Preface
Contents
Contributors
Chapter 1: The Single-Cell Gel Electrophoresis Genotoxin Sensitivity Assay
1 Introduction
2 Materials
2.1 Separation of Lymphocytes
2.2 Solutions of the Test Compounds
2.3 Components of the Activation Mix (S9 Mix)
2.4 Single-Cell Gel Electrophoresis (SCGE) Assay
3 Methods
3.1 Intervention Trials
3.2 Preparation of Slides
3.3 Collection and Storage of Lymphocytes
3.4 Preparation of S9 Mix
3.5 Incubation Mixtures
3.6 Determination of the Viability of the Cells
3.7 SCGE Assay
3.8 Evaluation
3.9 Statistics
4 Notes
References
Chapter 2: Skin Sensitization Tests: The LLNA and the RhE IL-18 Potency Assay
1 Introduction
1.1 The Local Lymph Node Assay (LLNA)
1.2 The RhE IL-18 Potency Assay
2 Materials
2.1 Local Lymph Node Assay
2.2 RhE IL-18 Potency Assay
3 Methods
3.1 LLNA
3.1.1 Determine Optimal Vehicle for Test Article (Solubility Testing)
3.1.2 Conduct Dermal Irritation Pre-screen to Determine Test Concentrations
3.1.3 Exposure
3.1.4 In Situ Labeling
3.1.5 Preparation of Lymph Node Cells
3.1.6 Analysis of Results
3.2 RhE IL-18 Potency Assay
3.2.1 Determine Chemical Solubility
3.2.2 Protocol Scheme
3.2.3 Exposure of the RhE to MTT and Isopropanol Extraction
3.2.4 Determination of the IL-18 Release Using In-House ELISA
3.2.5 Data Analysis
3.3 General Discussion
4 Notes
References
Chapter 3: Skin Equivalent Models: Protocols for In Vitro Reconstruction for Dermal Toxicity Evaluation
1 Introduction
2 Materials
2.1 Primary Culture of Human Skin Cells from Foreskin Biopsies
2.2 Skin Equivalents (SE) on Air-Liquid Interface
2.3 Reconstructed Human Epidermis (RHE) on Polyester Membrane
3 Methods
3.1 Primary Culture of Human Skin Cells from Foreskin Samples
3.1.1 Collection of Dermal and Epidermal Single Cells
3.1.2 Establishment of Epidermal Compartment
3.1.3 Establishment of Dermal Compartment
3.2 Skin Equivalents (SE)
3.2.1 Preparation of Dermal Compartment
3.2.2 Preparation of Epidermal Compartment
Keratinocytes Collection
Melanocytes Collection
Final Assembly of Epidermal Compartment
3.3 Reconstructed Human Epidermis (RHE)
3.3.1 Polyester Inserts
3.3.2 Keratinocytes
4 Notes
References
Chapter 4: Intravital Microscopy: A Tool to Investigate the Toxicity in the Immune System, Vessel Rheology, and Xenobiotic Dis...
1 Introduction
2 Materials
2.1 Anesthesia
2.2 Optical Microscopy and Camera
2.3 IVM Solutions
2.4 Mesentery Technique Surgical Materials
2.5 Dorsal Skinfold Chamber Surgical Materials
2.6 Blood-Brain Barrier (BBB)-Specific Materials
2.7 IVM BBB Solutions
3 Methods
3.1 Mesentery
3.2 Dorsal Skinfold Chamber
3.2.1 Dorsal Chamber Implantation
3.2.2 IVM of Dorsal Chamber Window
3.3 Blood-Brain Barrier
4 Notes
References
Chapter 5: Cell-Based Biosensor to Visualize Nitric Oxide Release from Living Cells for Toxicity Assessment
1 Introduction
2 Materials
2.1 Cell Culture and Transfection
2.2 Live Cell Imaging
3 Methods
3.1 Design of the Cell-Based Biosensor, Named Piccell
3.2 Construction of Piccell and Its Culture
3.3 Preparation of NO Solutions
3.4 Fluorescence Imaging of Piccell
3.5 Fluorescence Imaging of NO Release from Vascular Endothelial Cells
4 Notes
References
Chapter 6: In Vitro Contraction of Isolated Cauda Epididymal Duct Smooth Muscle as a Complimentary Approach to Physiological, ...
