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.

Altered immune responses have been observed following occupational, inadvertent, or therapeutic exposure to xenobiotics. Many pesticides are known to cause immunotoxicity. Exposure to mixtures of pesticides, either concurrently or sequentially, may result in potentiating this effect partly because one can effect the metabolism of the other. The objective of this study was to determine the effect of the insecticides endosulfan, permethrin and their mixtures on C57/BL6 male mice thymocytes in vitro and to ascertain the mechanism by which these effects take place. Permethrin, a broad-spectrum synthetic pyrethroid, is a widely used insecticide in agriculture and public health. Endosulfan is a highly toxic chlorinated hydrocarbon insecticide used worldwide. We examined the immunotoxic potential of these pesticides using a flow cytometric technique in combination with 7-Amino Actinomycin D (7AAD) to distinguish live, early apoptotic, and late apoptotic/necrotic cells. DNA ladder assay, a hallmark of apoptosis, was also used to determine the occurrence of apoptosis. Both endosulfan and permethrin were found to cause significant apoptotic death of thymocytes in a dose- and time- dependent manner. Thus, permethrin at 50, 100 or 300 µM was found to cause 5.5, 11.5 and 26.1% increases in early apoptotic cell death relative to control, respectively. Endosulfan at 25, 50 or 250 µM was found to cause 11.9, 15.7 and 68.0% early apoptotic cell death, respectively. For the mixture study, concentrations of 100 µM permethrin and 50 µM endosulfan were selected and found to cause 27.1% apoptosis. Thus, these pesticides in mixture have an additive immunotoxic effect. Increases in late-apoptotic/necrotic cells were found at these concentrations for either pesticide when exposed for 12 hours. DNA ladder assay confirmed the presence of DNA fragments and therefore the presence of significant apoptotic cell death. Apoptosis is a morphologically distinct form of cell death that can be mediated by a variety of pathological and physiological stimuli. Because permethrin and endosulfan were found to induce apoptosis in C57/BL6 mice thymocytes in vitro, the objective of the second half of this study was to elucidate the potential mechanism by which these pesticides regulate apoptosis in immune cells. Caspases are a family of cystine-dependent, aspartate-directed proteases that have an integral role in apoptotic cell death. Caspases, which are normally inactive in healthy cells, are activated during apoptosis and form an irreversible cascade. There are two subsets of caspases, initiator caspases (i.e. caspase 8 and 9) and effector caspases (i.e. caspases 3 and 6). Caspase 3, a downstream effector of apoptosis, is activated by many different pathways and is an apoptotic marker in cells. Caspase 8 is the apical caspase in the extrinsic pathway. Caspase 9 is the apical caspase in the intrinsic pathway, therefore we investigated mechanisms of pesticide induced apoptosis involving the thymocyte caspase system. Thymocytes from C57/BL6 mice were incubated with varying concentrations of pesticides for varying amounts of time. Active caspase 3 was then measured using EnzCheck Caspase 3 Assay Kit. Relative fluorescence for permethrin exposed cells after 12 hours incubation in the presence of pesticides at 150, 100, and 50 µM and 40 minutes in the presence of AFC-substrate was found to be 387, 386, and 297, respectively. Relative fluorescence for endosulfan exposed cells at 150, 100 and 50 µM was 188, 177, and 294. Caspase 3 activity increased as permethrin concentrations increased and decreased as endosulfan concentrations were increased. Then the extrinsic and intrinsic pathways of apoptosis were further investigated. Active caspase 8 was measured using the ApoAlert Caspase Fluorescent Assay Kit. Relative fluorescence for permethrin exposed cells after 7 hours incubation in the presence of pesticides at 100, 150, and 200 µM was found to be 35.5, 10.5, and 0, respectively. Relative fluorescence for endosulfan exposed cells after 7 hours incubation at 25, 50, 100 and 150 µM was found to be 32.8, 63.8, 69.5, and 55.5, respectively. A mixture study was then performed using endosulfan (50, 100, 150 µM) combined with permethrin (100 µM). All combinations were found to have more than an additive effect, therefore the extrinsic pathway seems to be involved. Caspase 9 activity was measured using Caspase 9/Mch6 Fluorometric Protease Assay Kit. Relative fluorescence for endosulfan exposed cells after 7 hours incubation at 25, 50, 100 and 150 µM was found to be 43, 73, 78.9, and 5.12, respectively. Relative Fluorescence for permethrin exposed cells at 100, 150 and 200 µM was found to be 34.5, 39, and 55.5, respectively. A mixture study was then performed using endosulfan (25, 50 µM) combined with permethrin (100 µM). Both combinations were found to have less than an additive effect. These results suggest that apoptosis caused by both endosulfan and permethrin exert their effects via the caspase pathway. The results also show that mixtures of pesticides have a less than additive effect on caspase 9 activation and more than an additive effect on caspase 8 activation, therefore the extrinsic pathway is predominantly involved in thymocyte apoptosis caused by mixtures of permethrin and endosulfan. / Master of Science

No description available.

