Carboxylesterases (CEs) are nonspecific hydrolytic enzymes and responsible for the metabolism of xenobiotics and endobiotics that contain ester bonds. There are two human CE isoforms found in liver, CES1 and CES2. In this study it is shown that the mere abundance of CES1 protein expression in human liver does not predict its biochemical activity. The human interindividual variation in CES1 activities may attribute to several mechanisms. One possibility is the presence of endogenous inhibitors in liver, arachidonic acid (AA) and 27-hydroxycholesterol (27-HC). CES1 is also expressed in human monocytes/macrophages and is proposed to catalyze the rate-limiting step of cholesterol ester mobilization in macrophages. It is of interest to determine whether CES1 can degrade the lipid mediators, 2-arachidonoylglycerol (2-AG), prostaglandin E2-1-glyceryl ester (PGE2-G), and prostaglandin F2α-1-glyceryl ester (PGF2α-G), in monocytes/macrophages and to determine if this metabolism is inhibited by organophosphate pesticide exposure.

No / The human epidermis holds an autocrine acetylcholine production and degradation including functioning membrane integrated and cytosolic butyrylcholinesterase (BuchE). Here we show that BuchE activities increase 9-fold in the presence of calcium (0.5 × 10-3 M) via a specific EF-hand calcium binding site, whereas acetylcholinesterase (AchE) is not affected. 45Calcium labelling and computer simulation confirmed the presence of one EF-hand binding site per subunit which is disrupted by H2O2-mediated oxidation. Moreover, we confirmed the faster hydrolysis by calcium-activated BuchE using the neurotoxic organophosphate O-ethyl-O-(4-nitrophenyl)-phenylphosphonothioate (EPN). Considering the large size of the human skin with 1.8 m2 surface area with its calcium gradient in the 10¿3 M range, our results implicate calcium-activated BuchE as a major protective mechanism against suicide inhibition of AchE by organophosphates in this non-neuronal tissue

Organophosphates (OPs) were initially designed as insecticides and later engineered as dangerous nerve agents that threaten our livelihood and safety. One of the treatments for OP exposure is the administration of an oxime to reactivate inhibited acetylcholinesterase (AChE). The currently approved oxime therapy in the US, pralidoxime (2-PAM), is unable to reactivate inhibited AChE in the brain due to its low ability to cross the blood-brain barrier (BBB) which has led our laboratory to develop novel substituted phenoxyalkyl pyridinium oximes (US Patent 9,227,937) that penetrate the BBB more effectively. Our lead oxime candidate, Oxime 20, has proven efficacious in reactivating inhibited AChE both in vitro and in vivo and is in the preliminary steps of drug development which require metabolism studies such as pharmacokinetics (PK), protein-binding (PB) and metabolite identification. PK parameters were explored for Oxime 20 and found to have an average half-life of 11.6 hours and an average Tmax of 0.083 hours in rats and an average half-life of 15 hours and an average Tmax of 0.11 hours in minipigs. As compared to 2-PAM, our oxime has displayed a 2-4 times longer half-life and a 3 times faster Tmax which allows it to be distributed at a faster rate and stay in circulation for longer. Furthermore, Oxime 20 was found to be >84% plasma protein-bound as compared to 2-PAM which was <8% protein-bound. These PB characteristics align with the PK parameters as highly protein-bound drugs tend to have a longer half-life than low protein-bound drugs. Finally, our oxime displayed a potentially safe metabolism in the presence of microsomes with the generation of two more polar metabolites as compared to Oxime 20, a hydroxyl metabolite and a carboxylic acid metabolite. With these findings, Oxime 20 continues to show promise and excellent characteristics for drug development and potentially will be the next suitable and effective option for treatment of OP exposure either by itself or in combination with 2-PAM.

