- •CONTENTS
- •Preface
- •Contributors
- •1 Introduction to Toxicology
- •1.1 Definition and Scope, Relationship to Other Sciences, and History
- •1.1.2 Relationship to Other Sciences
- •1.1.3 A Brief History of Toxicology
- •1.3 Sources of Toxic Compounds
- •1.3.1 Exposure Classes
- •1.3.2 Use Classes
- •1.4 Movement of Toxicants in the Environment
- •Suggested Reading
- •2.1 Introduction
- •2.2 Cell Culture Techniques
- •2.2.1 Suspension Cell Culture
- •2.2.2 Monolayer Cell Culture
- •2.2.3 Indicators of Toxicity in Cultured Cells
- •2.3 Molecular Techniques
- •2.3.1 Molecular Cloning
- •2.3.2 cDNA and Genomic Libraries
- •2.3.3 Northern and Southern Blot Analyses
- •2.3.4 Polymerase Chain Reaction (PCR)
- •2.3.5 Evaluation of Gene Expression, Regulation, and Function
- •2.4 Immunochemical Techniques
- •Suggested Reading
- •3.1 Introduction
- •3.2 General Policies Related to Analytical Laboratories
- •3.2.1 Standard Operating Procedures (SOPs)
- •3.2.2 QA/QC Manuals
- •3.2.3 Procedural Manuals
- •3.2.4 Analytical Methods Files
- •3.2.5 Laboratory Information Management System (LIMS)
- •3.3 Analytical Measurement System
- •3.3.1 Analytical Instrument Calibration
- •3.3.2 Quantitation Approaches and Techniques
- •3.4 Quality Assurance (QA) Procedures
- •3.5 Quality Control (QC) Procedures
- •3.6 Summary
- •Suggested Reading
- •4 Exposure Classes, Toxicants in Air, Water, Soil, Domestic and Occupational Settings
- •4.1 Air Pollutants
- •4.1.1 History
- •4.1.2 Types of Air Pollutants
- •4.1.3 Sources of Air Pollutants
- •4.1.4 Examples of Air Pollutants
- •4.1.5 Environmental Effects
- •4.2 Water and Soil Pollutants
- •4.2.1 Sources of Water and Soil Pollutants
- •4.2.2 Examples of Pollutants
- •4.3 Occupational Toxicants
- •4.3.1 Regulation of Exposure Levels
- •4.3.2 Routes of Exposure
- •4.3.3 Examples of Industrial Toxicants
- •Suggested Reading
- •5 Classes of Toxicants: Use Classes
- •5.1 Introduction
- •5.2 Metals
- •5.2.1 History
- •5.2.2 Common Toxic Mechanisms and Sites of Action
- •5.2.3 Lead
- •5.2.4 Mercury
- •5.2.5 Cadmium
- •5.2.6 Chromium
- •5.2.7 Arsenic
- •5.2.8 Treatment of Metal Poisoning
- •5.3 Agricultural Chemicals (Pesticides)
- •5.3.1 Introduction
- •5.3.3 Organochlorine Insecticides
- •5.3.4 Organophosphorus Insecticides
- •5.3.5 Carbamate Insecticides
- •5.3.6 Botanical Insecticides
- •5.3.7 Pyrethroid Insecticides
- •5.3.8 New Insecticide Classes
- •5.3.9 Herbicides
- •5.3.10 Fungicides
- •5.3.11 Rodenticides
- •5.3.12 Fumigants
- •5.3.13 Conclusions
- •5.4 Food Additives and Contaminants
- •5.5 Toxins
- •5.5.1 History
- •5.5.2 Microbial Toxins
- •5.5.3 Mycotoxins
- •5.5.4 Algal Toxins
- •5.5.5 Plant Toxins
- •5.5.6 Animal Toxins
- •5.6 Solvents
- •5.7 Therapeutic Drugs
- •5.8 Drugs of Abuse
- •5.9 Combustion Products
- •5.10 Cosmetics
- •Suggested Reading
- •6 Absorption and Distribution of Toxicants
- •6.1 Introduction
- •6.2 Cell Membranes
- •6.3 Mechanisms of Transport
- •6.3.1 Passive Diffusion
- •6.4 Physicochemical Properties Relevant to Diffusion
- •6.4.1 Ionization
- •6.5 Routes of Absorption
- •6.5.1 Extent of Absorption
- •6.5.2 Gastrointestinal Absorption
- •6.5.3 Dermal Absorption
- •6.5.4 Respiratory Penetration
- •6.6 Toxicant Distribution
- •6.6.1 Physicochemical Properties and Protein Binding
- •6.7 Toxicokinetics
- •Suggested Reading
- •7 Metabolism of Toxicants
- •7.1 Introduction
- •7.2 Phase I Reactions
- •7.2.4 Nonmicrosomal Oxidations
- •7.2.5 Cooxidation by Cyclooxygenases
- •7.2.6 Reduction Reactions
- •7.2.7 Hydrolysis
- •7.2.8 Epoxide Hydration
- •7.2.9 DDT Dehydrochlorinase
- •7.3 Phase II Reactions
- •7.3.1 Glucuronide Conjugation
- •7.3.2 Glucoside Conjugation
- •7.3.3 Sulfate Conjugation
- •7.3.4 Methyltransferases
- •7.3.7 Acylation
