Principles of Toxicology

Neurotoxicology II
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. Nissl substance: Arrays of rough endoplasmic reticulum, associated ribosomes and free ribosomes responsible for the substantial protein synthesis needs of neurons Materials travel along 5 different transport matrices

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 axons structure. Microtubules Tubular shaped 24 nm in diameter, and 100um in length. Made up of and tubulins Important structurally and for transport Neurofilaments

Types and Characteristics of Axonal Transport

Fast Axonal Transport - via microtubules (speeds in mm/d) Anterograde Kinesin Mitochondria 50-100 200-300 Larger organelles carrying lysosomebound materials as well as endogenous factors collected through endocytosis 250-400 Vesicle bound proteins, tubulovesicular structures, membrane-associated enzymes, neurotransmitters, neuropeptides and membrane lipids

Microtubules Microfilaments Microfilaments actin subunits intertwined to form 4-6 nm fibrils. Important in membrane and cellular cytoskeletons.

Retrograde Dynein

Slow Axonal Transport via neurofilaments slow components SC SCb SCa 2-8 0.2-1 Microfilaments / actin , tubulin, metabolic enzymes, Microfilaments, tubulin,

Kinesin and Dynein

Molecular motors that transport materials down and up the axon, respectively Require ATP to fuel their actions
Red arrows: connections between vesicle and microtubule
Organelle/ Vesicle

Wallerian Degeneration of Axons

Axotomy: severing of the axon from the cell body Degeneration of the distal axon segments, 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). New Schwann cells express NGF / NGF receptors, stimulating axonal growth. Remain in place to guide new axonal growth.
Microtubules
100 nm

Myosins may have a role in slow axonal transport, associated with microfilaments.

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. USEPA Health Effects Test Guidelines, OPPTS 870.6200, Neurotoxicity Screening Battery
http://www.epa.gov/opptsfrs/OPPTS_Harmonized/870_Health_Effects_Test_Guidelines/Series/

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

Functional Observation Battery (FOB) Thorough observations of general appearance, behavior and functional integrity In home cage and in open field Through Manipulative tests Motor Skills test Histopathology

FOB Endpoints II
(7) Forelimb and hindlimb grip strength (8) Quantitative measure of landing foot splay; (9) Sensorimotor responses to different stimuli, Pain perception (tailpinch, 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

Chemicals Toxic to the Neuron Neuronopathies

High metabolic rate, extensions of axons and dendrites, and excitable membrane all make neurons susceptable. Death often due to effects on sensitive characteristics. Neuronal death is irreversible, includes degradation of all cytoplasmic extensions (axons and dendrites) and associated myelin, whether by necrosis or induced apoptosis. Generally diffuse in toxic action but examples of extreme specificity occur.

Methyl Mercury, CH3Hg+

Entrance into brain via binding to cysteine and mimicking methionine 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 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 grain
http://www.project-syndicate.org/commentaries/commentary_text.php4?id=1323&lang=1&m=contributor

Minamata Disease Distribution of Lesions in Adult, Infantile and Fetal Brains

Visual Cortex

Adult

Cerebellum
Non-Fetal Infantile

Does Hg+2 cause neuron death due to sulfhydral binding?

Fetal

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 Parkinsons 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

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 Contaminant Lipophilic crosses BBB Doesnt cross BBB

Meperidine

MPTP is metabolized by monamine oxidase B to MPP+, which mimics dopamine and is taken up into substantia nigra Neurotoxicity due to inhibition of complex I of oxid. phosphoryl. and also, perhaps, oxidative damage.
Astrocyte Substantia Nigra

Do Graded Exposures to Neurotoxicants Lead to Graded Levels of Effect in Parkinsons Disease? Axonopathies
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 Parkinsons disease. Local concentration of early-onset Parkinsons disease being investigated at TTUHSC Perhaps agriculturally associated. Due to chemical transection of the axon and separation from the cell body Often due to disruption of axonal transport processes Long sensory and motor neurons and longer spinal cord tracts are most sensitive. Repairable in the periphery, but not in the CNS where inhibitory factors from myelin cells and astrocytes prevent axonal regrowth.

Axonopathic Chemicals

Hexane and 2,5-Hexanedione

O
CH3 CH2 CH2 CH2 CH2 CH3

CH3 C CH2 CH2 C CH3

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 They attack e- rich amines Similar mechanism with carbon disulfide The carbon atom is the electrophile in both linking steps CS2 pathology is similar to that of the hexanes

Axonal Degeneration
Normal axon

S=C=S

Wallerian degeneration with myelin ovoids

Subsequent oxidation and electrophilic attack lead to cross-linking

N-Hexane intoxication with swelling and accumulation of mitochondria and vesicles

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
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

Poisoned ~50,000, some recovered Generally resulted in a high-stepping, footslapping walk known as the Jake Walk Jake Leg Blues
http://www.ibiblio.org/moonshine/drink/jake.html

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 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.

Lead and Tellurium

Both cause a prominent demyelination in the periphery.

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