In dieser Arbeit wird die Entwicklung eines bifunktionellen Biosensors nach dem Vorbild eines Baukastensystems beschrieben. Das Ziel wird durch die Kombination verschiedenster molekularer Erkennungselemente erreicht. Solche molekularen Erkennungselemente im verwendeten System sind: • Propidium und die periphere anionische Bindungsstelle der Acetylcholinesterase (AChE) • Organophosphate und das aktive Zentrum der AChE • ein an die AChE gekoppeltes Hapten und das Epitop eines Antikörpers • ein an die AChE gekoppeltes Hapten, das als Ligand ein weiteres Enzym bindet Neben dem molekularen Erkennungselement wird ein Biosensor ebenso durch die Art des Transducers charakterisiert. Hier werden Quarzplättchen mit Goldelektroden zur Signalumwandlung eingesetzt. Die Verwendung solcher Sensoren mit einem EQCM-Gerät (electrochemical quartz crystal microbalance) ermöglicht es zwei Messsignale gleichzeitig aufzunehmen: die piezoelektrische Bestimmung einer Massebeladung und die amperometrische Detektion von Enzymaktivität auf der Sensoroberfläche. Für die Analytik stehen somit zwei verschiedene Assay-Varianten zur Verfügung: die Bestimmung der Inhibition der ACHE-Aktivität und ein Bindungstest über das Hapten. Die Basis beider Tests ist die Modifizierung der piezoelektrischen Kristalle mit Propidium – einem reversiblen Inhibitor der Acetylcholinesterase. Dies ermöglicht die Beladung des Sensors mit AChE über die Wechselwirkung mit der peripheren anionischen Bindungsstelle des Enzyms. Die Aktivität der so immobilisierten AChE und die Inhibition durch Organophosphate (Pestizide) werden amperometrisch bestimmt. Durch die chemische Kopplung eines Hapten an die Cholinesterase wird ein weiteres Erkennungselement eingeführt. Das eröffnet die Möglichkeit, an die auf dem Propidium-modifizierten Sensor immobilisierte, haptenisierte Cholinesterase einen Antikörper zu binden. Als Voraussetzung für elektrochemische Bestimmung der AChE-Aktivität wurde zunächst die Optimierung der amperometrischen Messmethode vorgenommen. Die Oxidatationspotentiale für die Detektion von Thiocholin wurden im Bereich von 150 mV bis 300 mV variiert. Dabei wurde für die nachfolgenden Untersuchungen eine Arbeitspotential von 200 mV (vs. Ag/AgCl) festgelegt, da hier das beste Verhältnis von gemessenem Oxidationsstrom und Langzeitstabilität der Propidium-modifizierten Sensoren erzielt wurde. Dieses Potential war deutlich geringer als die bisher publizierten Mediator-freien AChE-Biosensoren. Es wurde ein Vergleich verschiedener Organophosphate über ihre Inhibitionskonstanten durchgeführt, um diejenigen herauszufinden, die möglichst schnell mit dem aktiven Zentrum der Acetylcholinesterase reagieren. Das verwendete Messsystem beruht nicht auf der Vorinkubation der AChE und damit einer Einstellung des Inhibitionsgleichgewichts. Stattdessen wurde die Inhibition der AChE direkt im Fließsystem verfolgt. Daher war eine schnelle Inhibitionskinetik für einen empfindlichen Organophosphat-Nachweis erforderlich. Da einige Inhibitoren nur als Phosphothionat vorlagen, wurde die Überführung dieser Substanzen in die entsprechenden Oxo-Formen mittels N-Bromsuccinimid untersucht. Die NBS-Aktivierung wurde erfolgreich durchgeführt, die erwartete Inhibitionsstärke konnte jedoch aufgrund hydrolytischer Vorgänge nicht erreicht werden. Untersuchungen mit Diisopropylfluorophosphat (DFP) und Chlorpyriphos-oxon (CPO) konnten die Voruntersuchungen über die Inhibitionskinetik in Bezug auf die erreichten Nachweisgrenzen von 2E-06 M für DFP und 5E-08 M für CPO bestätigen. Für die chemische Modifizierung der Acetylcholinesterase wurde zunächst 2,4-Dichlorphenoxyessigsäure (2,4-D) als Hapten ausgewählt. 2,4-D wird als Herbizid eingesetzt und in der EU über die Gewässerschutzrichtlinie reguliert. 2,4-D konnte in unterschiedlichen molaren Verhältnissen von 2,6 : 1 bis 260 : 1 (2,4-D : AChE) nach Aktivierung mit einem Norbornendicarboximido-Derivat an die AChE gekoppelt werden. Dabei konnte die spezifische Aktivität der Acetylcholinesterase erhalten und die Bindung eines anti-2,4-D-Antikörpers ermöglicht werden. Zur Verstärkung des piezolelektrischen Signals der Antikörperbindung wurden die Immunoglobuline zunächst an Goldnanopartikel gekoppelt. Damit konnte eine Verstärkung um den Faktor 10 erreicht werden. Allerdings waren die Antikörper-modifizierten Goldnanopartikel nicht langzeitstabil. Daher wurden auch Silica-Nanopartikel als Matrix für die Antikörperkopplung getestet. Mit diesem System konnte eine Verstärkung um den Faktor von 5 bis 13 je nach Grad der Beladung den Nanopartikel mit Antikörper bestimmt werden. Die hohe unspezifische Bindung der Antikörper-Nanopartikel-Konjugate an den Propidium-modifizierten QCM-Sensor konnte keinen empfindlichen 2,4-D-Nachweis ermöglichen. Als Alternative wurde Kokain (Benzoylecgonin, BZE) als Hapten an die Aceytlcholinesterase gekoppelt. Da Kokain selbst auch als Inhibitor im aktiven Zentrum der AChE binden kann, wurden zwei verschiedene Strategien zur Konjugatsynthese verfolgt. Durch Zugabe von Kokain während der Kopplung sollte die kovalente