Les quantum dots (QDs) sont des cristaux semi-conducteurs de dimensions nanométriques. Ils peuvent être employés comme des marqueurs photosensibles du métabolisme cellulaire et peuvent être utiles dans différents domaines notamment en médecine mais il s’est rapidement avéré nécessaire de démontrer leur innocuité avant leur utilisation à grande échelle et leur diffusion dans l’environnement. Nous proposons un projet de thèse de doctorat sur le thème : La génotoxicité des quantum dots et le rôle du stress oxydant, implications sur l’environnement et la santé humaine. Il s’organise suivant trois axes: L’étude in vitro des propriétés génotoxiques et mutagènes des QDs Les QDs induisent des lésions primaires de l’ADN sur cellules CHO-K1 par le test des comètes qui sont associées à un stress oxydant. Ils sont plus actifs après irradiation par le spectre solaire. Ils induisent des mutations chromosomiques. L’étude in vivo des propriétés génotoxiques et mutagènes des QDs Les QDs induisent une augmentation significative des lésions de l'ADN chez le rat qui varie selon l’organe considéré (foie, rein, poumon, cerveau et testicule). Ils induisent une augmentation significative et une réponse dose-dépendante des micronoyaux indiquant nettement leur pouvoir clastogène/aneugène. Aucune variation significative des variables biochimiques mesurées n’est apparue. La mise en évidence de leurs effets sur l’environnement L'exposition aux QDs et au CdCl2 a entraîné une augmentation significative des lésions de l'ADN chez E. fetida et N. diversicolor. / Quantum dots (QDs) are semiconductor nanocrystals which can be employed as sensitive biomarkers of cellular metabolism and thus show their usefulness in various fields, including medicine and it soon became necessary to prove their safety before their widespread use and their distribution in the environment. The thesis project targeted on: Genotoxicity of quantum dots and the role of oxidative stress implications for the environment and human health. This study was organized in three main parts The in vitro study of the genotoxic and mutagenic properties of QDs QDs induced primary DNA lesions in CHO-K1 cells using the comet assay and were associated with oxidative stress. We demonstrated that the QDs were more active after irradiation by the solar spectrum. We showed the ability of QDs to induce chromosomal mutations. The main mechanism was probably that of the production of free radicals. The in vivo study of the genotoxic and mutagenic properties of QDs The comet assay shows that QDs induced an overall significant increase in DNA lesions of different organs (liver, kidneys, lungs, brain and testes). However, each organ had a specific susceptibility. QDs induced a significant increase in a dose-dependent manner of micronuclei. These results clearly indicated the in vivo clastogenic / aneugenic properties of QDs. No significant variation in the measured biochemical variables. The evidence of their effects on the environment Evaluation of genotoxicity was performed on coelomocytes of E. fetida and N. diversicolor resulting in a significant increase in DNA damage.

