Parkinson's disease (PD) is a debilitating motor disorder that primarily afflicts older individuals (> 50yrs). Although its cause is unknown, many factors are thought to contribute to the disease. There is growing epidemiological evidence supporting a link between pesticide exposure and PD. The present immunohistochemical study was undertaken to characterize the role of insecticide exposure in the etiology of idiopathic PD. The insecticides selected for study were the pyrethroid permethrin (PE) and the organophosphate chlorpyrifos (CP), both of which possess properties that could damage or disrupt the nigrostriatal pathway, which is the principal neurodegenerative target in PD. The present study examined possible alteration of the amount of dopamine re-uptake transporter protein (DAT), within the striatum of the C57BL/6 mouse, using DAT antibodies, following low (0.8, 1.5 & 3.0 mg/kg) and high (200 mg/kg) doses of PE, respectively. Possible nigrostriatal terminal degeneration was examined using antibodies to tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, following treatment with 50 mg/kg of CP alone or in combination with the high dose of PE. For both the high dose of PE alone and for the combined PE/CP treatment, glial fibrillary acidic protein (GFAP) antibodies were used to examine the possibility of non-degenerative tissue injury. Groups of matched treated/vehicle-control mice received three IP injections of the insecticide/dose of interest over a 2-week period. Counts of immunoreactive (IR) neuropil in the dorsolateral striatum were made from four pre-selected fields per striatal tissue section. Counts were compared between matched sections, processed on the same slide, from a treated mouse and its vehicle control. A mean difference score, across slides, for each treated/vehicle control pair was determined. All low dose PE groups showed a trend of decreased DAT IR neuropil, but only the 3.0mg/kg group showed a statistically significant reduction (p<.0078). The 200 mg/kg PE group showed a trend toward reduced TH IR neuropil that was not statistically significant, but a significant increase in GFAP IR (p = .048) was observed. No significant change in TH IR neuropil was observed for CP (50mg/kg) alone. A significant increase was observed for GFAP IR neuropil for the PE/CP (200/50 mg/kg) combination dose (p = .033). The combined insecticide treatment failed, however, to produce a significant change in TH IR within the striatum, compared to vehicle controls. These data suggest that the significant increases in GFAP IR neuropil, in the striatum, reflect some form of tissue insult, following exposure to a high dose of PE, or PE/CP in combination, that is insufficient to induce degeneration of dopaminergic terminals within the temporal interval investigated. Although such damage may be sufficient to account for previously reported decreases in maximal dopamine uptake observed with high doses of these compounds, the DAT IR data appear to indicate that this damage is unlikely to be a change in the amount of DAT in these high dose conditions. The decreases in striatal DAT IR neuropil observed for low doses of PE suggest an alteration in the normal integrity of the nigrostriatal pathway and in the route by which environmental toxins may enter dopaminergic neurons. / Master of Science

No description available.

Pesticides including organophosphates (OPs) and carbamates (CBs) are widelyused to control undesirable pests. These compounds are neurotoxic and inhibithydrolysis of the neurotransmitter acetylcholine by acetylcholinesterase. Public healthconcerns have increased with the escalating usage of pesticides. Reliable monitoringprograms are required to detect and quantify pesticide exposure, as well as to promotean understanding of their neurotoxic properties. In this dissertation, both theanticholinergic (Part I) toxicity and neurotoxicity in neuroblastoma cells (Part II) ofpesticides were explored using mass spectrometry (MS). The high sensitivity andhigh-throughput of this technique renders it well-suited for proteomics analysis.Part I describes the study of butyrylcholinesterase (BChE) inhibition resultingfrom OP and CB exposure. The main hypothesis of Part I is that the specialmodification of BChE can provide the origin and extent of pesticide exposure. A novelmethod for detection and quantification of pesticide exposure was designed using aproteomics approach and equine BChE (eBChE) as a model system. The methodologyfeatured detection and analysis of phosphorylated or carbamylated peptides at theactive site serine residue. The developed technique was successfully applied towardsthe study of human BChE (hBChE) inhibition in vitro and in serum samples. Aspecially designed affinity column enabled an isolation of BChE from serum. EnrichedBChE was subjected to enzymatic digestion by a novel on-bead double digestionprotocol. LC/MS/MS was employed to produce a calibration system for the analysis ofhBChE inhibition, which was then applied towards quantification of the enzyme.Part II describes a proteomic study of the neurotoxicity in neuroblastoma cellscaused from chlorpyrifos (CPF), an organophosphate pesticide. The concerns of CPFexposure to pregnant women, infants and children are increasing due todevelopmentally neurotoxic effects of this chemical. The main hypothesis of Part II isthat CPF can cause protein alterations and these altered proteins can be detected usingproteomics. Systematic studies at subcellular levels evaluated proteome changes inSH-SY5Y cells exposed to CPF. Two-dimensional gel electrophoresis (2DE) wasapplied with MALDI-TOF-MS to analyze differential protein expression. Thirty sevencommon unique altered proteins were identified, which play important roles inmetabolic pathway.