Phthalate esters (PEs) and organophosphate esters (OPEs) are common indoor pollutants frequently detected in environmental (dust, air), personal (hand wipes, diet) and human matrices (urine, serum etc.). In this thesis, mathematical models were used to establish links between intake and body burden for a comprehensive dataset based on a Norwegian study population. Also, the relative importance of different PE uptake pathways was assessed and discussed. Furthermore, the suitability of human nails as an alternative, non-invasive biomonitoring matrix for PEs was investigated. Additionally, information regarding alternative plasticizers to PEs was collected and presented extensively. Results showed that for PEs (paper II), daily intakes based on external exposure media agree with back-calculations using urinary metabolite concentrations, leading to the conclusion that human exposure for the general adult population is well understood and that the most important uptake routes were captured. Overall intake levels are comparable or lower than level presented in recent comprehensive studies and hazard quotients were well below 1 (low risk). As expected, diet was found to be the most important uptake route for all PEs. For lower molecular weight PEs, inhalation becomes a strong contributing pathway whereas for higher molecular weight PEs, dust ingestion was also important. Daily intake based on hand wipes was found to be much lower than the estimated total dermal intake based on air, dust and personal care products, questioning the relevance of hand wipes to represent total dermal exposure. Human nails were found to be unsuitable for replacing urine as a biomonitoring matrix for PEs as internal intake (from blood) cannot explain measured nail concentrations and uptake from air is too slow to reach observed concentrations within a realistic time frame (paper III). Hence, the kinetic links between intake and nail concentrations could not be established. Although exposure to traditional PEs is decreasing, use and body burden of some alternatives are increasing (paper I). Fortunately, most alternative plasticizers have favorable toxicological properties, resulting in low risk for humans. In contrast to PEs, OPEs still remain a group of poorly studied substances in terms of human exposure (paper IV). Due to lack of information regarding human metabolism, reliable links between intake and concentrations in serum and urine could not be established. Modelling results showed that concentrations in serum, and to some extent, urine, were underestimated for 2 compounds. It is likely that a combination of missing intake and suboptimal biomarkers were the cause for this under-prediction. Because of this, further studies regarding human metabolism should be performed for OPEs and potentially more specific biomarkers identified in the future. For PEs, there is a need for more comprehensive datasets to study exposure for high risk groups such as infants and children. Furthermore, dermal uptake remains poorly understood and the uptake of PEs into human nails should be studied in more detail to establish the kinetic links between exposure and body burden. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 4: Manuscript.</p>

A anatoxina-a(s) (antx-a(s)) é uma neurotoxina produzida por cianobactérias, cujo mecanismo de ação conhecido consiste na inibição irreversível da atividade da enzima acetilcolinesterase (AChE). Em razão da instabilidade da molécula e da inexistência de um padrão analítico, as informações referentes a presença dessa toxina nos reservatórios de água doce e, a completa elucidação dos processos envolvidos na sua toxicidade, são escassas. Na última década, a pesquisa referente a capacidade dos inseticidas organofosforados de induzir estresse oxidativo em humanos e animais tem sido bastante importante. Pelo fato da antx-a(s) ser o único organofosforado produzido por cianobactérias conhecido, este trabalho teve como objetivo investigar os mecanismos bioquímicos envolvidos na ação pró-oxidante de extratos aquosos contendo antx-a(s). A atividade das enzimas colinesterásicas AChE e butirilcolinesterase (BuChE) e, das enzimas antioxidantes catalase (CAT), glutationa peroxidase (GPx), glutationa redutase (GR) e superóxido dismutase (SOD) foram avaliadas em dois modelos experimentais: (i) Camundongos Swiss tratados por via intraperitoneal (ip) com uma dose sub-letal (20 mg.kg-1) de extrato contendo antx-a(s) e sacrificados após 24 h, 48 h, 7 e 14 dias e, (ii) sementes de alfafa regadas com extrato contendo antx-a(s) por um período de 7 dias. Também foi avaliado o efeito de extratos contendo antx-a(s) em coração de baratas da espécie Leurolestes circunvagans. A atividade enzimática das colinesterases avaliadas em camundongos tratados