Michael J. Hooper and Andrea Kirk- Neurotoxicology II
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Principles of Toxicology
Neurotoxicology IIII
Michael J. Hooper
and
Andrea Kirk
The Environmental Toxicology Department
Texas Tech University
Lubbock, Texas
Axonal Transport
The axon contains a large proportion of the volume and
surface area of a neuron.
Axon and axon terminus do not have advanced cellular
capacities such as organelle or protein synthesis /
degradation.
Essential materials are produced, packaged and sent out
into the axon from the cell body.
Materials travel along 5 different transport matrices
Nissl substance: Arrays of rough endoplasmic reticulum,
associated ribosomes and free ribosomes responsible for the substantial protein synthesis needs of neurons
Internal Structural Components of an Axon
Neurofilaments – Rope-like fibrils 8-12 nm in diameter, 10-100 um
long, that make up the bulk of the axon’s structure.
Microtubules – Tubular shaped 24 nm in diameter, and 100um in
length. Made up of α and β tubulins – Important structurally and for
transport
Microfilaments – actin
subunits intertwined toform 4-6 nm fibrils.
Important in membrane
and cellular cytoskeletons.
Neurofilaments
Microtubules
Microfilaments
Types and Characteristics of Axonal Transport
Fast Axonal Transport - via microtubules – (speeds in mm/d)
Anterograde 250-400 Vesicle bound proteins, tubulovesicular
structures, membrane-associated
enzymes, neurotransmitters,
neuropeptides and membrane lipids
Retrograde 200-300 Larger organelles carrying lysosome-
bound materials as well as endogenous
factors collected through endocytosis
Mitochondria 50-100
Slow Axonal Transport – via neurofilaments – “slow components – SC”
SCb 2-8 Microfilaments / actin , tubulin,
metabolic enzymes,
SCa 0.2-1 Microfilaments, tubulin,
‘Kinesin’
‘Dynein’
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Kinesin and Dynein
Molecular motors that
transport materials down and
up the axon, respectively
Require ATP to fuel their actions
Myosins may have a role in slow
axonal transport, associated with
microfilaments.Microtubules
Organelle/
Vesicle
100 nm
Red arrows: connections between
vesicle and microtubule
Wallerian Degeneration of Axons
Axotomy: severing
of the axon from the
cell body
Degeneration of the
distal axonsegments, removal
by macrophages,
target cell/organ
effects
Chromatolysis – Response of cell body to axotomy. Dissolution of Nissl
substance, dendrite “disconnection” and withdrawal, active nucleolus
production of RNA and increased ribosome production
Macrophages stimulate Schwann cells to replicate (via interleukin 1). NewSchwann cells express NGF / NGF receptors, stimulating axonal growth.
Remain in place to guide new axonal growth.
Axon can regrow and innervate the target tissue as long as cell survives
Functional Manifestations of Neurotoxicity
Observations of motor and behavioral skills can be used to diagnose
the occurrence of neurotoxicity.
Functional Observation Battery (FOB)
Motor Skills test
Histopathology
USEPA Health Effects Test Guidelines, OPPTS 870.6200,
Neurotoxicity Screening Battery
http://www.epa.gov/opptsfrs/OPPTS_Harmonized/870_Health_Effects_Test_Guidelines/Series/
Thorough observations of general appearance, behavior and
functional integrity
In home cage and in open field
Through Manipulative tests
Functional Observation Battery Endpoints
List of measures.
(1) Assessment of signs of autonomic function
(i) lacrimation and salivation
(ii) piloerection and exophthalmus.
(iii) urination and defecation, including polyuria and diarrhea.
(iv) Pupillary function
(v) Degree of palpebral closure, e.g., ptosis.
(2) Convulsions, tremors, abnormal motor movements
(3) Reactivity to general stimuli such as removal from the cage or
handling,
(4) Level of activity during observations
(5) Posture and gait abnormalities
(6) Ranking of gait abnormalities
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FOB Endpoints II
(7) Forelimb and hindlimb grip strength
(8) Quantitative measure of landing foot splay;
(9) Sensorimotor responses to different stimuli, Pain perception (tail-
pinch, tail-flick, or hot-plate) Sudden sound,(10) Body weight.