- •7.3.8 Phosphate Conjugation
- •Suggested Reading
- •8 Reactive Metabolites
- •8.1 Introduction
- •8.2 Activation Enzymes
- •8.3 Nature and Stability of Reactive Metabolites
- •8.4 Fate of Reactive Metabolites
- •8.4.1 Binding to Cellular Macromolecules
- •8.4.2 Lipid Peroxidation
- •8.4.3 Trapping and Removal: Role of Glutathione
- •8.5 Factors Affecting Toxicity of Reactive Metabolites
- •8.5.1 Levels of Activating Enzymes
- •8.5.2 Levels of Conjugating Enzymes
- •8.5.3 Levels of Cofactors or Conjugating Chemicals
- •8.6 Examples of Activating Reactions
- •8.6.1 Parathion
- •8.6.2 Vinyl Chloride
- •8.6.3 Methanol
- •8.6.5 Carbon Tetrachloride
- •8.6.8 Acetaminophen
- •8.6.9 Cycasin
- •8.7 Future Developments
- •Suggested Reading
- •9.1 Introduction
- •9.2 Nutritional Effects
- •9.2.1 Protein
- •9.2.2 Carbohydrates
- •9.2.3 Lipids
- •9.2.4 Micronutrients
- •9.2.5 Starvation and Dehydration
- •9.2.6 Nutritional Requirements in Xenobiotic Metabolism
- •9.3 Physiological Effects
- •9.3.1 Development
- •9.3.2 Gender Differences
- •9.3.3 Hormones
- •9.3.4 Pregnancy
- •9.3.5 Disease
- •9.3.6 Diurnal Rhythms
- •9.4 Comparative and Genetic Effects
- •9.4.1 Variations Among Taxonomic Groups
- •9.4.2 Selectivity
- •9.4.3 Genetic Differences
- •9.5 Chemical Effects
- •9.5.1 Inhibition
- •9.5.2 Induction
- •9.5.3 Biphasic Effects: Inhibition and Induction
- •9.6 Environmental Effects
- •9.7 General Summary and Conclusions
- •Suggested Reading
- •10 Elimination of Toxicants
- •10.1 Introduction
- •10.2 Transport
- •10.3 Renal Elimination
- •10.4 Hepatic Elimination
- •10.4.2 Active Transporters of the Bile Canaliculus
- •10.5 Respiratory Elimination
- •10.6 Conclusion
- •Suggested Reading
- •11 Acute Toxicity
- •11.1 Introduction
- •11.2 Acute Exposure and Effect
- •11.3 Dose-response Relationships
- •11.4 Nonconventional Dose-response Relationships
- •11.5 Mechanisms of Acute Toxicity
- •11.5.1 Narcosis
- •11.5.2 Acetylcholinesterase Inhibition
- •11.5.3 Ion Channel Modulators
- •11.5.4 Inhibitors of Cellular Respiration
- •Suggested Reading
- •12 Chemical Carcinogenesis
- •12.1 General Aspects of Cancer
- •12.2 Human Cancer
- •12.2.1 Causes, Incidence, and Mortality Rates of Human Cancer
- •12.2.2 Known Human Carcinogens
- •12.3 Classes of Agents Associated with Carcinogenesis
- •12.3.2 Epigenetic Agents
- •12.4 General Aspects of Chemical Carcinogenesis
- •12.5 Initiation-Promotion Model for Chemical Carcinogenesis
- •12.6 Metabolic Activation of Chemical Carcinogens and DNA Adduct Formation
- •12.7 Oncogenes
- •12.8 Tumor Suppressor Genes
- •12.8.1 Inactivation of Tumor Suppressor Genes
- •12.8.2 p53 Tumor Suppressor Gene
- •12.9 General Aspects of Mutagenicity
- •12.10 Usefulness and Limitations of Mutagenicity Assays for the Identification of Carcinogens
- •Suggested Reading
- •13 Teratogenesis
- •13.1 Introduction
- •13.2 Principles of Teratology
- •13.3 Mammalian Embryology Overview
- •13.4 Critical Periods
- •13.5 Historical Teratogens
- •13.5.1 Thalidomide
- •13.5.2 Accutane (Isotetrinoin)
- •13.5.3 Diethylstilbestrol (DES)
- •13.5.4 Alcohol
- •13.6 Testing Protocols
- •13.6.1 FDA Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use
- •13.6.3 Alternative Test Methods
- •13.7 Conclusions
- •Suggested Reading
- •14 Hepatotoxicity
- •14.1 Introduction
- •14.1.1 Liver Structure
- •14.1.2 Liver Function
- •14.2 Susceptibility of the Liver
- •14.3 Types of Liver Injury
- •14.3.1 Fatty Liver
- •14.3.2 Necrosis
- •14.3.3 Apoptosis
- •14.3.4 Cholestasis
- •14.3.5 Cirrhosis
- •14.3.6 Hepatitis
- •14.3.7 Oxidative Stress
- •14.3.8 Carcinogenesis
- •14.4 Mechanisms of Hepatotoxicity
- •14.5 Examples of Hepatotoxicants
- •14.5.1 Carbon Tetrachloride
- •14.5.2 Ethanol
- •14.5.3 Bromobenzene