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.

This thesis examines the toxicity of the herbicide glyphosate (two formulations ¡V technical grade and Roundup Biactive RB) and the insecticide chlorpyrifos CPF to a model freshwater food chain of a producer and consumer. The importance of studying the toxicity of low (environmentally realistic) concentrations of pesticides to non-target organisms is highlighted. An extensive literature review on the toxicity of glyphosate and chlorpyrifos to aquatic organisms is provided. The requirements for the maintenance of algal (Chlorella vulgaris, Chlorella pyrenoidosa and Pseudokirchneriella subcapitata) and Daphnia carinata cultures are discussed. The effects of two formulations of the herbicide glyphosate (technical grade and Roundup Biactive„µ) and the insecticide chlorpyrifos on the growth of Chlorella pyrenoidosa and Pseudokirchneriella subcapitata were studied, and the EC50 values determined. Hormesis was observed when P. subcapitata was exposed to concentrations of Roundup equal to 7% and 4% of its EC50 respectively. When exposed to chlorpyrifos concentrations 0.3-5 ƒÝg/L, hormesis was observed for both algal species with a maximum at 0.06% of EC50. The effects of sublethal concentrations of chlorpyrifos on population characteristics of Daphnia carinata were investigated in multiple-generation toxicity testing using individual culture. Exposure to chlorpyrifos affected survival and fecundity of animals in the first generation. In the second generation the most affected endpoint was time to the first brood with an indication of hormesis. LC50 tests were then conducted using animals of the third generation from each of the exposures in individual tests. Results of testing the third generation showed a constant significant decline in LC50 in the order of control daphnids through to ¡¥0.1 LC50¡¦ pre-exposed daphnids. The same experimental protocol was used in testing of glyphosate (technical grade and Roundup Biactive). Glyphosate was tested in two different media: sea salt solution and M4 medium, while Roundup Biactive was tested in M4 medium. Results indicated that glyphosate and Roundup Biactive had low toxicity to Daphnia. Hormesis was evident in sea salt medium exposures in the first and second generations of daphnids with glyphosate. When exposed to glyphosate and Roundup Biactive in M4 medium animals showed no indication of hormesis. It is hypothesized that glyphosate may have compensated for the lack of microelements in the sea salt medium, and possible mechanisms discussed.The modifying effect of glyphosate on the toxicity of cadmium to Daphnia carinata was studied using the same experimental design. Low concentrations of Roundup Biactive reduced the toxicity of cadmium, and the performance of daphnia was enhanced in terms of animal size, survival, fecundity, and the rate of natural increase in both generations in the presence of glyphosate. However when the third generation was tested for their sensitivity to Cd in the 48-h LC50 experiments there was no difference between RB-free and RB-spiked treatments in pair wise comparisons, indicating that no adaptation mechanisms were involved in the enhancement. The implications of these observed effects for environmental freshwater food chains subjected to pesticide exposure are discussed and recommendations on modifying pesticide use are provided.

