Biochemical ECOTOXICOLOGY Principles and Methods This pageintentionallyleftblank Biochemical ECOTOXICOLOGY Principles and Methods FRANÇOIS GAGNÉ Senior Research Scientist Biochemical Ecotoxicology at Environment Canada Québec, Canada With AdditionalContributions From CHANTALE ANDRÉ JOËLLE AUCLAIR ÉMILIE LACAZE BRIAN QUINN AMSTERDAM(cid:1)BOSTON(cid:1)HEIDELBERG(cid:1)LONDON NEWYORK(cid:1)OXFORD(cid:1)PARIS(cid:1)SANDIEGO SANFRANCISCO(cid:1)SINGAPORE(cid:1)SYDNEY(cid:1)TOKYO AcademicPressisanimprintofElsevier AcademicPressisanimprintofElsevier 32JamestownRoad,LondonNW17BY,UK 225WymanStreet,Waltham,MA02451,USA 525BStreet,Suite1800,SanDiego,CA92101-4495,USA Copyrightr2014ElsevierInc.Allrightsreserved. Nopartofthispublicationmaybereproduced,storedinaretrievalsystemortransmittedinanyformor byanymeanselectronic,mechanical,photocopying,recordingorotherwisewithoutthepriorwritten permissionofthepublisher. PermissionsmaybesoughtdirectlyfromElsevier’sScience&TechnologyRightsDepartmentinOxford,UK: phone(144)(0)1865843830;fax(144)(0)1865853333;email:[email protected], visittheScienceandTechnologyBookswebsiteatwww.elsevierdirect.com/rightsforfurtherinformation. Notice Noresponsibilityisassumedbythepublisherforanyinjuryand/ordamagetopersonsorpropertyasamatterof productsliability,negligenceorotherwise,orfromanyuseoroperationofanymethods,products,instructions orideascontainedinthematerialherein. Becauseofrapidadvancesinthemedicalsciences,inparticular,independentverificationofdiagnosesanddrug dosagesshouldbemade. BritishLibraryCataloguing-in-PublicationData AcataloguerecordforthisbookisavailablefromtheBritishLibrary LibraryofCongressCataloging-in-PublicationData AcatalogrecordforthisbookisavailablefromtheLibraryofCongress ISBN:978-0-12-411604-7 ForinformationonallAcademicPresspublications visitourwebsiteatelsevierdirect.com PrintedandboundinUnitedStatesofAmerica 14 15 16 17 10 9 8 7 6 5 4 3 2 1 CONTENTS ListofContributors xiii Dedication xv Preface xvii Acknowledgments xix Introduction xxi 1. Quantitative AssessmentsofBiochemicalAnalyses 1 1.1. GeneralOverview 1 1.2. ReplicationinDataAnalysis 5 1.3. ReplicationoftheExperiment 6 1.3.1. CalibrationorStandardization 7 1.3.2. UsingSpecificandGenericStandards 8 1.4. ReferenceSubstances 10 1.5. DefiningDetectionLimitsintheAbsenceofAnalyticalStandards 10 1.6. Reproducibility 12 1.6.1. ReproducibilityattheCalibrationLevel 12 1.6.2. ReproducibilityattheLeveloftheExperiment 13 1.7. UsingReferenceMaterialtoStandardizeMethodologies 13 1.8. NormalizationApproaches 15 1.8.1. ResidualExtractionMethod 16 1.8.2. StepwiseAdditionMethod 18 References 19 2. TissuePreparation and SubcellularFractionation Techniques 21 2.1. TypesofHomogenizationBuffers 22 2.1.1. pH 22 2.1.2. IonChelators 24 2.1.3. Antioxidants 24 2.1.4. ProteasesInhibitors 24 2.1.5. DNase/RNaseInhibitors 25 2.1.6. Osmolarity 26 2.1.7. CryoprotectiveAgents 26 2.2. HomogenizationMethods 27 2.2.1. TeflonTissuePestle 27 v vi Contents 2.2.2. Polytron 28 2.2.3. Ultrasounds 28 2.2.4. Freeze(cid:3)Thaw 29 2.2.5. SubcellularFractionationbyDifferentialCentrifugation 29 2.2.6. ConservationofBiologicalSamplesforBiomarkers 31 References 31 3. Preparation andMaintenanceof Live Tissues and PrimaryCultures for ToxicityStudies 33 ContributedbyBrianQuinn 3.1. GeneralIntroduction 33 3.2. ReagentsRequiredandSolutionPreparation 35 3.3. Procedure 37 3.3.1. AnimalCollectionandMaintenance 37 3.3.2. PreparationofPrimaryCellCultures 38 3.4. ApplicationtoToxicityTesting 41 3.4.1. CytotoxicityEvaluation 41 3.4.2. ChronicEffects(BiomarkerExpression) 44 3.5. Calculations/DataHandling 45 3.5.1. CalculatingCellDensityandViabilityUsingtheNeubauerHemocytometer 45 References 45 4. Measuring Effectsatthe Gene TranscriptionLevel 49 ContributedbyChantaleAndré 4.1. Introduction 49 4.2. Material 53 