- •14.5.4 Acetaminophen
- •14.6 Metabolic Activation of Hepatotoxicants
- •Suggested Reading
- •15 Nephrotoxicity
- •15.1 Introduction
- •15.1.1 Structure of the Renal System
- •15.1.2 Function of the Renal System
- •15.2 Susceptibility of the Renal System
- •15.3 Examples of Nephrotoxicants
- •15.3.1 Metals
- •15.3.2 Aminoglycosides
- •15.3.3 Amphotericin B
- •15.3.4 Chloroform
- •15.3.5 Hexachlorobutadiene
- •Suggested Reading
- •16 Toxicology of the Nervous System
- •16.1 Introduction
- •16.2 The Nervous system
- •16.2.1 The Neuron
- •16.2.2 Neurotransmitters and their Receptors
- •16.2.3 Glial Cells
- •16.3 Toxicant Effects on the Nervous System
- •16.3.1 Structural Effects of Toxicants on Neurons
- •16.3.2 Effects of Toxicants on Other Cells
- •16.4 Neurotoxicity Testing
- •16.4.1 In vivo Tests of Human Exposure
- •16.4.2 In vivo Tests of Animal Exposure
- •16.4.3 In vitro Neurochemical and Histopathological End Points
- •16.5 Summary
- •Suggested Reading
- •17 Endocrine System
- •17.1 Introduction
- •17.2 Endocrine System
- •17.2.1 Nuclear Receptors
- •17.3 Endocrine Disruption
- •17.3.1 Hormone Receptor Agonists
- •17.3.2 Hormone Receptor Antagonists
- •17.3.3 Organizational versus Activational Effects of Endocrine Toxicants
- •17.3.4 Inhibitors of Hormone Synthesis
- •17.3.5 Inducers of Hormone Clearance
- •17.3.6 Hormone Displacement from Binding Proteins
- •17.4 Incidents of Endocrine Toxicity
- •17.4.1 Organizational Toxicity
- •17.4.2 Activational Toxicity
- •17.4.3 Hypothyroidism
- •17.5 Conclusion
- •Suggested Reading
- •18 Respiratory Toxicity
- •18.1 Introduction
- •18.1.1 Anatomy
- •18.1.2 Cell Types
- •18.1.3 Function
- •18.2 Susceptibility of the Respiratory System
- •18.2.1 Nasal
- •18.2.2 Lung
- •18.3 Types of Toxic Response
- •18.3.1 Irritation
- •18.3.2 Cell Necrosis
- •18.3.3 Fibrosis
- •18.3.4 Emphysema
- •18.3.5 Allergic Responses
- •18.3.6 Cancer
- •18.3.7 Mediators of Toxic Responses
- •18.4 Examples of Lung Toxicants Requiring Activation
- •18.4.1 Introduction
- •18.4.2 Monocrotaline
- •18.4.3 Ipomeanol
- •18.4.4 Paraquat
- •18.5 Defense Mechanisms
- •Suggested Reading
- •19 Immunotoxicity
- •19.1 Introduction
- •19.2 The Immune System
- •19.3 Immune Suppression
- •19.4 Classification of Immune-Mediated Injury (Hypersensitivity)
- •19.5 Effects of Chemicals on Allergic Disease
- •19.5.1 Allergic Contact Dermatitis
- •19.5.2 Respiratory Allergens
- •19.5.3 Adjuvants
- •19.6 Emerging Issues: Food Allergies, Autoimmunity, and the Developing Immune System
- •Suggested Reading
- •20 Reproductive System
- •20.1 Introduction
- •20.2 Male Reproductive Physiology
- •20.3 Mechanisms and Targets of Male Reproductive Toxicants
- •20.3.1 General Mechanisms
- •20.3.2 Effects on Germ Cells
- •20.3.3 Effects on Spermatogenesis and Sperm Quality
- •20.3.4 Effects on Sexual Behavior
- •20.3.5 Effects on Endocrine Function
- •20.4 Female Reproductive Physiology
- •20.5 Mechanisms and Targets of Female Reproductive Toxicants
- •20.5.1 Tranquilizers, Narcotics, and Social Drugs
- •20.5.2 Endocrine Disruptors (EDs)
- •20.5.3 Effects on Germ Cells
- •20.5.4 Effects on the Ovaries and Uterus
- •20.5.5 Effects on Sexual Behavior
- •Suggested Reading
- •21 Toxicity Testing
- •21.1 Introduction
- •21.2 Experimental Administration of Toxicants
- •21.2.1 Introduction
- •21.2.2 Routes of Administration
- •21.3 Chemical and Physical Properties
- •21.4 Exposure and Environmental Fate
- •21.5 In vivo Tests
- •21.5.1 Acute and Subchronic Toxicity Tests
- •21.5.2 Chronic Tests
- •21.5.3 Reproductive Toxicity and Teratogenicity
- •21.5.4 Special Tests
- •21.6 In vitro and Other Short-Term Tests
- •21.6.1 Introduction
- •21.6.2 Prokaryote Mutagenicity
- •21.6.3 Eukaryote Mutagenicity
