The Mechanisms of Neuro-toxic Pesticides
Agricultural pesticides have become part of the chemical landscape that we all live in. To be able to make intelligent decision about the use and regulation of these chemicals, it’s important to understand how they work. Almost all modern pesticides are chemicals that interfere in some way with the nervous system. The characteristics of the chemical interaction with the nervous system function can shed light on the effectiveness of the pesticide and on its physiological effects at residual levels. We will start by looking at how some of the normal processes of the nervous system work, because it will be disruption of those processes that lead to toxic effect. Then we will look at the mode of action for three major classes of pesticides and how they specifically interfere with normal function. In a future article we will look at how the specific mechanisms of action can effect dose scaling relationships.
Normal Neuron Function – Neurons, action potentials, sodium and potassium voltage gated ions channels, and ion pumps
The nervous system of insects and humans share many common features, starting with the basic structure of the neuron.
There are many variations on the same theme in different parts of the organism. Terminal branches can attach to dendrites of other neurons at synapses, or through motor synapses to muscle cells. Individual neurons are connected in complex, interacting networks by the synaptic connections. Information processing involves summing the inputs from many neurons and generating an output. When the summed stimulus is high enough, the neuron will generate an electrical pulse that is sent along the axon and which will, in turn, stimulate multiple downstream neurons connected through synapses to the axon branch terminals.
Neuronal signalling is accomplished by way of “action potentials”, which are short electro-chemical pulse that travel along the neuron axon. The short pulse-like nature of the nerve signals are generated and maintained by way of “voltage-gated” ion channels and ion pumps. Ion pumps use the cellular energy store, ATP, to move sodium and potassium ions across the cell membrane, setting up a concentration gradient across the membrane that establishes a “resting potential” of about -70mV from the inside to the outside of the nerve cell. Once this gradient is established, then merely opening ion channels in the cell wall allows the sodium or potassium ions to move back across the membrane and move the potential closer to zero. Nature’s trick, that turns this process into a useful information processing network, is to open the ion channels which depolarize the neuron with a positive feedback action associated with the membrane potential. Once the membrane potential rises from its resting potential to a “threshold” the voltage gated channels open, steepening the rising edge into the action potential nerve pulse. The figure below is a nice schematic of the ‘anatomy’ of the action potential.
Signaling happens by way of the action potentials, which propagate along the axons and terminate at the synapse. There are several ways the action potential can be interact with cellular structures. We will concentrate on the acetylcholine mediated synaptic response because this is the target of several pesticide chemicals.
Normal Synapse Function – acetylcholine-mediated transmission
Acetylcholine (ACh) is a molecular neurotransmitter that conveys information across the synapse. In the figure above, the basic steps of the interaction are illustrated. Action potentials, those pulses of neural activity, cause synaptic vesicles containing ACh to release the ACh molecules into the synaptic cleft, the junction region between the two cells. The ACh quickly diffuses across the narrow junction region and is captured by acetylcholine receptors (AChRs) that are part of ion channel molecules. The AChRs that have captured an ACh molecule open the ion channel and allow Na+ ions to enter the post-synaptic neuron. The binding is transitory, however; the ion channels rapidly open and close as the ACh molecules latch and unlatch from the AChR channel. Meanwhile, another ACh receptor is also present in the synaptic junction called acetylcholinesterase (AChE). This molecule is an enzyme which rapidly breaks apart the acetylcholine into choline and acetate, effectively ridding the synaptic cleft of the neurotransmitter almost as fast as it is made available. The result of all of this chemical activity is that the AChRs, as an ensemble, are open only for a few milliseconds. During this time, ions flood into the post-synaptic dendrite, depressing the potential in the down stream neuron, making it more likely to generate its own action potential.
This simplified discussion leaves out many details. There are many more specialized molecules that are part of cell membranes. Often molecules that are specific for one important function also are involved in unrelated functions. Nerve cells can be specialized and synaptic details can vary. Nevertheless, the basic picture we are painting is valid across much of the animal kingdom. These same basic process happen in the nervous systems of humans and bees alike. Now let us move on to discuss ways to interrupt these normal processes for insecticidal effect.
