氧化石墨烯对嗜热四膜虫的毒性效应
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摘要
以嗜热四膜虫(Tetrahymena thermophila)作为受试生物,考察了纳米材料氧化石墨烯(GO)对其细胞生长率、乙酰胆碱酯酶(AchE)和氧化应激酶活性、生物膜损伤及细胞凋亡的影响,以探究GO的毒性效应.结果表明,GO浓度高于32mg/L时显著抑制嗜热四膜虫的细胞增殖(P<0.05),细胞存活率低于50%;在0～64mg/L实验范围内,随GO暴露浓度增加,细胞内活性氧自由基(ROS)和超氧化物歧化酶(SOD)水平呈先升后降的趋势,AchE活性受抑;GO抑制位于线粒体内膜的琥珀酸脱氢酶(SDH)活性,促进细胞质中乳酸脱氢酶(LDH)的释放;64mg/L GO导致四膜虫细胞出现明显凋亡现象.以上结果显示,中低浓度GO(0～8mg/L)暴露下,氧化应激机制对细胞毒性起主要贡献作用;高浓度GO(32和64mg/L)作用下,四膜虫凋亡现象的产生可能是GO抑制其生长作用导致的.
Effects of graphene oxide(GO) on the growth, acetylcholinesterase(AchE) activity, oxidative stress, membrane damage and apoptosis of Tetrahymena thermophila were investigated. The results showed that the concentrations higher than 32mg/L GO significantly inhibited the growth of T. thermophila(P<0.05), and the cell survival rate was less than 50%. With the increase of GO concentration(0～64mg/L), the levels of reactive oxygen species(ROS) and superoxide dismutase(SOD) in T. thermophila cells initially also increased but then decreased while the activity of AchE decreased. GO inhibited the activity of succinate dehydrogenase(SDH) and promoted the release of lactate dehydrogenase(LDH). Highest concentration of GO(64mg/L) caused cell apoptosis.Based on the results, it can be concluded that oxidative stress contributed to cytotoxicity at low and medium concentrations of GO(0～8mg/L) while at higher concentrations(32 and 64mg/L) of GO, apoptosis of Tetrahymena may be caused by GO inhibiting its growth.
Effects of graphene oxide(GO) on the growth, acetylcholinesterase(AchE) activity, oxidative stress, membrane damage and apoptosis of Tetrahymena thermophila were investigated. The results showed that the concentrations higher than 32mg/L GO significantly inhibited the growth of T. thermophila(P<0.05), and the cell survival rate was less than 50%. With the increase of GO concentration(0～64mg/L), the levels of reactive oxygen species(ROS) and superoxide dismutase(SOD) in T. thermophila cells initially also increased but then decreased while the activity of AchE decreased. GO inhibited the activity of succinate dehydrogenase(SDH) and promoted the release of lactate dehydrogenase(LDH). Highest concentration of GO(64mg/L) caused cell apoptosis.Based on the results, it can be concluded that oxidative stress contributed to cytotoxicity at low and medium concentrations of GO(0～8mg/L) while at higher concentrations(32 and 64mg/L) of GO, apoptosis of Tetrahymena may be caused by GO inhibiting its growth.
引文
[1]Vallabani N V,Mittal S,Shukla R K,et al.Toxicity of graphene in normal human lung cells(BEAS-2B)[J].J.Biomed.Nanotechnol.,2011,7(1):106-107.
[2]Chatterjee N,Eom H J,Choi J.A systems toxicology approach to the surface functionality control of graphene-cell interactions[J].Biomaterials,2014,35(4):1109-1127.
[3]Liu Y,Luo Y,Wu J,et al.Graphene oxide can induce in vitro and in vivo mutagenesis[J].Science Reports,2013,3(12):3469.
[4]Hu C,Wang Q,Zhao H,et al.Ecotoxicological effects of graphene oxide on the protozoan Euglena gracilis[J].Chemosphere,2015,128:184-190.