4.2.1. Reagents 53 4.2.2. Equipment 54 4.3. Procedure 54 4.3.1. ChoiceofDetectionChemistry 54 4.3.2. PrimerDesign 55 4.3.3. SelectingtheTargetNucleotideSequence(TargetandReferenceGenes) 55 4.3.4. PreparationofNucleicacidTemplate 56 4.3.5. AssayOptimization 62 4.4. DataAnalysis-RelativeQuantificationwithReferenceGeneNormalization 69 References 72 WebResources 73 Contents vii 5. Metal Metabolism and Detoxification 75 ContributedbyJoëlleAuclair 5.1. IntracellularFreeMetalAvailability 77 5.1.1. Introduction 77 5.1.2. Reagents 77 5.1.3. Procedure 77 5.1.4. DataCalculation 78 5.2. SubcellularMetalBindingFractions 78 5.2.1. ReagentsandEquipment 79 5.2.2. Procedure 79 5.2.3. DataAnalysisandCalculation 80 5.3. Metallothioneins 80 5.3.1. SilverSaturationAssay 80 5.3.2. SpectrophotometricAssay 82 5.4. EnzymeImmunoassay 84 5.4.1. Reagent 84 5.4.2. Procedure 85 5.4.3. DataCalculationandAnalysis 86 5.5. HeatShockProtein70DetectionandQuantification byWesternBlotAnalysis 86 5.5.1. ReagentsandMaterials 86 5.5.2. Procedure 91 5.5.3. Detection-ColorimetricAlkalinePhosphataseActivity 95 5.5.4. Troubleshooting 97 5.5.5. CaseStudy 99 References 100 6. OxidativeStress 103 6.1. AntioxidantEnzymes 104 6.1.1. SOD 104 6.1.2. Peroxidase 106 6.1.3. Catalase 107 6.1.4. MeasurementofAntioxidantCapacity 109 6.1.5. LipidPeroxidation(OxidativeDamage) 110 6.1.6. MeasurementofAge-RelatedPigmentsandLipofuscins 111 6.2. Applications 113 References 115 viii Contents 7. XenobioticBiotransformation 117 7.1. XenobioticBiotransformation 117 7.2. GenericAssayforCytochromeP450 119 7.2.1. Reagents 119 7.2.2. Procedure 120 7.2.3. DataCalculation 120 7.3. CytochromeP4501A1andBenzo(a)PyreneHydroxylaseActivities 120 7.3.1. Reagents 121 7.3.2. Procedure 121 7.3.3. DataCalculation 122 7.4. CytochromeP4503A4Activity 122 7.4.1. Reagents 122 7.4.2. Procedure 123 7.4.3. DataCalculation 123 7.5. GlutathioneS-TransferaseActivity 123 7.5.1. Reagents 124 7.5.2. Procedure 124 7.5.3. DataCalculation 124 7.6. UridineDiphosphate-GlucuronylTransferaseActivity 124 7.6.1. Reagents 125 7.6.2. Procedure 125 7.6.3. DataCalculation 125 7.7. Sulfotransferases 126 7.7.1. Reagents 126 7.7.2. Procedure 126 7.7.3. DataCalculation 126 7.8. MultidrugResistanceGlycoproteins 127 7.8.1. Reagents 127 7.8.2. Procedure 128 7.8.3. DataCalculation 129 References 129 8. Cellular Energy Allocation 131 8.1. EnergyReserves 132 8.1.1. TotalCarbohydrates 132 8.1.2. Lipids 133 8.1.3. AnalysisofLipidProfilesby1HNMR 135 8.1.4. ProteinContent 136 8.1.5. MitochondriaElectronTransportActivityand TemperatureDependence 141 Contents ix 8.1.6. CytochromecOxidaseActivity 142 References 144 9. Neuroendocrine Disruption 145 9.1. VitellogeninEvaluationasaMarkerofEstrogenicity 146 9.1.1. Reagents 147 9.1.2. Procedure 147 9.1.3. DataCalculation 148 9.1.4. Precautions 148 9.2. Estradiol-17βorSteroidBindingSites 150 9.2.1. Reagents 150 9.2.2. Procedure 151 9.2.3. DataCalculation 152 9.3. FluorometricAssayforCatecholaminesandIndolamines 152 9.3.1. Reagents 152 9.3.2. BiogenicAmineExtraction 153 9.3.3. DeterminationofIndolamines(Serotonin) 153 9.3.4. Determinationof5-HIAA 154 9.3.5. DeterminationofAdrenalineandDopamine 154 9.3.6. DataCalculation 154 9.4. CompetitiveImmunoassaysforDopamineandSerotonin 154 9.4.1. ReagentsandSolutions 155 9.4.2. Procedure 155 9.4.3. DataCalculationandAnalysis 157 9.5. MonoamineOxidaseActivity 158 9.5.1. Reagents 158 9.5.2. Procedure 159 9.5.3. DataAnalysis 159 9.6. NeurotransmitterDirectedAdenylateCyclaseActivityinSynaptosomes 160 9.6.1. Reagents 160 9.6.2. Procedure 160 9.6.3. DataCalculationandAnalysis 161 9.7. Acetylcholinesterase 162 9.7.1. Reagents 162 9.7.2. Procedure 163 9.7.3. DataAnalysis 163 9.8. SteroidAnalysisbyHigh-PerformanceThinLayerChromatography 164 9.8.1. Reagents 164 9.8.2. Procedure 164 9.8.3. DataAnalysis 166