Neurotoxicology:

Overview:Factors unique to nervous system

Examples of neurotoxicants

TOXC707 Advanced Toxicology (2007)

Cells of the Nervous System

◄ neurons◄ neuroglia (90% of cells)

oligodendrocytes (Schwann cells in PNS) astrocytes microglia gliosis is a marker of gross CNS toxicity

Increased expression of Glial Fibrillary Acidic Protein- (GFAP)

Dopamine: A Brain Neuromodulator

Frontal Cortex

Gyrus Cinguli

Sub. Nigra

Corpus Callosum

Tegmentum

Entorhinal Cortex

Basal Ganglia

Olfactory Tubercle

Nuc. Accumbens

Medial Forebrain Bundle

Hypothalamus

Pituitary

Midbrain}

Types of neuronal connections

Axo-somaticSynapse

Axons

Dendro-dendriticSynapse

Axo-axonalSynapse

Dendrites

Axo-dendriticSynapse

Perikarya

Major Components of Peripheral Nervous System

Peripheral neuron

Three views of myelinating Schwann cell

Nucleus

Compact membrane (Myelin)

Cytoplasmic channel(Schmidt-Lantermann)

Schwann cell cytoplasm

Unwrapped

Longitudinal

Cross-section

Processes of demyelination and remyelination

Susceptibility to neurotoxicants

◄ High metabolic rate and electrical excitability are dependent on membrane integrity and aerobic metabolism

◄ Extended length of axons poses logistical problems associated with transport from cell bodies to terminal fields

◄ Metabolism of some neurotransmitters may produce oxidative stress (e.g., dopamine)

◄ Inability to replace dead or dying cells

Dr. Mailman’s Pet Peeves

◄ Neurotoxin A toxic compound of natural origin

◄ Neurotoxicant A toxic compound

◄ Putative “Generally regarded as such; supposed” (American

Heritage) “Generally thought to be or to exist, whether or not this is

really true” (Cambridge) Does not mean hypothesized or speculated.

Consequences of neuronal characteristics

◄ axonal transport sensitive to toxicants◄ hexanes cause cross-linking of neurofilaments◄ diabetic neuropathy

Neurodevelopmental Toxicology

Unique aspects of the nervous system for neurotoxicology: Neurodevelopment

◄ Massive loss of neurons during vertebrate development has been known for more than a century. Beard (1889) – loss of neuronal populations in fish

(Rohon-Beard Neurons) Collin (1906) – death of many sensory and motor neurons

in the chick embryo

Clarke, Rogers & Cowan J. Comp. Neurol. 167: 125 (1976)

~50% of Post-mitotic neurons die during normal development

Apoptotic neuronal death in the developing substantia nigra

R. Burke. Cell Tiss. Res. 2004

Victor Hamburger: Peripheral Targets Regulate Cell Death

led to NGF discovery

Transcriptional regulation of apoptotic cell death

Summary

◄ There is massive death of neurons, neuroprogenitors, and oligodendroglia in normal vertebrate development.

◄ This is largely regulated by access to limiting supplies of exogenous survival-promoting trophic factors.

◄ Survival is promoted largely by activation of Akt as well as Erks, and involves blockade of death pathways at multiple points.

◄ Developmental neuron death is transcription dependent. ◄ Induction of death involves multiple pro-apoptotic signaling

pathways, some of which converge on induction of BH3-domain proteins.

Impact of neurodevelopment on toxicology

◄ The effects of toxicological insults may be temporally delayed, being expressed as a variety of alterations in development.

◄ The effects of toxicant exposure will be markedly affected not only by dose/concentration, but also by timing.

◄ Insults by the same dose/concentration at different times during development may result in markedly different sequelae.

◄ Extrapolation from animal models present an even greater challenge than usual because of species differences in developmental patterns.

◄ This will be discussed later re. Fetal Alcohol Syndrome and solvents.

Toxicant Access and Metabolism

Unique aspects of the nervous system for neurotoxicology: Blood-brain barrier

◄ The choroid plexus separates the blood from the cerebrospinal fluid, whereas the blood-brain barrier limits the influx of circulating substances into the immediate brain interstitial space.

