Pathogenesis of neuromuscular synaptic dysfunction and transmission failure in organophosphate toxicity

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Every year, across the world, hundreds of thousands of people die following poisoning with organophosphorus (OP) pesticides. Other populations are at risk from terrorist use of OP nerve agents (as recently used in the Middle East). These compounds interrupt signalling in the brain and muscles, causing paralysis and death when breathing fails. However, if patients reach hospital in time, they can be treated: first with antidotes and then with assisted breathing, by mechanical ventilation, in an intensive care unit. Unfortunately and for unknown reasons, some patients are unable to breathe for themselves for several weeks, leaving them dangerously at risk of lethal complications, like pneumonia. We are trying to uncover the mechanism linking OP poisoning to paralysis and cessation of breathing. We started by setting up animal models of poisoning, focusing on a particular agricultural OP pesticide, dimethoate EC40. This pesticide contains two essential ingredients: dimethoate itself and an organic solvent, cyclohexanone. These substances are broken down in the body by metabolism: dimethoate is converted into omethoate in the liver; and cyclohexanone is converted to cyclohexanol in the stomach and intestines. Our investigations have so far led us to a surprising discovery: the two metabolites, omethoate and cyclohexanol, are much more toxic to the connections between motor neurones and muscle cells, at their nerve-muscle ("neuromuscular") junctions, than either of the main ingredients in the pesticide itself. Acetylcholine is the neurotransmitter molecule that carries signals from the motor nerve endings to the muscle at these neuromuscular junctions. Our data strongly suggest that omethoate and cyclohexanol act aggressively together, directly affecting the protein receptors for acetylcholine, located on the muscle side of neuromuscular junctions. In this project, we will home in on and investigate this theory in three, concerted ways. First of all, we will inject the human DNA code for the acetylcholine receptor proteins into cells in tissue culture. The cells will turn the DNA into working receptors, allowing us to make recordings using an exquisite electrical technique called patch-clamping. In this way we can test and measure directly how omethoate, cyclohexanol or both together affect the way the receptor molecules respond to acetylcholine. Secondly, we will use a new type of tissue culture assay, to measure the progressive toxicity of omethoate and cyclohexanol on transmission at neuromuscular junctions over a period of 24 hours, the same period over which patients typically deteriorate. For these measurements, we will take advantage of nerves and muscles in a breed of mouse (called WldS) whose neuromuscular junctions are normally quite resistant to nerve damage. This feature gives us the time we need to find out why pesticide and its metabolites cause muscles to become progressively paralysed. This assay also gives us the option of rhythmically stimulating the nerve (as occurs naturally in muscles we use for breathing) to test whether this activity actually makes the pesticide poisoning worse. Finally, we will combine electrical recording with an exciting new technology, called confocal endomicroscopy, and apply these to direct observation of living neuromuscular junctions. We will use this approach to assess the benefits of different kinds of drugs that protect acetylcholine receptors, which should prevent pesticide and their breakdown products from damaging the receptors. We will test these drugs in our DNA-injected cells, in our WldS mouse tissue cultures, and lastly in anaesthetised pigs, because we have found that pigs react to OP pesticides in a very similar way to humans. Treatments that work will then be tested in clinical trials, in patients poisoned by OP pesticides that we currently look after in hospitals in Sri Lanka. An effective treatment will save tens of thousands of lives every year.