INTRODUCTION: Cytochrome P450 2B (CYP2B) is a drug-metabolizing enzyme subfamily found in both the brain and liver, which metabolizes clinical drugs, drugs of abuse (e.g. nicotine), toxicants and endogenous neurochemicals. Brain CYP2B’s role in the local metabolism of centrally acting substrates is important to investigate because of its ability to metabolize a variety of centrally active substrates. Additionally, CYP2B regulation by genetics, and exposure to xenobiotics, results in great inter-individual differences in the brain expression of this enzyme. METHODS: We investigated the time-course of rat brain CYP2B induction after chronic nicotine treatment. Using the rat model of brain CYP2B induction, combined with intracerebroventricular (ICV) inhibition of CYP2B, we assessed the effects of brain CYP2B in the response to the anaesthetic substrate, propofol. We also investigated the role of brain CYP2B-mediated activation of the pesticide chlorpyrifos on its neurotoxicity. RESULTS: Nicotine’s induction of rat brain CYP2B was long lasting, returning to basal levels by day 7, and was unaffected by nicotinic receptor blockade. Induction of CYP2B in rat brain, by chronic nicotine treatment, reduced the anaesthetic efficacy of propofol, through increased brain CYP2B-mediated metabolic inactivation. Inhibition of brain CYP2B, using mechanism based inhibitors of the enzyme, inhibited both basal and induced brain CYP2B activity, and prolonged propofol sleep time by reducing the local brain inactivation of the anaesthetic. Inhibition of rat brain, and not hepatic, CYP2B was able to effectively block local brain production of the toxic chlorpyrifos oxon, significantly attenuating the reductions in brain acetylcholinesterase activity and body temperature. Additionally, inhibition of brain CYP2B also significantly reduced the behavioural toxicity after chlorpyrifos exposure in a chlorpyrifos (CP) dose- and time-dependent manner. CONCLUSION: These studies indicate that rat brain CYP2B enzymes are active in vivo and play a meaningful role in the local metabolism of, and the response to, centrally acting substrates (i.e. propofol, chlorpyrifos). These data provide a first demonstration of the important role that brain CYP-mediated metabolism plays in the response to centrally acting substrates (i.e. clinical drugs, toxicants, endogenous neurochemicals), potentially contributing to the inter-individual variability seen in human responses to centrally active drugs and toxicants.

INTRODUCTION: Cytochrome P450 2B (CYP2B) is a drug-metabolizing enzyme subfamily found in both the brain and liver, which metabolizes clinical drugs, drugs of abuse (e.g. nicotine), toxicants and endogenous neurochemicals. Brain CYP2B’s role in the local metabolism of centrally acting substrates is important to investigate because of its ability to metabolize a variety of centrally active substrates. Additionally, CYP2B regulation by genetics, and exposure to xenobiotics, results in great inter-individual differences in the brain expression of this enzyme. METHODS: We investigated the time-course of rat brain CYP2B induction after chronic nicotine treatment. Using the rat model of brain CYP2B induction, combined with intracerebroventricular (ICV) inhibition of CYP2B, we assessed the effects of brain CYP2B in the response to the anaesthetic substrate, propofol. We also investigated the role of brain CYP2B-mediated activation of the pesticide chlorpyrifos on its neurotoxicity. RESULTS: Nicotine’s induction of rat brain CYP2B was long lasting, returning to basal levels by day 7, and was unaffected by nicotinic receptor blockade. Induction of CYP2B in rat brain, by chronic nicotine treatment, reduced the anaesthetic efficacy of propofol, through increased brain CYP2B-mediated metabolic inactivation. Inhibition of brain CYP2B, using mechanism based inhibitors of the enzyme, inhibited both basal and induced brain CYP2B activity, and prolonged propofol sleep time by reducing the local brain inactivation of the anaesthetic. Inhibition of rat brain, and not hepatic, CYP2B was able to effectively block local brain production of the toxic chlorpyrifos oxon, significantly attenuating the reductions in brain acetylcholinesterase activity and body temperature. Additionally, inhibition of brain CYP2B also significantly reduced the behavioural toxicity after chlorpyrifos exposure in a chlorpyrifos (CP) dose- and time-dependent manner. CONCLUSION: These studies indicate that rat brain CYP2B enzymes are active in vivo and play a meaningful role in the local metabolism of, and the response to, centrally acting substrates (i.e. propofol, chlorpyrifos). These data provide a first demonstration of the important role that brain CYP-mediated metabolism plays in the response to centrally acting substrates (i.e. clinical drugs, toxicants, endogenous neurochemicals), potentially contributing to the inter-individual variability seen in human responses to centrally active drugs and toxicants.