- •21.6.4 DNA Damage and Repair
- •21.6.5 Chromosome Aberrations
- •21.6.6 Mammalian Cell Transformation
- •21.6.7 General Considerations and Testing Sequences
- •21.7 Ecological Effects
- •21.7.1 Laboratory Tests
- •21.7.2 Simulated Field Tests
- •21.7.3 Field Tests
- •21.8 Risk Analysis
- •21.9 The Future of Toxicity Testing
- •Suggested Reading
- •22 Forensic and Clinical Toxicology
- •22.1 Introduction
- •22.2 Foundations of Forensic Toxicology
- •22.3 Courtroom Testimony
- •22.4.1 Documentation Practices
- •22.4.2 Considerations for Forensic Toxicological Analysis
- •22.4.3 Drug Concentrations and Distribution
- •22.5 Laboratory Analyses
- •22.5.1 Colorimetric Screening Tests
- •22.5.2 Thermal Desorption
- •22.5.6 Enzymatic Immunoassay
- •22.6 Analytical Schemes for Toxicant Detection
- •22.7 Clinical Toxicology
- •22.7.1 History Taking
- •22.7.2 Basic Operating Rules in the Treatment of Toxicosis
- •22.7.3 Approaches to Selected Toxicoses
- •Suggested Reading
- •23 Prevention of Toxicity
- •23.1 Introduction
- •23.2 Legislation and Regulation
- •23.2.1 Federal Government
- •23.2.2 State Governments
- •23.2.3 Legislation and Regulation in Other Countries
- •23.3 Prevention in Different Environments
- •23.3.1 Home
- •23.3.2 Workplace
- •23.3.3 Pollution of Air, Water, and Land
- •23.4 Education
- •Suggested Reading
- •24 Human Health Risk Assessment
- •24.1 Introduction
- •24.2 Risk Assessment Methods
- •24.2.2 Exposure Assessment
- •24.2.3 Dose Response and Risk Characterization
- •24.3 Noncancer Risk Assessment
- •24.3.1 Default Uncertainty and Modifying Factors
- •24.3.2 Derivation of Developmental Toxicant RfD
- •24.3.3 Determination of RfD and RfC of Naphthalene with the NOAEL Approach
- •24.3.4 Benchmark Dose Approach
- •24.3.5 Determination of BMD and BMDL for ETU
- •24.3.6 Quantifying Risk for Noncarcinogenic Effects: Hazard Quotient
- •24.3.7 Chemical Mixtures
- •24.4 Cancer Risk Assessment
- •24.5 PBPK Modeling
- •Suggested Reading
- •25 Analytical Methods in Toxicology
- •25.1 Introduction
- •25.2 Chemical and Physical Methods
- •25.2.1 Sampling
- •25.2.2 Experimental Studies
- •25.2.3 Forensic Studies
- •25.2.4 Sample Preparation
- •25.2.6 Spectroscopy
- •25.2.7 Other Analytical Methods
- •Suggested Reading
- •26 Basics of Environmental Toxicology
- •26.1 Introduction
- •26.2 Environmental Persistence
- •26.2.1 Abiotic Degradation
- •26.2.2 Biotic Degradation
- •26.2.3 Nondegradative Elimination Processes
- •26.3 Bioaccumulation
- •26.4 Toxicity
- •26.4.1 Acute Toxicity
- •26.4.2 Mechanisms of Acute Toxicity
- •26.4.3 Chronic Toxicity
- •26.4.5 Abiotic and Biotic Interactions
- •26.5 Conclusion
- •Suggested Reading
- •27.1 Introduction
- •27.2 Sources of Toxicants to the Environment
- •27.3 Transport Processes
- •27.3.1 Advection
- •27.3.2 Diffusion
- •27.4 Equilibrium Partitioning
- •27.5 Transformation Processes
- •27.5.1 Reversible Reactions
- •27.5.2 Irreversible Reactions
- •27.6 Environmental Fate Models
- •Suggested Reading
- •28 Environmental Risk Assessment
- •28.1 Introduction
- •28.2 Formulating the Problem
- •28.2.1 Selecting Assessment End Points
- •28.2.2 Developing Conceptual Models
- •28.2.3 Selecting Measures
- •28.3 Analyzing Exposure and Effects Information
- •28.3.1 Characterizing Exposure
- •28.3.2 Characterizing Ecological Effects
- •28.4 Characterizing Risk
- •28.4.1 Estimating Risk
- •28.4.2 Describing Risk
- •28.5 Managing Risk
- •Suggested Reading
- •29 Future Considerations for Environmental and Human Health
- •29.1 Introduction
- •29.2 Risk Management
- •29.3 Risk Assessment
- •29.4 Hazard and Exposure Assessment
- •29.5 In vivo Toxicity
- •29.6 In vitro Toxicity
- •29.7 Biochemical and Molecular Toxicology
- •29.8 Development of Selective Toxicants