1 Introduction
2 Materials
3 Methods
3.1 Initializing and Calibrating the Device
3.2 Distal Epididymal Cauda Isolation
3.3 Potassium Chloride Challenge
3.4 Control Curve: Concentration-Response Curve to NE (In Vitro Assay)
3.5 Drug Effect on Concentration- Response Curve for NE (In Vitro Assay, See Note 14)
3.6 Data Quantification (See Note 15)
3.7 Data Interpretation of the Delta Value
3.8 Data Interpretation of the Area Under the Curve Value
4 Notes
References
Chapter 7: Early Life Stage Assays in Zebrafish
1 Introduction
2 Materials
3 Methods
3.1 Spawning
3.2 Eggs Selection
3.3 Exposure Conditions
3.4 Analytical Measurements
3.5 Morphological Observations
3.6 Representative Results
4 Notes
References
Chapter 8: Assessment of Urothelial Cytotoxicity at Morphological and Molecular Levels: Urinary Bladder as Target Organ
1 Introduction
2 Materials
2.1 Urinary Bladder Processing for SEM Analysis
2.2 Collection of Urinary Bladder Cells for Total RNA Isolation (See Note 2)
3 Methods
3.1 Urinary Bladder Processing for SEM Analysis
3.2 Collection of Urinary Bladder Cells for Total RNA Isolation
4 Notes
References
Chapter 9: Evaluation of Development of the Rat Uterus as a Toxicity Biomarker
1 Introduction
2 Materials
2.1 Experimental Protocol and Exposure
2.2 Dissection of Uterine Horns
2.3 Tissue Processing
2.4 Histomorphometrical Analysis
2.5 Immunohistochemistry and Quantification
3 Methods
3.1 Neonatal Exposure Protocol
3.2 Histomorphometrical Analysis
3.3 Immunohistochemistry
3.4 Quantification of Cell Proliferation
3.5 Quantification of Protein Expression by Image Analysis
4 Notes
References
Chapter 10: Acetylcholinesterase (AChE) Activity in Embryos of Zebrafish
1 Introduction
2 Materials
2.1 General Materials
2.2 For Sample Preparation
2.3 For Protein Determination
2.4 For AChE Determination
3 Methods
3.1 Sample Preparation
3.2 Protein Determination
3.3 AChE Activity
4 Notes
References
Chapter 11: Ecotoxicity Assays Using Freshwater Planarians
1 Introduction
2 Materials
3 Methods
3.1 Laboratory Culture of Planarians
3.2 Acute Survival Test
3.3 Planarian Locomotor Activity Assay
3.4 Postexposure Feeding Assay
3.5 Planarian Head Regeneration Assay
4 Notes
References
Chapter 12: Toxicogenomic Analysis
1 Introduction
2 Materials
2.1 Total RNA Extraction
2.2 Total RNA Yield and Quality Analysis
2.3 IlluminaTruSeq Stranded Total RNA Sample Preparation
2.4 Library Validation and Quantification
2.5 Library Normalization and Sample Pool Preparation
2.6 Cluster Generation and Sequencing
2.7 Real-Time qPCR Validation
2.8 Protein Extraction
2.9 Protein Quantification
2.10 SDS Polyacrylamide Gel
2.11 In Situ Protein Tryptic Hydrolysis
2.12 Peptide Purification in Tip C18´´
2.13 Liquid Chromatographyin Tandem´´ with Mass Spectrometry (LC-MS/MS)
3 Methods
3.1 Total RNA Extraction
3.2 Total RNA Yield and Quality Analysis
3.3 Illumina TruSeq Stranded Total RNA Sample Preparation
3.3.1 Ribosomal RNA Removal
3.3.2 Depleted RNA Cleanup
3.3.3 RNA Fragmentation
3.3.4 First cDNA Strand Synthesis
3.3.5 Second cDNA Strand Synthesis
3.3.6 cDNA Cleanup
3.3.7 cDNA Adenylation, Adaptor Ligation, and Cleanup
3.3.8 Library Amplification by PCR and Cleanup
3.4 Library Validation and Quantification
3.5 Library Normalization and Sample Pool Preparation
3.6 Cluster Generation and Sequencing
3.7 Analysis
3.7.1 Real-Time qPCR Validation
3.8 Protein Extraction
3.9 Protein Quantification
3.10 12% Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
