Non-Cancer Toxicology April 21, 2017 Non-Cancer Toxicology Introduction to the Health Effects of Chemicals Neurotoxicology Steven G. Gilbert, PhD, DABT & www.asmalldoseof.org www.toxipedia.org A Small Dose of Toxicology - Overview

April 21, 2017 Introduction “You cannot reach your full genetic potential with a damaged nervous system.” S.G. Gilbert A Small Dose of Toxicology - Overview

What is Neurotoxicity An adverse change in the chemistry, structure or function of the nervous system during development or at maturity, following exposure to a chemical or physical agent.

Nervous System Sensitivity Even minor changes in the structure or function of the nervous system may have profound consequences for neurological, behavioral, and related body functions.

Examples of non-cancer toxicity Developmental alterations Immunological impairment Neurobehavioral effects Kidney/liver dysfunction Mutagenesis Reproductive impairment Metabolic inhibition

General Mechanisms of Non-Cancer Toxicity Toxicant Delivery to target tissue T O X I C Y Exposure Interaction with target molecule Cell dysfunction and/or Injury Tissue damage

Are there general mechanisms? Interference with receptor-ligand interactions Interference with membrane functions Interference with cellular energy production Binding to macromolecules Perturbation of calcium regulation Toxicity from selective cell loss Non-lethal genetic alterations

Animal Cell Rough Endoplasmic Reticulum Lysosomes Peroxisomes Mitochondria Membrane Protein Nucleus Golgi Ribosomes

Interference with Membrane Function Cell Transport Protein Hydrophilic head Lipid bilayer Hydrophobic tails Channel Pump

Neuro Insulation

“LEAD MAKES THE MIND GIVE WAY” Dioscorides - GREEK 2ND BC April 21, 2017 Ancient Awareness “LEAD MAKES THE MIND GIVE WAY” Dioscorides - GREEK 2ND BC A Small Dose of Toxicology - Overview

April 21, 2017 Current Awareness “The upsurge of interest in recent years in academia, industry, and government on the effects of toxic chemicals on the nervous system has created a new discipline of neurotoxicology.” Peter S. Spencer & Herbert H. Schaumberg, in Experimental and Clinical Neurotoxicology, 1980 A Small Dose of Toxicology - Overview

Historical Events 1930’s – Ginger-Jake Syndrome April 21, 2017 Historical Events 1930’s – Ginger-Jake Syndrome During prohibition, an alcohol beverage was contaminated with TOCP (triorthocresyl phosphate) causing paralysis in 5,000 with 20,000 to 100,000 affected. 1950’s – Mercury poisoning Methylmercury in fish cause death and sever nervous system damage in infants and adults. A Small Dose of Toxicology - Overview

Lead – damages developing brain Alcohol – Fetal alcohol syndrome April 21, 2017 Case Studies Lead – damages developing brain Alcohol – Fetal alcohol syndrome MPTP – similar to Parkinson’s disease A Small Dose of Toxicology - Overview

Lead In Homes

Nervous Systems Effects April 21, 2017 Nervous Systems Effects Lead Neurotoxicity Developmental Neurotoxicity Reduced IQ Impaired learning and memory Life-long effects A Small Dose of Toxicology - Overview

H H H C C OH H H Alcohol (ethanol) Ethyl Alcohol April 21, 2017 A Small Dose of Toxicology - Overview

Alcohol Vulnerability of Developing Nervous System April 21, 2017 Alcohol Vulnerability of Developing Nervous System FAS – Fetal Alcohol Syndrome FAE – Fetal Alcohol Effects What is a save level of consumption during pregnancy? A Small Dose of Toxicology - Overview

ALCOHOL Vulnerability of Developing Nervous System April 21, 2017 ALCOHOL Vulnerability of Developing Nervous System FAS – Fetal Alcohol Syndrome FAE – Fetal Alcohol Effects What is a safe level of ethanol consumption during pregnancy? http://www.fetalalcohol.com/what-is-fase.htm http://www.alumni.ca/~syed4s0/discussion.htm A Small Dose of Toxicology - Overview

FAS Child

MPTP N CH3 1-methyl-4-phenyl-1,2,3,6-tetrahydrophyridine April 21, 2017 MPTP N CH3 1-methyl-4-phenyl-1,2,3,6-tetrahydrophyridine A Small Dose of Toxicology - Overview

