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Organophosphorus (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.

Com o advento da agricultura ampliou-se a produção alimentar e os bens de consumo, no entanto, os riscos ambientais também foram maximizados em função da adoção de técnicas produtivas baseadas no uso intensivo de insumos agrícolas. Esta problemática é mundial, embora mais evidenciada nos países em desenvolvimento e que tem, na produção agrícola, a base de sua economia. O Brasil enquadra-se nesta situação e desde 2009 é considerado o maior consumidor de agrotóxicos do mundo, criando um cenário de risco ambiental e de saúde humana. Os efeitos ambientais, base deste estudo, estão relacionados não somente à perda de espécies não-alvo, uma vez que os agrotóxicos não são seletivos, mas também as alterações em nível ecossistêmico, a qual se relaciona com as perdas das funções e dos serviços gerados pelos sistemas naturais. Adiciona-se a esta complexidade, a forma de ação de cada agrotóxico, a distribuição dos mesmos nos diferentes compartimentos (ar, solo e água), o período de permanência de cada um, as relações sinérgicas decorrentes das interações entre diferentes produtos, a formação de subprodutos no processo de degradação, entre outros fatores, como as diferenças existentes entre o ingrediente ativo e a formulação comercial, na qual existem os chamados ingredientes inertes em sua composição, os quais podem ser muito mais tóxicos para espécies e ecossistemas. Considerando esta abordagem, a presente pesquisa foi desenvolvida com base na realidade de um local de referência, o município de Bom Repouso (MG/BR), no qual a intensificação da produção de morango e batata tem trazido uma série de riscos sociais e ambientais. Semelhante a outras regiões produtivas do país, o uso de agrotóxicos é recorrente, amplo e irrestrito, com destaque para as formulações comerciais Kraft®36EC e Score®250EC, as quais, juntamente com seus respectivos ingredientes ativos (abamectina e difenoconazol), foram avaliadas por meio de testes de toxicidade com espécies de diferentes níveis tróficos representativas de um ecossistema aquático, gerando informações que foram avaliadas em nível de espécie e de ecossistema, simulando o cenário de aplicação dos produtos no local de referência. Os resultados obtidos permitiram concluir sobre as diferenças de sensibilidade das espécies e quais seriam as mais indicadas para se avaliar os efeitos tóxicos de ambos os agrotóxicos; os efeitos diferenciados entre a formulação comercial e os ingredientes ativos; bem como as respostas em termos de espécies e de ecossistemas, demonstrando a necessidade de que ambas as análises sejam consideradas na avaliação de risco ecológico. / With the advent of agriculture, food production and consumer goods has expanded, however, environmental risks were also maximized due to the adoption of production techniques based on intensive use of agricultural inputs. This worldwide problem is more evident in developing countries which have agricultural production as the basis of its economy. Brazil is into this situation and since 2009 it is considered the largest consumer of the world\'s pesticides, creating a scenario of environmental risk and human health. The environmental effects from this study are related not only to loss of non-targeted species, since pesticides are nonselective, but also changes in the ecosystem level, which relates to the loss of functions and services generated by natural systems. Add to this complexity, the mode of action of each pesticide, their distribution in the different compartments (air, soil and water), the period of stay of each, the synergistic relationships resulting from interactions between different products, the formation of by-products in the process of degradation, among other factors, such as differences between the active ingredient and the commercial formulation in which there are so-called ingredients inert in the composition, which may be more toxic to species and ecosystems. Considering this approach, this research was developed based on the reality of a reference site, the municipality of Bom Repouso (MG/BR), in which the intensification of production of strawberry and potato has brought a series of social and environmental risks. Similar to other productive regions of the country, the use of pesticides is recurrent, widespread and unrestrained, highlighting the commercial formulations Kraft®36EC and Score®250EC, which, along with their active ingredients (abamectin and difenoconazole) have been evaluated by toxicity tests with representative species of different trophic levels of an aquatic ecosystem, generating information that have been evaluated at the species level and ecosystem, simulating the application scenario of the products on the reference location. The results showed differences on the sensitivity of species and what are the most appropriate to evaluate the toxic effects of both pesticides; the differential effects between the commercial formulation and the active ingredients; as well as the responses in terms of species and ecosystems, demonstrating the need for both to be considered in ecological risk assessment.

