The human nervous system is unfathomably sophisticated in how it detects signals from both the external and internal environment, makes sense of this information, and executes an appropriate action to address the stimulus. Sensory nervous tissues transmit millions of signals every minute and the body integrates this information to activate the proper tissue or organs. Due to its unique structure and complexity, the nervous system is especially sensitive to toxic insult. The ability of a substance to negatively impact the structure or function of the nervous system is defined as “neurotoxicity” (Blake, 2010). The class of compounds known as acetylcholinesterase inhibitors can have relevant pharmacological applications in the case of reversible inactivators. However, the mode of action of many other acetylcholinesterase inhibitors is irreversible giving them neurotoxic potential.
In vertebrates, the nervous system can be divided into the central nervous system (CNS) and the peripheral nervous system (PNS). The central nervous system consists of the brain and spinal cord. The peripheral nervous system consists of the nervous tissues outside the brain and spinal cord such as ganglia and peripheral nerves and can be more specifically separated into the somatic nervous system (SNS) and the autonomic nervous system (ANS). The SNS is comprised of afferent nerves that carry sensory information from the skin, eyes, and organs to the CNS and efferent nerves that transmit a signal stemming from the CNS to activate skeletal muscle and cause a voluntary motor response. The autonomic nervous system is more of an involuntary system that innervates organs and glands not under direct control, such as the heart, GI tract, and adrenal glands. The ANS can be subdivided into sympathetic and parasympathetic nerves. The sympathetic subdivision governs roles involved in expending energy such as the “fight or flight” response while the parasympathetic subdivision is involved in conserving energy during “rest and digest” functions (Hall, 2016).
The general design of the nervous system centers on the basic functional unit of the nervous system: the neuron. Incoming signals are received at synapses on the dendrites and outgoing signals travel away from the cell body, or soma, down a single axon and branch out where they synapse with the dendrites of another neuron, at neuromuscular junctions, or organs. A typical neuron has a segmented myelin sheath around the axon, protecting and insulating the axon. The microstructure of the nervous system also encompasses glial cells, which include astrocytes, microglial cells, oligodendrocytes, and Schwann cells. Astrocytes provide metabolic and protective support. Microglia such as macrophages are the immune cells of the nervous system. Oligodendrocytes and Schwann cells are the myelinating cells of the CNS and PNS respectively (Blake, 2016).
Agents can interfere with the nervous system in a number of ways targeting specific structural components of neurons such as myelin or a specific segment of the axon while other effects can cause total neuronal cell death. There are two kinds of demyelinating neurotoxicants; those that are directly detrimental to the myelin-producing cells oligodendrocyrtes and Schwann cells and those that damage the myelin sheath without affecting the myelinating cells. For example, diphtheria toxin can cause demyelination by impairing Schwann cells while hexachlorophene damages myelin membranes. Toxic insult to glial cells can indirectly affect neurons by disturbing the balance glial cells help maintain within the nervous system. Other neurotoxicants do not cause structural damage to tissue itself but rather interfere with ion channels or neurotransmitter release at the synapses thereby interrupting action potential propagation.
Some neurotoxicants alter synaptic processes by disrupting various receptors within the nervous system either by activating or inhibiting their function, or by modifying the extent to which neurotransmitter is made available to such receptors. Anything that interrupts the balance between excitation and inhibition can be toxic to the nervous system (Blake, 2016). Whether by targeting the initial presynaptic synthesis of neurotransmitters, their postsynaptic receptors, or the enzyme that breaks down acetylcholine, neurotransmitter release and availability within the nervous system is susceptible to many agents. Several pesticide compounds have the ability to alter the availability of the neurotransmitter acetylcholine within the synaptic cleft by inhibiting the enzyme acetylcholinesterase, exemplifying this type of neurotoxicity.
The stimulatory neurotransmitter acetylcholine is produced in the presynaptic terminal from choline and acetyl CoA and packaged into synaptic vesicles. These synaptic vesicles fuse with the outer membrane of the axon terminal where it releases acetylcholine into the synaptic cleft or neuromuscular junction. Acetylcholine is taken up by cholinergic receptors on postsynaptic cells, triggering a change in the permeability of the membrane to sodium and potassium ions. Depolarization occurs resulting in the propagation of a unidirectional electrical impulse known as an “action potential,” ultimately leading to contraction of muscle (in the case of a neuromuscular junction). Agricultural compounds such as DDT and pyrethrins interact with these voltage-gated ion channels hindering their ability to close, as well as the neuronal ATPase pump that restores resting potential across the membrane (Bonner, 2016).
Back in the synaptic cleft, acetylcholine’s nerve transmission activity ceases milliseconds after it has been released from the nerve terminal when acetylcholine is rapidly degraded into choline and an acetate ion, catalyzed by acetylcholinesterase. This is a ravenous enzyme with the power to hydrolyze over ten times the amount of acetylcholine that is actually exocytosed into the synaptic cleft by nerve terminals within one millisecond. Choline is transported back in to the axon terminal and is recycled to make more acetylcholine. Drugs such as edrophonium, neostigmine and prostigmine are inhibitors of acetylcholinesterase and can be used clinically to diagnose and treat myasthenic disroders. Two classes of agricultural of compounds used in pesticide control known as organophosphates and carbamates both adversely affect the nervous system by interfering with acetylcholine signaling.