com extrato contendo antx-a(s) manteve-se inibida 58% até 48 h após tratamento (n=9, p<0,001), voltando a níveis normais (comparada ao controle) a partir do sétimo dia (n=10, p>0,05). Em contraposição, as alterações na atividade das enzimas antioxidantes avaliadas iniciaram-se após 48 h do tratamento, quando foi observada uma diminuição na atividade das enzimas CAT e GPx (n=9, p<0,001). No sétimo dia, enquanto a atividade das enzimas CAT e GR apresentou-se aumentada, a atividade da enzima GPx manteve-se significantemente diminuída (n=10, p<0,001). A SOD não mostrou diferença estatística significante em nenhum dos tratamentos. A atividade de todas as enzimas avaliadas voltou a normalidade após 14 dias. O desequilíbrio encontrado na atividade das enzimas antioxidantes em camundongos tratados com extrato contendo antx-a(s), também foi observado em sementes de alfafa. Neste último modelo de exposição, além da diminuição da atividade da enzima GPx (n=5, p<0,01) houve um aumento na atividade da enzima glutationa transferase (GST) com relação ao controle (n=5, p<0,05). Com relação ao efeito do extrato contendo antx-a(s) sobre a preparação de coração semi-isolado de baratas, observou-se um efeito taquicardíaco significante (aumento da frequência em quase 20%) nos animais tratados com 2,5x103 &#181;g de extrato contendo antx-a( s).g-1 (n=9, p<0,05). A partir destes resultados, pôde-se concluir que o extrato contendo antx-a(s) apresentou capacidade pró-oxidante em ambos os modelos avaliados (camundongo e semente) induzindo alterações na atividade das enzimas antioxidantes. Assim também, este extrato mostrou exercer um efeito cardiotóxico em baratas. / Anatoxin-a(s) (antx-a(s)) is a cyanobacterial neurotoxin whose principal mechanism of action is the irreversible inhibition of the enzymatic activity of acetylcholinesterase (AChE). Because of the molecule´s instability and the lack of an analytical standard, the occurrence of this toxin in freshwater reservoirs and the knowledge of the whole events related to its toxicity are scarce. In the last decade, research related to organophosphates insecticides capability to induce oxidative stress in humans and animals has been profuse. Considering that antx-a(s) is the unique organophosphate produced by cyanobacteria currently known; the main of this work was to investigate the biochemical mechanism related to the pro-oxidant capacity of antx-a(s)-containing extracts. In order to achieve the objective, there was determined the activity of cholinesterasic and antioxidant enzymes such as acetylcholinesterase (AChE), butirylcholinesterase (BuChE), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and superoxide dismutase (SOD). Two experimental models were used: (i) Swiss mice were treated intraperitoneally (ip) with 20 mg.kg-1 of antx-a(s)-containing extract and sacrificed after 24 h, 48 h, 7 and 14 days of treatment and, (ii) alfalfa seeds were irrigated with antx-a(s)-containing extract for 7 days. Additionally, the cardiac effect of antx-a(s)-containing extract on Leurolestes circunvagans cockroach was evaluated. The AChE and BuChE activity in mice treated with antx-a(s)-containing extract stayed inhibited more than 55% during 48 h (n=9, p<0,001). Normal activity of both enzymes was observed after seven days of treatment. By the other hand, changes in the antioxidant enzymes activity only began after the second day of treatment. Initially, both CAT and GPx showed lower activity than the control group after 48 h. Among those enzymes, GPx showed the highest decreased activity (n=9, p<0,001). After seven days, while the antx-a(s)- containing extract promoted increasing of CAT and GR activity, GPx activity remained deeply decreased (n=10, p<0,01). SOD activity did not show any statistical difference related to the control during all the treatments. Activity of all the evaluated enzymes was completely recovered after fourteen days. A similar unbalance on the antioxidant enzymes activity was provoked by the extract containing antx-a(s) in seeds irrigated with the extract. The seeds\' GPx activity showed low level (n=5, p<0,01), while GST activity was higher than the control (n=5, p<0,05). Regarding the cardiac effect of antx-a(s)-containing extract on semi-isolated cockroach heart, significant tachycardia (increase of almost 20% of the heartbeat frequency) was observed on the animals treated with 2,5x103 &#181;g.g-1 (n=9, p<0,05). From the obtained results, it can be concluded that the antx-a(s) extract demonstrated its pro-oxidant capacity in both experimental models evaluated (mice and seeds). This fact was proved through the unbalance on the activity of the antioxidant enzymatic defense system. Furthermore, the antx-a(s)-containing extract provoked a cardiotoxic effect on cockroach.