Algumas áreas do canal de Piaçaguera, localizado na Baixada Santista, SP-Brasil, apresenta alto nível contaminação por hidrocarbonetos policíclicos aromáticos (HPA) entre outros contaminantes nos sedimentos. O objetivo do presente estudo foi verificar diferentes biomarcadores de exposição em bile e de efeitos genotóxicos em sangue de peixes, coletados in situ, que poderiam ser utilizados como ferramenta de monitoramento. O canal de Bertioga foi selecionado como região de referência. Embora não seja uma região sem nenhuma interferência de contaminantes, os sedimentos apresentam baixos valores para HPA, metais e atividade mutagênica. A espécie Mugil curema foi selecionada por ser freqüentemente encontrada em ambas as regiões de estudo. Observamos que os peixes coletados no canal de Piaçaguera apresentaram maior porcentagem de micronúcleos e danos ao DNA (ensaio cometa) no sangue quando comparados com a região de referência. Para o teste de micronúcleo, realizamos leituras de 1000 e 4000 células/indivíduo e não foram observadas diferenças estatísticas, sugerindo que o número de 1000 células poderia ser suficiente para gerar dados confiáveis para a espécie Mugil curema. A avaliação de mutagenicidade da bile foi realizada pelo teste Salmonella/microssoma combinado à extração de bile utilizando Blue rayon (BR) com as linhagens TA98, TA100 e YG1041 com e sem S9 (ativação metabólica). As maiores respostas mutagênicas foram observadas para os peixes coletados no canal de Piaçaguera. A mutagenicidade com a linhagem YG1041 na presença de S9 foi mais elevada quando comparado à mutagenicidade observada na ausência de S9 e também em comparação com a sua linhagem parental TA98, indicando a provável presença de compostos da classe das aminas aromáticas na bile como responsáveis pelos efeitos observados. A quantificação de metabólitos equivalentes de HPA foi realizada por HPLC/fluorescência na bile bruta e embora os resultados não se correlacionem com a mutagenicidade, altos níveis também foram encontrados para os peixes coletados no canal de Piaçaguera em comparação com Bertioga. O protocolo de amplificação do gene ras para a espécie Mugil curema foi estabelecido e os genes foram seqüenciados para verificação da presença de mutações. Não foram observadas mutações nos poucos peixes analisados e mais estudos são necessários para verificar a utilização deste biomarcador. O teste de micronúcleo, ensaio cometa e análise de mutagencidade em bile extraída por Blue rayon parecem ser ferramentas biológicas adequadas para monitoramento da qualidade ambiental de estuários contaminados. / Some areas of the Piaçaguera channel at Baixada Santista, SP, Brazil present high levels of polycyclic aromatic hydrocarbons (PAHs) among other contaminants in the sediment. The aim of the present study was to verify if biomarkers of exposure in bile and effect in blood of fishes collected in the field could be used as a monitoring tool. We selected Bertioga channel as a reference area. Although it is not a pristine area, the sediment presents low values of PAHs, metals and mutagenic activity. We selected the fish species Mugil curema, because they are frequently found in both areas during the entire year. We observed that the fish collected at the Piaçaguera channel showed a higher percentage of micronuclei and DNA damage (comet assay) in blood when compared to the Bertioga channel. The micronucleus readings were done in 1000 and 4000 cells/fish and no statistical difference was observed, suggesting that the number of 1000 cells would be sufficient to generate reliable data for the species Mugil curema , in the studied area. The mutagenicity of the bile was performed using the Salmonella/microsome assay combined with Blue rayon (BR) extraction with TA98, TA100 and YG1041 strains with and without rat liver S9. We observed that the mutagenic responses were higher in fish collected in Piaçaguera. The mutagenicity in YG1041 with S9 in fishes collected in the Piaçaguera channel was higher when compared to the mutagenicity observed in the absence of S9 and also higher than the response with the parental strain TA98. The results suggest that mutagenic polycyclic compounds, probably from the class of the aromatic amines are causing the observed effect. PAH metabolites-equivalent were also determined using HPLC/fluorescence in crude bile, and although the results did not correlate with the mutagenicity, higher levels were observed in fish collected in the Piaçaguera channel when compared to Bertioga. A protocol for amplification of the ras gene for the Mugil curema species was established and gene sequenced. No mutations were observed in the liver of the few fishes analyzed, and more studies are required to verify the utility of this biomarker in this fish. The micronuclei, comet assay and mutagenicity in bile extracted with blue rayon seem to be suitable biological tools to monitor the environmental quality of contaminated estuaries.

