Toxicology of the Peripheral Nervous System

• Systems Toxicology: Focus on the toxicology of the Peripheral Nervous System (PNS)

Learning Objectives

• Review the anatomy, physiology, and biochemistry of the nervous system.
• Recognize toxicants that affect the PNS.
• Obtain knowledge of Absorption, Distribution, Metabolism, and Excretion (ADME) and mechanisms of action of PNS toxicants.
• Describe clinical signs, diagnosis, and treatment of specific PNS toxicants.

Nervous System Overview

• The central nervous system (CNS) consists of the brain and spinal cord and receives input from the periphery.
• The peripheral nervous system (PNS) has afferent and efferent divisions.
• The afferent division carries sensory and visceral stimuli, and the efferent division is further divided into somatic and autonomic nervous systems.
• The somatic nervous system controls motor neurons and skeletal muscles.
• The autonomic nervous system (ANS) includes the sympathetic and parasympathetic systems.
• The sympathetic and parasympathetic systems impact smooth muscle, cardiac muscle, glands, and effector organs.

PNS Overview

• The parasympathetic nervous system uses acetylcholine (ACh) at both pre- and post-ganglionic neurons, acting on muscarinic (M) and nicotinic (N) receptors.
• The sympathetic nervous system uses ACh at preganglionic neurons, acting on nicotinic (N) receptors, and norepinephrine (NE) at postganglionic neurons, acting on adrenergic receptors (α, β).
• Equine sweat glands are predominantly under beta-adrenergic control.

Autonomic Nervous System (ANS)

• The Autonomic Nervous System (ANS) affects cardiac and smooth muscles, gland cells, and nerve terminals.

General Mechanisms of Toxicity of ANS Toxicants/Toxins

• Direct toxicity: Impairs neurotransmission, potentially causing structural damage such as neuronopathy, axonopathy, or myelinopathy.
• Indirect toxicity: Alters metabolism or blood supply, leading to secondary neuronal dysfunction, which may involve hypoxia, hypoglycemia, or disruption of ion homeostasis.

Direct Neurotoxic Injury

• Normal Neuron: Contains cell bodies and dendrites, myelinating cells encircling the axon, and synapses for neurotransmission.
• Neuronopathy: Results from the death of the entire neuron.
• Axonopathy: Injury primarily occurs at the axon.
• Myelinopathy: Disruption of the myelin sheath or selective injury to myelinating cells.
• Impairment of neurotransmission occurs by blocking excitation or causing excessive stimulation.

Key Steps in Neurotransmission

• Neurotransmission involves synthesis of neurotransmitters (NT).
• NT storage occurs in presynaptic cells.
• Calcium-dependent NT release is stimulated.
• Neurotransmitters interact with postsynaptic receptors.
• Neurotransmitter action is terminated by reuptake, metabolism, and diffusion.

Nomenclature of ANS Toxicants

• Cholinergic: Includes cholinomimetic and parasympathomimetic agents.
• Anticholinergic: Includes cholinolytic and parasympatholytic agents.
• Adrenergic: Includes adrenomimetic and sympathomimetic agents.
• Antiadrenergic: Includes adrenolytic and sympatholytic agents.

Locations and Responses of Cholinergic Receptors

• Nicotinic receptors include NN and NM subtypes, which are ligand-gated.
  • NN receptors are located in autonomic ganglia and cause depolarization and firing of post-ganglionic neurons.
  • NN receptors are located in the adrenal medulla and cause secretion of catecholamines, particularly epinephrine (~80%).
  • NM receptors are found at the neuromuscular junction, causing end-plate depolarization and skeletal muscle contraction.
• Muscarinic receptors are G-protein coupled and include M1-M5 subtypes.
  • M3 receptors in the eye cause sphincter muscle contraction, miosis, ciliary muscle contraction, and accommodation (near vision).
  • M1 and M3 receptors in the stomach and instestine increase tone, motility, and secretion.
  • M1 and M3 receptors in the sweat, lacrimal, and salivary glands cause secretions
  • M2 receptors in the heart decrease the SA node rate and AV node conduction velocity.
  • M3 receptors in bronchial muscle cause contraction and secretion.
  • M1, M3, and M5 receptors are excitatory.
• M2 and M4 receptors are inhibitory.