Organophosphates were first synthesized in 1820. In 1944, parathion was introduced as a major insecticidal agent and by 1992 over 250,000 insecticidal organophosphates had been synthesized, 200 of which are currently being sold on the market. Organophosphorous insecticides came to replace the organochlorine insectesticides including DDT that were banned in the 1970s after it was discovered that organochlorine insecticides were accumulating in the environment. While organophospates may degrade more easily and naturally in the environment, they possess greater acute toxicity and thus are a greater threat to humans, especially to people involved in the agricultural use of pesticides.
Organophosphates function by inhibiting acetylcholinesterase, the enzyme responsible for breaking down acetylcholine. These compounds bind to the active site of acetylcholinesterase, phosphorylating a serine amino acid residue in the catalytic site of the enzyme. This phosphorylation event inactivates acetylcholinesterase, which can either remain stably inhibited for hours to days until spontaneous hydrolysis of the phosphate group reestablishes functionality of the enzyme or it can “age,” becoming resistant to reactivation. The process of “aging” refers to the loss of a side chain, specifically an alkyl group, attached to the phosphate moiety. It is the size of these alkyl groups that determines when spontaneous hydrolysis occurs. Longer alkyl groups increase the rate of spontaneous hydrolysis, yet when one or more of these alkyl groups is lost during aging, spontaneous hydrolysis becomes impossible and the enzyme is permanently inhibited.
When acetylcholine is not broken down by acetylcholinesterase, the neurotransmitter accumulates in the synapse resulting in overstimulation of acetylcholine receptors in the CNS and PNS. Impulses are continually transmitted at neuromuscular junctions where muscular effects include fatigue, weakness and cramps. Parasympathetic nerve endings in smooth muscle, cardiac muscle, and glands, as well as sympathetic and parasympathetic autonomic ganglia are over-stimulated. This can result in bronchoconstriction, increased bronchial secretions, increased salivation, lacrimation, sweating, nausea, vomiting, diarrhea and constriction of the pupils. Respiratory failure is usually the cause of death. Organophosphates and other acetylcholinesterase inhibitors can also impact the blood brain barrier by increasing the permeability of the blood brain barrier to themselves and other agents.
Such acute neurotoxicity, known as the “cholinergic toxidrome,” lead to organophosphates being developed as a chemical warefare agents such as sarin and diisopropyl fluorophosphates. Sarin nerve gas is a highly toxic antagonist that phosphorylates acetylcholinestease so that the enzyme is no longer capable of hydrolyzing acetylcholine. When nerve impulses are continually transmitted, excessive neurotransmitter collects in the synapse and the muscle contracts repetitively. This overstimulation induces seizures or laryngospasm that can lead to death. Diisopropyl fluorophosphate, another potent nerve gas poison, is capable of inhibiting acetylchoinesterase for weeks making it especially deadly (Hall, 2016).
Most of the symptoms of organophosphate toxicity are sympathetic and can be antagonized by atropine or 2-PAM, also known as pralidoxime, an oxime that reverses binding of acetylcholinesterase inhibitors. Pralidoxime attacks the phosphorus moiety of an organophosphorylated acetylcholinesterase complex to break apart the phosphate-ester bond. This won’t work if aging has occurred, as oximes have no effect on dealkylated enzymes. Atropine reduces the effects of acetylcholine by preventing it from binding to muscarinic receptors only so it counteracts some of the hyperstimulation from organophosphates. Atropine has no impact on nicotinic receptors so effects of organosphate toxicity on skeletal muscle and other sympathetic responses persist. For this reason atropine is usually administered in combination with pralidoxime.
Carbamate insecticides also inhibit acetylcholinesterase but there are important features that differentiate carbamate poisoning from organophosphate poisoning. While signs and symptoms of carbamate poisoning mimic those of organophosphate poisoning, toxic carbamate doses are widely separated from lethal doses. Furthermore, carbamates inactivate the acetylcholinesterase enzyme by carbamylating the active site as opposed to the phosphorylation mechanism by which organophosphates act. Carbamylation of the active site is drastically less stable than phosphorylation of the active site. In fact, the half-life of carbamylated acetylcholinesterase is 15-30 minutes in contrast to organophosphorylated acetylcholinesterase, which reactivates with a half-life of one to two hours. Therefore reactivation of the acetylcholinesterase enzyme from carbamate poisoning is faster which reduces recovery time following exposure. Oxime-based antidotes that counteract toxicity by organophosphates have no effect on carbamate-mediated toxicity and may actually exacerbate carbamate poisoning by increasing the stability of the carbamylated acetylcholinesterase.
The vast complexity, sensitivity, and adaptability of the nervous system pose it at risk for toxic insult by many different agents that can interfere with many different neurological processes. Organophosphates are one of the most common sources of human poisonings due to their common application in agriculture as insecticides around the globe. The neurotoxicity of organophosphates is due to their functionality as acetylcholinesterase inhibitors. When acetylcholinesterase inhibitors inactivate the enzyme acetylcholinesterase so that it can no longer degrade acetylcholine, neurotransmitter signaling is potentiated. Such compounds can be classified as reversible inhibitors or irreversible inhibitors, the latter of which are mostly associated with neurotoxic potential.