Com o advento da agricultura ampliou-se a produção alimentar e os bens de consumo, no entanto, os riscos ambientais também foram maximizados em função da adoção de técnicas produtivas baseadas no uso intensivo de insumos agrícolas. Esta problemática é mundial, embora mais evidenciada nos países em desenvolvimento e que tem, na produção agrícola, a base de sua economia. O Brasil enquadra-se nesta situação e desde 2009 é considerado o maior consumidor de agrotóxicos do mundo, criando um cenário de risco ambiental e de saúde humana. Os efeitos ambientais, base deste estudo, estão relacionados não somente à perda de espécies não-alvo, uma vez que os agrotóxicos não são seletivos, mas também as alterações em nível ecossistêmico, a qual se relaciona com as perdas das funções e dos serviços gerados pelos sistemas naturais. Adiciona-se a esta complexidade, a forma de ação de cada agrotóxico, a distribuição dos mesmos nos diferentes compartimentos (ar, solo e água), o período de permanência de cada um, as relações sinérgicas decorrentes das interações entre diferentes produtos, a formação de subprodutos no processo de degradação, entre outros fatores, como as diferenças existentes entre o ingrediente ativo e a formulação comercial, na qual existem os chamados ingredientes inertes em sua composição, os quais podem ser muito mais tóxicos para espécies e ecossistemas. Considerando esta abordagem, a presente pesquisa foi desenvolvida com base na realidade de um local de referência, o município de Bom Repouso (MG/BR), no qual a intensificação da produção de morango e batata tem trazido uma série de riscos sociais e ambientais. Semelhante a outras regiões produtivas do país, o uso de agrotóxicos é recorrente, amplo e irrestrito, com destaque para as formulações comerciais Kraft®36EC e Score®250EC, as quais, juntamente com seus respectivos ingredientes ativos (abamectina e difenoconazol), foram avaliadas por meio de testes de toxicidade com espécies de diferentes níveis tróficos representativas de um ecossistema aquático, gerando informações que foram avaliadas em nível de espécie e de ecossistema, simulando o cenário de aplicação dos produtos no local de referência. Os resultados obtidos permitiram concluir sobre as diferenças de sensibilidade das espécies e quais seriam as mais indicadas para se avaliar os efeitos tóxicos de ambos os agrotóxicos; os efeitos diferenciados entre a formulação comercial e os ingredientes ativos; bem como as respostas em termos de espécies e de ecossistemas, demonstrando a necessidade de que ambas as análises sejam consideradas na avaliação de risco ecológico. / With the advent of agriculture, food production and consumer goods has expanded, however, environmental risks were also maximized due to the adoption of production techniques based on intensive use of agricultural inputs. This worldwide problem is more evident in developing countries which have agricultural production as the basis of its economy. Brazil is into this situation and since 2009 it is considered the largest consumer of the world\'s pesticides, creating a scenario of environmental risk and human health. The environmental effects from this study are related not only to loss of non-targeted species, since pesticides are nonselective, but also changes in the ecosystem level, which relates to the loss of functions and services generated by natural systems. Add to this complexity, the mode of action of each pesticide, their distribution in the different compartments (air, soil and water), the period of stay of each, the synergistic relationships resulting from interactions between different products, the formation of by-products in the process of degradation, among other factors, such as differences between the active ingredient and the commercial formulation in which there are so-called ingredients inert in the composition, which may be more toxic to species and ecosystems. Considering this approach, this research was developed based on the reality of a reference site, the municipality of Bom Repouso (MG/BR), in which the intensification of production of strawberry and potato has brought a series of social and environmental risks. Similar to other productive regions of the country, the use of pesticides is recurrent, widespread and unrestrained, highlighting the commercial formulations Kraft®36EC and Score®250EC, which, along with their active ingredients (abamectin and difenoconazole) have been evaluated by toxicity tests with representative species of different trophic levels of an aquatic ecosystem, generating information that have been evaluated at the species level and ecosystem, simulating the application scenario of the products on the reference location. The results showed differences on the sensitivity of species and what are the most appropriate to evaluate the toxic effects of both pesticides; the differential effects between the commercial formulation and the active ingredients; as well as the responses in terms of species and ecosystems, demonstrating the need for both to be considered in ecological risk assessment.