3.11 In Situ Protein Tryptic Hydrolysis
3.12 Peptide Cleanup Using Tips Packaged with C18 Reverse-Phase Matrix
3.13 Liquid Chromatography ``in Tandem´´ with Mass Spectrometry (LC-MS/MS)
3.14 Peptides/Proteins Identification in the Database
4 Notes
References
Chapter 13: Strategy Combining Nonanimal Methods for Ocular Toxicity Evaluation
1 Introduction
2 Materials
2.1 STE Test
2.2 HET-CAM Test Method
2.3 BCOP Test Method
3 Methods
3.1 STE Test
3.1.1 Preparation of the SIRC Cell Culture
3.1.2 SIRC Cell Exposure to Test and Control Materials
3.1.3 Data Processing
3.2 HET-CAM Test
3.2.1 Preparing the CAM and Exposure to Test Materials
3.2.2 Data Processing
3.3 BCOP Test + Corneal Histomorphometric Analysis
4 Notes
References
Chapter 14: Immunocytochemistry Analysis of HepG2 Cell 3D Culture Encapsulated as Spheroids in Alginate Beads
1 Introduction
2 Materials
2.1 3D HepG2 Cell Encapsulation in Alginate Beads (See Note 1)
2.2 Immunocytochemistry Analysis of 3D HepG2 Cells Encapsulated in Alginate Beads
3 Methods
3.1 3D HepG2 Cells Encapsulation in Alginate Beads
3.2 Immunocytochemistry of 3D HepG2 Cells Encapsulated in Alginate Beads
4 Notes
References
Chapter 15: Oxygen Consumption Evaluation: An Important Indicator of Metabolic State, Cellular Function, and Cell Fate Along N...
1 Introduction
2 Materials
2.1 Equipment
2.1.1 For Cell Culture
2.1.2 For Blood Sampling
2.1.3 Brain Dissection
2.1.4 Brain Slices
2.1.5 Oxygen Consumption
2.2 Solutions
3 Methods
3.1 Oxygraph Calibration
3.2 Oxygen Consumption Rate (OCR) Measurement (O2/nmol/mL/min)
3.2.1 OCR Measurement in Cell Suspension-from Adherent Cells (Astrocytes)
3.2.2 OCR Measurement in Cell Suspension-from Adherent Cells (Neurons)
3.2.3 OCR Measurement in Cell Suspension-from Non-adherent Cells (Lymphoblasts)
3.2.4 OCR Measurement in Cell Suspension-from Non-adherent Cells (Peripheral Blood Mononuclear Cells (PBMCs))
3.2.5 OCR Measurement in Brain Tissue-from 12-Days-Old Cortex from Brain Rats
3.2.6 OCR Measurement in Brain Tissue-from 30-Days-Old Cortex from Brain Rats
3.2.7 OCR Measurement in Brain Slices (See Note 27)
3.3 Analysis of Oxygen Consumption
3.4 Internal Experiment Calibration (mg of Protein, Brain Slice Size, and/or Tissue Weight)
4 Notes
References
Chapter 16: Mitochondrial Bioenergetic Assays as a Standard Protocol for Toxicological and Metabolic Assessment
1 Introduction
2 Materials
3 Methods
3.1 Hepatic Mitochondrial Isolation (See Note 3)
3.2 Mitochondrial Membrane Potential (DeltaΟ) Evaluation (See Note 10)
3.3 Mitochondrial Oxygen Consumption Measurement
3.4 Mitochondrial Calcium-Induced Swelling Quantification
3.5 Mitochondrial Calcium Flux Evaluation
3.6 Mitochondrial Hydrogen Peroxide Generation Quantification
4 Notes
References
Chapter 17: In Vitro Apoptotic Cell Death Assessment
1 Introduction
1.1 Homeostasis and Cell Death
1.2 Principles of the Apoptosis In Vitro Assays
1.2.1 Exposure of Phosphatidylserine in the Outer Membrane
1.2.2 Cell Cycle Analyses
1.2.3 Nuclear Fragmentation
1.2.4 Apoptotic Protein Quantification
2 Materials
2.1 Apoptosis Analyses by Flow Cytometry with Annexin-V
2.2 Cell Cycle Analyses
2.3 Nuclear Fragmentation Analyses
2.4 Detection of Cytosolic and Mitochondrial Proteins by Western Blotting
3 Methods
3.1 Apoptosis Analyses by Flow Cytometry with Annexin-V
3.2 Cell Cycle Analyses
3.3 Nuclear Fragmentation
3.4 Detection of Cytosolic and Mitochondrial Proteins by Western Blotting
4 Notes
5 Conclusion
References
Index