Dunaliella tertiolecta, uma alga verde fitoplanctônica de ampla distribuição no ambiente marinho, foi escolhida como organismo teste para estudar a possibilidade de ser utilizada no ensaio cometa, um teste de detecção de danos no DNA em células individualizadas muito utilizado na ecotoxicologia. Essas algas foram facilmente lisadas pela solução de lise alcalina iônica, seus cometas foram corados eficientemente pelo brometo de etídio e pela prata, e a análise por índice de danos apresentou boa correlação com momento de cauda, comprimento de cauda e porcentagem de DNA na cauda. As algas foram expostas a concentrações crescentes de 4-nitroquinolina-N-óxido (4NQO) e benzo[a]pireno (BAP) por 1, 2 e 4 h no escuro. Após somente 1 h de exposição, observou-se um aumento significativo de danos no DNA das algas expostas a 0,25 µM de 4NQO, demonstrando a sensibilidade das mesmas em relação a células de animais. Os dados obtidos da exposição ao BAP não foram consistentes e necessitam de verificação. A metabolização de BAP em compostos tóxicos pelas algas e o efeito das condições de luminosidade antes e durante as exposições são discutidos. Os resultados indicam que D. tertiolecta pode ser utilizada em laboratório para avaliação de genotoxicidade na água através do ensaio cometa. / D. tertiolecta, a phytoplanktonic green algae with ubiquitous distribution in the marine environment, was chosen as a test organism to study the possibility of being used in the comet assay, a frequently used test in ecotoxicology to detect DNA damage in single cells. These algae were easily lised by the alkaline ionic lysis solution, their comets were efficiently stained by ethidium bromide and silver and the damage index was well correlated to tail moment, tail length and percentage of DNA in the tail. The algae were exposed to increasing concentrations of 4-nitroquinoline-N-oxide (4NQO) and benzo[a]pyrene (BAP) for 1, 2 and 4 h in the dark. After 1 h exposure, a significant increase in the DNA damage of algae exposed to 0,25 µM of 4NQO was observed, demonstrating their sensitivity in relation to cells from animals. The data of BAP exposure were not consistent and need further verification. The metabolization of BAP to toxic compounds by algae and the light conditions before and during exposure are discussed. The results indicate that D. tertiolecta can be used in laboratory to evaluate water genotoxicity with comet assay.

O composto estudado neste trabalho foi o corante comercial CI Disperse Blue 291 (DB291), que contém o aminoazobenzeno 2-[(2-bromo-4,6-dinitrofenil)azo]-5-(dietilamino)-4-metoxiacetanilida. Esse produto é um azo-corante disperso usado largamente pelas indústrias têxteis para o tingimento de poliéster e pode ser encontrado em ambientes aquáticos oriundo da descarga de efluentes industriais, podendo levar à exposição de humanos por meio da ingestão de água ou alimentos contaminados. Portanto, faz-se importante a avaliação toxicológica do DB291. Este produto apresentou atividade mutagênica para linhagens de Salmonella typhimurium que possuem alta expressão das enzimas nitrorredutase e Ο-acetiltransferase. Tais enzimas também são expressas pelas bactérias da flora intestinal humana e de roedores e, desempenham importante papel na biotransformação de substâncias presentes na luz intestinal. O objetivo deste trabalho foi investigar a atividade do corante DB291 na indução de lesões pré-neoplásicas no cólon de ratos, avaliada pelo teste do cometa e pelo desenvolvimento de focos de criptas aberrantes (FCAs). Resultados com 2, 8, 16 e 24 semanas de tratamento demonstraram que o DB291 não foi capaz de induzir FCAs em ratos tratados por gavage com a dose de 50mg/kg de peso corpóreo, três vezes por semana em dias alternados. Entretanto, resultados com o teste do cometa demonstraram que o corante foi capaz de causar danos ao DNA das células da mucosa do cólon de ratos tratados por via intra-retal. Estes resultados sugerem que o DB291 possui atividade genotóxica in vivo. Considerando a resposta genotóxica para o teste do cometa, a alta atividade mutagênica no teste Salmonella microssoma e o recente relato de que o DP291 causou danos em células de fígado humano (HepG2), faz-se necessário a realização de testes de carcinogênese de longa