◄ Blood brain barrier limits influx of circulating substances from capillaries into interstitial space

◄ Brain capillaries, unlike those in other tissues, are not fundamentally porous. Tight junctions between adjacent capillary endothelial cells Processes from adjacent cells (astrocytic end feet). A microperoxidase (molecular mass 1800 daltons) that is readily transverses

capillaries in other tissues will not pass through capillaries in the brain. ◄ Carrier-mediated transport systems exist for entry of certain required

molecules (e.g., hexoses, carboxylic acids, amino acids (separate ones for neutral, basic, and acidic amino acids), amines, and inorganic ions

Breaching the barrier

◄ Generalizations for healthy brain Large molecules (large peptides and proteins) are excluded Polar molecules are excluded; nonpolar lipid-soluble molecules can

penetrate more easily e.g., increased absorption of dimethyl mercury vs. inorganic mercury

(Minamata disease) e.g., MPP+ (toxic metabolite of MPTP) does not cross the BBB

Specific transport systems may facilitate toxicant passage e.g., elemental mercury forms complex with cysteine and is recognized by

amino acid transporters as methionine

◄ Alterations in BBB substances that alter membrane function (organic solvents) brain edema bacterial meningitis

Unique aspects of the nervous system for neurotoxicology: Toxicant metabolism

◄ Although some xenobiotic metabolic capacity exists in brain, the relative concentration is low compared to the liver or other tissues.

◄ Detoxification mechanisms in CNS have much lower capacity and diversity than in periphery.

◄ Can be important for specific toxicants. 2,4,5‑trihydroxyphenylalanine is activated MPTP is activated

Unique aspects of the nervous system for neurotoxicology: Plasticity

◄ The nervous system has a unique capacity to accommodate to change.

◄ These changes may sometimes mask, or even be caused by, neurotoxic insult.

◄ Interesting phenomena include: Desensitization Sensitization Up- and down-regulation Long-term potentiation and other types of synaptic plasticity Sprouting

Neurotransmission

Neurotransmission

◄ Relies on separation of positive and negative charges across membrane

◄ Ionic gradient depends on ATP-linked Na+/K+ pump at rest, interior more negatively charged following sufficient stimulus in dendritic region, unidirectional impulse

flow along axon occurs role of ion channels

voltage gated sodium voltage-gated potassium channels

◄ Electrochemical neurotransmission vs. electrical transmission

Synapse

◄ Specialized structure for releasing and sensing small amounts of neurotransmitters

◄ Neurotransmitters vs. neuromodulators◄ Importance to toxicologists

toxicants may act directly at synaptic loci toxicants may indirectly alter synaptic function

Synaptic Structure

1

4

2

Na+

transmitter synthesis Precursor

5

6

3

Second messenger events

8

PresynapticCell

PostsynapticCell

ImpulseFlow

etc.

mRNA

Nucleus

(in perikaryon)

7

adenylate cyclaseG protein

GTPcAMPATP

GDPGTPcAMPATP

GDP

Synaptic targets for toxicants

◄ Neurotransmitter synthesis◄ Neurotransmitter storage◄ Neurotransmitter inactivation or degradation◄ Neurotransmitter receptor binding◄ Receptor-linked second messenger events◄ Pumping or transport of ions◄ Downstream cellular function (e.g., nucleic acid

synthesis)

Mechanisms of toxicity:Receptors

Receptors and Signal Transduction

2 Ion

R R

ligand 1

E2

R E1

ligand

ligand

nucleus

R

R

3 ligand

E

R R

4

P

P

P

P

R R

Toxicants acting directly on receptors

◄ morphine and codeine alkaloids of the opium poppy that causes acute analgesic, antitussive, euphoric,

emetic/antiemetic effects◄ mescaline

derivative of peyote cactus mescaline is believed to cause central actions via interactions with serotonin receptors

◄ ergot alkaloids LSD (ergot-contaminated grain and medieval European cities and the Salem witch

trials??). ◄ methylxanthines

caffeine; theophylline are found in coffee and tea Adenosine receptor ligands plus phosphodiesterase inhibitors

◄ reserpine – VMAT2 ligand blocks vesicular monamine transporter in dopamine and serotonin neurons initial effect is massive release later effect is long term depletion

Cholinergic toxins

◄ Snake neurotoxins -bungarotoxin

blocks nicotinic acetylcholine receptor (binds irreversibly)◄ Belladonna alkaloids

atropine; scopolamine derived from “deadly nightshade” (Belladonna sp.) competitively blocks muscarinic cholinergic receptors also used as antidote for muscarinic agonists/ACh overactivity

◄ nicotine nAChR agonist

◄ Cholinesterase inhibitors solanine and chaconine (Solanum sp., tomoato, potato) Huperzine A physostigmine (eserine) from Calabar bean

Clostridium Indirect Actions

◄ Tetanus Cl. tetani produces 70,000 KDa protein called tetanospasmin Blocks inhibitory synaptic input on spinal motor neurons, resulting in

spastic paralysis. moved through nerve cells via retrograde axonal transport until it

binds, or is fixed, to gangliosides in the brain stem or cord. Ricin also retrogradely transported.

