Pesticides are among the most pervasive environmental contaminants and they are an important potential risk for human health. Agricultural workers are constantly exposed to pesticide spray, drift and residues in the soil and foliage. Many agricultural pesticides are readily absorbed by the body, through contact with the skin, the respiratory track, the eyes, and the gastrointestinal system. Multiple studies have reported a strong association between pesticide exposure and various health outcomes including cancer. Oxidative stress and DNA damage have been proposed as mechanisms linking pesticide exposure to health effects and neurological diseases. The focus of the present translational study is to examine the relationship between human exposure to the organophosphate pesticide azinphos methyl (AZM) and oxidative stress by measuring biomarkers of oxidative stress in biological fluids (i.e., urine, serum) and peripheral blood lymphocytes (PBLs) of agricultural workers. The findings from these field studies will be validated in vitro by examining cultures of human lymphocytes treated with AZM for similar biomarkers of oxidative stress. Since the collection of PBLs from study participants is highly invasive and not suitable for studies involving younger subjects, we also examined buccal cells for biomarkers of oxidative stress (i.e., DNA damage) as a more universal source of human tissue to assess oxidative stress in pesticide exposed individuals. We demonstrated in this study that AZM induces oxidative stress and causes DNA damage in human tissues. Agricultural workers who had been exposed to AZM showed elevated serum levels of lipid peroxides, increased urinary levels of 8-OH-dG, and lymphocytes from these individuals showed increased DNA damage and associated changes in oxidative DNA repair enzymes. Biomarkers of oxidative stress were also elevated in human lymphocytes treated with physiologically relevant concentrations of AZM. In cultures of human lymphocytes, AZM caused a concentration-dependent loss of viability and associated increases in ROS and a reduction in intracellular GSH. We also demonstrated that viable leukocytes from the oral cavity can be readily obtained from humans and these buccal cells can be used to assess DNA damage following exposure to occupational and environmental genotoxicants. We also noted that oral leukocytes are especially sensitive to cryopreservation with DMSO and thus, these cells must be cryoprotected with 5% DMSO to preserve the viability of these cells for subsequent biochemical studies. In summary, these in vivo and in vitro studies demonstrated that AZM induces oxidative stress in a dose-dependent matter and that oral lymphocytes are a good source of human tissue for assessing DNA damage and possibly other biochemical changes. The possible health implications of the variations in these biomarkers of oxidative stress and DNA damage are undetermined. Yet the findings from these studies have provided a strong foundation for determining the mechanism by which pesticide induce oxidative stress, to explore the putative relationship between pesticide-induced oxidative stress and disease (e.g. cancer, neurodegenerative disorders) and determine whether tissue damage in humans is brought about by direct or by indirect action of organophosphate pesticides. / Graduation date: 2010

Made available in DSpace on 2015-03-03T13:58:19Z (GMT). No. of bitstreams: 1 AngelicaMSL_DISSERT.pdf: 779404 bytes, checksum: 28160c948fb7d8a8df206e8b5a2b131e (MD5) Previous issue date: 2011-09-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Violacein is a violet pigment isolated from many gram-negative bacteria, especially from Chromobacterium violaceum, a betaproteobacterium found in the Amazon River in Brazil. It has potential medical applications as an antibacterial, fungicide, anti-tryptanocidal, anti-ulcerogenic and anti-cancer drug, among others. Furthermore, its pro-oxidant activity has been suggested, but only in two specific tumor lineages. Thus, in the present study, the prooxidant effects of violacein were investigated in both normal and tumor cells, seeking to evaluate the cell responses. The evaluation of violacein cytotoxicity using the Trypan blue dye exclusion method indicated that CHO-K1 cells were more resistant than tumor HeLa cells. The oxidative stress induced by violacein was manifested as an increase in intracellular SOD activity in CHO-K1 and MRC-5 cells at a specific concentration range. Nevertheless, a decrease was detected specifically at 6-12 ?M in HeLa and MRC-5 