Fixierung des Kokain-Derivats BZE-DADOO im aktiven Zentrum verhindert werden (Konjugat B). In der Tat konnten mit dieser Synthesestrategie 67% der spezifischen Cholinesterase-Aktivität erhalten werden, während im Kokain-freien Ansatz (Konjugat A) nur 2% der Ausgangsaktivität wiedergefunden wurden. Das BZE-AChE-Konjugat ermöglichte auch die Untersuchung der Bindungskinetik der anti-BZE-Antikörper. Dabei konnte eine Assoziationsgeschwindigkeitskonstante ka von 12911 l/(mol•s) berechnet werden. Dieser Wert ist trotz der vergleichsweise geringen Oberflächenbeladung vergleichbar mit den in der Literatur angegebenen Werten. Die Dissoziationsgeschwindigkeitskonstante ist mit 2,89E−3 1/s um den Faktor 30 höher als der Literaturwert. Diese Abweichung ist auf Unterschiede im Bindungsmodell zurückzuführen. Mit beiden BZE-AChE-Konjugaten konnte ein kompetetiver Immunoassay mit Kokain im Fließsystem durchgeführt werden. Dabei zeigte sich für beide Konjugate ein ähnlicher Testmittelpunkt: IC50 = 4,40E−8 mol/l für Konjugat A bzw. IC50 = 1,77E−8 mol/l für Konjugat B. Diese Werte sind vergleichbar zu bereits publizierten Kokainassays im Fließsystem. Wie vorstehend beschrieben, bindet Kokain als Inhibitor auch im aktiven Zentrum von Cholinesterasen. Diese Eigenschaft wurde genutzt, um ein zweites Enzym – Butyrylcholinesterase (BChE) – an die BZE-AChE zu binden. Die Spezifität dieser Bindung konnte durch die Abwesenheit einer Affinität der BChE zum Propidium und durch die Blockierbarkeit der Bindung von BChE und BZE-AChE durch Kokain nachgewiesen werden. Damit konnte erfolgreich die Kombination mehrere molekularer Erkennungselemente demonstriert werden. Die Propidium-Plattform ermöglicht den Aufbau einer Architektur aus verschiedenen Cholinesterasen, die über unterschiedliche Bindungsstellen wechselwirken. Sowohl freie als auch BZE-modifizierte AChE können über die Affinität zum Propidium auf dem EQCM-Sensor immobilisiert werden. Mit Kokain als Substrat der Butyrylcholinesterase kann Benzoylecgonin nicht nur als Epitop für die Bindung eines Antikörpers, sondern auch als Erkennungselement für die BChE genutzt werden. Auf der anderen Seite erschwert die geringe Affinität der BChE im Gegensatz zum anti-BZE-Antikörper den Einsatz dieses Systems für analytische Zwecke. Durch die Verwendung anderer Ligand-Enzym-Kombinationen läßt sich das in dieser Arbeit vorgestellte Konzept noch weiter ausbauen und ermöglicht damit eine Entwicklung ausgehend von „einfachen“ molekularen Erkennungselementen (MRE) hin zu „multifunktionellen“ Erkennungselementsystemen. In dieser Arbeit konnte demonstriert werden, dass der Aufbau solch komplexe Systeme möglich ist, ohne Abstriche in Bezug auf die Empfindlichkeit der einzelnen Assays hinzunehmen. / This work describes the development of a bifunctional biosensor following a modular assembly approach. This aim is reached through the combination of various molecular recognition elements. The system presented herein uses the following recognition elements: • propidium and the peripheral anionic site of the acetylcholinesterase (AChE) • an organophosphate and the active site of the AChE • a hapten – covalently coupled to the AChE – and the epitope of an antibody • a hapten – covalently coupled to the AChE – binding as a ligand to another enzyme A biosensor is not only characterized by the molecular recognition element, but also by the type of signal transducer. This work is based on an electrochemical quartz crystal microbalance (EQCM) device that uses gold-plated quartz sensors for the signal transduction. This allows monitoring two distinct signals at the same time: the piezoelectric determination of a mass loading and the amperometrical detection of enzymatic activity on the sensor surface. Thus two different assay systems are provided: the determination of the inhibition of the AChE activity and ligand binding assay via the hapten. Both tests are based on the modification of the piezoelectric crystals with propidium – a reversible AChE inhibitor. This allows the deposition of AChE on the sensor surface via the interaction with the enzyme’s peripheral anionic site. The enzymatic activity of the in-situ immobilized AChE and the inhibition by organophosphates (pesticides) are measured amperometrically. Another recognition element is introduced by the chemical coupling of a hapten to the cholinesterase. This provides the opportunity bind an antibody to the haptenized cholinesterase that is immobilized on the propidium-modified sensor. Preliminary experiments were focussed on the improvement of the amperometric determination the AChE activity. The applied potential for the oxidation of thiocholine was changed over a range from 150 to 300 mV. The best results for the measured oxidation current and the long-term stability of the propidium-modified sensors were obtained at 200 mV (vs. Ag/AgCl). This potential was used throughout all subsequent experiments. This potential was also found to be lower as compared to mediator-free AChE-biosensors published hitherto. Different organophosphates were