Environmental Toxicology and Toxicogenomics: Principles, Methods, and Applications

Description

This detailed book provides an accessible compendium of up-to-date methods in the fields of environmental toxicology, molecular toxicology, and toxicogenomics. Organized into four major sections, the volume examines methods utilizing model animal species, such as nematode, fruit fly, mice, chicken, and amphibians, methods using plants to study chemical toxicity, applying the Ames assay to chemical mutagenicity study, as well as methods for environmental chemical analysis. Although this book is divided into these parts, the methods can be used across species. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.  Authoritative and practical, Environmental Toxicology and Toxicogenomics: Principles, Methods, and Applications serves as a valuable resource for the scientific community, particularly for young scientists and graduate and undergraduate students, inspiring more research in the vitally important field of environmental toxicity, molecular toxicology, and toxicogenomics. Preface Contents Contributors Part I: Animal Toxicity Chapter 1: Detection of Caenorhabditis elegans Germ Cell Apoptosis Following Exposure to Environmental Contaminant Mixtures: A... 1 Introduction 2 Materials 2.1 Buffers 2.2 Growth Media for C. elegans 2.3 C. elegans Strains 2.4 C. elegans Germ Line Apoptosis Assay 2.5 Apoptosis Gene Expression Assay 3 Methods 3.1 NGM Agar Plates for C. elegans Maintenance and Synchronization 3.2 E. coli OP50 Stocks and Seeding of NGM Agar 3.3 Maintenance and Synchronization of C. elegans 3.4 Preparation and Treatment of C. elegans 3.5 Expression of Apoptosis-Associated Genes and Cytochrome P450 Genes 3.6 Assessment of Apoptotic Germ Cells in MD701 Worms 3.7 Assessment of Apoptotic Germ Cells in TJ1 Worms 4 Notes References Chapter 2: High-Throughput Measurement for Toxic Effects of Metal Mixtures in Caenorhabditis elegans 1 Introduction 2 Materials 2.1 C. elegans Strains 2.2 Solutions Preparation 2.3 Preparation of Reagents for COPAS Biosort 2.4 General Equipment 3 Methods 3.1 Preparation of Age-Synchronized L1 Growth-Arrested Larvae 3.2 Preparing Food Source for Liquid-Based Test 3.3 General Experimental Design 3.4 Prepare Worms for Sorting Using COPAS Biosort 3.5 Optimizing COPAS Biosort for Worm Sorting and Dispensing 3.6 High-Throughput Assays for Growth, Brood Size, and Feeding 3.6.1 Growth 3.6.2 Brood Size 3.6.3 Feeding 3.7 Chou-Talalay Combination Index (CI) Method for Assessing the Toxicity of Metal Mixtures 4 Notes References Chapter 3: Evaluations of Environmental Pollutant-Induced Mitochondrial Toxicity Using Caenorhabditis elegans as a Model System 1 Introduction 2 Materials 2.1 Strains 2.2 Equipment and Instruments 2.3 Chemicals 2.4 Mitochondrial Staining and Microscopy 2.5 ATP Production Assessment 2.6 Oxygen Consumption Measurement 2.7 Mitochondrial Gene Expression and Modifications Assessment 3 Methods 3.1 Exposure and Survival Assay 3.2 Mitochondrial ROS Detection 3.3 Mitochondrial Morphology Analysis 3.4 Mitochondrial Function Evaluation 3.4.1 ATP Production Assessment 3.4.2 Oxygen Consumption Measurement 3.5 Mitochondrial Gene Expression and Modifications Assessment 3.5.1 RT-PCR 3.5.2 Functional Assessment Using Genetic-Modified Strains 4 Notes References Chapter 4: Methods to Assay the Behavior of Drosophila melanogaster for Toxicity Study 1 