[5]涂海峰,刘成,王宇擎,等.氧化石墨烯对邻苯二甲酸二丁酯藻毒性的影响[J].环境科学,2018,39(8):3927-3936.Tu H F,Liu C,Wang Y Q,et al.Effect of graphene oxide on algal toxicity of dibutyl phthalate[J].Environmental Science,2018,39(8):3927-3936.
[6]Zhao J,Cao X,Wang Z,et al.Mechanistic understanding toward the toxicity of graphene-family materials to freshwater algae[J].Water Research,2016,111:18-27.
[7]Hazeem L J,Bououdina M,Dewailly E,et al.Toxicity effect of graphene oxide on growth and photosynthetic pigment of the marine alga Picochlorum sp.during different growth stages[J].Environmental Science and Pollution Research,2017,24(4):4144-4152.
[8]Souza J P,Baretta J F,Santos F,et al.Toxicological effects of graphene oxide on adult zebrafish(Danio rerio)[J].Aquatic Toxicology,2017,186:11-18.
[9]Lv X,Yang Y,Tao Y,et al.A mechanism study on toxicity of graphene oxide to Daphnia magna:Direct link between bioaccumulation and oxidative stress[J].Environmental Pollution,2018,234:953-959.
[10]吕小慧,陈白杨,朱小山.氧化石墨烯的水环境行为及其生物毒性[J].中国环境科学,2016,36(11):3348-3359.Lv X H,Chen B Y,Zhu X S.Fate and toxicity of graphene oxide in aquatic environment[J].China Environmental Science,2016,36(11):3348-3359.
[11]段伟艳,杜永祥,孟范平,等.氧化石墨烯对双壳类动物文蛤的亚致死毒性研究[J].中国环境科学,2017,37(7):2755-2764.Duan W Y,Du Y X,Meng F P,et al.The sublethal toxicity of graphene oxide to bivalve Meretrix meretrix[J].China Environmental Science,2017,37(7):2755-2764.
[12]石柳,王栋,张瑛,等.氧化石墨烯对大型溞的生物毒性效应研究[J].生态毒理学报,2017,12(3):416-424.Shi L,Wang D,Zhang Y,et al.The toxic effects of graphene oxide on crustacean Daphnia magna[J].Asian Journal of Ecotoxicology,2017,12(3):416-424.
[13]Mesaric T,Gambardella C,Milivojevic T,et al.High surface adsorption properties of carbon-based nanomaterials are responsible for mortality,swimming inhibition,and biochemical responses in Artemia salina larvae[J].Aquatic Toxicology,2015,163:121-129.
[14]傅诚杰,俞婷,缪炜,等.四膜虫:毒理学与生态毒理学研究中的优良模式生物[J].动物学杂志,2005,(1):108-113.Fu C J,Yu T,Miao W,et al.Tetrahymena:a good model organism for toxicology and ecotoxicology[J].Chinese Journal of Zoology,2005,(1):108-113.
[15]Szabo T,Berkesi O,Dekany I.DRIFT study of deuterium-exchanged graphite oxide[J].Carbon,2005,43(15):3186-3189.
[16]Garza K M,Soto K F,Murr L E.Cytotoxicity and reactive oxygen species generation from aggregated carbon and carbonaceous nanoparticulate materials[J].International Journal of Nanomedicine,2008,3(1):83-94.
[17]Weckx J,Clijsters H.Oxidative damage and defense mechanisms in primary leaves of Phaseolus vulgaris as a result of root assimilation of toxic amounts of copper[J].Physiologia Plantarum,1996,96(3):506-512.
[18]胡俊杰,劳志朗,吴康铭,等.氧化石墨烯的环境行为和毒性效应研究进展[J].生态环境学报,2017,26(12):2169-2176.Hu J J,Lao Z L,Wu K M,et al.Research progress in environmental behavior and toxicity of graphene oxide[J].Ecology and Environmental Sciences,2017,26(12):2169-2176.