Tez (Tıpta Uzmanlık) - Süleyman Demirel Üniversitesi, Tıp Fakültesi, Biyokimya Anabilim Dalı, 2005. / Kaynakça var.

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Resumo: O objetivo deste trabalho foi procurar conhecer os efeitos de três substâncias químicas, sulfluramida, fipronil e clorpirifós, sobre a espécie de formiga não-alvo Ectatomma brunneum (Hymenoptera: Formicidae: Ectatomminae), respondendo às seguintes questões: as marcas comerciais de iscas formicidas granuladas utilizadas neste trabalho são atrativas a esta espécie de formiga não-alvo ou simplesmente repelentes? Sendo atrativas, qual o comportamento exercido pelas operárias? Elas ingerem as iscas no local ou carregam para o interior do ninho? Há devolução? Em condições de múltipla escolha alimentar, esta atratividade permanece ou ocorre somente na ausência do alimento convencional? No que acarreta o contato direto e indireto das formigas com as iscas? Ocorre mortalidade significativa? Qual dos ingredientes ativos é mais prejudicial? Os produtos são seletivos? Os resultados obtidos através dos testes em laboratório revelaram que, em condições normais de exposição, as operárias de E. brunneum não foram repelidas pelas iscas formicidas. No entanto, não houve atratividade suficiente para induzir o carregamento ou ingestão. Quando esse material foi umedecido, a atratividade aumentou significativamente, permitindo que porções das iscas fossem ingeridas na própria arena de forrageamento ou carregadas para o interior do ninho e distribuídas aos outros indivíduos, não sendo registrada, em nenhum dos tratamentos, a devolução total ou parcial das porções carregadas. Após 48 horas de exposição, os três ingredientes ativos foram tóxicos, de modo que a população foi reduzida em 35,56% no tratamento com clorpirifós, 31,11% com sulfluramida e 30% com fipronil ao final de 20 dias de observação. As iscas à base de fipronil foram mais atrativas que as de sulfluramida e clorpirifós...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This work aimed at contributing to a better knowledge on the effects of three chemical substances, sulfluramid, fipronil and chlorpyrifos, on the non-target species of ant Ectatomma brunneum (Hymenoptera: Formicidae: Ectatomminae), answering the following questions: are the brands of granulated insecticide baits employed in this work attractive to this species of non-target ant or are they just repellent? If attractive, what is the behavior exhibited by the workers? Do they ingest the baits in the spot or take them into the nest? Is there any devolution? Under conditions of multiple food choice, does this attractiveness remain or does it happen only in the lack of the conventional food? What is the outcome of direct or indirect contact between the ants and the baits? Is there significant mortality? Which of the active ingredients is the most harmful? Are the products selective? The results obtained from the tests in laboratory showed that, under conditions of normal exposure, the workers of E. brunneum were not repelled by the insecticide baits. Nevertheless, there was not enough attractiveness as to lead to transporting or ingesting. When the material was moistened, the attractiveness increased significantly, allowing portions of bait to be ingested at the very foraging arena or to be taken into the nest and distributed to the other individuals. Total or partial devolution of the portions carried was not observed in any of the treatments. After a 48 hour exposure the three active ingredients proved to be toxic, for the population was reduced in 35.56% in the treatment with chlorpyrifos, 31.11% with sulfluramid and 30% with fipronil at the end of 20 days of observation. The fipronil based baits were more attractive than the ones based on sulfluramid and chlorpyrifos...(Complete abstract click electronic access below) / Orientador: Edilberto Giannotti / Coorientador: Marcos Aparecido Pizano / Banca: Evoneo Berti Filho / Banca: Nivar Gobbi / Mestre