浅论利用排泄行为参数评价重金属暴露对于秀丽线虫代谢状态的毒害【摘要】目的：研究重金属暴露对秀丽线虫排泄行为的影响。方法：用于分析的CdCl2、CrCl2、HgCl2和Pb(NO3)2溶液浓度分别为50、100、150和200从molL-1。参数平均排泄循环长度用于评价重金属暴露秀丽线虫的代谢状态改变。线性回归用于分析平均排泄循环长度与暴露重金属浓度之间、暴露动物中平均排泄循环长度和咽泵运动速率之间的关联。结果：所有分析浓度的汞和铅暴露以及浓度为100ImolL-1到200pmolL-1的镉和铭暴露均可以显着增加线虫的平均排泄循环长度。线虫平均排泄循环长度具有伴随重金属暴露浓度改变而变化的特点，且平均排泄循环长度显着相关于所暴露重金属的浓度。在重金属暴露的线虫中，咽泵运动速率还显着相关于平均排泄循环长度。此外，汞和铅暴露对于线虫排泄行为的影响远比铭和镉的暴露严重。结论：参数平均排泄循环长度可以有效评价重金属暴露对线虫代谢状态的毒害，而且在重金属暴露线虫中可以观察到平均排泄循环长度与咽泵运动速率之间的显着相关性。【关键词】重金属；排泄行为；代谢状态；秀丽线虫Caenorhabditiselegans,afreelivingsoilnonparasiticnematode,survivesinterrestrial,benthic,andaquaticenvironments,andhasathin,translucentbodyapproximatelytomminlength,andashortlifecycle.C.eleganscanshowarapidresponsewhiletheyareexposedtoaseverestressortoxicsolution.Byvirtueoftheseproperties,ithasbeenchosenasanimportantbioindicatorfortheecologicalriskassessmentinsoilandwater1-8.Sofar,C.eleganshasbeenshowntobeeffectiveinthedetectionofenvironmentalcontaminationandthestudyofbiomedicalandenvironmentaltoxicologybymonitoringtheadverseeffectsonmortality2,7,9-10,lifespan11-14,reproduction15-17,development12-13,18,feeding16,19-20,behavior19-22,andbehavioralplasticity13,23asecologicallyrelevantendpoints.Itiswellknownthatmetalcontaminationhasposedenvironmentalandhumanhealththreatthroughouttheworld.Amongtheadverseeffectsfrommetalexposure,thealteredmetabolismstatecanbeusuallydetectedinexposedorganisms24-26,andthemetabolismstatecanbeassessedbytheendpointsoffeedinganddefecation24,26-27.Feedinganddefecationhavealreadybeenacceptedasendpointstoassessthedietarymetaltoxicityinotherorganisms24,26,28-29.InC.elegans,themetabolismstatecanalsobeevaluatedbypharyngealpumpinganddefecation16,20,30.Inaddition,thefeedinghasbeenexploredasanecologicallyrelevantendpointinevaluatingthemetaltoxicityasmeasuredbyachangeinopticaldensity(OD)ofbacteriasuspensionsovertheexposureperiod16,20.Nevertheless,itisstillunclearwhethertheendpointofdefecationbehaviorcanalsobeexploredtoevaluatethetoxicityonmetabolismstateinmetalexposednematodes.Thedefecationsystemistheresultofastereotypedmotorprogrammodulatedbyenvironmentalfactorsandsensoryinputssuchastemperature,food,andmechanicalstimulationinC.elegans31.Theobjectivesofthisstudyweretostudythepossibleadverseeffectsofmetalexposureatdifferentconcentrationsondefecationbehavior,andtoevaluatetheassociationsbetweenpharyngealpumpinganddefecationinmetalexposednematodes.1MaterialsandmethodsReagentsThemetalconcentrationsusedinthisstudywerereferredtoourpreviousdescription21.FourconcentrationsofCdCl2,CrCl2,HgCl2,andPb(NO3)2solutionswereusedinthecurrentwork,andtheywere50昏molL-1,10011mol-L-1,150/Imol-L-1,and200imol-L-1,respectively.Metalconcentrationsofexposedsolutionswereanalyzedbyatomicabsorptionspectrophotometry(AAS;PyeLnicammodelSP9,Cambridge,UK).AllthechemicalswereobtainedfromSigmaAldrich(St.Louis,MO,USA).StrainpreparationNematodesusedinthepresentstudywerewildtypeN2,originallyobtainedfromtheCaenorhabditisGeneticsCenter(fundedbytheNIHNationalCenterforResearchResource,USA).Theyweremaintainedonnematodegrowthmedium(NGM)platesseededwithEscherichiacoliOP50at20Casdescribed32.Gravidnematodeswerewashedofftheplatesintocentrifugetubes,andwerelysedwithableachingmixture(mol-L-1NaOH,2%HOCl).AgesynchronouspopulationsofL4larvalanimalswereobtainedbythecollectionasdescribed33.Thecollectednematodeswerewashedwithdoubledistilledwatertwice,followedbywashingwithmodifiedKmediumonce(50mmolL-1NaCl,30mmolL-1KCl,10mmolL-1NaOAc,pH)34.Exposureswereperformedin12wellsteriletissuecultureplates.The24hofexposurewasperformedonL4larvalnematodesin20Cincubatorinthepresenceoffood.Aftermetalexposure,thetreatednematodeswereusedforassayofmeandefecationcyclelength,andpumpingrate.MeandefecationcyclelengthThemethodwasperformedasdescribed30.MeandefecationcyclewasscoredunderDICopticswithaZeissaxioscope.Individualnematodeswereobservedforafixednumberofcycles,andacycleperiodwasdefinedastheintervalbetweentheinitiationoftwosuccessiveposteriorbodywallmusclecontractionsteps.Atleast30replicateswereperformedforstatisticalpurposes.PumpingrateThemethodwasperformedasdescribed35.Pharyngealpumpingwascountedfor1minunderDICopticswithaZeissaxioscope.NematodeswereplacedonNGM/OP50platesat20Candleftundisturbedfor1hbeforemeasuring.Allcontrolortreatednematodesremainedonfoodduringtheperiodofobservations.Atleast30replicateswereperformedforstatisticalpurposes.StatisticalanalysisAlldatainthisarticlewereexpressedasmeans士GraphsweregeneratedusingMicrosoftExcel(MicrosoftCorp.,Redmond,WA).Onewayanalysisofvariance(ANOVA)followedbyaDunnettsttestwasusedtodeterminethesignificanceofthedifferencesofdefecationbehaviors.Theprobabilitylevelsofandwereconsideredstatisticallysignificant.Associationsfortheconcentrationofexposedmetalswithmeandefecationcyclelengthwereassessedwithlinearregressionanalysis.Inaddition,associationsbetweenpumpingrateandmeandefecationcyclelengthwerealsoevaluatedwithlinearregressionanalysis.2ResultsEffectsofmetalexposureonde