The application of pesticides in agriculture production systems is a complex process and involves a series of factors that dynamically interact to impact overall pesticide application efficacy. Spray droplet formation, target impaction and deposition, plant uptake, and subsequent biological response are all functions of pesticide active ingredient, nozzle selection, application pressure, and carrier volume. Smaller spray droplets with a lower kinetic energy result in greater spray droplet retention on the leaf surface relative to larger droplets. Consequently, larger spray droplets with higher kinetic energy are poorly retained on the leaf surface and yield minimal coverage of leaf surface tissue. While smaller droplets maximize target coverage and spray droplet retention, larger spray droplets minimize off-target movement and agrichemical transport. Consequently, application factors that maximize pesticide efficacy and minimize off-target movement are often incongruous elements. Therefore, the objective of this research was to evaluate the impact of carrier volume and spray droplet size on the efficacy of various pesticide applications in Mississippi. These data indicate that 1) for thrips and tarnished plant bug control, acephate and sulfoxaflor are superior dicamba application partners relative to dimethoate and thiamethoxam, respectively, insecticide efficacy did not vary due to spray droplet size when applied without dicamba, and maximum efficacy was achieved with a carrier volume of 187 L ha-1; 2) dicamba + acephate and dicamba + thiamethoxam or sulfoxaflor are efficacious options to control Palmer amaranth relative to dicamba applied alone, and maximum Palmer amaranth control was achieved with a carrier volume of 187 L ha-1; 3) cotton defoliation efficacy is positively and negatively correlated with carrier volume and spray droplet size, respectively, and maximum efficacy was achieved with cotton defoliation programs consisting of two-applications, each with a carrier volume of 187 L ha-1 and 200 µm droplets; 4) soybean harvest aid efficacy is primarily a function of the harvest aid applied and that the impact of carrier volume and spray droplet size varies across harvest aids. However, when paraquat is applied, a carrier volume between 47 and 187 L ha-1 should be utilized with droplets of 200-500 µm to maximize harvest aid efficacy.

44 pp. / Updated December 15, 2003

Although they are currently effectively controlled by chemicals, sweet potato whiteflies have the potential to once again be a dominant pest in Arizona vegetables. We need to explore alternatives such as biological control so that we are not reliant solely on pesticides. We have been examining dispersal by the whitefly parasitoid Eretmocerus sp. in order to find more effective ways to deploy these agents in the field. We have learned in the laboratory that these wasps are efficient flyers since they are capable of moving into strong winds for more than 30 min. We also now know that most flight takes place within a short distance of the release point. We also have a better understanding of differences between male and female flight. This, along with other information we have obtained, will lead to our ability to effectively release these and parasitoids of other insect pests as well.

A new insect management technology known as "particle film" technology (Surround) was evaluated for it potential for control early fall pests of head lettuce in Yuma, AZ. Surround did appear to slightly reduce larval population of cabbage looper, beet armyworm and Heliothis, but not enough to be considered a commercially effective treatment. Additionally, Surround appeared to antagonize Success’s activity towards cabbage loopers. Against whiteflies, Surround did appear to reduce ovipositioning, but resultant impact on the nymph population could not be adequately assessed. Overall, Surround does not appear to have good fit in the pest management system in leafy vegetables in Yuma, AZ.

An insect pest monitoring network spanning the major growing areas in Yuma was implemented in 1998-1999. This project was designed to measure the relative activity and movement of adult populations during the growing season and provide important information to growers and PCA’s. A series of pheromonebaited and yellow sticky traps were placed in 11 locations among the growing areas in the Yuma, Gila and Dome Valleys in Yuma county. Numbers of adults / trap /night were recorded weekly from September-April. Seasonal differences in insect species activity and abundance among locations were observed, but difficult to precisely explain because of the lack of historical trap data. Information gathered from the trapping network will provide historical baseline data for pest activity on an area wide basis from which relationships between insect trap captures and seasonal factors that influence their activity and abundance may be explained. The results of the monitoring network during the 1998-1999 growing season for is provided in this report.