◄ Botulism Cl. botulinum produces a series of neurotoxins Bind to presynaptic cholinergic nerve terminals

◄ Gas gangrene Cl. perfringens

Amino acid receptors

◄ strychnine blocks glycine receptors in spinal cord predominantly effects due to blockade of normal inhibitory influence of

glycine receptor complex

◄ monosodium glutamate (MSG) sodium salt of amino acid glutamate can be actively transported into brain

Ion channel ligands

◄ Alteration in sodium channel activity tetrodotoxin (isolated from puffer fish) and saxitoxin (dinoflaggelate

phytoplankton) binds to voltage-dependent sodium channel and blocks increases in

conductance disrupts generation of action potentials

veratridine steroidal alkaloid (found in Veratrum and Zygadenus species) depolarizes

nerve membranes. grayanotoxins (plant alkaloids from leaves of Ericaceae family)

causes reversible increase in Na+ channel permeability

◄ Ouabain inhibits Na+K+ ATPase by high affinity binding to a site on the

enzyme interferes with maintenance of electrical potential across membrane

Agents that disrupt calcium homeostasis

◄ In addition to effects on Na+ channels, pyrethroid insecticides target Ca2+/Mg2+ ATPase and calmodulin inhibition of these enzymes increases intracellular calcium excessive intracellular calcium is linked to a variety of deleterious

effects

◄ Among other effects, a variety of heavy metals (lead, mercury, aluminum) are associated with increased intracellular calcium actions may derive from competition for binding sites on various types

of calcium binding proteins

Agents that alter intracellular signaling

◄ Mercury has ubiquitous effects e.g., interferes with synthesis of tubulin and other proteins mechanism my be its ability to couple to cysteine and other thiol-

containing groups, promoting binding to many proteins

◄ Aluminum competes with iron for cellular uptake due to similar coordination

chemistry participates in redox cycling and oxygen radical formation promotes aggregation of certain proteins

has been linked to pathogenesis of Alzheimer’s disease (AD) presence of aluminum in neurofibrillary tangles may be a consequence, not

a cause of AD

Agents that cause hypoxia

◄ Any agent that derives CNS of oxygen is neurotoxic◄ Neuronal subpopulations with very metabolic activity are

particularly susceptible (e.g., hippocampus, neocortex)◄ Sequelae of hypoxia are similar to excitotoxicity◄ Anoxic hypoxia (compromised oxygen supply to brain despite

adequate blood flow) carbon monoxide

◄ Ischemic hypoxia (block of blood supply to brain) any agent causing cardiovascular failure (digitalis glycosides)

◄ Cytotoxic hypoxia (interference with cellular respiration) cyanide azide

Agents that affect membranes

◄ Organic and inorganic lead damage membranes probably occurs via disruption of ion channels results in ultrastructural damage to mitochondria,

breakdown of active transport, damage to myelin-containing membranes

◄ Copper can participate in formation of reactive oxygen species and lipid peroxidation

◄ Solvents and vapors are lipid soluble and can alter membrane fluidity

Indirect effects on neurotransmission

◄ activation of neurotransmitter release latrotoxin (black widow venom) releases vesicle-bound

neurotransmitters amphetamine, methamphetamine, ephedrine release

catecholamines methylmercury neurotransmitter release occurs secondary

to altered calcium homeostasis

◄ inhibition of neurotransmitter reuptake or metabolism organophosphates inhibit acetylcholinesterase

Agents that interfere with oxidative phosphorylation

◄ Classical inhibitors of oxidative phosphorylation dinitrophenol cyanide hydrogen sulfide

◄ Lead, mercury and other metals indirectly compromise oxidative phosphorylation by mitochondrial insult

◄ MPTP directly inhibits oxidative phosphorylation

Agents that damage myelin

◄ Some demyelinating agents do not cross BBB and demyelinate only in periphery

◄ Other agents of capable of CNS and PNS effects Hexachlorophene Isoniazid Tellurium Organotins

Protection from oxidative damage

◄ Brain has highest rate of oxidative activity of any organ◄ Endogenous oxygen-derived radicals are thought to be

important in pathogenesis of many neurodegenerative diseases◄ Both neurons and glia contain protective mechanisms; neurons

benefit from secreted enzymes manufactured in glia e.g., glutathione is distributed ubiquitously--chelates transition metals

and prevents redox cycling events glutathione peroxidase and superoxide dismutase are present in

astrocytes catalase is found in oligodendrocytes

◄ The cytochrome P450 isozymes found in brain purported to have a role in Parkinson’s disease

Translational Medicine (Buzzzzzzz):

How can we assess neurotoxicity?

Discussion