(11) 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.
(12) Additional measures. Such as:
(a) Count of rearing activity on the open field.
(b) Ranking of righting ability.
(c) Body temperature.(d) Excessive or spontaneous vocalizations.
(e) Alterations in rate and ease of respiration, e.g., rales or
dyspnea.
(f) Sensorimotor responses to visual or proprioceptive stimuli.
Manifestations of Toxic Effect
Where and How Do Toxic Effects Occur?
Neuropathies
Neuronopathy
Axonopathy
Myelinopothy
Neurotransmission-Associated
Neuronopathies
Neuronal death is irreversible, includes degradation of all
cytoplasmic extensions (axons and dendrites) and associated
myelin, whether by necrosis or induced apoptosis.
High metabolic rate, extensions of axons and dendrites, and
excitable membrane all make neurons susceptable.
Death often due to effects on sensitive characteristics.
Generally diffuse in toxic action but examples of extreme
specificity occur.
Chemicals Toxic to the Neuron
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Methyl Mercury, CH3Hg+
Entrance into brain via binding to cysteine and mimicking methionine
Major Incidents:
Minamata Bay, Japan (1950s-1960s) – Hg+2 used as catalyst in
acetaldehyde and vinyl chloride manufacture. Waste
transformed to MeHg by bacteria in sediments of bay.
Exposure via fish from bay
Iraq (1972) – Consumption of MeHg treated seed grainhttp://www.project-syndicate.org/commentaries/commentary_text.php4?id=1323&lang=1&m=contributor
In adults – damage to the visual cortex and granular cells of the
cerebellum. Leads to blindness and ataxia
In Children – widespread neuronal death, leading to mental retardation
and paralysis. Particularly bad with in utero exposure
Mechanism unclear – Many effects including inhibition of: protein
and nucleic acid synthesis inhibition, glycolysis and
respiration. Increases oxidative damage, alters Ca+2
homeostasis. Likely combination of effects…
Minamata Disease -
Distribution of Lesions in
Adult, Infantile and
Fetal BrainsAdult
Non-Fetal
Infantile
Fetal
Cerebellum
Visual Cortex
Does Hg+2 cause neuron death
due to sulfhydral binding?
The answer is still not clear…
MPTP
1-Methyl-4-Phenyl-1,2,3,6-TetrahydroPyridine
Unintended contaminant in the production of meperidine, a
synthetic heroin analogue
Led to Parkinson’s disease-like symptoms in those that used it
masked faces tremors rigidity
difficulty in initiating and terminating movement
Affected the substantia nigra
Meperidine Synthesis and MPTP Contamination
MPTP
Contaminant
Meperidine (Demerol) is a synthetic
morphine analog that was
produced (illegally) as a heroin
alternative and sold to heroin
users.
MPTP was a contaminant in the
production process.
MPTP is metabolized by
monamine oxidase B to MPP+,
which mimics dopamine and is
taken up into substantia nigra
Meperidine
Doesn’t cross
BBB
Lipophilic –crosses BBB
Substantia
Nigra
AstrocyteNeurotoxicity due to inhibition of
complex I of oxid. phosphoryl.
and also, perhaps, oxidative
damage.
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Do Graded Exposures to Neurotoxicants
Lead to Graded Levels of Effect in Parkinson’s Disease?
MPTP exposures did not always lead to frank symptoms.
Those without symptoms developed early-onset Parkinsonism.
Suggestive that there is an accumulated decrement that does
not display symptoms until ~80% of S.N. is affected.
Herbicide, pesticide and metal exposures have been implicated
as life-time risk factors in Parkinson’s disease.
Local concentration of early-onset Parkinson’s disease being
investigated at TTUHSC – Perhaps agriculturally associated.