- •Glossary
- •Index
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16.4NEUROTOXICITY TESTING
A large number of the chemicals used in industry today remain poorly characterized with respect to their toxic effects on the nervous system. In order to determine potential risks to human and environmental well-being, existing neurotoxicants must be identified, and the approximately 2000 new chemicals introduced each year must be screened for their potential neurotoxic effects. Often, a tiered approach is used, with the first tier consisting of general screening tests to identify acute hazards. The Environmental Protection Agency (EPA) has proposed screening guidelines for tests in rodents that include a functional observational battery (FOB, see below) to evaluate sensory, motor, and autonomic effects, tests that identify changes in motor activity, and neuropathological assessment. Interpretation of the outcome of tier 1 screening may lead to more selective testing and examining the effects of repeated exposures in the second tier. Specialized tests for behavioral effects, developmental neurotoxicity, or delayed organophosphate effects may be required. If necessary, a third tier of testing characterizes mechanisms of neurotoxicant-induced injury. Complete and comprehensive evaluation of potential neurotoxicant effects requires that data from different types of sources be considered; this can range from molecular interactions to whole animal and human exposure analysis. Below are examples of techniques commonly used for testing neurotoxic effects.
16.4.1In vivo Tests of Human Exposure
Historically the first indication of neurotoxic potential by a chemical has often followed accidental human exposure in the workplace. Case reports of incidents involving individuals, or clusters of individuals, are useful for documentation but generally provide a limited amount of information about the specific details of an exposure. Procedures included in most case reports include a patient medical history and clinical neurological exam, sometimes supplemented with psychiatric or neurophysiological tests, and/or neuroimaging. Although the specific tests involved vary depending on the clinician, most basic clinical neurological exams rely heavily on evaluation of mental status (level of consciousness, orientation, mood, etc.) and sensorimotor function (gait, coordination, muscle tone, sensitivity to touch, reflexes).
Human epidemiological studies generally represent a deeper investigation into the causal relationship between an exposure and neurotoxicological effects. Some of the methods used to identify neurotoxic effects in epidemiological studies include behavioral assessments, neurophysiological evaluations, and neuroimaging techniques. Neurobehavioral assessments examine a variety of psychological and cognitive functions such as mood, attention, memory, perceptual and visuospatial ability, and psychomotor performance. In an effort to standardize neurotoxicological testing of human behavioral effects, particularly for studies involving worksite exposure, the World Health Organization (WHO) and the US National Institute for Occupational Safety and Health (NIOSH) devised a the Neurobehavioral Core Test Battery (NCBT). The NCBT (Table 16.1) consists of seven tests that were shown previously to be sensitive indicators of neurotoxicant exposure. The battery is designed to be administered one on one by an examiner. Although this battery has a relatively narrow
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TOXICOLOGY OF THE NERVOUS SYSTEM |
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Table 16.1 The WHO Neurobehavioral Core Test Battery (NBCT) |
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Domain |