evaluated with regard to their inhibition constants to find those which react with active site of the acetylcholinesterase as fast as possible. The assay format used herein monitors the inhibition of the AChE directly in the flow-system. That is, it is not based preincubation of the enzyme with the inhibitor and therefore no inhibition equilibrium is reached. This approach requires fast inhibition kinetics in order to detect the organophosphates highly sensitively. Some of the inhibitors were only available in the phosphothionate form. Thus was necessary to convert these compounds to their respective oxon-forms by N-bromosuccinimide (NBS). The NBS-activation was performed successfully, though the expected inhibition potential could not be reached due to hydrolytic processes. Experiments with diisopropylfluorophosphate (DFP) und chlorpyriphos-oxon (CPO) could confirm the previous experiments on the inhibtion kinetics. Lower limits of detection of 2E-06 M for DFP and 5E-08 M for CPO could be reached with this approach. Initially 2,4-dichlorphenoxyacetic acid (2,4-D) was chosen as a hapten for the chemical modification of the acetylcholinesterase. The use of 2,4-D as a herbicide is regulated by the water protection directive of the European Union. 2,4-D was coupled to AChE in different molar ratios from 2,6 : 1 to 260 : 1 (2,4-D : AChE) after activation with a norbornendicarboximido derivative. The chosen coupling method allowed to recover the complete specific activity of the acetylcholinesterase and to bind a specific anti-2,4-D-antibody. Furthermore, the coupling of the immunoglobulins to gold nanoparticles was tested to enhance the piezoelectric signal of the antibody binding. An amplification factor of 10 was reached with this system. However the antibody-coated gold nanoparticles show a very poor long-term stability. Therefore also silica nanoparticles were tested as a matrix for the coupling of the antibodies. This approach yielded an amplification factor from 5 to 13 depending amount of antibodies bound to the nanoparticles. Unfortunately the high non-specific binding of the antibody nanoparticle conjugates did not allow a sensitive 2,4-D detection assay. Cocaine (benzoylecgonine, BZE) was coupled as a hapten to Acetylcholinesterase in an alternative approach. Two different strategies for the synthesis of the conjugate were pursued, since cocaine can bind also bind as an inhibitor for the AChE’s active site. The addition of excess cocaine during the coupling reaction should the covalent binding of the cocaine derivative BZE-DADOO at the active site (conjugate B). Indeed over two thirds of the original specific cholinesterase activity could be recovered with this strategy, while the cocaine-free batch (conjugate A) showed only 2% of the original activity. Furthermore the BZE-AChE conjugate allowed the evaluation of the binding kinetics of the anti-BZE-antibody. The association rate constant ka was calculated to 12911 l/(mol•s). Despite the low surface coverage this value is still comparable to other published results. The dissociation rate constant kd of 2,89E−3 1/s is thirty times higher than values found in the literature. This deviation is due to differences in the applied binding model. Both BZE-AChE conjugates could be applied in a competitive immunoassay for cocaine in the flow system. It was shown that for both conjugates a similar half maximal inhibitory concentration was reached: : IC50 = 4,40E−8 mol/l for conjugate A and IC50 = 1,77E−8 mol/l for conjugate B, respectively. These values are comparable to other published assay for cocaine in a flow system. As described earlier, cocaine is also able to bind to the active site of cholinesterases. This feature was used to examine the interaction of a second enzyme – butyrylcholinesterase (BChE) – with the BZE-AChE. Evidence for the specificity of this interaction was provided by two further experiments, i.e. BChE has no affinity towards Propidium and the binding of BChE towards BZE-AChE could be blocked by excess cocaine. Thus the successful integration of recognition elements on the molecular level could be demonstrated. The propidium-modifies sensor allowed the construction of a scaffold of cholinesterases that interact via different recognition sites. Unmodified and BZE-coupled AChE can be immobilized on the EQCM-sensor via the interaction with propidium. With cocaine being a substrate BChE this compound cannot only be used to capture anti-BZE-antibodies, but also as a recognition element for BChE. The affinity of the BChE towards is relatively low as compared to the antibody’s binding strength, thus making it difficult to employ this system for analytical purposes. Still the concept presented herein can be extended by other ligand-enzyme-combinations. On the basis of “simple” molecular recognition elements this enables the development of “multifunctional” recognition element systems. This work could show that the construction of such complex systems is possible without cutting back with regard to the sensitivity of the individual assays.