Axonopathies
Due to chemical transection of the axon and separationfrom the cell body
Repairable in the periphery, but not in the CNS where
inhibitory factors from myelin cells and astrocytesprevent axonal regrowth.
Long sensory and motor neurons and longer spinal cord
tracts are most sensitive.
Often due to disruption of axonal transport processes
Axonopathic Chemicals Hexane and 2,5-Hexanedione
CH3
CH2
CH2
CH2
CH2
CH3
CH3
C CH2
CH2
C CH3
O O
Peripheral neuropathy occurs with daily occupational
n-hexane exposure or from repeated intentional
inhalation of hexane-containing glues
The diketone is the ultimate toxic moiety. It is highly reactive
with amines in all tissues, forming pyrrols.
Stability of neurofilaments makes them a preferred target
Neurofilament aggregates form in distal axon, proximal to
nodes of Ranvier. Occur proximally with continued
exposure.
Leads to degeneration of axon and myelin cells
Presents clinically first as stockings-and-gloves sensory loss,
progressing to more proximal sensory and motor loss
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Pyrrol Formation and Crosslinking
Carbonyl Cs
are
electrophilic
S=C=S
They attack
e- rich amines
Subsequent
oxidation and
electrophilic
attack lead to
cross-linking
Similar
mechanism
with carbon
disulfide
The carbon
atom is the
electrophile in
both linking
steps
CS2
pathology issimilar to
that of the
hexanes
Axonal Degeneration
Wallerian degeneration with
myelin ovoids
N-Hexane intoxication with
swelling and accumulation of mitochondria and vesicles
Normal axon
Acrylamide
Monomer of material that forms polyacrylamide gels, also
used as a soil stabilizer, waterproofer and in paper mfg.
Causes a dying-back neuropathy starting at the synapse
Affects fast axonal transport, distal buildup of mitos and vesicles
Recent studies have shown it to be present in
carbohydrates cooked at high temperatures
Microtubule-Associated Neurotoxicity
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Organophosphate-Induced Delayed Neuropathy
A variety of OPs cause a progressive
neuropathy that is delayed in onset from 7
to 10 days
Associated with the inhibition of “Neuropathy
Target Esterase” activity, rather than
acetylcholinesterase
Repairable in the periphery, but not in the long
central tracts.
Initial human occurrence in 1930 – adulterated
ginger extract drunk for its 70% alcohol content
(during prohibition)
Contained tri-ortho-cresyl phosphate
Poisoned ~50,000, some recovered
Generally resulted in a high-stepping, foot-
slapping walk known as the Jake Walk
Jake Leg Blueshttp://www.ibiblio.org/moonshine/drink/jake.html
Tri-o-cresyl Phosphate
Myelinopathies
In absence of healthy myelin, neuronal transmission
slows or stops
Separation of the myelin lamellae – intramyelinic edema –
can progress to demylenation
Remyelination limited in the CNS but occurs in the PNS
Hexachlorophene
Anti-bacterial used on newborns and premature infants,
leading to neruotoxicity
Lipophilic, absorbed through skin and on to nervous system
Uncouples ox-phos and leads to intramyelinic edema, vacuole
formation and “spongiosis” of the brain
High acute dose causes axonal degeneration and loss of
photoreceptors in the retina
Generalized weakness, confusion and seizures
can progress to coma and death
Lead and Tellurium
Both cause a prominent demyelination in the periphery.
Tellurium causes a shift in the profile of lipids formed, with
decreases in critical myelin components
Lead affects myelin membrane structure and fluidity – leads
to encephalopathy in children exposed to high doses
and peripheral myelonopathy/neuropathy in adults
A spectrum of effects is seen in lead-exposed children, from
the encephalopathy at high doses to incremental
decreases in IQ for moderate and low doses.
More on this in the metals section.
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Neurotransmission-Associated Neuropathy
Function and performance affected by toxicants
NT synthesis, storage, release
NT breakdown / recycling
NT receptor function: Agonists
antagonists, inhibitors
Ion channel blockers,
modifiers, openers