duração para avaliação segura do seu potencial carcinogênico, não somente em cólon, mas em outros órgãos tais como fígado e bexiga. / The commercial disperse dye product CI Disperse Blue 291 , which contain the aminoazobenzene 2-[(2-bromo-4,6-dinitrophenyl)azo]-5-(diethylamino)-4-methoxyacetanilide (CAS registry no. 56548-64-2) is used for polyester fibers dyeing. It can be released in the aquatic environment through the discharge of industrial effluents. Humans can be exposed through the consumption of water and food contaminated with this product therefore its toxicological properties are important to be evaluated. This product showed elevated mutagenic activity with nitroreductase and Ο-acetyltransferase overproducing Salmonella strains. These enzymes are also expressed by human intestinal microflora, making intestines a possible target organ to the development of cancer after exposure to this product. The aim of this study was to investigate the effects of the commercial disperse dye product containing the CI Disperse Blue 291 on rat colon carcinogenesis, evaluated by the single cell gel assay (comet assay) and by aberrant crypt foci development. Results within different experimental periods showed the DB291 were not able to induce preneoplastic lesions in the colon of rats orally treated with 50mg/kg b.w., three times a week. The DB291 induced damages in the DNA of the rats colon mucosa, evaluated by the comet assay. These data indicate that the OB291 showed genotoxic activity in the colon mucosa cells. Considering these results, the mutagenic activity with Salmonella test and the recent data that the OB291 presents toxicity to human liver cells (HepG2), further long time carcinogenesis assays are needed to security evaluation of its carcinogenic potential, not only in colon, but also another organs like liver and kidney.

Dunaliella tertiolecta, uma alga verde fitoplanctônica de ampla distribuição no ambiente marinho, foi escolhida como organismo teste para estudar a possibilidade de ser utilizada no ensaio cometa, um teste de detecção de danos no DNA em células individualizadas muito utilizado na ecotoxicologia. Essas algas foram facilmente lisadas pela solução de lise alcalina iônica, seus cometas foram corados eficientemente pelo brometo de etídio e pela prata, e a análise por índice de danos apresentou boa correlação com momento de cauda, comprimento de cauda e porcentagem de DNA na cauda. As algas foram expostas a concentrações crescentes de 4-nitroquinolina-N-óxido (4NQO) e benzo[a]pireno (BAP) por 1, 2 e 4 h no escuro. Após somente 1 h de exposição, observou-se um aumento significativo de danos no DNA das algas expostas a 0,25 µM de 4NQO, demonstrando a sensibilidade das mesmas em relação a células de animais. Os dados obtidos da exposição ao BAP não foram consistentes e necessitam de verificação. A metabolização de BAP em compostos tóxicos pelas algas e o efeito das condições de luminosidade antes e durante as exposições são discutidos. Os resultados indicam que D. tertiolecta pode ser utilizada em laboratório para avaliação de genotoxicidade na água através do ensaio cometa. / D. tertiolecta, a phytoplanktonic green algae with ubiquitous distribution in the marine environment, was chosen as a test organism to study the possibility of being used in the comet assay, a frequently used test in ecotoxicology to detect DNA damage in single cells. These algae were easily lised by the alkaline ionic lysis solution, their comets were efficiently stained by ethidium bromide and silver and the damage index was well correlated to tail moment, tail length and percentage of DNA in the tail. The algae were exposed to increasing concentrations of 4-nitroquinoline-N-oxide (4NQO) and benzo[a]pyrene (BAP) for 1, 2 and 4 h in the dark. After 1 h exposure, a significant increase in the DNA damage of algae exposed to 0,25 µM of 4NQO was observed, demonstrating their sensitivity in relation to cells from animals. The data of BAP exposure were not consistent and need further verification. The metabolization of BAP to toxic compounds by algae and the light conditions before and during exposure are discussed. The results indicate that D. tertiolecta can be used in laboratory to evaluate water genotoxicity with comet assay.