Introduction 2 Materials 2.1 Equipment 2.2 Chemicals and Solution 2.3 Lab Suppliers 3 Methods 3.1 Survival Assay 3.2 Climbing Ability (Mobility Assay) 3.3 Reproductive Behavior Assay 3.4 Lifespan Assay 4 Notes References Chapter 5: Investigating the Joint Effects of Pesticides and Ultraviolet B Radiation in Xenopus laevis and Other Amphibians 1 Introduction 2 Materials 3 Methods 3.1 Adult X. laevis Husbandry 3.2 Obtain X. laevis Eggs and Larvae 3.3 Obtain Eggs and Larvae of Other Amphibians 3.4 Experimental Design 3.5 Prepare UVB Exposure 3.6 Prepare Exposure Solutions 3.7 Assign Animals to Treatments and Daily Observation 3.8 Experimental Conditions 3.9 Experimental Endpoints 4 Notes References Chapter 6: Identification of Stable Reference Genes for Toxicogenomic and Gene Expression Analysis 1 Introduction 2 Materials 2.1 Cell Lines 2.2 Chemicals and Regents 2.3 Instruments 2.4 Other Lab Suppliers 3 Methods 3.1 Cell Culture and Treatment 3.2 RNA Extraction 3.3 Housekeeping Gene Selection, Primer Design and Preparation 3.3.1 Housekeeping Gene Selection 3.3.2 Primer Design 3.3.3 Primer Preparation 3.4 Reverse Transcription PCR (RT-PCR) 3.5 Primer Efficiency Test 3.6 Primer Specificity Test 3.7 qRT-PCR 3.8 Identifying the Most Stable Reference Gene 4 Notes References Chapter 7: Semi-Quantitative RT-PCR: An Effective Method to Explore the Regulation of Gene Transcription Level Affected by Env... 1 Introduction 2 Materials 2.1 RNA Extraction 2.2 Reverse Transcription 2.3 PCR Amplification 3 Methods 3.1 Treatment of Experimental Apparatus 3.2 RNA Extraction and Detection (see Note 4) 3.3 Reverse Transcription (see Note 4) 3.4 PCR Amplification and Electrophoresis Detection 4 Notes References Chapter 8: Employing Multiple New Neurobiological Methods to Investigate Environmental Neurotoxicology in Mice 1 Introduction 2 Materials 2.1 Animals 2.2 Equipment and Instruments 2.2.1 Equipments and Instruments of Chemogenetics 2.2.2 Equipments and Instruments of Optogenetics 2.2.3 Equipments and Instruments of Fiber Photometry 2.2.4 Equipments and Instruments of In Vivo Two-Photon Imaging 2.2.5 Equipments and Instruments of In Vivo Electrophysiological Recording 2.2.6 Equipments and Instruments of In Vivo Miniscope Calcium Imaging 2.3 Viruses 2.4 Chemicals 3 Methods 3.1 Methods of Chemogenetics 3.2 Methods of Optogenetics 3.3 Methods of Fiber Photometry 3.4 Methods of In Vivo Two-Photon Imaging 3.5 Methods of In Vivo Electrophysiological Recording 3.6 Methods of In Vivo Miniscope Calcium Imaging 4 Notes References Chapter 9: Analyses of Epigenetic Modification in Environmental Pollutants-Induced Neurotoxicity 1 Introduction 2 Materials 2.1 Analyses of Genome-Wide DNA Methylation 2.2 Analyses of Genome-Specific DNA Methylation 2.3 Analyses of Genome-Wide RNA Methylation 2.4 Analyses of Gene-Specific RNA Methylation 2.5 Non-coding RNA 2.6 Histone Modification 2.6.1 Stock Solutions, Working Solutions, and Buffers 2.6.2 Other Reagents, Materials, and Equipment 3 Methods 3.1 Analyses of Genome-Wide DNA Methylation 3.2 Analyses of Genome-Specific DNA Methylation 3.3 Analyses of Genome-Wide RNA Methylation 3.3.1 RNA Isolation and Fragmentation 3.3.2 RNA Immunoprecipitation Reaction 3.3.3 Elution of m6A-Positive RNA 3.3.4 Extraction and Cleanup Step of the RIP 3.3.5 Library Construction and Sequencing 3.4 Analyses of Gene-Specific RNA Methylation 3.4.1 Site Prediction 3.4.2 mRNA Isolation