<p>Background: Organophosphate (OP) pesticides are well-known developmental neurotoxicants that have been linked to abnormal cognitive and behavioral endpoints through both epidemiological studies and animal models of behavioral teratology, and are implicated in the dysfunction of multiple neurotransmitters, including dopamine. Chemical similarities between OP pesticides and organophosphate flame retardants (OPFRs), a class of compounds growing in use and environmental relevance, have produced concern regarding whether developmental exposures to OPFRs and OP pesticides may share behavioral outcomes, impacts on dopaminergic systems, or both. Methods: Using the zebrafish animal model, we exposed developing fish to two OPFRs, TDCIPP and TPHP, as well as the OP pesticide chlorpyrifos, during the first 5 days following fertilization. From there, the exposed fish were assayed for behavioral abnormalities and effects on monoamine neurochemistry as both larvae and adults. An experiment conducted in parallel examined how antagonism of the dopamine system during an identical window of development could alter later life behavior in the same assays. Finally, we investigated the interaction between developmental exposure to an OPFR and acute dopamine antagonism in larval behavior. Results: Developmental exposure to all three OP compounds altered zebrafish behavior, with effects persisting into adulthood. Additionally, exposure to an OPFR decreased the behavioral response to acute D2 receptor antagonism in larvae. However, the pattern of behavioral effects diverged substantially from those seen following developmental dopamine antagonism, and the investigations into dopamine neurochemistry were too variable to be conclusive. Thus, although the results support the hypothesis that OPFRs, as with OP pesticides such as chlorpyrifos, may present a risk to normal behavioral development, we were unable to directly link these effects to any dopaminergic dysfunction.</p> / Dissertation

Oximas têm sido extensivamente usadas como antídoto para envenenamento por organofosforados e como desontaminante. Micelas e vesículas, utilizadas como catalisadores e transportadores de drogas, constituem agentes potenciais para tratamento e descontaminação. Neste trabalho descrevemos a reação de p-Nitrofenildifenilfosfato (PNPDPP), um substrato modelo para organofosforado, com: acetofenoxima (I); ácido 10- fenil-10-hidroxiiminodecanóico (II); 4-(9-carboxinonanil)-1-(9-carboxi-1-hidroiimino nonanil) benzeno (III); cloreto de N-dodecilpiridina (IV); cloreto N-metilpiridina 2-aldoxima (V), na presença de micelas catiônicas e zwitteriônicas de cloreto de hexadeciltrimetilamônio, CTAC e N-Hexadecil-N,N-dimetil-1-propano sulfonato, HPS, respectivamente, e vesículas catiônicas de dioctadecildimetilamônio, DODAC. O pKa aparente, pKap, das oximas em agregados de anfifilicos, a constante de velocidade de segunda ordem de oximólise em micelas ou vesículas, km, e as constantes de velocidade observadas para a oximólise de PNPDPP, kobs, foram determinadas espectrofotometricamente, a pH constante, variando-se a concentração dos anfifílicos. Os resultados foram analisados usando as teorias: modelo de pseudofase (PP) e modelo de pseudofase com considerações de troca iônica (PIE), descrita na literatura pelo nosso grupo. As constantes de segunda ordem para oximólise de PNPDPP em água, kox, determinadas foram 6,5 M^-1 min^-1 (I, II e III) e 2,8 M^-1 min^-1 (IV e V). O kobs máximo em micelas e vesículas, kobsmax, e o kobs em água, kw, no mesmo pH, foram utilizadas para calcular o fator de aceleração máxima, AF, para cada anfifílico (AF = kobsmax/kw). Os agregados catalisam a decomposição de PNPDPP e os valores de AF (e km) foram da ordem de 10^4 (32 min^-1), 10^4 (125 min^-1) e 10^6 (80 min^-1) para a reação da oxima IV com CTAC, HPS e DODAC, respectivamente. A análise quantitativa da dependência da concentração de agregados anfifílicos na oximólise mostrou um considerável aumento da constante de velocidade da reação produzido por micelas e vesículas (maior que 8 x 10^6 vezes). Esse efeito é parcialmente devido a: concentração local dos reagentes, efeitos nos pKas dos nucleófilos e, mais importante, mudança na reatividade intrínseca das oximas. / Oximes have been extensively used as antidotes and decontaminants of organophosphates. Micelles and vesicles, catalysts and drug transport agents, constitute potential vehicles for Oxime treatment. Here we describe the reaction of p-nitrophenyldiphenylphosphate (PNPDPP) with: acetophenoxime (I); 10-phenyl-10-hydroxyiminodecanoic acid (II); 4-(9-carboxynonanyl)-1-(9-carboxy-1-hydroyiminononanyl) benzene (III); N-dodecylpyridinium chloride (IV); N-methylpyridinium 2-aldoxime chloride (V), in the presence of cationic and zwitterionic micelles, hexadecyltrimethylammonium chloride, CTAC and N-Hexadecyl-N,N-dimethyl-1-propanesulfate, HPS, respectively, and cationic vesicles of dioctadecyldimethylammonium, DODAC. The apparent pKa, pKap, of the oximes in the amphiphile aggregates, the second order rate constants of oximolysis in micelles and vesicles, km, and the observed rate constants for PNPDPP oximolysis, kobs, were determined spectrophotometrically at constant varying amphiphilic concentrations. The results were analyzed using the pseudo-phase theory (PP) and pseudo-phase / ion exchange (PIE). The second order rate constant for (uncatalyzed) oximolysis of PNPDPP were 6.5 M^-1 min^-1 (I, II and III) and 2.77 M^-1 min^-1 (IV and V). From the maximum value of kobs in micelles and vesicles, kobsmax, and the value of kobs in water, kox, at the same pH, the maximum acceleration factor, AF, were calculated (AF = kobsmax / kw). The amphiphiles catalyzed the oximolysis of PNPDPP and the values of AF (and km) were ca 10^4 (32 min^-1), 10^4 (125 min^-1) and 10^6 (80 min^-1) for the reactions of Oxime IV in CTAC, HPS and DODAC, respectively. Quantitative analysis of the amphiphile concentration-dependence of rates demonstrated that the considerable rate increase produced by micelles and vesicles on the rate of oximolysis (up to 8 x 10^6 fold) is partly due to reagent concentration in the aggregate, effects on the pKas of the nucleophiles and, more importantly, catalysis.