Algumas áreas do canal de Piaçaguera, localizado na Baixada Santista, SP-Brasil, apresenta alto nível contaminação por hidrocarbonetos policíclicos aromáticos (HPA) entre outros contaminantes nos sedimentos. O objetivo do presente estudo foi verificar diferentes biomarcadores de exposição em bile e de efeitos genotóxicos em sangue de peixes, coletados in situ, que poderiam ser utilizados como ferramenta de monitoramento. O canal de Bertioga foi selecionado como região de referência. Embora não seja uma região sem nenhuma interferência de contaminantes, os sedimentos apresentam baixos valores para HPA, metais e atividade mutagênica. A espécie Mugil curema foi selecionada por ser freqüentemente encontrada em ambas as regiões de estudo. Observamos que os peixes coletados no canal de Piaçaguera apresentaram maior porcentagem de micronúcleos e danos ao DNA (ensaio cometa) no sangue quando comparados com a região de referência. Para o teste de micronúcleo, realizamos leituras de 1000 e 4000 células/indivíduo e não foram observadas diferenças estatísticas, sugerindo que o número de 1000 células poderia ser suficiente para gerar dados confiáveis para a espécie Mugil curema. A avaliação de mutagenicidade da bile foi realizada pelo teste Salmonella/microssoma combinado à extração de bile utilizando Blue rayon (BR) com as linhagens TA98, TA100 e YG1041 com e sem S9 (ativação metabólica). As maiores respostas mutagênicas foram observadas para os peixes coletados no canal de Piaçaguera. A mutagenicidade com a linhagem YG1041 na presença de S9 foi mais elevada quando comparado à mutagenicidade observada na ausência de S9 e também em comparação com a sua linhagem parental TA98, indicando a provável presença de compostos da classe das aminas aromáticas na bile como responsáveis pelos efeitos observados. A quantificação de metabólitos equivalentes de HPA foi realizada por HPLC/fluorescência na bile bruta e embora os resultados não se correlacionem com a mutagenicidade, altos níveis também foram encontrados para os peixes coletados no canal de Piaçaguera em comparação com Bertioga. O protocolo de amplificação do gene ras para a espécie Mugil curema foi estabelecido e os genes foram seqüenciados para verificação da presença de mutações. Não foram observadas mutações nos poucos peixes analisados e mais estudos são necessários para verificar a utilização deste biomarcador. O teste de micronúcleo, ensaio cometa e análise de mutagencidade em bile extraída por Blue rayon parecem ser ferramentas biológicas adequadas para monitoramento da qualidade ambiental de estuários contaminados. / Some areas of the Piaçaguera channel at Baixada Santista, SP, Brazil present high levels of polycyclic aromatic hydrocarbons (PAHs) among other contaminants in the sediment. The aim of the present study was to verify if biomarkers of exposure in bile and effect in blood of fishes collected in the field could be used as a monitoring tool. We selected Bertioga channel as a reference area. Although it is not a pristine area, the sediment presents low values of PAHs, metals and mutagenic activity. We selected the fish species Mugil curema, because they are frequently found in both areas during the entire year. We observed that the fish collected at the Piaçaguera channel showed a higher percentage of micronuclei and DNA damage (comet assay) in blood when compared to the Bertioga channel. The micronucleus readings were done in 1000 and 4000 cells/fish and no statistical difference was observed, suggesting that the number of 1000 cells would be sufficient to generate reliable data for the species Mugil curema , in the studied area. The mutagenicity of the bile was performed using the Salmonella/microsome assay combined with Blue rayon (BR) extraction with TA98, TA100 and YG1041 strains with and without rat liver S9. We observed that the mutagenic responses were higher in fish collected in Piaçaguera. The mutagenicity in YG1041 with S9 in fishes collected in the Piaçaguera channel was higher when compared to the mutagenicity observed in the absence of S9 and also higher than the response with the parental strain TA98. The results suggest that mutagenic polycyclic compounds, probably from the class of the aromatic amines are causing the observed effect. PAH metabolites-equivalent were also determined using HPLC/fluorescence in crude bile, and although the results did not correlate with the mutagenicity, higher levels were observed in fish collected in the Piaçaguera channel when compared to Bertioga. A protocol for amplification of the ras gene for the Mugil curema species was established and gene sequenced. No mutations were observed in the liver of the few fishes analyzed, and more studies are required to verify the utility of this biomarker in this fish. The micronuclei, comet assay and mutagenicity in bile extracted with blue rayon seem to be suitable biological tools to monitor the environmental quality of contaminated estuaries.