MPTP Effects 1980s – Designer Drug April 21, 2017 MPTP Effects 1980s – Designer Drug Caused effects similar to Parkinson’s disease Damaged neurons that secrete dopamine A Small Dose of Toxicology - Overview

April 21, 2017 MPTP MPTP – 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine: a contaminant in “synthetic heroin” Parkinson’s Disease-like symptoms in drug addicts Loss of neurons in the substantia nigra which synthesize and secrete dopamine MPTP-->MPP+ ---> oxidative stress A Small Dose of Toxicology - Overview

MPTP – Real People http://www.swmed.edu/stars/resources/neurodisslides.html

http://www.swmed.edu/stars/images/neurodisslides/Slide26.JPG

PD Brain http://www.swmed.edu/stars/images/neurodisslides/Slide29.JPG

Nervous System Biology April 21, 2017 Nervous System Biology CNS – Central Nervous System PNS – Peripheral Nervous System Blood brain barrier Neuronal cells Neurotransmitters & receptors 10-100 billion cells with 1015 connections A Small Dose of Toxicology - Overview

Nervous System – CNS & PNS April 21, 2017 Nervous System – CNS & PNS Central Nervous System (CNS) Brain & Spinal Cord Peripheral Nervous System (PNS) Afferent (sensory) Nerves – Carry sensory information to the CNS Efferent (motor) Nerves – Transmit information to muscles or glands A Small Dose of Toxicology - Overview

Peripheral Nervous System Central Nervous System PNS Peripheral Nervous System CNS Central Nervous System Autonomic Somatic Sympathetic Parasympathetic

Central Nervous System Central Nervous System (CNS) (Brain and Spinal Cord) Peripheral Nervous System (PNS) Afferent (sensory) Nerves (Carry sensory information to the CNS) Efferent (motor) Nerves (Transmit information to muscles or glands) Autonomic Somatic Sympathetic Parasympathetic

Peripheral Nervous System April 21, 2017 Peripheral Nervous System Peripheral Nervous System (PNS) Efferent (motor) Nerves – Transmit information to muscles or glands Somatic Nervous System Stimulates Skeletal muscles Autonomic Nervous System Stimulates Glands and Organs (e.g. heart) Sympathetic - Adrenergic – stress response Parasympathetic - Cholinergic – basic functions A Small Dose of Toxicology - Overview

Cells of the Nervous System April 21, 2017 Neurons Information conductors Supporting Cells (Glia cells) Astrocytes (CNS – blood brain barrier) Oligodendrocytes (CNS – link cells) Schwann cells (PNS – wrap cells) A Small Dose of Toxicology - Overview

Blood-brain Barrier Not an absolute barrier Anatomic Characteristics April 21, 2017 Blood-brain Barrier Not an absolute barrier Caffeine (small) Methylmercury cysteine complex Lipids (brain is a ball of fat) Anatomic Characteristics Capillary endothelial cells are tightly joined – no pores between cells Capillaries in CNS surrounded by astrocytes Low protein concentration in CNS fluid Active ATP-dependent transporter – moves chemicals into the blood A Small Dose of Toxicology - Overview

Neuronal Cells Myelin (Schwann cell) Synapse Axon Dendrite Nucleus April 21, 2017 Neuronal Cells Myelin (Schwann cell) Synapse Axon Dendrite Nucleus Cell Body A Small Dose of Toxicology - Overview

Neurotransmission Dopamine Transmitter Cell Dopamine Receptor Cell (Excitatory Neuron) Synaptic Vesicles Synaptic Cleft Dopamine Receptor Cell (Post-synaptic receptor) Dopamine Receptor

Neuronal Transmission April 21, 2017 +40 -40 -70 EPSP Action Potential Excitatory Synapse + + - + + - - + - - - - - - - + + + + + + - K+ Na+ Cl- Inhibitory Synapse +40 -40 -70 No Action Potential Action Potential IPSP A Small Dose of Toxicology - Overview

Exposure Issues Inhalation (e.g. solvents, nicotine) April 21, 2017 Exposure Issues Inhalation (e.g. solvents, nicotine) Ingestions (e.g. lead, alcohol) Skin (e.g. pesticides, nicotine) Physical (e.g. load noise) A Small Dose of Toxicology - Overview

