High-Throughput Screening Assays in Toxicology

✍ Scribed by Hao Zhu; Menghang Xia

• Publisher: Humana
• Year: 2022
• Tongue: English
• Leaves: 198
• Edition: 2nd ed. 2022
• Category: Library

✦ Synopsis

This second edition volume expands on the previous edition by exploring the latest advancements in high throughput screening (HTS) in toxicity studies by using in vitro, ex vivo, and in vivo models. This volume also covers the application of artificial intelligence (AI) and data science to curate, manage, and use HTS data. Written in the highly successful Methods in Molecular Biology series format, 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. Cutting-edge and thorough, High Throughput Screening Assays in Modern Toxicology, Second Edition is a valuable resource for scientists pursuing chemical toxicology research. This book will aid scientists and researchers in translating new HTS techniques into standardized chemical toxicology assessment tools that can refine, reduce, and replace animal testing.

✦ Table of Contents

Preface
Acknowledgments
Contents
Contributors
Part I: In Vitro Toxicological High Throughput Screening Methods
Chapter 1: Cell-Based Assays to Identify ERR and ERR/PGC Modulators
1 Introduction
2 Materials
2.1 Equipment
2.2 Solutions
3 Methods
3.1 Optimization of Cell-Based ERR and PGC/ERR Assays
3.2 Data Analysis
4 Notes
References
Chapter 2: Mitochondrial Membrane Potential Assay
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents/Supplies
3 Methods
3.1 Cell Culture and Maintenance
3.2 Quantitative High-Throughput Screening (qHTS) Protocol of MMP and Cell Viability Multiplex Assay in HepG2 and A16 Cells
3.3 Optimization of MMP Assay in NoSpin HepaRG Cells
3.4 Imaging Based MMP Assay
4 Notes
References
Chapter 3: Cell-Based hERG Channel Inhibition Assay in High-Throughput Format
1 Introduction
2 Materials
2.1 Cell Lines and Cell Culture Condition
2.2 Assay Reagents and Chemicals
2.3 Supplies and Equipment
3 Methods
3.1 Cell Culture
3.2 Thallium Flux Assay (See Table 2 for Simplified Protocol)
4 Notes
References
Chapter 4: Identifying CAR Modulators Utilizing a Reporter Gene Assay
1 Introduction
2 Materials
2.1 Equipment
2.2 Solutions and Vectors
3 Methods
3.1 Generation of hCAR-CYP2B6-HepG2 Cell Line
3.2 Optimization of a Quantitative High-Throughput Screen
3.3 Quantitative High-Throughput Screen for 10K Compounds
4 Notes
References
Chapter 5: Study Liver Cytochrome P450 3A4 Inhibition and Hepatotoxicity Using DMSO-Differentiated HuH-7 Cells
1 Introduction
2 Materials
2.1 Equipment
2.2 Cells and Culture Media
2.3 Reagents and Solutions
2.4 Reagent Preparation for CYP3A4 Reversible Inhibition Assay
2.5 Reagent Preparation for CYP3A4 Time-Dependent Inhibition Assay
2.6 Reagent Preparation for Hepatotoxicity Assay
3 Methods
3.1 Cell Culture
3.2 CYP3A4 Reversible Inhibition Assay
3.3 CYP3A4 Time-Dependent Inhibition Assay
3.4 Hepatotoxicity Assay
4 Notes
References
Chapter 6: Acetylcholinesterase Inhibition Assays for High-Throughput Screening
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents and Supplies
3 Methods
3.1 Cell-Based Assays
3.2 Human Recombinant AChE-Based Assays
3.3 Human Recombinant AChE-Based Assays with Liver Microsomes
3.4 Data Analysis
3.5 Confirmation of Km and Vmax for Colorimetric Recombinant AChE-Based Assay (Optional)
4 Notes
References
Chapter 7: Cell-Based Assays to Identify Modulators of Nrf2/ARE Pathway
1 Introduction
2 Materials
2.1 Cell Line and Cell Culture Condition
2.2 Assay Reagents and Chemicals
2.3 Supplies and Equipment
3 Methods
3.1 Cell Culture
3.2 Preparation of Positive Control Plate
3.3 ARE-bla Assay
3.4 ARE-luc Assay
4 Notes
References
Part II: In Vitro Toxicological High Content Screening Methods
Chapter 8: Cell-Based Imaging Assay for Detection of Phospholipidosis
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents/Supplies
3 Method
3.1 HepG2 Cell Thawing
3.2 Cell Propagation of HepG2 Cells
3.3 HepG2 Cell Plating
3.4 HepaRG Cell Thawing
3.5 HepaRG Cell Plating
3.6 Compound Treatment
3.7 Fixation and Staining
3.8 Image Readout
3.9 Image Analysis
4 Notes
References
Chapter 9: GFP-LC3 High-Content Assay for Screening Autophagy Modulators