Submitted by Edineia Teixeira (edineia.teixeira@unioeste.br) on 2017-08-21T19:00:07Z No. of bitstreams: 2 Adir S Cembranel 2017.pdf: 757851 bytes, checksum: 5f897e15d7a5141b3530d4f771bca6d8 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-08-21T19:00:07Z (GMT). No. of bitstreams: 2 Adir S Cembranel 2017.pdf: 757851 bytes, checksum: 5f897e15d7a5141b3530d4f771bca6d8 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-05-05 / Recognizing residual concentrations of pesticides in the environment is important to understand the threats that these compounds represent to the environment and to human health, especially when natural resources are used for human consumption. Thus, this research aimed to determine the concentrations of organochlorine and organophosphorus pesticides present in the sediments of an urban lake located in the Southern region of Brazil, as well as the potential ecological risk and possible sources of contamination according to the use and occupation of the soil through time. Samples of sediment on the surface of the lake were collected every three months, from April 2010 to June 2013, covering the four hydrological stations, in five different collection points, accounting for 65 samples. The methodology used in the extraction, identification and quantification of pesticides was the QuEChERS method. The reading was performed in Gas Chromatograph attached to a mass spectrum. The analyses indicated that organochlorine pesticide contamination dates back to past agricultural activity. Concentrations of organophosphorus pesticides indicate recent and continuous occurrence from urban activities. In general, concentrations of organochlorines rarely indicate a potential ecological risk to organisms in the benthic environment. However, for organophosphates, especially Disulfoton, the risk factor indicates a significant potential adverse effect on organisms in the environment. / O reconhecimento de concentrações residuais de pesticidas no ambiente é importante para compreender as ameaças destes compostos ao meio ambiente e à saúde humana, sobretudo, quando recursos naturais são utilizados para consumo humano. Desta forma, esta pesquisa buscou determinar as concentrações de pesticidas organoclorados e organofosforados presentes nos sedimentos do lago urbano situado na região Sul do Brasil, bem como o potencial risco ecológico e as possíveis fontes de contaminação de acordo com o uso e a ocupação do solo ao longo do tempo. As amostras de sedimentos superficiais do lago foram coletadas a cada três meses, no período de abril de 2010 a junho de 2013, abrangendo as quatro estações hidrológicas, em cinco diferentes pontos de coleta, totalizando 65 amostras. A metodologia utilizada na extração, identificação e quantificação dos pesticidas foi o método QuEChERS. A leitura foi realizada em Cromatógrafo Gasoso acoplado a espectro de massa. As análises indicaram que a contaminação por pesticidas organoclorados se refere às atividades agrícolas no passado. As concentrações dos pesticidas organofosforados indicam ocorrência recente e contínua, proveniente das atividades urbanas. De modo geral, as concentrações dos organoclorados indicam raro potencial risco ecológico aos organismos em ambiente bentônico. Entretanto, para os organofosforados, especialmente o Disulfoton, o coeficiente de risco indica potencial significativo de efeito adverso aos organismos no ambiente.

A anatoxina-a(s) (antx-a(s)) é uma neurotoxina produzida por cianobactérias, cujo mecanismo de ação conhecido consiste na inibição irreversível da atividade da enzima acetilcolinesterase (AChE). Em razão da instabilidade da molécula e da inexistência de um padrão analítico, as informações referentes a presença dessa toxina nos reservatórios de água doce e, a completa elucidação dos processos envolvidos na sua toxicidade, são escassas. Na última década, a pesquisa referente a capacidade dos inseticidas organofosforados de induzir estresse oxidativo em humanos e animais tem sido bastante importante. Pelo fato da antx-a(s) ser o único organofosforado produzido por cianobactérias conhecido, este trabalho teve como objetivo investigar os mecanismos bioquímicos envolvidos na ação pró-oxidante de extratos aquosos contendo antx-a(s). A atividade das enzimas colinesterásicas AChE e butirilcolinesterase (BuChE) e, das enzimas antioxidantes catalase (CAT), glutationa peroxidase (GPx), glutationa redutase (GR) e superóxido dismutase (SOD) foram avaliadas em dois modelos experimentais: (i) Camundongos Swiss tratados por via intraperitoneal (ip) com uma dose sub-letal (20 mg.kg-1) de extrato contendo antx-a(s) e sacrificados após 24 h, 48 h, 7 e 14 dias e, (ii) sementes de alfafa regadas com extrato contendo antx-a(s) por um período de 7 dias. Também foi avaliado o efeito de extratos contendo antx-a(s) em coração de baratas da espécie Leurolestes