基于代谢组学指标的土壤亚致死剂量汞对蚯蚓的毒性研究
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摘要
将赤子爱胜蚓(Eisenia fetida)暴露于亚致死剂量(1.0、5.0、25.0 mg·kg~(-1))汞污染土壤中4周,以蚯蚓个体(致死率、体重增长率)及小分子代谢物(代谢组)为指标研究其对汞的动态毒性响应,并采用最小二乘判别分析(OPLSA-DA)对暴露组及对照组的代谢物进行分类,进而识别潜在的标记物.结果表明,蚯蚓对汞的吸收尚未达到稳态,蚯蚓体内代谢物的响应依赖于暴露剂量及暴露时间.暴露1周时,蚯蚓体重略有增长但不显著;最低暴露剂量(1.0 mg·kg~(-1))导致蚯蚓体内亮氨酸、异亮氨酸、色氨酸、组氨酸、酪氨酸、5-氧脯氨酸、2-脱氧肌苷显著低于对照水平,柠檬酸、肌苷酸与腺苷在5.0、25.0 mg·kg~(-1)剂量下显著高于对照水平;暴露4周时,最高暴露剂量(25 mg·kg~(-1))显著抑制了蚯蚓的生长;汞添加组的蚯蚓体内谷氨酸、酪氨酸、马来酸、2-脱氧肌苷水平显著低于对照.上述代谢物对汞的动态变化表明它们可作为潜在生物标记物,用于诊断土壤汞污染.对代谢途径分析发现,1.0～25.0 mg·kg~(-1)汞即可破坏蚯蚓正常氨基酸代谢、三羧酸循环,扰乱能量代谢,对蚯蚓产生氧化损伤.本研究结果表明,相比个体水平的受试终点,代谢组学指标比个体水平指标能更敏感地响应较低剂量汞,是土壤汞污染生态毒性效应诊断的有效指标.另外,本研究结果可为土壤汞污染的风险评估及相关环境标准的修订提供大量基础数据.
In this study, earthworms(Eisenia fetida) were exposed to soil with sublethal doses of Hg(with concentration of 1.0, 5.0 and 25.0 mg·kg~(-1) soil) for 4 weeks. Dynamic responses of the individuals(mortality and weight growth rate) and the small molecular metabolites(the metabolomics) were studied. The supervised orthogonal partial lease squares discriminant method(OPLS-DA) was used to analyze the metabolites in the exposed groups and the control group, by which the potential biomarkers can be identified. Experimental results indicate that Hg accumulation in earthworms has not reached a steady state for 4 weeks exposure, and the responses of the metabolites depend on the exposure dose and the exposure time. After 1 week, the weight of earthworms increased insignificantly. For the lowest exposure dose(1.0 mg·kg~(-1)), the concentration of leucine, isoleucine, tryptophan, histidine, tyrosine, 5-oxyproline and 2-deoxyinosine in the exposure group were significantly lower than those in the control group. While, for 5.0,25.0 mg·kg~(-1) dose, the concentration of citrate, inosine and adenosine in the exposure group were significantly higher than those in the control group. After 4 weeks, the worms′ growth was significantly inhibited at the highest exposure dose(25.0 mg·kg~(-1)). In addition, the concentration of glutamic acid, tyrosine, maleic acid and 2-deoxyinosine in the exposure group were significantly lower than those of the control. The dynamic responses of above metabolites suggest that they can be used as potential biomarkers for the diagnosis of Hg-contaminated soil. Analysis on metabolic pathways revealed that 1.0～25.0 mg·kg~(-1) Hg could disrupt the normal amino acid metabolism, the tricarboxylic acid cycle and the energy metabolism, thus leading to oxidative damage to earthworms. The results of this study suggest that metabolomics are more sensitive and effective than individual endpoints in response to lower-dose Hg. In addition, our results provide a large amount of basic data for the risk assessment of Hg-contaminated soil and the revision of environmental standards.