Insecticides targeting axonal voltage-gated ion channels
Two major classes of insecticides target the voltage gated ion channels shown in our cartoon. The organochlorines (e.g. DDT, dieldrin, chlordane) and pyrethroids (e.g. deltamethrin) act by opening these voltage gated ion channels. The molecules hold open the channels and allow ions into the axon that depolarizes the neuron. In the depolarized state the neuron is non functional, characterized by paralysis. In between the normal state and paralysis there is a range where the depolarization of the neuron is only partial. Partial depolarization leaves the neuron susceptible to “false triggering”. A small stimulus that would normally not trigger an action potential will produce one more easily as the resting potential gradually climbs to the threshold required to launch an action potential. Organisms in this state typically exhibit twitching and uncontrolled movements as the uncontrolled nerve impulses trigger muscles to move.
Nothing is static at the molecular scale. As organic molecules interact with one another, they can latch onto each other either very loosely or with tenacity depending upon the exact shape of the molecules involved and type of binding that happens. Binding that occurs via the covalent sharing of electrons is usually very strong, essentially permanent and irreversible. In contrast, many biological molecules interact through polar or Van Der Walls forces that are much weaker. Such interactions may last for a fleeting amount of time before thermal fluctuations pull them apart. Weak binding is reversible and can be characterized by a dissociation time, how long it takes to break the bond due to random and thermal fluctuations.
When dealing with pesticide chemicals, stronger bonds mean the insecticide is spending more time at the active site, so its potency is higher. Frequently it is just how tenacious the binding that determine the potency of the insecticide.
Chemical scavengers known as cytochrome P450 enzymes are always on the lookout for foreign chemicals which these enzymes break down into smaller parts in the process of metabolizing and eliminating unwanted molecules. Often, within a few hours much of a foreign chemical will be metabolized and eliminated from the organism. Bound molecules are not as easily digested by the cytochrome P450s so once toxins are bound to their site of action, they are more immune to detoxification.
Insecticides targeting the acetylcholine pathway
There are several classes of pesticides that disrupt the acetylcholine pathway. We will start by looking at the neonicotinoids because they have the simplest mechanism, similar to the “direct action” of the pyrethroids discussed above.
The neonicotinoids bind strongly to the AChRs. Binding causes he ion channels to open so Na+ ions can flow into the neuron. Unlike the normal acetylcholine response where the channel is only open for about a millisecond, when the neonicotinoid binds the receptors never close. Hence, it takes only a relatively few open channels to eventually depolarize the neuron. If the ion pumps cannot keep up with the leakage through the nicotinoid-bound AChRs the cell will depolarize. Partial depolarization will make the neuron more excitable; complete depolarization leads to paralysis.
This situation is more complicated with acetylcholinesterase inhibitors such as the organophosphate and carbaryl insecticides. For these chemicals, the insecticide does not directly bind to neuronal receptors that open ion channels. Instead the chemicals bind to the acetylcholinesterase (AChE) enzymes which rid the synaptic junction of the ACh neurotransmitter than is released with normal activity. However, without the AChE to clear the junction, the ACh continues to bind with AChR ion channels. The figure below shows schematically what happens with these AChE inhibitors.
Insecticide molecules bind to the acetylcholinesterace (AChE) sites in the synaptic junction, preventing the naturally released ACh for being removed and recycled from the junction. The acetylcholine continues to activate receptors, keeping their channels open thereby depolarizing the post synaptic neuron. Again, poisoning symptoms begin with an over-excitable nervous system, characterized by uncontrolled twitching, similar to the other classes of neurotoxins we have looked at.
Neurotoxins are among the most potent biological chemicals known. The chemicals are targeted to interact with specific receptor molecules that are crucial for nervous system function. This means that very few pesticide molecules are required to have a large biological effect. Chemicals used as pesticides need to effectively poison target species while remaining benign to non-target organisms and humans. However, much of the cellular machinery is shared across the animal kingdom, so differentiating between target and non-target organisms is a challenge. Often only space and time are used to separate target and non-targets creatures from chemical exposure. The environmental effects of pesticide chemicals depends upon the success of various strategies to limit harmful exposure to non-target species. In many cases dilution is the solution, but as industrial agriculture and residential uses of potent chemicals become even more widespread, minute residual levels of toxins is inevitable. Next time we will see why this is more likely to be a problem with some classes of chemicals more than others.