General Clinical Signs of Cholinergic Stimulation

• DUMBBELLS
  • Diarrhea, Dyspnea
  • Urination
  • Miosis, Muscle weakness
  • Bradycardia, Bronchoconstriction/Bronchorrhea
  • Emesis
  • Lacrimation
  • Lethargy
  • Salivation
• Other signs:
  • Anxiety/agitation, Seizures, Coma, Muscle fasciculations, Flaccid paralysis, Excitation, and/or depression

Muscarinic Toxicants (Parasympathomimetics)

• Muscarinic alkaloids: Muscarine, Pilocarpine, Slaframine
• Muscarine choline esters: Acetylcholine, Methacholine, Carbachol, Bethanechol

Slaframine (Slobber Syndrome, Clover Poisoning, Salivary Syndrome)

• Sources: An indolizidine alkaloid mycotoxin produced in red clovers (Trifolium spp.) infected with the fungus Rhizoctonia leguminicola
  • Also associated with other legumes like white clover, soybean, kudzu, cowpea, blue lupine, and alfalfa.
• Risk factors: Encountered mostly in wet weather and high humidity.

Slaframine ADME

• Rapidly absorbed and produces effects.
• It is activated by liver microsomal flavoprotein oxidase to a ketoimine metabolite.
• The ketoimine metabolite is chemically and physiologically similar to acetylcholine and causes toxicosis.
• Species affected: cattle, horse, sheep, goats, swine, chickens.

Slaframine Toxic Dose

• Few studies have been conducted:
  • LD50 = 81.6 mg/kg in day old male broiler chicks
  • 300 mg/kg in cats causes salivation
  • 60 µg/kg reduces feed intake and body weight in cattle
  • 250-300 mg/kg caused death in guinea pigs
• MOT: Parasympathomimetic
  • Cholinergic stimulation of exocrine and endocrine glands
  • High affinity for GI tract muscarinic (M3) receptors

Slaframine Clinical Signs

• Natural cases are reported only in horses and ruminants, lasting several hours to >3 days.
• Excessive salivation (slobbers), lacrimation, anorexia, diarrhea, frequent urination, bloating, stiffness, respiratory distress, bradycardia, and decreased milk production can occur.
• Death is rare

Slaframine Dx

• Clinical signs (↑ salivation) in livestock animals consuming legumes, particularly red clovers
• Detection of plant lesions (black patches)
• The diagnosis involves eliminating exposure to contaminated feed.
• Toxin detection in the plant via chemical analysis
• Guinea pig bioassay of salivary response

Slaframine DDx

• The clinical syndromes that cause salivation include:
  • Vesicular stomatitis
  • Foot and mouth disease
  • Ulcerative stomatitis
  • Mechanical or chemical irritation of the mouth
  • Dental problems, Glossitis, Oral foreign body

Slaframine Tx

• Slaframine toxicosis is not life-threatening and animals usually recover once the forage is removed.
• Thus, treatment is not usually needed.
• Atropine reverses muscarinic signs if given early but is ineffective once salivation is excessive.
• Antihistamines may alleviate some clinical signs.

Anticholinesterase Toxicants

• Organophosphates and Carbamates
• Anatoxin-a(s) [blue green algae]
• Pharmaceuticals: Physostigmine, neostigmine, endrophonium, pyridostigmine

Organophosphates (OPs) and Carbamates (CMs) Sources

• OPs: Pesticides used on yards, gardens, homes, or directly on animals; parasiticides in vet medicine (wormers); chemical weapons of mass destruction (nerve gases: soman, sarin, tabun, VX, VR).
• CMs are derivatives of carbamic acid with similar uses as the OPs.

Organophosphates and Carbamates Structure

• OPs and CMs bear structural similarity to acetylcholine.

Organophosphates and Carbamates examples

• OPs: Chlorpyrifos (Dursban), Coumaphos (CoRal), Diazinon, Fenthion (Pro-Spot), Malathion, Parathion, Disulfoton, Terbufos, Phorate.
• Carbamates: Aldicarb (Temik), Carbaryl (Sevin), Carbofuran (Furadan), Methiocarb, Methomyl, Propoxur (Baygon).

Exposure and Susceptibility to OPs and CMs

• Most exposures are food-related (80% of all food animal exposures).
• Exposure can also come from improper/careless use, dose miscalculation, use on stressed animals, or malicious poisoning.
• All species are susceptible, but cats, fish, and birds are more susceptible.

Toxicity and Risk of OPs and CMs

• Variable from low to highly toxic, with LD50 ≤ 1mg/kg for most toxic ones.
  • Metabolism and formulation affect the probability of poisoning.
• Those that are very sensitive include cats specifically, and bulls are also very susceptible to chlorpyrifos.
• Young animals are poisoned at lower doses.

ADME of OPs and CMs

• Absorbed readily via skin, GI, and respiratory tracts.
• OPs undergo oxidation and hydrolysis by esterases in plasma and liver, followed by conjugation reactions.
• Excreted via urine and feces.
• Many OPs undergo “lethal synthesis” in the liver.