[19]Nogueira P F M,Nakabayashi D,Zucolotto V.The effects of graphene oxide on green algae Raphidocelis subcapitata[J].Aquatic Toxicology,2015,166:29-35.
[20]Lu K,Dong S,Petersen E J,et al.Biological uptake,distribution,and depuration of radio-labeled graphene in adult zebrafish:Effects of graphene size and natural organic matter[J].ACS Nano,2017,11(3):2872-2885.
[21]Schallreuter K U,Elwary S,Gibbons N,et al.Activation/deactivation of acetylcholinesterase by H2O2:More evidence for oxidative stress in vitiligo[J].Biochemical and Biophysical Research Communications,2004,315(2):502-508.
[22]蔡文超,黄韧,李建军,等.生物标志物在海洋环境污染监测中的应用及特点[J].水生态学杂志,2012,(2):137-146.Cai W C,Huang R,Li J J,et al.Applications and characteristics of biomarkers on monitoring of marine environmental pollution[J].Journal of Hydroecology,2012,(2):137-146.
[23]Soares J C,Pereira T C B,Costa K M,et al.Developmental neurotoxic effects of graphene oxide exposure in zebrafish larvae(Danio rerio)[J].Colloids and Surfaces B-Biointerfaces,2017,157:335-346.
[24]周梦媛,赵群芬,吴雅菁,等.Al2O3纳米颗粒对梨形四膜虫细胞毒性机理研究[J].生物学杂志,2012,29(6):47-52.Zhou M Y,Zhao Q F,Wu Y J,et al.Cytotoxic mechanism of aluminium oxide nanoparticles to Tetrahymena pyriformis[J].Journal of Biology,2012,29(6):47-52.
[25]Zhou H,Zhang B,Zheng J,et al.The inhibition of migration and invasion of cancer cells by graphene via the impairment of mitochondrial respiration[J].Biomaterials,2014,35(5):1597-1607.
[26]王娟,王妹梅,王晶晶,等.线粒体在纳米毒性效应中的作用及其机制研究进展[J].应用与环境生物学报,2015,21(4):579-589.Wang J,Wang M M,Wang J J,et al.Review on the role of mitochondria in nano-toxicology[J].Chinese Journal of Applied and Environmental Biology,2015,21(4):579-589.
[27]Zhang Y,Ali S F,Dervishi E,et al.Cytotoxicity effects of graphene and single-wall carbon nanotubes in neural phaeochromocytomaderived PC12cells[J].ACS Nano.2010,4(6):3181-3186.
[28]Zhang X,Hu W,Li J,et al.A comparative study of cellular uptake and cytotoxicity of multi-walled carbon nanotubes,graphene oxide,and nanodiamond[J].Toxicology Research,2012,1(1):62-68.
[29]Hu W,Peng C,Lv M,et al.Protein corona-mediated mitigation of cytotoxicity of graphene oxide[J].ACS Nano,2011,5(5):3693-3700.
[30]Ou L,Song B,Liang H,et al.Toxicity of graphene-family nanoparticles:a general review of the origins and mechanisms[J].Particle and Fibre Toxicology,2016,13(1):57.
[31]Kang Y,Liu J,Wu J,et al.Graphene oxide and reduced graphene oxide induced neural pheochromocytoma-derived PC12cell lines apoptosis and cell cycle alterations via the ERK signaling pathways[J].International Journal of Nanomedicine,2017,12:5501-5510.
[32]Wang A,Pu K,Dong B,et al.Role of surface charge and oxidative stress in cytotoxicity and genotoxicity of graphene oxide towards human lung fibroblast cells[J].Journal of Applied Toxicology,2013,33(10):1156-1164.
[2]Chatterjee N,Eom H J,Choi J.A systems toxicology approach to the surface functionality control of graphene-cell interactions[J].Biomaterials,2014,35(4):1109-1127.
[3]Liu Y,Luo Y,Wu J,et al.Graphene oxide can induce in vitro and in vivo mutagenesis[J].Science Reports,2013,3(12):3469.