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Analysis |
Test |
Task |
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Psychomotor |
Motor speed, |
Pursuit aiming |
Follow a pattern of small |
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performance |
motor steadiness |
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circles, placing a dot |
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in each circle around a |
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pattern; subject’s score |
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is number of taps in |
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circle within one |
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minute. |
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Manual dexterity, |
Santa Ana |
Perform skillful |
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hand–eye |
Dexterity Test |
movements with hands |
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coordination |
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and arms. |
Perceptual coding |
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Wechsler Digit |
Each number in a list is |
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and perceptual |
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Symbol Test |
associated with a |
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motor speed |
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simple symbol. On a |
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list of random digits |
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with blank spaces |
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below them, write the |
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correct symbols in |
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blank spaces as fast as |
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possible. |
Attention and |
Attention and |
Simple reaction |
Test reactions of hands |
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short-term |
response speed |
time |
or feet from visual and |
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memory |
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auditory signals. |
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Visual perception |
Benton Visual |
Recall and reproduce |
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and memory |
Retention Test |
figures. |
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Auditory memory |
Wechsler Digit |
Recall digits in series |
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Span Test |
forwards and |
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backwards |
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immediately after |
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hearing them. |
Mood and affect |
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Profile of Mood |
Evaluate, by |
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States |
questionnaire, anger, |
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tension, confusion, |
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depression, etc. |
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focus, primarily on the effects most commonly seen in CNS toxicity, it also provides suggestions for the selection of further testing depending on the exposure setting. The NCBT has been widely used because of its ease of administration, relatively low cost, and its large base of control data. A broader battery of cognitive and psychomotor tests that is often used is the Neurobehavioral Evaluation System (NES). The NES consists of a combination of automated (computerized) and hand-administered tests. The sensitivity of the NES to effects caused by neurotoxicants in industrial settings has been validated internationally.