circunvagans. A atividade enzimática das colinesterases avaliadas em camundongos tratados com extrato contendo antx-a(s) manteve-se inibida 58% até 48 h após tratamento (n=9, p<0,001), voltando a níveis normais (comparada ao controle) a partir do sétimo dia (n=10, p>0,05). Em contraposição, as alterações na atividade das enzimas antioxidantes avaliadas iniciaram-se após 48 h do tratamento, quando foi observada uma diminuição na atividade das enzimas CAT e GPx (n=9, p<0,001). No sétimo dia, enquanto a atividade das enzimas CAT e GR apresentou-se aumentada, a atividade da enzima GPx manteve-se significantemente diminuída (n=10, p<0,001). A SOD não mostrou diferença estatística significante em nenhum dos tratamentos. A atividade de todas as enzimas avaliadas voltou a normalidade após 14 dias. O desequilíbrio encontrado na atividade das enzimas antioxidantes em camundongos tratados com extrato contendo antx-a(s), também foi observado em sementes de alfafa. Neste último modelo de exposição, além da diminuição da atividade da enzima GPx (n=5, p<0,01) houve um aumento na atividade da enzima glutationa transferase (GST) com relação ao controle (n=5, p<0,05). Com relação ao efeito do extrato contendo antx-a(s) sobre a preparação de coração semi-isolado de baratas, observou-se um efeito taquicardíaco significante (aumento da frequência em quase 20%) nos animais tratados com 2,5x103 &#181;g de extrato contendo antx-a( s).g-1 (n=9, p<0,05). A partir destes resultados, pôde-se concluir que o extrato contendo antx-a(s) apresentou capacidade pró-oxidante em ambos os modelos avaliados (camundongo e semente) induzindo alterações na atividade das enzimas antioxidantes. Assim também, este extrato mostrou exercer um efeito cardiotóxico em baratas. / Anatoxin-a(s) (antx-a(s)) is a cyanobacterial neurotoxin whose principal mechanism of action is the irreversible inhibition of the enzymatic activity of acetylcholinesterase (AChE). Because of the molecule´s instability and the lack of an analytical standard, the occurrence of this toxin in freshwater reservoirs and the knowledge of the whole events related to its toxicity are scarce. In the last decade, research related to organophosphates insecticides capability to induce oxidative stress in humans and animals has been profuse. Considering that antx-a(s) is the unique organophosphate produced by cyanobacteria currently known; the main of this work was to investigate the biochemical mechanism related to the pro-oxidant capacity of antx-a(s)-containing extracts. In order to achieve the objective, there was determined the activity of cholinesterasic and antioxidant enzymes such as acetylcholinesterase (AChE), butirylcholinesterase (BuChE), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and superoxide dismutase (SOD). Two experimental models were used: (i) Swiss mice were treated intraperitoneally (ip) with 20 mg.kg-1 of antx-a(s)-containing extract and sacrificed after 24 h, 48 h, 7 and 14 days of treatment and, (ii) alfalfa seeds were irrigated with antx-a(s)-containing extract for 7 days. Additionally, the cardiac effect of antx-a(s)-containing extract on Leurolestes circunvagans cockroach was evaluated. The AChE and BuChE activity in mice treated with antx-a(s)-containing extract stayed inhibited more than 55% during 48 h (n=9, p<0,001). Normal activity of both enzymes was observed after seven days of treatment. By the other hand, changes in the antioxidant enzymes activity only began after the second day of treatment. Initially, both CAT and GPx showed lower activity than the control group after 48 h. Among those enzymes, GPx showed the highest decreased activity (n=9, p<0,001). After seven days, while the antx-a(s)- containing extract promoted increasing of CAT and GR activity, GPx activity remained deeply decreased (n=10, p<0,01). SOD activity did not show any statistical difference related to the control during all the treatments. Activity of all the evaluated enzymes was completely recovered after fourteen days. A similar unbalance on the antioxidant enzymes activity was provoked by the extract containing antx-a(s) in seeds irrigated with the extract. The seeds\' GPx activity showed low level (n=5, p<0,01), while GST activity was higher than the control (n=5, p<0,05). Regarding the cardiac effect of antx-a(s)-containing extract on semi-isolated cockroach heart, significant tachycardia (increase of almost 20% of the heartbeat frequency) was observed on the animals treated with 2,5x103 &#181;g.g-1 (n=9, p<0,05). From the obtained results, it can be concluded that the antx-a(s) extract demonstrated its pro-oxidant capacity in both experimental models evaluated (mice and seeds). This fact was proved through the unbalance on the activity of the antioxidant enzymatic defense system. Furthermore, the antx-a(s)-containing extract provoked a cardiotoxic effect on cockroach.