In this study, earthworms(Eisenia fetida) were exposed to soil with sublethal doses of Hg(with concentration of 1.0, 5.0 and 25.0 mg·kg~(-1) soil) for 4 weeks. Dynamic responses of the individuals(mortality and weight growth rate) and the small molecular metabolites(the metabolomics) were studied. The supervised orthogonal partial lease squares discriminant method(OPLS-DA) was used to analyze the metabolites in the exposed groups and the control group, by which the potential biomarkers can be identified. Experimental results indicate that Hg accumulation in earthworms has not reached a steady state for 4 weeks exposure, and the responses of the metabolites depend on the exposure dose and the exposure time. After 1 week, the weight of earthworms increased insignificantly. For the lowest exposure dose(1.0 mg·kg~(-1)), the concentration of leucine, isoleucine, tryptophan, histidine, tyrosine, 5-oxyproline and 2-deoxyinosine in the exposure group were significantly lower than those in the control group. While, for 5.0,25.0 mg·kg~(-1) dose, the concentration of citrate, inosine and adenosine in the exposure group were significantly higher than those in the control group. After 4 weeks, the worms′ growth was significantly inhibited at the highest exposure dose(25.0 mg·kg~(-1)). In addition, the concentration of glutamic acid, tyrosine, maleic acid and 2-deoxyinosine in the exposure group were significantly lower than those of the control. The dynamic responses of above metabolites suggest that they can be used as potential biomarkers for the diagnosis of Hg-contaminated soil. Analysis on metabolic pathways revealed that 1.0～25.0 mg·kg~(-1) Hg could disrupt the normal amino acid metabolism, the tricarboxylic acid cycle and the energy metabolism, thus leading to oxidative damage to earthworms. The results of this study suggest that metabolomics are more sensitive and effective than individual endpoints in response to lower-dose Hg. In addition, our results provide a large amount of basic data for the risk assessment of Hg-contaminated soil and the revision of environmental standards.
引文
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Effects of heavy metals on survival and respiration rate of tubificid worms: Part I—Effects on survival[J].Environmental Pollution,13(1):65-72
Brown S A E,Simpson A J,Simpson M J et al.2009.1H NMR metabolomics of earthworm responses to sub-lethal PAH exposure[J].Environmental Chemistry,6(5):432-440
Chen T,Liu Y,Li M H,et al.2016.Integrated 1H NMR-based metabolomics analysis of earthworm responses to sub-lethal Pb exposure[J].Environmental Chemistry,13(5):792-803
Colacevich A,Sierra M J,Borghini F,et al.2011.Oxidative stress in earthworms short- and long-term exposed to highly Hg-contaminated soils[J].Journal of Hazardous Materials,194(5):135-143
Dang F,Zhao J,Zhou D.2016.Uptake dynamics of inorganic mercury and methylmercury by the earthworm Pheretima guillemi[J].Chemosphere,144:2121-2126
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Hocher B,Adamski J.2017.Metabolomics for clinical use and research in chronic kidney disease[J].Nature Reviews Nephrology,13(5):269-284
胡文友,黄标,马宏卫,等.2014.南方典型设施蔬菜生产系统镉和汞累积的健康风险[J].土壤学报,51(5):1045-1055
Kaddurah-Daouk R,Krishnan K R.2009.Metabolomics: A global biochemical approach to the study of central nervous system diseases[J].Neuropsychopharmacology,34(1):173-186
Kim S H,Jung M Y,Lee Y M.2011.Effect of heavy metals on the antioxidant enzymes in the marine ciliate Euplotes crassus[J].Toxicology and Environmental Health Sciences,3(4):213-219
Kim S,Yoon D,Lee M,et al.2016.Metabolic responses in zebrafish(Danio rerio) exposed to zinc and cadmium by nuclear magnetic resonance-based metabolomics[J].Chemistry and Ecology,32(2):136-148