Lethal Synthesis of OPs and CMs

• CYP450 or flavin monooxygenase catalyzes metabolism in the liver.
  • Parathion is converted to paraoxon.
  • Malathion is converted to malaoxon.

Mechanism of Toxicity: Anticholinesterase Action of OPs and CMs

• Acetylcholine (ACh) transfers impulses at cholinergic nerve synapses and neuromuscular junctions.
• ACh is rapidly catabolized by acetylcholinesterase (AChE) and other ChEs to choline and acetic acid.
• Anticholinesterases bind to AChE and other ChEs, impairing ACh catabolism.

Effect of Organophosphates (OPs)

• After OP binding, the enzyme is phosphorylated and unavailable to participate in the catabolism of ACh
• Most OPs bind irreversibly to ChEs
• The OP-ChE bond is enhanced through 'aging' caused by the loss of an alkyl group, such as a methyl, ethyl, or propyl group.

Effect of Carbamates (CMs)

• It is similar to OPs, except that the ChEs undergo carbamylation
• Because carbamates are poor substrates for ChEs
  • They have lower affinity for the enzyme binding site than OPs
  • This results in reversible ChE inhibition because CMs spontaneously dislodge from the enzyme

Interaction of OPs and CMs with AChE (Summary)

• Acetylation: Rapid hydrolysis at 150 μSec
• Carbamylation: t½ = 15-30 min
• Phosphorylation: t½ = days/irreversible
• The AChE hydrolysis rate is ACh > CMs > OPs.

Consequence of ChE Inhibition

• The phosphorylation or carbamylation of AChE ceases its normal function, impairing ACh catabolism.
  • It builds up in nerve synapses and NMJs, continuously stimulating nervous, glandular, GI tract, and muscular cholinergic receptors.

AChE Inhibition = ACh Accumulation

• It results in an increased cholinergic response.

Summary: Organophosphate (OP) and Carbamate (CM) Mechanism of Toxicity

• OPs and CMs inhibit AChE, leading to an accumulation of ACh, which stimulates muscarinic and nicotinic receptors, resulting in a cholinergic crisis.

Clinical Signs: Cholinergic crisis

• Signs of acute poisoning appear within 30 minutes, usually within 6 hours of exposure.
  • Muscarinic, nicotinic, and CNS symptoms depend on individuals
  • Muscarinic signs are usually the first to appear and are preceded by apprehension/uneasiness, with DUMBBELLS being present.

Nicotinic Signs (Neuromuscular)

• Skeletal muscle stimulation
• Twitching of facial muscles, eyelids, tongue, and general musculature.
• Tremors followed by convulsions and seizures.
• Tachycardia and mydriasis
• Weakness, paresis, or paralysis, including respiratory paralysis and potential death.

CNS Signs

• Stimulation, then severe depression of CNS with coma and terminal death
• Anxiety, restlessness, stiffness, and ataxia.
• Food animals often exhibit hyperactivity and rarely seizures.
• Dogs and cats have clonic-tonic seizures and hyperactivity.
• It can centrally mediate respiratory paralysis → death

Other Syndromes of OP Toxicity: Intermediate Syndrome (IMS)

• It appears in dogs/cats 24-96h after ingestion of highly lipophilic OPs.
  • After repetitive exposure to low doses of OPs, or prolonged dermal exposure.
  • The OPs include chlorpyrifos, diazinon, malathion, parathion, phosmet, and bromophos.
• The CM carbofuran can cause IMS.
  • Pathogenesis is unclear, but it is due to decreased AChE and nicotinic ACh receptor mRNA expression.

Intermediate Syndrome Clinical Signs

• Clinical Signs: Anorexia, generalized muscular weakness, paralysis, tremors, seizures, depression, and death.
• There is severe AChE inhibition without muscarinic receptor-associated hypersecretory activity.
• The defect is at the neuromuscular and postsynaptic level.
• Some OPs preferentially distribute to muscles and have a higher affinity for nicotinic ACh receptors.

OP-induced Delayed Polyneuropathy (OPIDP)

• It is caused by OPs, e.g., by TOCP, *EPN, or leptophos.
• Toxicity occurs after 1-4 weeks of exposure to the toxic agent; chickens are the most sensitive animals.
• Characterized by distal degeneration of long and large-diameter motor and sensory axons of both peripheral cord nerves.
  • The degeneration of axons and myelin sheaths is due to the inhibition of neuropathy target esterase (NTE).
  • Signs are Weakness, ataxia, and limb paralysis.

Dx of OPs or CMs

• It is determined by a history of access to or treatment.
• Clinical signs are observed
• Atropine test - no atropinization
• ChE activity in heparinized whole blood is depressed.
  • Measure brain ChE activity in dead animals.
• Chemical residues are tested in stomach contents, vomitus, hair, and bait by GC-MS.