Alzheimer’s disease (AD) is a devastating neurodegenerative disease that affects millions of peoples’ lives worldwide. While the consequences of AD are recognizable, the etiology is unclear. Gene-environment interactions have been implicated in the development of the disease, and exposure to organophosphorus (OPs) compounds is one of the environmental factors associated with AD. Evidence links exposure to levels of OPs encountered in agriculture, horticulture, and other work places with neurodegenerative disease, psychiatric illness, and sensorimotor deficits. Unfortunately, the mechanisms underlying these effects have yet to be established. Here, we set out to examine the long-term consequences of exposure to a commonly applied OP insecticide, chlorpyrifos (CPF), in an attempt to identify a causal link between occupational exposures and chronic illnesses. We exposed a transgenic rodent model of AD, TgF344-AD, to levels of CPF representing occupational exposures and examined ensuing behaviors and neuropathologies. We observed a sex-specific, biphasic response in CPF-exposed animals, including acute neurotoxicities, followed by intermediate recovery, and finally, chronic cognitive impairments. CPF exposure exacerbated neuronal damage in brain regions critical to the impaired behaviors, and neuroinflammatory pathways were identified as facilitators of this damage. This work emphasizes the long-term consequences of early life repeated exposures to OPs and identifies dysregulated microglia as a potential deleterious modifier of disease. Additionally, we investigated the efficacy of a neuroprotective compound, (-)-P7C3-S243 in TgF344-AD rats. P7C3 compounds exert protection by preventing young hippocampal neurons from dying prematurely and also enhancing flux of nicotinamide adenine dinucleotide (NAD), thereby aiding in neuron survival under conditions that normally cause axon degeneration and cell death. These compounds have proven effective in preclinical models of Parkinson’s disease, amyotrophic lateral sclerosis, and traumatic brain injury. Thus, we sought to investigate the neuroprotective efficacy of P7C3 compounds in AD, as well. (-)-P7C3-S243 was administered to wild-type and transgenic male and female rats daily for 9 and 18 months, and classic hallmarks of the disease were assessed. Transgenic rats developed a spectrum of AD pathologies and behaviors, as expected, and (-)-P7C3-S243 ameliorated early depression-like behaviors, late learning and memory deficits, and progressive neuronal damage in this model, without influencing amyloid plaque deposition, tauopathies, or neuroinflammation. This data suggests that targeting neuronal cell death pathways is a promising treatment strategy in AD. Taken together, the research presented here expands our current understanding of pathways of regulation in Alzheimer’s disease—organophosphates are capable of exacerbating the severity of AD, while P7C3 compounds are promising therapeutic candidates for neuronal death in the disease. Given the overlapping molecular pathways of modulation in CPF-induced toxicity and (-)-P7C3-S243 neuroprotection in AD, future studies will investigate the efficacy of (-)-P7C3-S243 in cognitive deficits induced by CPF exposure. Ultimately, this body of work highlights the plasticity of neuronal cell death and cognitive impairment in AD, thus indicating a better understanding of these pathways could facilitate vastly improved intervention strategies in Alzheimer’s disease.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A importÃncia dos praguicidas como agente causal de intoxicaÃÃes e Ãbitos à evidenciado pelo nÃmero de ocorrÃncias registradas, principalmente nos paÃses em desenvolvimento. No Brasil, os praguicidas ocupam a terceira posiÃÃo dentre os agentes responsÃveis por intoxicaÃÃes agudas. Eles agem por inibiÃÃo enzimÃtica (acetilcolinesterase) de modo irreversÃvel, podendo provocar aparecimento de sinais e sintomas colinÃrgicos caracterÃsticos. Podem causar ainda o aparecimento da sÃndrome intermediÃria e da polineuropatia tardia. Este estudo teve como objetivos traÃar o perfil epidemiolÃgico dos pacientes intoxicados no estado do CearÃ, no perÃodo de fevereiro a julho de 2004, atendidos no Centro de AssistÃncia ToxicolÃgica do Cearà (CEATOX); avaliar o tratamento proposto; verificar os sinais e sintomas das sÃndromes colinÃrgica e intermediÃria e analisar os fatores de risco associados à evoluÃÃo dos pacientes. Foi realizado um levantamento de dados preliminares referentes a janeiro de 2002 atà junho de 2003. A coleta dos dados foi realizada a partir dos prontuÃrios mÃdicos, fichas de notificaÃÃo do CEATOX e entrevista com o paciente ou responsÃvel. Foram analisados 19 pacientes, sendo 10 (52,6%) do sexo masculino e 9 (47,4%) do feminino. A mÃdia de idade foi 27,8Â11,4 anos. A ocupaÃÃo trabalhador agrÃcola representou 42,1% (8/19). As tentativas de suicÃdio representaram 84,2% (16/19) dos casos. Houve 4 (21,1%) Ãbitos. As principais manifestaÃÃes clÃnicas foram diarrÃia (78,9%), miose (57,9%) e sialorrÃia(52,6%). Fez-se dosagem da AChE na admissÃo dos pacientes intoxicados, sendo mÃdia e desvio-padrÃo 1,06Â1,50 UI/mL. As demais dosagens foram feitas no decorrer do internamento de cada paciente. Dentre as complicaÃÃes clÃnicas, as do trato respiratÃrio foram as mais importantes (57,2%). Atropina foi administrada em 16 (84,2%) dos pacientes. A mÃdia de internaÃÃo foi 8,6Â8,5dias. Houve associaÃÃo estatÃstica quando se comparou ventilaÃÃo, tempo