O uso indiscriminado de inseticidas pode causar a contaminação do ambiente, afetando o solo, o ar e as águas superficiais e subterrâneas. Um dos organofosforados mais utilizados no Brasil é o Malathion, com ampla utilização na agricultura e uso domiciliar, bem como em programas de saúde pública no controle de vetores de doenças. Em ambientes sujeitos a pressão seletiva de agrotóxicos, os alelos conferem resistência a inseticidas tendem a aumentar sua frequência. Dados obtidos no estudo com Zaprionus indianus confirmam a presença de um locus marcador para ambientes expostos ao Malathion, conferindo a essa espécie numerosas possibilidades de uso como bioindicador para monitorar o uso excessivo. 0 presente estudo, teve como objetivo: ampliar o conhecimento sobre a ocorrência e a sobrevivência de Z. indianus, submetidas ao bioensaio com Malathion, analisar a atividade enzimática e a inibição por Malathion. Os indivíduos coletados em Uberlândia, apresentaram o maior número e a maior diferenciação da proporção sexual. Não houve diferença significativa estatisticamente no padrão de resistência ao Malathion, quanto a sobrevivência dos indivíduos de Z. indianus, entre as localidades estudadas. O conjunto de dados obtidos nesse trabalho reforça a premissa que Z. indianus, aparece em grande abundância dentro das comunidades de drosofilídeos coletadas em áreas do Bioma Cerrado. Apresenta também, aspectos enzimáticos na inibição por Malathion, os quais poderão auxiliar no direcionamento de estudos futuros, ligados à avaliação de risco ecológico pela contaminação de inseticidas no ambiente urbano. / The indiscriminate use of insecticides can cause contamination of the environment, affecting soil, air and surface and groundwater. One of the most organophosphorus insecticide widely used in Brazil is the malathion one, which is employed in agriculture and domestic, as well as in control of disease’s vector into public health. In environments subject to selective pressure of pesticides, alleles confer resistance to insecticides tend to increase their frequency. Data obtained in the study with Zaprionus indianus confirm the presence of a marker locus for environments exposed to Malathion, giving this species numerous possibilities of use as a bioindicator to monitor overuse. The objective of this study was to increase knowledge about the occurrence and survival of Zaprionus indianus, submitted to the bioassay with Malathion, to analyze the enzymatic activity and inhibition by Malathion. The individuals collected in Uberlândia, presented the greatest number and the greatest differentiation of the sexual proportion. There was no statistically significant difference in the pattern of resistance to Malathion, as the survival of individuals of Z. indianus, among the studied localities. The set of data obtained in this work reinforces the premise that Z. indianus appears in great abundance within the communities of drosofilídeos collected in areas of the Cerrado Biome. It also presents enzymatic aspects in inhibition by Malathion, which may help in the direction of future studies, linked to the ecological risk assessment by the contamination of insecticides in the urban environment.