DDx of OPs or CMs

• Tremorgenic mycotoxicosis
• Amitraz toxicosis
• Pyrethrin/pyrethroid toxicosis
• Pancreatitis
• Garbage intoxication
• Blue-green algae toxicosis
• Muscarinic mushrooms
• Cationic surfactants

Tx: Acute Syndrome

• Initiate ASAP and stabilize the patient.
• Decontaminate with emesis for recent oral exposure, administer activated charcoal and a cathartic.
• Lavage the gastric or enterogastric regions for large amounts of OP/CM before emesis has occurred or if emesis is contraindicated.
• For dermal exposure: wash the animal with water and mild hand dishwashing detergent.

Antidotes and Supportive Care of OPs or CMs

• Give Antidotes such as atropine, which blocks muscarinic ACh receptors.
  • Primary goal addresses is bradycardia and secretions.
• Pralidoxime (2-PAM) Reactivates AChE before aging, and is ineffective for CMs.
• Treatment includes diazepam or short-acting barbiturates for convulsions.
• Ensure artificial respiration to mitigate effects of respiratory paralysis.

Tx: Intermediate Syndrome

• Atropine is not indicated (no muscarinic signs).
• Treatment is mainly supportive
  • With parenteral or pharyngostomy feedings
• Correct dehydration and electrolyte imbalance
• Bathe the animal if the exposure is dermal
• surprisingly, Give 2-PAM

Anatoxin-a(s)

• Produced by certain species of blue-green algae (cyanobacteria).
• Water blooms generally occur in stagnant eutrophic ponds, lakes, and ditches.
• Blooms thrive in warm temperatures and calm weather.
• Animals are at risk when blooms are concentrated along the shoreline by wind.
• Susceptible species: include dogs, cattle, swine, and waterfowl.

Algal Bloom

• Indicates rapid increase in algal growth due to high nutrients and concentrations along the shoreline of water body.

Anatoxin-a(s) ADME

• Cyanobacteria is ingested with water
• Cell lysis in acidic stomach causes toxin release
• Toxins absorb in the small intestine causing toxicosis.
• MOT: Anatoxin-a(s) naturally inhibits AChE irreversibly.
• Results in Acute onset, nicotinic signs present in lethal cases.

Anatoxin-a(s) Dx

• Includes known exposure and clinical signs
• Determination of blood AChE activity remains unchanged in the brain
• Perform Algae ID water, and any animal samples
• Detect toxins with HPLC-LCMS in water sources and gastrointestinal contents.

Anatoxin-a(s) Tx

• Administer Atropine as 2-PAM can be ineffective
• Provide symptomatic and supportive care
• Perform an animal decontamination followed by activated charcoal
• Remove animals from contaminated sources and treat the contaminated water with copper sulfate.

Anticholinergic Toxicants

• Cholinergic Blockers: Antimuscarinic, Antiparasympathetic, Cholinolytic, Parasympatholytic

Signs of Parasympathetic Cholinergic Blockade

• Mydriasis
• Tachycardia, Hypertension, Hyposalivation, Thirst/dry mouth, and decreased GI motility.
• Rapid pulse, Constipation, Urinary retention, Hyperthermia, Seizures
• Altered level of consciousness (coma, delirium)

Commonly Described Signs of Parasympathetic Cholinergic Blockade

• Hot as a hare/pistol (hyperthermia)
• Dry as a bone (dry skin)
• Red as a beet (flushed skin)
• Blind as a bat (mydriasis)
• Mad as a hatter (delirium)
• Full as a flask (urine retention)

Anticholinergic Drugs

• Commonly-used includes
  • Atropine
  • Scopolamine
  • ipratropium
  • trimethaphan
• It is due to competitive antagonsim of ACh.
• Autonomic ganglia
• No effect on NMJs

Plants with Anticholinergic Effects

• Many members of the Solanaceae (nightshades/potato) family. These range from 88 genera with well-over 2300 individual species.
• Key is tropical alkaloids
  • Includes: Atropine, hyosyamine, and scopolamine.

Toxicity of Anticholinergic Plants

• The mechanism of Action includes antagonism of ACh at muscarinic receptors. It affects ruminents, horses, cattle, etc.
• General parasympatholytic symptoms include, Thirst, dry skin, paralysis, and a lack of coordination.

Identifying Toxicity of ACH Plants

• presence of plants in animals, pasture or hay or the animals' GI tracts with compatible clinical signs suggests toxicity.

Treat toxicity of ACH by:

• Controlling all clinical signs, e.g. prescribing barbituates or sedatives that control activity.
• Remove animals as it can be source of symptoms.