de UTI e tipo de intoxicaÃÃo com o surgimento de complicaÃÃes clÃnicas (p<0,05) (teste do qui-quadrado e exato de Fisher). A regressÃo logÃstica nÃo mostrou significÃncia estatÃstica quando analisou risco de Ãbito a partir das variÃveis em estudo. Os resultados mostraram que se faz necessÃrio uma reestruturaÃÃo no protocolo de atendimento e tratamento dos pacientes intoxicados com IOF, bem como sua divulgaÃÃo no hospital Instituto Dr. Josà Frota e em outras unidades hospitalares. Os resultados demonstram ainda a situaÃÃo crÃtica quanto ao uso inadequado de praguicidas organofosforados no estado do Cearà e a necessidade de orientaÃÃo ao consumidor e adequada vigilÃncia sanitÃria quanto ao uso e comercializaÃÃo dos mesmos. / The importance of the pesticides causal agent of intoxication and deaths is clearly shown by the number of registered cases, mainly in developed countries. In Brazil, pesticides places the third more important group of substances responsible for acute intoxication. They act through enzymatic inhibition (acetylcholinesterase) in an irreversible way, leading to characteristic cholinergic signs and symptoms. They are also able to cause the intermediary syndrome and the delayed polyneuropathy. This study aimed to set out the poisoning patientsâ epidemiological outline in Cearà state, observed from February to July 2004, who received medical care at Centro de AssistÃncia ToxicolÃgica do Cearà (CEATOX); evaluating the proposed treatment; verifying the signs and symptoms related to the cholinergic and intermediary syndromes; analyzing the risk factors matched to patients recovery. Some preliminary data were obtained from January 2002 to June 2003 concerning to these poisoning events. The data collection was performed from medical recording, CEATOX notification files and patients or their companion interviews. 19 patients were analyzed, 10 (52,6%) men and 9 (47,4%) women. The average age was 27,8  11,4. Agriculturists represented 42,1% (8/19) of all cases. Attempted suicide constituted 84,2% (16/19). There were 4 (21,1%) deaths. The main clinical observations were diarrhea (78,9%), myosis (57,9%) and sialorrhea (52,6%). The AChE levels were measured immediately after admission and expressed in terms of mean  standard deviation (1,06  1,50 UI/mL). The other AChE measurements were performanced during the permanence of the patients in the hospital. Among clinical complications, those ones related to the respiratory tract were the most relevant (57,2%). Atropine was administered to 16 (84,2%) patients. The average time of admission was 8,6  8,5 days. There was statistical association when were compared ventilation, time spent in the ICU and type of intoxication with clinical complications (p<0,05) (Chi-square test and Fisherâs test). The logistic regression did not show statistical differences when the death risk was analyzed comparing to the studied variables. The results stress the need to adopt more suitable measures with a restructured protocol in handling patients intoxicated with IOF, giving ample publicity at the hospital units of Dr. Josà Frota Institute and others. The data obtained also point out the critical situation in Cearà State in the use, misure or inappropriate vigilance by the sanitary authorities.

Oximas têm sido extensivamente usadas como antídoto para envenenamento por organofosforados e como desontaminante. Micelas e vesículas, utilizadas como catalisadores e transportadores de drogas, constituem agentes potenciais para tratamento e descontaminação. Neste trabalho descrevemos a reação de p-Nitrofenildifenilfosfato (PNPDPP), um substrato modelo para organofosforado, com: acetofenoxima (I); ácido 10- fenil-10-hidroxiiminodecanóico (II); 4-(9-carboxinonanil)-1-(9-carboxi-1-hidroiimino nonanil) benzeno (III); cloreto de N-dodecilpiridina (IV); cloreto N-metilpiridina 2-aldoxima (V), na presença de micelas catiônicas e zwitteriônicas de cloreto de hexadeciltrimetilamônio, CTAC e N-Hexadecil-N,N-dimetil-1-propano sulfonato, HPS, respectivamente, e vesículas catiônicas de dioctadecildimetilamônio, DODAC. O pKa aparente, pKap, das oximas em agregados de anfifilicos, a constante de velocidade de segunda ordem de oximólise em micelas ou vesículas, km, e as constantes de velocidade observadas para a oximólise de PNPDPP, kobs, foram determinadas espectrofotometricamente, a pH constante, variando-se a concentração dos anfifílicos. Os resultados foram analisados usando as teorias: modelo de pseudofase (PP) e modelo de pseudofase com considerações de troca iônica (PIE), descrita na literatura pelo nosso grupo. As constantes de segunda ordem para oximólise de PNPDPP em água, kox, determinadas foram 6,5 M^-1 min^-1 (I, II e III) e 2,8 M^-1 min^-1 (IV e V). O kobs máximo em micelas e vesículas, kobsmax, e o kobs em água, kw, no mesmo pH, foram utilizadas para calcular o fator de aceleração máxima, AF, para cada anfifílico (AF = kobsmax/kw). Os agregados catalisam a decomposição de PNPDPP e os valores de AF (e km) foram da ordem de 10^4 (32 min^-1), 10^4 (125 min^-1) e 10^6 (80 min^-1) para a reação da oxima IV com CTAC, HPS e DODAC, respectivamente. A análise quantitativa da dependência da concentração de agregados anfifílicos na oximólise mostrou um considerável aumento da constante de velocidade da reação produzido por micelas e vesículas (maior que 8 x 10^6 vezes). Esse efeito é parcialmente devido a: concentração local dos reagentes, efeitos nos pKas dos nucleófilos e, mais importante, mudança