michael j. hooper and andrea kirk- neurotoxicology ii

8/3/2019 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’



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



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



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.



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



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



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.



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

michael j. hooper and andrea kirk- neurotoxicology ii

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