cells. Interestingly, the increase in SOD activity was not followed by a concomitant increase in catalase activity. Regarding to oxidative stress biomarkers, increased protein carbonylation and lipid hydroperoxides levels were detected respectively in CHO-K1 and MRC-5 cells treated with violacein at 1.5-3 ?M and 3 ?M, which may be an evidence that this compound causes oxidative stress specifically in these conditions. Additionally, it is believed that the decline in cell viability observed in MRC-5 cells and HeLa treated with violacein at 6-12 M is due to mechanisms not related to oxidative stress. Moreover, the results suggested that violacein might cause oxidative stress by increasing endogenous levels of O2 -, since the occurrence of an expressive change in SOD activity. In addition, in order to evaluate the antioxidant activity of violacein in the absence of a biological system, the total antioxidant and iron chelating activity were evaluated, so that antioxidant activities were detected at 30 and 60 ?M of violacein. Altogether, the results indicate that although oxidative stress is triggered by incubation with violacein, it did not seem to be high enough to cause serious damage to cell biomolecules in HeLa cells and only at specific concentrations in CHOK-1 and MRC-5 cells. Comparing the results obtained in cell culture and the in vitro antioxidant activity evaluation, the results confirmed that violacein presents opposing oxidant features when in presence or absence of a biological system and the antioxidant character only occurs at high concentrations of the pigment. / A violace?na ? um pigmento violeta isolado de v?rias esp?cies de bact?rias gram-negativas, especialmente da Chromobacterium violaceum, uma betaproteobact?ria encontrada no rio Amazonas, no Brasil. Diversas atividades biol?gicas j? foram descritas para este pigmento e dentre elas destacam-se a antibacteriana, antif?ngica, tripanocida, antileishmaniose, anti-?lcerog?nica, antiviral e antitumoral. Apesar de uma atividade antioxidante in vitro ter sido sugerida, a atividade pr?-oxidante tamb?m j? foi observada especificamente em duas linhagens tumorais e parece ser dependente de mecanismos espec?ficos para cada linhagem. Nesse sentido, os efeitos citot?xicos e pr?oxidantes da violace?na foram investigados em c?lulas normais e tumorais buscando-se avaliar a ocorr?ncia de diferentes respostas celulares. A an?lise da citotoxicidade da violace?na indicou que c?lulas CHO-K1 foram mais resistentes ao composto em rela??o ?s tumorais HeLa. Quanto ?s enzimas do aparato antioxidante, observou-se um aumento significativo na atividade da SOD intracelular nas linhagens CHO-K1 e MRC-5. Por?m, houve uma diminui??o na atividade enzim?tica especificamente nos tratamentos com 6 e 12 M nas linhagens MRC-5 e HeLa. Interessantemente, o aumento na atividade da SOD n?o foi acompanhado pelo aumento concomitante na atividade da catalase. Em rela??o aos biomarcadores de estresse oxidativo, n?veis elevados de prote?nas carboniladas e hidroper?xidos de lip?dio foram observados em c?lulas CHO-K1 e MRC-5 quando tratadas respectivamente com 1,5-3 ?M e 3 ?M de violace?na, indicando que o pigmento apresenta efeitos pr?-oxidantes especificamente nessas concentra??es. Adicionalmente, acredita-se que a acentuada queda na viabilidade celular observada em c?lulas MRC-5 e HeLa tratadas com 6-12 ?M de violace?na se deve a outros mecanismos n?o relacionados ? gera??o de estresse oxidativo propriamente dita. Os resultados obtidos em cultura de c?lulas sugerem tamb?m que a violace?na induz estresse oxidativo por eleva??o dos n?veis end?genos de O2 ??, visto a ocorr?ncia de uma significativa altera??o nos n?veis de atividade de SOD. Em adi??o, com o objetivo de avaliar o car?ter antioxidante in vitro da violace?na na aus?ncia de um sistema biol?gico celular, a capacidade antioxidante total e a atividade de quela??o f?rrica do pigmento foram avaliadas, de forma que atividades antioxidantes foram detectadas a 30 e 60 ?M de violace?na. Frente aos resultados obtidos, apesar do desencadeamento do estresse oxidativo ap?s a incuba??o com violace?na, este parece n?o ser suficiente para causar danos significativos aos componentes e estruturas celulares em c?lulas HeLa e apenas em concentra??es espec?ficas de pigmento para CHO-K1 e MRC-5, dentro das condi??es avaliadas. Por fim, os resultados confirmam que violace?na apresenta car?teres oxidantes opostos quando na presen?a ou aus?ncia de um sistema biol?gico, al?m de que o car?ter antioxidante s? se d? em concentra??es elevadas do pigmento