What causes neurotoxicity? April 21, 2017 What causes neurotoxicity? Wide ranged of agents – chemical and physical A Small Dose of Toxicology - Overview

Types Of Neurotoxicity April 21, 2017 Types Of Neurotoxicity Neuronopathy Cell Death. Irreversible – cells not replaced. MPTP, Trimethytin Axonopathy Degeneration of axon. Reversible. Hexane, Acrylamide Myelinopathy Damage to myelin (e.g. Schwann cells) Lead, Hexachlorophene Transmission Toxicity Disruption of neurotransmission Organophosphate pesticides, Cocaine, DDT A Small Dose of Toxicology - Overview

Neurotoxic Injury Neuron Myelin Axon Synapse Normal Axonopathy April 21, 2017 Neurotoxic Injury Normal Axonopathy Transmission Neuronopathy Myelinopathy Neuron Myelin Axon Synapse A Small Dose of Toxicology - Overview

Examples of Neurotoxicology Diseases Parkinson's, Alzheimer's, MS, ALS.. Environmental Lead, Methylmercury, PCBs Occupational Solvents, Pesticides Drugs - Clinical Vincristine, cisplatin Drugs - Social Alcohol, cocaine, nicotine

Neurotoxic Effects Cognitive Effects Motor Effects Sensory Effects - memory, learning, confusion Motor Effects - weakness, convulsion, paralysis Sensory Effects - vision, auditory, touch, balance Mood and Personality Effects - sleep, depression, irritability, excitability General Effects - loss of appetite, fatigue

Classification of Neurotoxicant Mechanism of action Temporary inhibition of nerve function Agents which alter membrane function Agents with interfere with synaptic transmission

Classification of Neurotoxicant Mechanism of action Permanent inhibition of nerve function Agents which cause Anoxia Anoxic anoxia (e.g. CO2 asphyxiation) Ischemic anoxia (e.g. blood clot) Cytotoxic anoxia (e.g. cyanide) Agents which damage myelin formation Oligodendroglia (CNS) Schwann cells (PNS) Agents which damage peripheral axons Agents which damage nerve cell body Agents which cause localized CNS lesions

Functional Observational Battery (FOB) 1. Autonomic function (lacrimation and salivation, piloerection and exophthalmus, urination and defecation, pupillary function, palpebral closure) 2. Convulsions, tremors, or abnormal motor movements 3. Reactivity to general stimuli such as removal from the cage or handling (no reaction to hyperreactivity) 4. Arousal level (from coma to hyperalertness) 5. Posture and gait abnormalities (home cage and open field) 6. Forelimb and hindlimb grip strength 7. Landing foot splay 8. Sensorimotor responses to stimuli (a tail-pinch, tailflick, hot-plate, acoustic startle response ) 9. Body weight 10. Any unusual or abnormal behaviors, excessive or repetitive actions (stereotypies), emaciation, dehydration, hypotonia or hypertonia, altered fur appearance, red or crusty deposits around the eyes, nose, or mouth, and any other observations that may facilitate interpretation of the data.

Functional Observational Battery (FOB) 1. Count of rearing activity on the open field. 2. Ranking of righting ability. 3. Body temperature. 4. Excessive or spontaneous vocalizations. 5. Alterations in rate and ease of respiration, e.g., rales or dyspnea. 6. Sensorimotor responses to visual or proprioceptive stimuli.

Other measures Memory and learning tests (e.g. radial maze, Morris water maze) Conditioned stimulus/responses Other supportive techniques: Histopathology/Quantitative Stereology EEG, EMG, Functional MRI: CBV-fMRI of rat brain induced by electrical stimulation of both forepaws. T. Reese, A. Sauter, N. Beckmann, M. Rudin et al. Novartis Pharmaceutical, Basel, Switzerland http://www.bruker-biospin.de/MRI/applications/bio29.html

WHAT IS THIS? O CH3 CH3 N N N O N CH3 7 1 3 April 21, 2017 A Small Dose of Toxicology - Overview

Physiological Sensitivity April 21, 2017 Physiological Sensitivity Dependence on oxygen Little anaerobic capacity CO – less available oxygen Cyanide – inability to use oxygen Dependence on glucose Sole energy source High metabolic rate A Small Dose of Toxicology - Overview