Neurobehavioral examinations are useful for identifying neurotoxicant-mediated deficits, but it is often difficult to localize the site of toxic action from such tests. For example, sensorimotor tests of reaction time, manual dexterity, hand-eye coordination, and finger tapping can indicate either neuromuscular or psychomotor damage.
NEUROTOXICITY TESTING |
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The results of these tests thus should be interpreted in the context of other experiments. For example, electrophysiological techniques can help to focus an investigation to the site of the lesion, and characterize electrical dysfunction within the damaged nerves. Electrophysiological nerve conduction studies can distinguish between proximal and distal axonal lesions in peripheral nerves and can be performed noninvasively (i.e., with skin surface electrodes). Characteristic changes in the velocity, duration, amplitude, waveform, or refractory period of peripheral nerves may be detected, depending on the agent. Evoked potentials represent another useful electrophysiological endpoint. These procedures measure the function of an entire system, such as the visual, auditory, or motor systems. The specific pathway is stimulated by an evoking stimulus, such as a flash of light or electrical nerve stimulation. The evoked potentials are read as changes in ongoing electroencephalograms (EEGs) in response to the stimulation. Thus the activity of the entire neural circuit is evaluated in the brain after peripheral stimulation. Evoked potentials can be very sensitive indicators of changes in neural activity when performed in a carefully controlled environment, and when interpreted in light of behavioral or other physiological findings.
An increasingly popular method of documenting brain pathology is the use of neuroimaging methods. Computerized axial tomography (CAT) and magnetic resonance imaging (MRI) can produce images of the brain that can show structural changes in the volume or density of a specific region or ventricle. Other techniques, such as positron emission tomography (PET) and single photon emission computerized tomography (SPECT), use radioactive tracer molecules to determine functional biochemical changes in processes like glucose utilization or receptor binding. The number of cases so far analyzed with neuroimaging techniques is still relatively small, and thus specific toxicant-mediated effects are not well characterized. Nevertheless, this growing field promises to contribute significantly to neurotoxicity studies in the future.
16.4.2In vivo Tests of Animal Exposure
The primary approach currently used to detect and characterize potential neurotoxicants involves the use of animal models, particularly rodents. Behavioral and neurophysiological tests, often similar to the ones used in humans, are typically administered. The sensitivity of these measures to neurotoxicant exposure is widely accepted. Although it is often not possible to test toxicant effects on some higher behavioral functions in animals (e.g., verbal ability, cognitive flexibility), there are other neurobehavioral outcomes such as memory loss, motivational defects, somatosensory deficits, and motor dysfunction that can be successfully modeled in rodents. These behaviors are based on the ability of the nervous system to integrate multiple inputs and outputs, thus they cannot be modeled adequately in vitro. Although the bulk of neurotoxicity data has been collected in rodents, birds and primates are also used to model human behavioral outcomes.