Lankadurai B P,Simpson A J,Simpson M J.2012.1H NMR metabolomics of Eisenia fetida responses after sub-lethal exposure to perfluorooctanoic acid and perfluorooctane sulfonate[J].Environmental Chemistry,9(6):502-511
Lankadurai B P,Nagato E G,Simpson A J,et al.2015.Analysis of Eisenia fetida earthworm responses to sub-lethal C60 nanoparticle exposure using 1H-NMR based metabolomics[J].Ecotoxicology & Environmental Safety,120:48-58
Lock K,Janssen C.2001.Ecotoxicity of mercury to Eisenia fetida,Enchytraeus albidus and Folsomia candida [J].Biology and Fertility of Soils,34(4):219-221
刘岩.2016.基于1H NMR代谢组学方法研究敌敌畏对金鱼的毒性及作用机制[D].南京:南京理工大学
Mckelvie J R,Wolfe D M,Celejewski M,et al.2010.Correlations of Eisenia fetida metabolic responses to extractable phenanthrene concentrations through time[J].Environmental Pollution,158(6):2150-2157
Nagato E G,Simpson A J,Simpson M J.2016.Metabolomics reveals energetic impairments in Daphnia magna exposed to diazinon,malathion and bisphenol-A[J].Aquatic Toxicology,170:175-186
OECD.1984.Proposal for Updating Guideline 207:Earthworm,Acute Toxicity Tests2 OECD Guideline for Testing of Chemicals 207.European Committe,Pairs[EB/OL].Pairs:European Committee.2015-06-12.https: //www.oecd.org/env/ehs/testing/Draft-Updated-Test-Guildeline-222-Earthworm-reproduction-Test.pdf
Tang H,Yan Q,Wang X,et al.2016.Earthworm(Eisenia fetida) behavioral and respiration responses to sublethal mercury concentrations in an artificial soil substrate[J].Applied Soil Ecology,104:48-53
Tang R,Ding C,Dang F,et al.2018.NMR-based metabolic toxicity of low-level Hg exposure to earthworms[J].Environmental Pollution,239:428-437
唐浩,刘钊钊,李银生,等.2017.土壤汞污染胁迫对蚯蚓体内几种抗氧化酶活性的影响[J].上海交通大学学报(农业科学版),35(3):17-23
Yang X X,Song Y F,Ackland M L,et al.2012.Biochemical responses of earthworm Eisenia fetida exposed to cadmium-contaminated soil with long duration[J].Bulletin of Environmental Contamination & Toxicology,89(6):1148-1153
杨道丽.2013.汞与溴苯腈复合污染对赤子爱胜蚓及球肾白线蚓的生态毒理效应研究[D].上海:上海交通大学
Zhao L,Huang Y,Hannah-Bick C,et al.2016.Application of metabolomics to assess the impact of Cu(OH)2 nanopesticide on the nutritional value of lettuce(Lactuca sativa): Enhanced Cu intake and reduced antioxidants[J].Nanoimpact,3-4:58-66
Zhu J,Li Z G,Yang D L,et al.2011.Acute toxicological effects of Hg pollution on earthworm Eisenia Foetida[J].Journal of Shanghai Institute of Technology,11(2):95-99
张成,陈宏,王定勇,等.2014.三峡库区消落带土壤汞形态分布与风险评价[J].环境科学,35(3):1060-1067
张文新,陈勇,齐誉,等.2017.新疆玛河流域土壤和蔬菜汞分布特征与生态、人体健康风险评价[J].环境化学,36(11):2441-2450
Baylay A J,Spurgeon D J,Svendsen C,et al.2012.A metabolomics based test of independent action and concentration addition using the earthworm Lumbricus rubellus[J].Ecotoxicology,21(5):1436-1447
Berntssen M H G,Aatland A,Handy R D,et al.2003.Chronic dietary mercury exposure causes oxidative stress,brain lesions,and altered behaviour in Atlantic salmon(Salmo salar) parr[J].Aquatic Toxicology,65(1):55-72
Blouin M,Hodson M E,Delgado E A,Baker G,et al.2013.A review of earthworm impact on soil function and ecosystem services[J].European Journal of Soil Science,64(2):161-182
Brand?o F,Cappello T,Raimundo J,et al.2015.Unravelling the mechanisms of mercury hepatotoxicity in wild fish(Liza aurata) through a triad approach: bioaccumulation,metabolomic profiles and oxidative stress[J].Metallomics,7(9):1352-1363
Effects of heavy metals on survival and respiration rate of tubificid worms: Part I—Effects on survival[J].Environmental Pollution,13(1):65-72
Brown S A E,Simpson A J,Simpson M J et al.2009.1H NMR metabolomics of earthworm responses to sub-lethal PAH exposure[J].Environmental Chemistry,6(5):432-440
Chen T,Liu Y,Li M H,et al.2016.Integrated 1H NMR-based metabolomics analysis of earthworm responses to sub-lethal Pb exposure[J].Environmental Chemistry,13(5):792-803
Colacevich A,Sierra M J,Borghini F,et al.2011.Oxidative stress in earthworms short- and long-term exposed to highly Hg-contaminated soils[J].Journal of Hazardous Materials,194(5):135-143