Interference with Energy Production Intermembrane Space H+ H+ H+ +++ +++++++ +++++++++++++ +++ +++ I III IV V - - - - - - - - - - - - - - - - - - - - - - - - - - - - - O2 + 4H+ 2H2O NADH + H+ NAD+ 3 ATP Matrix H+

Energy Production Inhibitors Carbon monoxide, nitrites Affect oxygen delivery to tissues Nitrophenols, organotins, mercurials Uncouple oxidative phosphorylation Rotenone, antimycin A, cocaine Inhibit electron transport

Binding to macromolecules Interference with enzyme function Methotrexate (DHFR inhibitor) Hydroxyurea (Ribonucleotide reductase) Formation of lipid hydroperoxides, free radical generation Carbon tetrachloride, paraquat, ozone Nucleic acids (DNA, RNA) Cyclophsophamide, MNU, MMS

Binding to macromolecules Oxidative stress Depletion of GSH (DEM, BSO, metals) Oxidation of protein thiols (metals) S Hg Hg S Active Inactive

Physiological Sensitivity April 21, 2017 Physiological Sensitivity Structure Long cell requires extensive intracellular transport Blood-Brain Barrier Developmental stage (lead and alcohol) A Small Dose of Toxicology - Overview

Reversibility of Damage April 21, 2017 Reversibility of Damage Neurons CANNOT divide and replace themselves Neurons CAN repair limited axonal damage Most Recovery Redundancy of Function Plasticity of Organization A Small Dose of Toxicology - Overview

Classification of neurotoxicants by mechanism of action Permanent inhibition of nerve function Agents which cause Anoxia Anoxic anoxia Ischemic anoxia Cytotoxic anoxia Agents which damage myelin formation Oligodendroglia (CNS) Schwann cells (PNS) Agents which damage peripheral axons Agents which damage nerve cell body Agents which cause localized CNS lesions

Neurological and Behavioral Effects of Exposure to Toxic Substances Motor Effects - Convulsions, weakness, tremor, twitching, lack of coordination, unsteadiness, paralysis, reflex abnormalities, activity changes Sensory Effects - Equilibrium changes, vision disorders, pain disorders, tactile disorders, auditory disorders Cognitive Effects - Memory problems, confusion, speech impairment, learning impairment Mood and personality effects - Sleep disturbances, excitability, depression, irritability, restlessness, nervousness, tension, delirium, hallucinations General effects - Loss of appetite, depression of neuronal activity, narcosis stupor, fatigue, nerve damage

Normal Receptor-Ligand Interaction 1 Ligand Outside Cell Receptor 2 Cell Membrane Inside Cell Ligand binds to receptor 3 Signal Protein Positive Response

Inactivation of Receptor by Toxicant 1 Toxicant 2 Toxicant inactivates receptor 3 No Response

Competition For Receptor Ligand 1 Toxicant 2 Toxicant out competes normal ligand Ligand cannot bind receptor 3 No Response

Who Is Vulnerable? Young or Old Male or Female April 21, 2017 Who Is Vulnerable? Young or Old Male or Female Genetics - Individual Diff. Species A Small Dose of Toxicology - Overview

Vulnerability / Sensitivity April 21, 2017 Fetal Nervous System Developing Nervous System Mature Nervous System Aging Nervous System A Small Dose of Toxicology - Overview

A Small Dose of ™ Neurotoxicity April 21, 2017 A Small Dose of ™ Neurotoxicity A Small Dose of Toxicology - Overview

Additional Information April 21, 2017 Additional Information Web Sites U.S. National Institute of Health - National Institute of Neurological Disorders and Stroke (NINDS). Online. Available HTTP: <http://www.ninds.nih.gov/> (accessed: 10 April 2003). Natural Resources Defense Council (NRDC). Online. Available HTTP: <http://www.nrdc.org/health/kids/cfqpa0599.asp> (accessed: 10 April 2003). NRDC site provides information on children’s health and neurotoxicology. Other Chapters Mercury, lead, pesticides A Small Dose of Toxicology - Overview

Authorship Information April 21, 2017 Authorship Information This presentation is supplement to “A Small Dose of Toxicology” For Additional Information Contact Steven G. Gilbert, PhD, DABT E-mail: smdose@asmalldoseof.org Web: www.asmalldoseof.org A Small Dose of Toxicology - Overview