Pathogenesis of neuromuscular synaptic dysfunction and transmission failure in organophosphate toxicity

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.

Organophosphorus (OP) toxicity is a global health problem affecting industrialised and developing countries. While OP nerve agents are a worldwide threat, OP pesticides kill more than 200,000 people per annum. One major cause of death is secondary, delayed neuromuscular junction (NMJ) failure (the 'intermediate syndrome', IMS), for which there is no effective treatment. Patients therefore require mechanical ventilation over days and weeks. We have set up pig and mouse models of OP poisoning, focusing on agricultural dimethoate to better understand its pathogenesis. Preliminary data indicate that toxicity arises from additive or synergistic effects of the pesticide metabolites, specifically omethoate and the solvent metabolite cyclohexanol. Electrophysiological analysis indicates that these metabolites produce a prolonged, use-dependent hyperactivation of acetylcholine receptors (AChR) that is partially mitigated by pretreatment with the nicotinic AChR antagonist rocuronium. We will test our AChR hypothesis: a) by measuring separate and combined effects of omethoate and cyclohexanol on the conductance, open-channel probability, and desensitisation of human nicotinic AChRs expressed in HEK cells, using outside-out patch recording and administration of ACh by a concentration-jump technique; b) by using a novel ex-vivo organ culture paradigm to examine the role of activity and the mitigating effects of potential treatments against toxicity of pesticide ingredients and metabolites; c) by measuring the protective benefits of rocuronium and other blockers that we identify as candidates ex-vivo, on neuromuscular transmission, combining novel methods of confocal microendoscopy and electrophysiology and applying them to our pig model of IMS in vivo. The results will inform our ongoing clinical studies in Sri Lanka. Rapid translation of our findings from the present study into clinical practice could potentially save thousands of lives annually.

This project fits with the MRC's Strategic Aim 3 'Going Global: Accelerating Progress in International Health Research' that aims to support global health research addressing inequalities in health. Our fundamental research has a high chance of being translated into clinical practice and of improving global health and advancing quality of life through UK-part funded collaborative clinical research facilities in Sri Lanka. The work also fits with the UK government's view that "Health is Global". Beneficiaries include:

1. Patients (Within 1-5 years of commencement of the project). Pesticide self-poisoning kills about 350,000 people annually, the majority in poor rural Asian communities. OP pesticides are responsible for about two-thirds of these deaths and for the majority of costs (due to the sustained ventilation they require). Each case places substantial burdens on health care systems and immense distress to families and communities. Preventing the onset of neuromuscular dysfunction after OP poisoning and/or shortening the duration of ventilation would save lives, improve quality of life, and reduce economic costs and societal distress.

2. MRC and UK Government (Within 1-4 years of commencement of the project). From support of the Strategic Aim of "Going Global" and "Health is Global"; thus, increasing effectiveness of public services and policy.

3. Victims of terrorism at home and overseas (Within 1-5 years of conclusion of the project). Recent events in the Middle East underscore how civilian populations are at high risk of terrorist attack with OP nerve agents or highly toxic OP pesticides, such as parathion or phorate. A treatment to reduce the risk of prolonged respiratory failure that could be given by paramedics soon after an attack, alongside atropine and ventilatory support during resuscitation, would be a major advance in therapy, representing benefits for health and, advancing quality of life.

4. Biomedical and Clinical Researchers (Within 1-4 years of commencement of the project). New treatments for neuromuscular dysfunction will come from basic research into the mechanisms of the effect of OPs and the solvents on the NMJ. The work proposed here should reveal new information about NMJ function and investigative approaches that will be useful to biomedical researchers, including our Project Partners, and clinical researchers studying neurological disease. The proposed work also fits into a comprehensive on-going programme of collaborative studies in Sri Lanka, Edinburgh, and Australia that aims to reduce global deaths from pesticide poisoning by 10-20%, through improved medical management, reducing exposure to pesticides in agricultural communities, and encouraging removal of the most toxic pesticides from agricultural practice. The results of the proposed work will slot into this multi-faceted approach, enhancing creative output, health and quality of life.

5. Pharmaceutical Companies (Within 2-4 years of commencement of the project). Novel approaches to protecting NMJs should be of interest to pharmaceutical companies seeking therapeutic targets for OP toxicity and/or neuromuscular diseases. Although OP pesticide poisoning in rural Asia has been under-researched, there is a great deal of on-going research, especially in the USA, on OP nerve agent therapeutics. Our research aims and objectives, towards better understanding of the pathogenesis of the NMJ dysfunction in IMS, should identify drugs that will increase therapeutic options, thereby contributing to global economic importance and competitiveness of UK industry.

6. General public (Within 1-5 years of commencement of the project). Education, engagement and communication arising from the project will increase awareness of the consequences of environmental toxicity, provide cautionary advice or reassurance, thereby increasing effectiveness of public services and quality of life.