Dang F,Zhao J,Zhou D.2016.Uptake dynamics of inorganic mercury and methylmercury by the earthworm Pheretima guillemi[J].Chemosphere,144:2121-2126
Driscoll C T,Mason R P,Chan H M,et al.2013.Mercury as a global pollutant: sources,pathways,and effects[J].Environmental Science & Technology,47(10):4967-4983
Fu Z,Wu F,Amarasiriwardena D,et al.2010.Antimony,arsenic and mercury in the aquatic environment and fish in a large antimony mining area in Hunan,China[J].Science of the Total Environment,408(16):3403-3410
Hocher B,Adamski J.2017.Metabolomics for clinical use and research in chronic kidney disease[J].Nature Reviews Nephrology,13(5):269-284
胡文友,黄标,马宏卫,等.2014.南方典型设施蔬菜生产系统镉和汞累积的健康风险[J].土壤学报,51(5):1045-1055
Kaddurah-Daouk R,Krishnan K R.2009.Metabolomics: A global biochemical approach to the study of central nervous system diseases[J].Neuropsychopharmacology,34(1):173-186
Kim S H,Jung M Y,Lee Y M.2011.Effect of heavy metals on the antioxidant enzymes in the marine ciliate Euplotes crassus[J].Toxicology and Environmental Health Sciences,3(4):213-219
Kim S,Yoon D,Lee M,et al.2016.Metabolic responses in zebrafish(Danio rerio) exposed to zinc and cadmium by nuclear magnetic resonance-based metabolomics[J].Chemistry and Ecology,32(2):136-148
Lankadurai B P,Simpson A J,Simpson M J.2012.1H NMR metabolomics of Eisenia fetida responses after sub-lethal exposure to perfluorooctanoic acid and perfluorooctane sulfonate[J].Environmental Chemistry,9(6):502-511
Lankadurai B P,Nagato E G,Simpson A J,et al.2015.Analysis of Eisenia fetida earthworm responses to sub-lethal C60 nanoparticle exposure using 1H-NMR based metabolomics[J].Ecotoxicology & Environmental Safety,120:48-58
Lock K,Janssen C.2001.Ecotoxicity of mercury to Eisenia fetida,Enchytraeus albidus and Folsomia candida [J].Biology and Fertility of Soils,34(4):219-221
刘岩.2016.基于1H NMR代谢组学方法研究敌敌畏对金鱼的毒性及作用机制[D].南京:南京理工大学
Mckelvie J R,Wolfe D M,Celejewski M,et al.2010.Correlations of Eisenia fetida metabolic responses to extractable phenanthrene concentrations through time[J].Environmental Pollution,158(6):2150-2157
Nagato E G,Simpson A J,Simpson M J.2016.Metabolomics reveals energetic impairments in Daphnia magna exposed to diazinon,malathion and bisphenol-A[J].Aquatic Toxicology,170:175-186
OECD.1984.Proposal for Updating Guideline 207:Earthworm,Acute Toxicity Tests2 OECD Guideline for Testing of Chemicals 207.European Committe,Pairs[EB/OL].Pairs:European Committee.2015-06-12.https: //www.oecd.org/env/ehs/testing/Draft-Updated-Test-Guildeline-222-Earthworm-reproduction-Test.pdf
Tang H,Yan Q,Wang X,et al.2016.Earthworm(Eisenia fetida) behavioral and respiration responses to sublethal mercury concentrations in an artificial soil substrate[J].Applied Soil Ecology,104:48-53
Tang R,Ding C,Dang F,et al.2018.NMR-based metabolic toxicity of low-level Hg exposure to earthworms[J].Environmental Pollution,239:428-437
唐浩,刘钊钊,李银生,等.2017.土壤汞污染胁迫对蚯蚓体内几种抗氧化酶活性的影响[J].上海交通大学学报(农业科学版),35(3):17-23
Yang X X,Song Y F,Ackland M L,et al.2012.Biochemical responses of earthworm Eisenia fetida exposed to cadmium-contaminated soil with long duration[J].Bulletin of Environmental Contamination & Toxicology,89(6):1148-1153
杨道丽.2013.汞与溴苯腈复合污染对赤子爱胜蚓及球肾白线蚓的生态毒理效应研究[D].上海:上海交通大学
Zhao L,Huang Y,Hannah-Bick C,et al.2016.Application of metabolomics to assess the impact of Cu(OH)2 nanopesticide on the nutritional value of lettuce(Lactuca sativa): Enhanced Cu intake and reduced antioxidants[J].Nanoimpact,3-4:58-66
Zhu J,Li Z G,Yang D L,et al.2011.Acute toxicological effects of Hg pollution on earthworm Eisenia Foetida[J].Journal of Shanghai Institute of Technology,11(2):95-99
张成,陈宏,王定勇,等.2014.三峡库区消落带土壤汞形态分布与风险评价[J].环境科学,35(3):1060-1067
张文新,陈勇,齐誉,等.2017.新疆玛河流域土壤和蔬菜汞分布特征与生态、人体健康风险评价[J].环境化学,36(11):2441-2450