and Fragmentation 3.4.3 Immunoprecipitation Reaction 3.4.4 RT-PCR 3.4.5 Data Analysis 3.5 Non-coding RNAs 3.6 Histone Modification 4 Notes References Chapter 10: The Application of Omics Technologies in the Research of Neurotoxicology 1 Introduction 2 Materials 2.1 Transcriptomics 2.2 Proteomics 2.3 Metabolomics 3 Methods 3.1 Transcriptomics 3.1.1 Sample Pretreatment 3.1.2 Total RNA Extraction and Quality Control 3.1.3 Illumina HiSeq Sequencing and Bioinformatics Analysis 3.2 Proteomics 3.2.1 Protein Extraction (See Note 5) 3.2.2 Sample Pretreatment 3.2.3 Protein Identification and Data Analysis 3.3 Metabolomics 3.3.1 Sample Pretreatment 3.3.2 Metabolites Separation and Analysis 3.3.3 Data Analysis and Study of Regulated/Diagnostic Functions 4 Notes References Chapter 11: Flow Cytofluorometric Analysis of Molecular Mechanisms of Premature Red Blood Cell Death 1 Introduction 2 Materials 2.1 RBC Samples 2.2 Blood/RBC Wash Buffer 2.3 Alsever´s Solution 2.4 Ringer Buffer 2.5 Ca2+-Free Ringer Buffer 2.6 Hyperosmotic Ringer Buffer 2.7 High-KCl Ringer Buffer 2.8 Chloride-Free Ringer Buffer 2.9 High-Ca2+ Ringer Buffer 2.10 Energy-Depleted Ringer Buffer 2.11 Hank´s Balanced Salt Solution (HBSS) 2.12 Inhibitors 2.13 Annexin-V-FITC 2.14 Fluo4/AM 2.15 H2DCFDA 2.16 A Confocal Microscope 2.17 A Centrifuge 2.18 A Spectrophotometer 2.19 A Flow Cytometer 3 Methods 3.1 RBC Isolation 3.2 Toxicant Treatment 3.3 Hemolysis (See Fig. 2) 3.4 Detection of PS Externalization, FSC, and SSC (Fig. 3) 3.5 Intracellular Calcium 3.6 Measurement of ROS 3.7 Signal Transduction Analysis 3.8 Confocal Microscopy 4 Notes References Chapter 12: Practical Methods and Technologies in Environmental Epidemiology 1 Introduction 2 Materials 2.1 Questionnaires 2.2 Sociodemographic Data 2.3 Environmental Exposure 2.3.1 Population Exposure 2.3.2 Individual Exposure 2.4 Health Effects 2.4.1 Population Health 2.4.2 Individual Health 3 Methods 3.1 Time Series Study 3.1.1 Design and Implementation Determination of the Study Purposes Data Collection Data Analysis Time Series Analysis Strategy Analysis Content 3.2 Case-Crossover Studies 3.2.1 Determine the Study Purpose 3.2.2 Determine the Hazard Period 3.2.3 Choose an Appropriate Control Form 3.2.4 Selection of Cases 3.2.5 Selection of Research Factors 3.2.6 Collection of Data 3.2.7 Statistical Analysis of Data and Interpretation of Results Data Analysis 1:1 Paired Data Analysis Normal Frequency Analysis 3.3 Panel Study 3.3.1 Basic Principles and Concepts Exposure Measurement Health Measurement Confounding Factors Crowd Selection Analysis Strategy and Basic Model 3.4 Cross-Sectional Study 3.4.1 Establish the Study Purposes 3.4.2 Select the Study Population and Determine the Survey Methods 3.4.3 Estimate the Sample Size 3.4.4 Confirm the Sampling Methods. 3.4.5 Variables Selection 3.4.6 Questionnaire Design 3.4.7 Establish the Study Purposes 3.4.8 Data Analysis 3.5 Case-Control Study 3.5.1 Determine the Study Purposes 3.5.2 Select the Study Population 3.5.3 Choice for Matching Methods 3.5.4 Calculations of Sample Size 3.5.5 Ascertain the Exposure Variables 3.5.6 Data Collection 3.5.7 Data Analysis 3.6 Cohort Study 3.6.1 Determine the Purposes, Exposure Factors, and Outcomes of the Study 3.6.2 Selection of the Fields of the Study and the Cohort 3.6.3 Calculations of Sample Size 3.6.4 Data Collection 3.6.5 Data Analysis 4 Notes References Chapter 13: In Ovo Early-in-Life Inhalation Exposure to Gas/Aerosol with a Chicken Embryo Model 1 Introduction 2 Materials 3 Methods 3.1 Determine Air Cell Area 3.2 Air Cell Infusion 4 Notes References Chapter 14: DNA Damage in Liver Cells of the Tilapia Fish Oreochromis mossambicus Larva Induced by the Insecticide Cyantranili... 