[4]Hu C,Wang Q,Zhao H,et al.Ecotoxicological effects of graphene oxide on the protozoan Euglena gracilis[J].Chemosphere,2015,128:184-190.
[5]涂海峰,刘成,王宇擎,等.氧化石墨烯对邻苯二甲酸二丁酯藻毒性的影响[J].环境科学,2018,39(8):3927-3936.Tu H F,Liu C,Wang Y Q,et al.Effect of graphene oxide on algal toxicity of dibutyl phthalate[J].Environmental Science,2018,39(8):3927-3936.
[6]Zhao J,Cao X,Wang Z,et al.Mechanistic understanding toward the toxicity of graphene-family materials to freshwater algae[J].Water Research,2016,111:18-27.
[7]Hazeem L J,Bououdina M,Dewailly E,et al.Toxicity effect of graphene oxide on growth and photosynthetic pigment of the marine alga Picochlorum sp.during different growth stages[J].Environmental Science and Pollution Research,2017,24(4):4144-4152.
[8]Souza J P,Baretta J F,Santos F,et al.Toxicological effects of graphene oxide on adult zebrafish(Danio rerio)[J].Aquatic Toxicology,2017,186:11-18.
[9]Lv X,Yang Y,Tao Y,et al.A mechanism study on toxicity of graphene oxide to Daphnia magna:Direct link between bioaccumulation and oxidative stress[J].Environmental Pollution,2018,234:953-959.
[10]吕小慧,陈白杨,朱小山.氧化石墨烯的水环境行为及其生物毒性[J].中国环境科学,2016,36(11):3348-3359.Lv X H,Chen B Y,Zhu X S.Fate and toxicity of graphene oxide in aquatic environment[J].China Environmental Science,2016,36(11):3348-3359.
[11]段伟艳,杜永祥,孟范平,等.氧化石墨烯对双壳类动物文蛤的亚致死毒性研究[J].中国环境科学,2017,37(7):2755-2764.Duan W Y,Du Y X,Meng F P,et al.The sublethal toxicity of graphene oxide to bivalve Meretrix meretrix[J].China Environmental Science,2017,37(7):2755-2764.
[12]石柳,王栋,张瑛,等.氧化石墨烯对大型溞的生物毒性效应研究[J].生态毒理学报,2017,12(3):416-424.Shi L,Wang D,Zhang Y,et al.The toxic effects of graphene oxide on crustacean Daphnia magna[J].Asian Journal of Ecotoxicology,2017,12(3):416-424.
[13]Mesaric T,Gambardella C,Milivojevic T,et al.High surface adsorption properties of carbon-based nanomaterials are responsible for mortality,swimming inhibition,and biochemical responses in Artemia salina larvae[J].Aquatic Toxicology,2015,163:121-129.
[14]傅诚杰,俞婷,缪炜,等.四膜虫:毒理学与生态毒理学研究中的优良模式生物[J].动物学杂志,2005,(1):108-113.Fu C J,Yu T,Miao W,et al.Tetrahymena:a good model organism for toxicology and ecotoxicology[J].Chinese Journal of Zoology,2005,(1):108-113.
[15]Szabo T,Berkesi O,Dekany I.DRIFT study of deuterium-exchanged graphite oxide[J].Carbon,2005,43(15):3186-3189.
[16]Garza K M,Soto K F,Murr L E.Cytotoxicity and reactive oxygen species generation from aggregated carbon and carbonaceous nanoparticulate materials[J].International Journal of Nanomedicine,2008,3(1):83-94.
[17]Weckx J,Clijsters H.Oxidative damage and defense mechanisms in primary leaves of Phaseolus vulgaris as a result of root assimilation of toxic amounts of copper[J].Physiologia Plantarum,1996,96(3):506-512.
