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dc.contributor.advisorEddleston, Michael
dc.contributor.advisorRibchester, Richard
dc.contributor.authorDissanayake, Kosala Nimanthi
dc.date.accessioned2016-06-01T14:20:20Z
dc.date.available2016-06-01T14:20:20Z
dc.date.issued2015-07-04
dc.identifier.urihttp://hdl.handle.net/1842/15837
dc.description.abstractOrganophosphorus (OP) pesticide toxicity is a global health problem. Respiratory failure due to neuromuscular transmission dysfunction accounts for about 300,000 deaths annually in rural Asia. However, the clinical manifestation is complex, and described in terms of acute, intermediate, and chronic syndromes. The underlying mechanism of toxicity is still unclear. OP pesticides contain inhibitors of acetylcholinesterase (AChE), for example dimethoate, emulsified in an organic solvent, typically cyclohexanone. A hypothesized mechanism is initial excitotoxicity through inhibition of acetylcholinesterase followed by failure of neuromuscular synaptic transmission. I tested this electrophysiologically in vitro by measuring properties of spontaneous miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) in isolated sciatic nerve/flexor digitorum brevis muscles from mice, bathed in HEPES-buffered mammalian physiological saline (MPS). Muscle action potentials were abolished with μ-conotoxin (2μM). First, we tested the effects of plasma taken from Göttingen minipigs instilled orally (isofluorane anaesthesia) with a formulated pesticide (2.5ml/kg) whose active ingredient is dimethoate dissolved in cyclohexanone. This plasma abolished evoked synaptic transmission and increased spontaneous MEPP frequency within 60-180 minutes of bath application. However plasma from minipigs instilled with dimethoate alone produced no failure of transmission. Plasma contained either pesticide or dimethoate significantly increased the half decay time of EPPs. However, pesticide-plasma also contained the metabolites omethoate (100μM) and cyclohexanol (5 mM). We found that bath application of omethoate alone caused a potent dose-dependent increase in EPP decay time. Cyclohexanol (5 mM) also increased EPP decay time but it also decreased both the excitability of axons and MEPP amplitude. In combination, omethoate and cyclohexanol produced greater disruption of neuromuscular transmission than either dimethoate or cyclohexanone, alone or in combination and this was particularly evident in isometric tension recordings, in which prolonged after-contraction and slow relaxation were observed during and immediately following tetanic stiumuation in the presence of omethoate and cyclohexanol. Voltage-clamp recordings of endplate currents (EPC) partially supported the EPP observations. Surprisingly, cyclohexanol-treated preparations showed no significant increase in EPC and MEPC decay time. However, there was some evidence of activity-dependent decline in MEPC amplitude in cyclohexanol while quantal content in these preparations showed evidence of an increase suggesting a homeostatic response in evoked transmitter release with cyclohexanol treatment. Analysis of presynaptic currents in cyclohexanol treated preparations also revealed preliminary evidence of sensitivity to cyclohexanol compared to control preparations. Finally, I tested the effects NMJ transmission of 24hr exposure to OP pesticide and its metabolites using a novel organ culture system, utilising a mouse mutant (WldS) with a slow nerve degeneration phenotype. After incubation of 24 hrs with MPS + pesticides and metabolites, these muscles showed significant reduction in function (response to nerve stimuli with EPP/action potential ± MEPPs) compared to control cultures. Together, the data indicate that failure of neuromuscular transmission by pesticide-plasma cannot be explained solely by dimethoate-mediated inhibition of acetylcholinesterase. Rather, a combination of metabolic breakdown products exerts potent, harmful presynaptic and postsynaptic effects. Either blocking the metabolic conversion of the constituents of OP pesticides, or transiently blocking their effects on receptors may therefore be an effective strategy for treatment of OP pesticide toxicity.en
dc.language.isoenen
dc.publisherThe University of Edinburghen
dc.relation.hasversionBrown R, Hynes-Allen A, Swan AJ, Dissanayake KN, Gillingwater TH, Ribchester RR (2015) Activity-dependent degeneration of axotomized neuromuscular synapses in Wld(S) mice. Neuroscience 290:300-320.en
dc.relation.hasversionEddleston M, Street JM, Self I, Thompson A, King T, Williams N, Naredo G, Dissanayake K, Yu LM, Worek F, John H, Smith S, Thiermann H, Harris JB, Eddie Clutton R (2012) A role for solvents in the toxicity of agricultural organophosphorus pesticides. Toxicology 294:94-103.en
dc.subjectneuromuscular junctionen
dc.subjectpesticideen
dc.subjecttoxicityen
dc.titleEvaluation of neuromuscular transmission in organophosphorus pesticide toxicityen
dc.typeThesis or Dissertationen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD Doctor of Philosophyen


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