1 Introduction 2 Materials 2.1 Test Organism 2.2 Chemicals 3 Methods 3.1 Fish Culture and Treatment 3.2 Acute Toxicity Test 3.3 Chronic Toxicity Test 3.4 Specific Growth Rate (%/Day) 3.5 Preparation of Primary Liver Cells 3.6 Micronucleus Test 3.7 Single Cell Gel Electrophoresis 3.8 Real-Time Fluorescence Quantitative PCR (qPCR) 3.9 Data Analysis 4 Notes References Part II: Plant Toxicity Chapter 15: Impact of Nanoparticles on Plant Growth, Development, and Biomass 1 Introduction 2 Materials 2.1 Chemicals and Regents 2.2 Plant Species 2.3 Instruments 2.4 Other Lab Suppliers 3 Methods 3.1 Preparation of Culture Medium 3.2 Seed Sterilization 3.3 Seed Plant and Culture 3.4 Seed Germination, Observation, and the Study on Seedling Growth and Development 3.5 Measurement of Root and Leaf Respiration 3.6 Measurement of Both Fresh and Dry Weight as Well as the Water Content 3.7 Data Analysis 4 Notes References Chapter 16: Biochemical and Physiological Toxicity of Nanoparticles in Plant 1 Introduction 2 Materials 2.1 Chemicals and Regents 2.2 Plant Species 2.3 Instruments 2.4 Other Lab Suppliers 3 Methods 3.1 Preparation of Culture Containers 3.2 Preparation of Hoagland Medium 3.3 Preparation of Seed Germination 3.4 Seedling Treatment 3.5 Measurement of Root and Leaf Respiration 3.6 Measurement of Plant Photosynthesis 3.6.1 Chlorophyll Fluorescence Determination Using Handy PEA 3.6.2 Oxygen Release Determination Using Chlorolab 2 3.7 Measurement of Root Vigor (See Note 11) 3.8 Measurement of Oxidative Stress 3.9 Data Analysis 4 Notes References Chapter 17: Determination of Oxidative Stress and Antioxidant Enzyme Activity for Physiological Phenotyping During Heavy Metal... 1 Introduction 2 Materials 2.1 Enzymes Extraction Buffer 2.2 Solutions for SOD Assay 2.3 Solution for POD Assay 2.4 Solution for CAT Assay 2.5 Solution for APX Assay 2.6 Solution for TBARS 2.7 Solution for H2O2 3 Methods 3.1 Enzymes Extraction 3.2 Superoxide Dismutase Activity (SOD) 3.3 Dynamic Study of Peroxidase Activity (POD) 3.4 Catalase Activity (CAT) 3.5 Determination of APX Activity 3.6 Lipid Peroxidation 3.7 Determination of H2O2 4 Notes References Chapter 18: Comprehensive Phytotoxicity Assessment Protocol for Engineered Nanomaterials 1 Introduction 2 Materials 2.1 Nanoparticle Characterization 2.2 Nanoparticle Purification Via Tangential Flow Filtration 2.3 Soil Preparation 2.4 Plantation 2.5 Symbiont/AMF Preparation and Inoculation 2.6 Measurement of Malondialdehyde Concentration 2.7 Measurement of Hydrogen Peroxide 2.8 Measurement of Superoxide Dismutase 2.9 Measurement of Catalase Activity 2.10 Measurement of Guaiacol Peroxidase Activity 2.11 Measurement of Ascorbate Peroxidase Activity 2.12 Measurement of Scavenging of Hydrogen Peroxide 2.13 Measurement of Root Parameters 2.14 Measurement of Biouptake 2.15 Localization of ENMs in Seed 2.16 Measurement of Crop Yield or Seed Biomass 3 Methods 3.1 Nanoparticle Synthesis and Characterization 3.2 Nanoparticle Purification Via Tangential Flow Filtration 3.3 Experimental Setup, Soil Preparation, and Exposure Conditions 3.4 Planting and Crop Management 3.5 