na reatividade intrínseca das oximas. / Oximes have been extensively used as antidotes and decontaminants of organophosphates. Micelles and vesicles, catalysts and drug transport agents, constitute potential vehicles for Oxime treatment. Here we describe the reaction of p-nitrophenyldiphenylphosphate (PNPDPP) with: acetophenoxime (I); 10-phenyl-10-hydroxyiminodecanoic acid (II); 4-(9-carboxynonanyl)-1-(9-carboxy-1-hydroyiminononanyl) benzene (III); N-dodecylpyridinium chloride (IV); N-methylpyridinium 2-aldoxime chloride (V), in the presence of cationic and zwitterionic micelles, hexadecyltrimethylammonium chloride, CTAC and N-Hexadecyl-N,N-dimethyl-1-propanesulfate, HPS, respectively, and cationic vesicles of dioctadecyldimethylammonium, DODAC. The apparent pKa, pKap, of the oximes in the amphiphile aggregates, the second order rate constants of oximolysis in micelles and vesicles, km, and the observed rate constants for PNPDPP oximolysis, kobs, were determined spectrophotometrically at constant varying amphiphilic concentrations. The results were analyzed using the pseudo-phase theory (PP) and pseudo-phase / ion exchange (PIE). The second order rate constant for (uncatalyzed) oximolysis of PNPDPP were 6.5 M^-1 min^-1 (I, II and III) and 2.77 M^-1 min^-1 (IV and V). From the maximum value of kobs in micelles and vesicles, kobsmax, and the value of kobs in water, kox, at the same pH, the maximum acceleration factor, AF, were calculated (AF = kobsmax / kw). The amphiphiles catalyzed the oximolysis of PNPDPP and the values of AF (and km) were ca 10^4 (32 min^-1), 10^4 (125 min^-1) and 10^6 (80 min^-1) for the reactions of Oxime IV in CTAC, HPS and DODAC, respectively. Quantitative analysis of the amphiphile concentration-dependence of rates demonstrated that the considerable rate increase produced by micelles and vesicles on the rate of oximolysis (up to 8 x 10^6 fold) is partly due to reagent concentration in the aggregate, effects on the pKas of the nucleophiles and, more importantly, catalysis.

Master of Science / Department of Biology / Brett K. Sandercock / Monitoring programs indicate that numerous shorebird populations are subject to on-going declines. The U.S. Shorebird Conservation Plan lists twenty-seven shorebird species as species of high concern and seven as highly imperiled, including the Buff-breasted Sandpiper (Tryngites subruficollis). One hypothesis for ongoing population declines is exposure to toxic chemicals and pollutants. The purpose of this project was to characterize plasma cholinesterases (ChEs) of migratory shorebirds and address potential exposure to organophosphorus (OP) and carbamate (CB) pesticides. Consumption or contact with these pesticides can cause mortality and a variety of sub-lethal effects. Buff-breasted Sandpipers and other upland shorebirds are particularly likely to encounter agrochemicals due to their habitat use at the non-breeding grounds. I sampled migratory shorebirds over three seasons, during north- and southbound migration in 2006 and 2007 in Texas, Kansas, and Nebraska and during the non-breeding season in 2007 in Argentina, Uruguay, and Paraguay. I collected blood samples and footwashings from reference sites, where OP and CB pesticides were not used, and agricultural sites, where these two insecticides were recommended for control of crop pests. I assessed several variables known to affect plasma ChE activity including body size, date of capture, time of capture, condition, sex, and region. Small-bodied species had higher levels of ChE activity in plasma than large-bodied species. Plasma ChE activities varied with date of capture in 3 of 5 species sampled in North America. Sex differences were significant in 1 of 4 species tested. Plasma acetylcholinesterase (AChE) activity was higher among White-rumped Sandpipers sampled in North America but there was no difference between regions among Buff-breasted Sandpipers. Time of capture and individual condition did not affect plasma ChE activity. Estimates of exposure to ChE inhibitors were addressed in five species. Plasma AChE and butyrylcholinesterase (BChE) activities of Buff-breasted Sandpipers were lower at agricultural sites in South America but BChE activity was higher at agricultural sites in North America. There were no differences between sites in four other species tested. A meta-analysis across all species indicated that in 4 of 6 comparisons habitat type had a negative effect on AChE activity consistent with exposure to ChE inhibitors but there was a regional positive effect of agricultural habitat on BChE activity in North America. Comparison of body mass between sites suggested that use of habitats with potential pesticide application did not affect mass gain. Project results suggest that 1 of 5 shorebird species tested was exposed to ChE-inhibiting pesticides at the non-breeding grounds and future monitoring is necessary to assess potential effects at the population level. This study highlights the importance of complete sampling and addressing variability in plasma ChEs before making estimates of exposure to OP and CB pesticides. It provides the first estimates of migratory shorebird exposure to OP and CB pesticides, a potential conservation issue. Future research should include continued monitoring of Buff-breasted Sandpiper ChE levels and habitat use. Other sources of anthropogenic declines such as habitat loss and illegal hunting should be investigated for species that did not show evidence of exposure.