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents/Supplies
3 Method
3.1 Thawing Cells
3.2 Culturing Cells
3.3 Plating Cells
3.4 Compound Treatment
3.5 Fixation and Staining
3.6 Image Readout
3.7 Image Analysis
4 Notes
References
Part III: Three-Dimensional Cell System for Toxicological High Throughput Screening
Chapter 10: Generation of iPSC-Derived Brain Organoids for Drug Testing and Toxicological Evaluation
1 Introduction
2 Materials
2.1 iPSC Culture
2.2 Forebrain Organoid Differentiation
2.3 Midbrain Organoids Differentiation
2.4 Choroid Plexus Organoids Differentiation
3 Methods
3.1 iPSC Culture
3.2 Forebrain Organoid Differentiation
3.3 Midbrain Organoid Differentiation
3.4 Choroid Plexus Organoid Differentiation
4 Notes
References
Part IV: In Vivo Toxicological High Throughput Screening Methods
Chapter 11: Zebrafish Behavioral Assays in Toxicology
1 Introduction
2 Experimental Design
3 Behavioral Assays
3.1 Spontaneous Tail Flexions and the Embryo Photomotor Response (EPR)-17-24 hpf
3.2 Larval Photomotor Response (LPR) ~120 hpf
3.3 Larval Startle Response (LSR) ~120 hpf
3.4 Touch Response (~24-120 hpf)
3.5 Strobe Light Test (5 dpf)
3.6 Optokinetic and Optomotor Response Assays (>5 dpf to Adult)
3.7 Social Preference (>5 dpf to Adult)
3.8 Color Avoidance (10 dpf)
3.9 Shoaling Assay (Juvenile and Adults)
3.10 Free Swim Assay (Juvenile and Adults)
3.11 Mirror Stimulus Test (Juvenile and Adults)
3.12 Schooling Assay (Adults)
3.13 Predator Avoidance Assay (Adults)
3.14 Active Avoidance Test (Adult)
3.15 Startle Tap Response (Adult)
3.16 Novel Tank Dive Test (Adult)
4 Assay and Analysis Considerations
5 Conclusions
References
Part V: In Silico High Throughput Screening Toxicity Data Analysis
Chapter 12: High-Throughput Screening Assay Profiling for Large Chemical Databases
1 Introduction
2 Materials
3 Methods
3.1 Programmatic Access to PubChem HTS Data Via PUG-REST
3.2 The Chemical In Vitro-In Vivo Profiling (CIIPro) Portal
4 Conclusions
5 Notes
References
Chapter 13: A Quantitative High-Throughput Screening Data Analysis Pipeline for Activity Profiling
1 Introduction
2 qHTS Data Pipeline
2.1 Plate Level Data Processing, Curve Fitting and Classification
2.2 Assay Performance Measured by Reproducibility
2.3 Identification of Autofluorescence and Cytotoxicity Artifacts
2.4 Compound Activity Assignment
3 Data Sharing
References
Chapter 14: CurveP Method for Rendering High-Throughput Screening Dose-Response Data into Digital Fingerprints
1 Introduction
2 Methods
2.1 User-Controlled Parameters of CurveP
2.2 Algorithmic Description of CurveP
2.3 CurveP Implementations
3 Application Notes
4 Concluding Remarks
References
Chapter 15: Accounting for Artifacts in High-Throughput Toxicity Assays
1 Introduction
2 Materials
2.1 Protocols
2.2 Computer Programs
3 Methods
3.1 Raw Read Normalization (See Note 1)
3.2 Readout Response Direction (See Note 2)
3.3 Noise Threshold Identification (See Note 3)
3.4 Other Parameters in Curvep (See Note 4)
3.5 Response Masking (See Note 5)
3.6 Artifacts at the Substance Level (See Note 6)
3.7 Artifacts at the Compound Level (See Note 7)
4 Notes
References
Chapter 16: Automatic Quantitative Structure-Activity Relationship Modeling to Fill Data Gaps in High-Throughput Screening
1 Introduction
2 Materials
3 Methods
3.1 Prepare the QSAR Modeling Workflow
3.2 Prepare the Training and Test Sets
3.3 Set up a Project Environment Variable
3.4 Install the Anaconda Environment
3.5 Run the QSAR Model Training Workflow
3.6 Filling Test Compound Data Gaps with Predictions
4 Conclusions
5 Notes
References
Chapter 17: Use In Silico and In Vitro Methods to Screen Hepatotoxic Chemicals and CYP450 Enzyme Inhibitors
1 Introduction
2 Materials
2.1 In Silico Program
2.2 Structure Data File
2.3 Cytotoxicity Assay
2.4 Human Recombinant Enzyme Assay
2.5 Human Liver Microsome Assay
2.6 Equipment
3 Methods
3.1 In Silico Prediction of Absorption
3.2 In Silico Prediction of Metabolism
3.3 In Silico Prediction of Hepatotoxicity
3.4 In Silico Prediction of CYP Inhibitors
3.5 In Vitro Screening of Hepatotoxicants
3.6 In Vitro Screening of CYP Inhibitors
3.6.1 Human Recombinant Enzyme Assays
3.6.2 Human Liver Microsomes Assay
3.7 Literature Search and Next Steps
References
Index