Symbiont/AMF Preparation and Inoculation 3.6 Determination of Endpoints of Oxidative Stress Responses 3.6.1 Measurement of Malondialdehyde Concentration 3.6.2 Measurement of Hydrogen Peroxide 3.6.3 Measurement of Superoxide Dismutase 3.6.4 Measurement of Catalase Activity 3.6.5 Measurement of Guaiacol Peroxidase Activity 3.6.6 Measurement of Ascorbate Peroxidase Activity 3.6.7 Measurement of Scavenging of Hydrogen Peroxide 3.7 Measurement of Root Parameters 3.8 Measurement of Biouptake 3.9 Localization of ENPs in Seed 3.10 Measurement of Crop Yield or Seed Biomass 4 Notes References Chapter 19: Detection of Cadmium Toxicity in Plant 1 Introduction 2 Materials 2.1 Reagents 2.2 Equipment 2.3 Plant Species (See Note 2) 2.4 Soil Type (See Note 3) 3 Methods 3.1 Detection of Cd in Soil 3.2 Detection of Cd in Plant Tissue 3.3 The Cd Uptake in Plant Tissues 3.4 Calculation of Cd Translocation from Soil to Plant 4 Notes References Part III: Microbe Toxicity Chapter 20: Mutagenicity Evaluation of Nanoparticles by the Ames Assay 1 Introduction 2 Materials, Medium, and Nanoparticle Preparations 2.1 Tested Metal Oxide Nanoparticles 2.2 Bacterial Strains 2.3 Control Reagents 2.4 Working S9 Mix 2.5 Preparation of Medium 2.6 Other Supplies 3 Methods 4 Data Analysis 5 Notes References Part IV: Extraction and Detection Analytical Methods Chapter 21: Dispersive Solid-Phase Extraction of Multiresidue Pesticides in Food and Water Samples 1 Introduction 2 Materials 3 Method 3.1 Determination of Pesticides in Fruit and Vegetables 3.2 Determination of Pesticides in Food with High Fat Content (Cornmeal) (Fig. 3) 3.3 Ground and Surface Water Sample (Fig. 4) 3.4 Determination of Pesticides in Soil Using DPX (Fig. 5) 4 Notes References Chapter 22: Quantitative Analysis of Multiresidue Pesticides Using Gas Chromatography-Mass Spectrometry 1 Introduction 2 Materials 3 Method 4 Notes References Chapter 23: Determination of the N-Nitroso Compounds in Mouse Following RDX Exposure 1 Introduction 2 Materials 2.1 Chemicals and Regents 2.2 Animals and Foods 2.3 Instruments 2.4 Other Lab Suppliers 3 Methods 3.1 Preparation of RDX-Spiked Food 3.2 Animal Treatments (See Note 7) 3.3 Extract RDX and its Derivatives 3.4 Detect the Concentrations of RDX and its Derivatives 3.5 Date Analysis 4 Notes References Chapter 24: Determination of Metal Content in Drosophila melanogaster During Metal Exposure 1 Introduction 2 Materials 2.1 Equipment 2.2 Chemicals and Solutions 2.3 Native Polyacrylamide Gel 2.4 Lab Suppliers 3 Methods 3.1 ALP Activity Assay 3.2 Aconitase Activity Assay 3.3 Ferrozine-Based Colorimetric Assay 3.4 ICP-MS Analysis 3.5 Perl´s Prussian Blue Staining for Iron Detection in Midgut 3.6 Determining Iron Contents in Intact Iron-Loaded Ferritin Extracted from Tissues 4 Notes References Chapter 25: Microplastics: A Review of Methodology for Sampling and Characterizing Environmental and Biological Samples 1 Introduction 2 Sampling 2.1 Trawl Sampling 2.2 Grab Sampling 2.3 Sampling of Biota 2.4 Methods to Investigate Human Routes of Exposure to Microplastics 3 Sample Preparation/Separation 3.1 Separation by Density 3.2 Separation by Size 3.3 Separation by Dissection 3.4 Digestion 4 Microscopy Detection and Quantitation 5 Spectroscopy Identification 5.1 Fourier Transform Infrared (FTIR) Microscopy 5.2 Attenuated Total Reflection (ATR)-FTIR Spectroscopy 5.3 Raman Spectroscopy 6 Conclusion References Index