The primary aim of the work described in this thesis is to establish a foundation for the applicability of a biochemical biomarker, cholinesterase (ChE) activity in food animal species, as an instrument for evaluating exposure to pollutants as well as predicting high-level effects on public health. Secondary aims are to increase the awareness of pesticide users of anti-ChE exposure, to decide whether poisoning episodes involve anti-ChE by measuring residual effects in tissues, and to identify sources of contamination in food animal tissues. The ChE are specialized carboxylic ester hydrolases that break down esters of choline. They are classified as either acetylcholinesterase (AChE) or butyrylcholinesterase (BChE). Both AChE and BChE activities were found to be higher in cattle than in sheep and higher in erythrocytes than in plasma and serum. The anticoagulant heparin significantly affects AChE activity in plasma compared with EDTA. Of the different tissue tested, the mean of ChE activities was found to be highest in tissue from the liver, followed by lung, muscle, kidney and heart for sheep and cattle. In pigs, the ChE activities tested higher in kidney, liver, lung, muscle and heart. The effect of freezing on ChE activities in liver and muscle tissues was significant inhibition after 6 months at -80 °C, whereas decreased after 3 months at -20 °C. A technique to improve the purification of AChE in sheep tissue was developed. BW284c51 strongly reduced acetylthiocholine iodide (AcTChI) and propionylthiocholine iodide (PrTChI) hydrolysis and slightly affected that of butyrylthiocholine iodide (BuTChI) in the liver, while iso-OMPA had no significant effect for muscle BuTChI of sheep and pigs. Histochemical study of liver tissue found AChE localised mainly in the cytoplasm of the cell lining in the sinusoids. The optimal pH values of AChE and BChE in liver and muscle ranged between 7.8 and 8.5. Both AChE and BChE activities increased when increase the time course and temperature. The half maximal inhibitory concentration (IC50) was found to be higher for carbaryl than dichlorvos (DDVP) and diazinon (DZN). Very little residual AChE activity was seen in the liver, but more was found in muscles. In general, the rate constants of inhibition (ki) values for liver and muscles were increased in different pHs according to the rank order of 8.5 > 7.5 > 6.5, while in plasma it was decreased in different temperatures as follows: 20 °C > 30 °C > 40 °C. The final experiments were carried out at the rate of spontaneous reactivation (ks) of inhibited AChE by DDVP and DZN from liver and muscle was found to be higher in sheep compared to cattle and pig, while the aging of phosphorylated AChE (ka) was found to be higher in cattle compared to sheep and pig. In addition, this study indicated that the developed bispyridinium symmetric (K048) oxime seems to be promising reactivated to DDVP-inhibited AChE for sheep and pigs while HI-6 was effective in cattle.

Uma das classes químicas de herbicidas que merece particular atenção é a dos bipiridílicos, representada pelo paraquat e diquat. Atualmente, muitos países têm banido ou restringido o uso destes herbicidas devido à grande quantidade de casos de intoxicação acidental, suicídio e envenenamento (tentativa de homicídio) ocorridos no passado. Em contrapartida, o paraquat ainda é bastante utilizado em cerca de 130 nações, com prevalência em países subdesenvolvidos ou em vias de desenvolvimento. O presente trabalho tem por objetivo o estudo e o desenvolvimento de métodos analíticos semi-quantitativo e quantitativo em amostras biológicas (plasma e urina) e aplicação no diagnóstico e investigação da intoxicação aguda por paraquat e diquat. Análises semi-quantitativas de triagem foram realizadas pela técnica enzimática-colorimétrica. Análises confirmatórias foram realizadas pela técnica de cromatografia em fase gasosa acoplada à espectrometria de massa (GC-MS). Após o desenvolvimento e validação dos métodos, as análises foram aplicadas em amostras de pacientes suspeitos de intoxicação aguda por paraquat/diquat atendidos no Hospital Regional do Vale do Ribeira (Pariquera-Açu) e no Hospital de Clínicas da Universidade Estadual de Campinas (SP). Foram avaliadas a efetividade do teste rápido semi-quantitativo e a correlação das concentrações plasmáticas e urinárias do paraquat/diquat obtidas com o método por GC-MS com o grau de intoxicação e o prognóstico de sobrevida dos pacientes após o diagnóstico e tratamento. / One of the chemical classes of herbicides that deserve particular attention is that of bipyridyl, represented by paraquat and diquat. Currently, many countries have banned or restricted these herbicides since large number of accidental intoxication, poisoning and suicide attempt cases occurred in the past. In spite of this, paraquat is still used in more than 130 countries, with prevalence in developing countries. The aim of this work was the study and the development of a screening test and a quantitative analytical method in biological samples (plasma and urine) and their application in diagnosis and prognosis of acute paraquat and diquat intoxication. Semi-quantitative analyses were performed by an enzymatic-colorimetric technique. Confirmatory analyses were performed by a gas chromatographic-mass spectrometric (GC-MS) method. After the development and the validation of both methods, analyses were applied to samples from patients suspected of acute paraquat/diquat poisoning attended in Hospital Regional do Vale do Ribeira (Pariquera-Açu) and Hospital de Clínicas da Universidade Estadual de Campinas (SP). In this present paper, we also evaluated the effectiveness of the rapid test and the correlation of paraquat/diquat plasma and urinary concentrations obtained with the GC-MS method with the degree of intoxication and prognosis of survival of patients after diagnosis and treatment.