[18]胡俊杰,劳志朗,吴康铭,等.氧化石墨烯的环境行为和毒性效应研究进展[J].生态环境学报,2017,26(12):2169-2176.Hu J J,Lao Z L,Wu K M,et al.Research progress in environmental behavior and toxicity of graphene oxide[J].Ecology and Environmental Sciences,2017,26(12):2169-2176.
[19]Nogueira P F M,Nakabayashi D,Zucolotto V.The effects of graphene oxide on green algae Raphidocelis subcapitata[J].Aquatic Toxicology,2015,166:29-35.
[20]Lu K,Dong S,Petersen E J,et al.Biological uptake,distribution,and depuration of radio-labeled graphene in adult zebrafish:Effects of graphene size and natural organic matter[J].ACS Nano,2017,11(3):2872-2885.
[21]Schallreuter K U,Elwary S,Gibbons N,et al.Activation/deactivation of acetylcholinesterase by H2O2:More evidence for oxidative stress in vitiligo[J].Biochemical and Biophysical Research Communications,2004,315(2):502-508.
[22]蔡文超,黄韧,李建军,等.生物标志物在海洋环境污染监测中的应用及特点[J].水生态学杂志,2012,(2):137-146.Cai W C,Huang R,Li J J,et al.Applications and characteristics of biomarkers on monitoring of marine environmental pollution[J].Journal of Hydroecology,2012,(2):137-146.
[23]Soares J C,Pereira T C B,Costa K M,et al.Developmental neurotoxic effects of graphene oxide exposure in zebrafish larvae(Danio rerio)[J].Colloids and Surfaces B-Biointerfaces,2017,157:335-346.
[24]周梦媛,赵群芬,吴雅菁,等.Al2O3纳米颗粒对梨形四膜虫细胞毒性机理研究[J].生物学杂志,2012,29(6):47-52.Zhou M Y,Zhao Q F,Wu Y J,et al.Cytotoxic mechanism of aluminium oxide nanoparticles to Tetrahymena pyriformis[J].Journal of Biology,2012,29(6):47-52.
[25]Zhou H,Zhang B,Zheng J,et al.The inhibition of migration and invasion of cancer cells by graphene via the impairment of mitochondrial respiration[J].Biomaterials,2014,35(5):1597-1607.
[26]王娟,王妹梅,王晶晶,等.线粒体在纳米毒性效应中的作用及其机制研究进展[J].应用与环境生物学报,2015,21(4):579-589.Wang J,Wang M M,Wang J J,et al.Review on the role of mitochondria in nano-toxicology[J].Chinese Journal of Applied and Environmental Biology,2015,21(4):579-589.
[27]Zhang Y,Ali S F,Dervishi E,et al.Cytotoxicity effects of graphene and single-wall carbon nanotubes in neural phaeochromocytomaderived PC12cells[J].ACS Nano.2010,4(6):3181-3186.
[28]Zhang X,Hu W,Li J,et al.A comparative study of cellular uptake and cytotoxicity of multi-walled carbon nanotubes,graphene oxide,and nanodiamond[J].Toxicology Research,2012,1(1):62-68.
[29]Hu W,Peng C,Lv M,et al.Protein corona-mediated mitigation of cytotoxicity of graphene oxide[J].ACS Nano,2011,5(5):3693-3700.
[30]Ou L,Song B,Liang H,et al.Toxicity of graphene-family nanoparticles:a general review of the origins and mechanisms[J].Particle and Fibre Toxicology,2016,13(1):57.
[31]Kang Y,Liu J,Wu J,et al.Graphene oxide and reduced graphene oxide induced neural pheochromocytoma-derived PC12cell lines apoptosis and cell cycle alterations via the ERK signaling pathways[J].International Journal of Nanomedicine,2017,12:5501-5510.
[32]Wang A,Pu K,Dong B,et al.Role of surface charge and oxidative stress in cytotoxicity and genotoxicity of graphene oxide towards human lung fibroblast cells[J].Journal of Applied Toxicology,2013,33(10):1156-1164.