Parkinson's disease (PD) is a slow-progressive neurodegenerative disorder affecting 5-6 million people around the globe. The disease is manifested by the rapid deterioration of dopaminergic cells in the substantia nigra portion of the brain; however, the pathological mechanism of selective dopaminergic neuronal death is unknown. A reduction in levels of 3,4-dihydroxyphenylacetaldehyde (DOPAL) is biologically critical as this aldehyde has been shown to be toxic to dopaminergic cells and is a highly reactive electrophile. Investigating neuronal protein targets is essential in determining the cause of toxicity. An essential protein-GAPDH (e.g., glyceraldehyde-3-phosphate dehydrogenase) is an abundantly expressed enzyme known for its glycolytic activity, and recent research has implicated its role in oxidative stress-mediated neuronal death. This work positively shows GAPDH as a target for DOPAL modification, and, for the first time, DOPAL is identified as a potent inhibitor for GAPDH enzymatic activity. LC-MS and other chemical probes (ie. thiol and amine modifiers) show that DOPAL modifies specific –Lys, -Arg, and –Cys residues in the cofactor binding-domain of GAPDH. The enzyme inhibition is also time and DOPAL dose-dependent. DOPAL has a unique structure, containing two reactive functional groups: an aldehyde and catechol ring. In-house syntheses of DOPAL analogues, containing the catechol group and lacking the aldehyde, and vice versa have been tested on GAPDH and do not inhibit or modify GAPDH. Therefore, both the catechol and aldehyde groups of DOPAL are specific to binding with GAPDH and are necessary to achieve modification and enzyme inhibition. In addition to finding a novel enzyme inhibited and modified by DOPAL, this work has also confirmed linking DOPAL levels to a fungicide associated with PD risk. This benzimidazole fungicide, benomyl was shown to inhibit ALDH2 in the SH-SY5Y neuroblastoma cell line via an increase in DOPAL and a decrease in DOPAC. The ratios of DOPAL and DOPAC, the product of ALDH, were measured by HPLC-ECD, and found that benomyl does inhibit ALDH2 in this dopaminergic cell model. The cytotoxicity of benomyl, DA, DOPAL and the combination of DA or DOPAL with benomyl was assessed by MTT assay. Surprisingly, the only toxic combination was the combination of DA or DOPAL with benomyl. In fact, this toxicity appears to be synergistic, as none of the single treatments are significantly toxic to the cells. This synergistic effect also affects GAPDH aggregation. The cell morphology is also drastically different in the presence of the combined treatments, compared to individual treatment of DA, DOPAL or benomyl; cells start to ebb and show apoptotic-like features at just 2h. A second class of pesticides, named chlorpyrifos and chlorpyrifos-oxon were tested for toxicity in PC6-3These compounds were toxic to these cells due to DOPAL accumulation reaching high levels in the 100 µM range. Exposure to environmental toxins such as pesticides and fungicides has long been linked to PD risk, but only recently to DOPAL levels. This work provides a novel mechanism by which fungicide exposure may stimulate PD pathogenesis.

The acute nervous system toxicity of organophosphate (OP) pesticides is well described. However, the reported long-term effects of OP pesticides on the nervous system are inconsistent. This inconsistency may be due to imprecise estimates of pesticide exposure, variability of central nervous system (CNS) and peripheral nervous system (PNS) assessment, small samples, and poor control of confounding. The primary goal of this research was to examine the association between long-term OP pesticide use on CNS and PNS function among pesticide applicators. An additional goal was to examine the association between high pesticide exposure events (HPEEs), which typically do not result in acute toxicity, and CNS function. Study participants were recruited from among applicators enrolled in the Agricultural Health Study (AHS) in Iowa and North Carolina. In 2006-2008, 701 male pesticide applicators completed a battery of neurobehavioral (NB) and neurological tests. Information about individual pesticide use was obtained from previous AHS interviews and a questionnaire administered during NB testing. Associations between pesticide use and neurological outcomes were estimated with linear and logistic regression models while controlling for covariates. When associations were examined between agent-specific pesticide use and nine NB tests, significantly poorer performance was observed on four tests and significantly better performance on five tests. Additionally, for some pesticides, we observed differential associations by state, suggesting that regional differences in pesticide practices may influence neurotoxicity. Overall, our results did not provide strong evidence that OP pesticide use was associated with adverse NB test performance. A history of at least one HPEE was reported by 23 percent of participants. Significant adverse associations were observed between HPEEs and two of the nine NB tests. Participants with HPEEs were, on average, 4.9 seconds slower on a test of visual scanning/processing, and 2.2 seconds slower on a test of visual scanning/motor speed. Overall, small but meaningful associations were observed between HPEEs and adverse CNS function. When associations were examined between pesticide use and PNS function, five of six neurological physical examination outcomes were associated with ever-use of one or more OP pesticides. Odds ratios ranged from 1.9 to 3.1. However, mostly null associations were observed between OP pesticide use and electrophysiological tests, hand strength, sway speed and vibrotactile threshold. This study provides some evidence that long-term exposure to OP pesticides is associated with impaired PNS function. In summary, our results suggest that exposure to a few individual OP pesticides as well as HPEEs may contribute to adverse neurological function. The observed exposure-effect associations were present after adjustment for confounding and were independent of past-diagnosed pesticide poisoning. We believe this research contributes important new evidence to an inconsistent literature. Reducing pesticide exposure and preventing HPEEs among pesticide applicators remain important public health goals.