As mentioned above, a useful screening tool for neurotoxicant exposure is a battery of observational tests of function known as an FOB. FOBs, like the one developed by the EPA, are used to detect overt changes in behavior and physiology of animals exposed to neurotoxicants. In the typical exam, an observer documents cageside observations regarding the appearance and activity of the animal. Then the animal is handled and examined for obvious signs such as lacrimation, salivation, or piloerection. Pupillary light responses and temperature are recorded, and the ease of handling the animal
296 TOXICOLOGY OF THE NERVOUS SYSTEM
is also noted. The animal is then placed in an open field, such as the top of a laboratory cart, and observed for a set period of time, during which the observer records exploratory behaviors, excretion rate, and whether or not there are any motor abnormalities. A number of manipulations are then performed to assess hearing, sensitivity to touch, righting reflex, coordination, and grip strength. The FOB used in the EPA guidelines has shown remarkable consistency in detecting chemical effects in diverse testing laboratories and situations. A test of motor activity can be administered along with the FOB, consisting of quantitative evaluations of the animal’s movement in either an open field or a maze. Motor activity tests reflect integrative abilities of the nervous system to process sensory input, association, and motor output. A number of agents, such as toluene, triadimefon, and chlorinated hydrocarbons, increase or decrease motor activity in a toxicant-specific manner, unrelated to their general effects on the health of the animal.
More in-depth behavioral tests are required if dose-related toxicant effects are noted in screening tests. These tests may also be required as part of more selective toxicological screening, such as for developmental neurotoxicity. Focused tests of neuromotor function and activity, sensory functions, memory, attention, and motivation help to identify sites of toxicant-mediated lesioning, aid in the classification of neurotoxicants, and may suggest mechanisms of action. Some of these tests, like the schedule-controlled operant behavior tests for cognitive function, require animal training and extensive operator interaction with the animals.
16.4.3In vitro Neurochemical and Histopathological End Points
In vitro methods for studying neurotoxicant effects are a valuable supplement to whole animal and human testing, allowing the researcher to supplement findings, test hypotheses, and reduce the number of animals used for toxicity testing. Much of the neurochemical and histopathological data on neurotoxicant effects in humans and animals is gathered concomitantly with, or immediately after, performing behavioral tests. This may involve collection of bodily fluids or samples from living subjects for the purpose of analyses such as red blood cell or plasma acetylcholinesterase or NTE activity, determination of hormone or neurotransmitter concentration, or detection of the presence of toxicant or metabolite in the cerebrospinal fluid.
Postmortem tissues can provide a wealth of information about the location, timing, extent, and mechanism of neurotoxicant-induced damage. For example, changes in the gross morphology and weight of brain or nerves may be seen at higher levels of toxicant exposure. Microscopically, fixed and stained tissues reveal the type of damage to target cells, such as axonopathic or demyelinating lesions. Degenerative changes in cells may be indicative of the injurious process, and whether cells are dying by necrosis or apoptosis. Typical stains such as cresyl violet (Nissl stain) and silver (Golgi stain) are useful for cell morphology and counting. Other stains are selective for damaged cells, like the specialized silver impregnation techniques that are frequently used to identify neurotoxicant-mediated degeneration of neurons.
Tissue sections may also be processed for immunohistochemical staining. A frequently used immunochemical marker for neuropathologic insult is glial fibrillary acidic protein (GFAP). GFAP is produced in large amounts by reactive astrocytes that proliferate in response to tissue injury. Stress proteins, apoptotic signals, and immediate early genes are also utilized as markers of neuronal activity and injury. Other protein markers
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