[39] ZHANG H M,TANG B P,WANG Y Q.The interaction of lysozyme with caffeine,theophylline and theobromine in solution[J].Molecular Biology Reports,2010,37(7):3127-3136.
[40] MU Y,LIN J,LIU R T.Interaction of sodium benzoate with trypsin by spectroscopic techniques[J].Spectrochimica Acta Part a-Molecular and Biomolecular Spectroscopy,2011,83(1):130-135.
[41] WU T Q,WU Q,GUAN S Y,et al.Binding of the environmental pollutant naphthol to bovine serum albumin[J].Biomacromolecules,2007,8(6):1899-1906.
[42] VARLAN A,HILLEBRAND M.Study on the interaction of 2-carboxyphenoxathiin with bovine serum albumin and human serum albumin by fluorescence spectroscopy and circular dichroism[J].Revue Roumaine De Chimie,2010,55(1):69-77.
[43] KOSHLAND D E.Correlation of structure and function in enzyme action[J].Science,1963,142(359):1533-1541.