Flame retardants are substances, which addition in various materials (furniture, plastics, electronics equipment, textiles, etc) could save a lot of lives and injuries caused by fires. On the other side, the migration of flame retardants from products during their whole life cycle results in their ubiquitous presence in the environment and reflects negative effects on ecosystems and human health. Global consumption of organophosphate flame retardants (OFR) as alternative substitutes of polybrominated diphenyl ethers has increased sharply in recent years. Studies on the presence and sources of OFR in surface water, ground water, sediments, snow, rainwater, indoor and outdoor air and analyses of OFR in these compartments have also increased in the last decade. In this doctoral thesis an analytical method was developed to determine six OFR (tris(2-chloroethyl) phosphate (TCEP), tris(2-chlorisopropyl) phosphate (TCPP), tris(1,3-dichloro-2-propyl) phosphate (TDCP), tris(2-butoxyethyl) phosphate (TBEP), tri(n-butyl) phosphate (TnBP) and triphenyl phosphate (TPP)) in soil. The method consists of a combination of Twisselmann extraction and solid-phase microextraction (SPME), followed by gas chromatography-mass spectrometry (GC-MS). To develop the method, spiked soils were extracted using a Twisselmann extractor after freeze-drying. The extracts were evaporated to dryness, redissolved, and filtered. A volume of 7 mL was then analysed by SPME, followed by GC-MS. The effects of different parameters on analyte recoveries during sample preparation e.g. solvent for Twisselmann extraction, solvent for redissolving the extract, addition of copper, and filtration of the extract were systematically investigated. Under optimum conditions, 10 g of soil were extracted using toluene, and the extract was redissolved in methanol/water (1:14) and filtered. It was not necessary to add copper. For TnBP, TBEP, TCPP, and TCEP, recoveries ranged from 77.0 % to 89.6 %. Those for TPP and TDCP were much lower, at 31.5 % and 42.0 %, respectively (addition level 22.9-45.8 ng/g). The variability of recoveries under these conditions was between 0.3 and 16.2 % (n = 3). Limits of detection (LOD) were 0.002-3 ng/g. When ultrasonication was used instead of Twisselmann extraction in the developed method, recoveries were three to four times lower (27.4 % to 30.6 %), but the variability of recoveries was below 3 % (n = 3). The method was applied to quantify OFR in soils collected from different sampling locations (urban, semi-urban and rural) in Germany. The results indicated for the first time that atmospheric deposition leads to soil contamination by OFR. Since it has been shown in animal experiments (F344/N rats and B6C3F1 mice) that chlorinated OFR were carcinogen and also have negative effects on human health (Matthews et al., 1991, 1993, Johnson, 1999), the further studies were focused on sources of chlorinated OFR. Therefore, the influence of dry and wet deposition processes as a source of chlorinated OFR in soils was systematically investigated. Soil samples were collected in 2010/11 during a period of snow falling to snow melting, a period of rainfall and a dry period. Snow and rainwater samples were also collected from the soil sampling site. Concentrations of TCEP were between 236 and 353 ng/L in snow and 78 and 234 ng/L in rain. TCPP concentrations were between 226 and 284 ng/L in snow and 371 and 385 ng/L in rain. In soil samples, concentrations ranged from 5.07 to 23.48 ng/g dry weight (dwt) for TCEP and 5.66 to 19.82 ng/g dwt for TCPP. Concentrations of TDCP in rainwater and snow samples were rather low (46 and 100 ng/L, respectively); concentrations of TDCP were below the limit of detection in soil samples. Snow melting caused enhanced soil concentrations of TCEP and TCPP. However a greater effect of snow melting was observed for TCEP than for TCPP. No significant correlation between precipitation amounts and soil concentrations was observed for both compounds. The influence of wet deposition to the soil contents of TCEP and TCPP may be covered by volatilisation or by the migration of both compounds to deeper soil zones with seepage water, based on their volatility and high water solubility, respectively. Snow was found to be even a more efficient source of chlorinated OFR in soil than rainwater. During dry weather, the soil concentrations of both compounds seemed to be driven mainly by concentrations in air, which are driven by source emission strengths and photochemical degradation in the atmosphere. Rainwater concentrations of OFR were used to assess air concentrations from the scavenging ratios at equilibrium conditions and the potential for the accumulation of OFR in soil based on the air-soil exchange was estimated. Calculated values of median air concentrations were 0.0034 ng/m3 for TCEP and 0.99 ng/m3 for TCPP. Total OFR specific loads were 3756 ng m-2 day-1 within the first 24 hours and 3028 ng m-2 day-1 within the next 24 h. Fugacity calculations (0.011 to 0.103 for TCPP and 0.005 to 0.073 for TCEP) indicated net deposition from air to soil for both compounds.

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