La fitness et a fortiori la survie d'une population dépendent de la stratégie et des performances de reproduction façonnées par son environnement. Par conséquent, les biomarqueurs traduisant une altération de la fonction de reproduction présentent un intérêt particulier. L'atteinte de la fertilité mâle au sein de la faune sauvage a notamment été adressée comme une problématique majeure susceptible de représenter une menace pour le maintien des populations. Toutefois, peu de méthodologies sont aujourd'hui disponibles chez des espèces pertinentes pour aborder cette problématique dans le cadre de programmes de biosurveillance environnementale, notamment chez les crustacés en dépit de leur représentativité au sein du règne animal et de leurs indispensables fonctions au sein des écosystèmes. Dans ce contexte, les présents travaux avaient pour objectif de proposer une ou plusieurs méthodologies basées sur la mesure de marqueurs de fonctionnalité et d'intégrité spermatique chez des crevettes Palaemonidae. Les investigations se sont portées sur deux espèces, une estuarienne, Palaemon longirostris, et une côtière, Palaemon serratus, pour leur complémentarité vis-à-vis du continuum estuaire - littoral. Compte tenu des nombreuses spécificités structurelles et fonctionnelles des spermatozoïdes de crustacés, I nombre de marqueurs transposables vers ces espèces s'est finalement avéré limité. Aussi, après une brève prospection, l'effort de recherche a rapidement été recentré sur la mesure de l'intégrité de l'ADN. Dès lors, la démarche scientifique a été construite de sorte à évaluer, point par point, la pertinence de la méthodologie développée dans une perspective d'application de l'outil dans le cadre de la surveillance environnementale. Une étape préliminaire d'optimisation et de validation méthodologique du test Cornet a démontré que, contrairement à une grande majorité de type spermatique, l'adaptation de ce test sur les spermatozoïdes de Palaemonidae ne nécessite aucune modification particulière du protocole. La dynamique de la réponse biologique en termes d'apparitions, de rémanence et d'effets possibles sur la fitness a été évaluée en conditions contrôlées au laboratoire. Ainsi, des expositions ex vivo et in vivo ont été conduites en utilisant une variété de génotoxiques modèles présentant différents modes d'actions. Les résultats ont attesté de la sensibilité, de la reproductibilité et du caractère intégrateur de la réponse. En revanche, aucun lien entre un ADN spermatique endommagé et une altération du succès de reproduction pré-éclosion n'a pu être établi. Parallèlement, une approche in situ a été conduite en vue de caractériser la valeur basale de la réponse mesurée. Différentes stratégies ont dû être adoptées en fonction des contraintes propres au milieu de vie de chacune des deux espèces. Un référentiel et une valeur seuil, communs aux deux espèces, ont pu être définis, soulignant le potentiel de transférabilité inter-espèces de l'outil. La méthodologie ainsi finalisée, a été éprouvée dans le cadre de plusieurs campagnes de suivi de différentes populations indigènes de l'estuaire et de la baie de Seine en 2015 et 2016. Les résultats se sont révélés très cohérents au regard de la pression de contamination et de la dynamique hydro-sédimentaire de la baie de Seine. En définitive, l'intégrité de l'ADN spermatique chez les Palaemonidae est opérationnelle en l'état pour un déploiement in situ en tant que biomarqueur d'exposition à un stress génotoxique. De futurs études devront néanmoins être conduites (1) pour mieux discerner les implications de ces dommages spermatiques en termes d'impact sur le recrutement des nouvelles cohortes et (2) éprouver la transférabilité de la méthodologie à d'autres espèces de crevettes et sur une plus large échelle géographique. / The environment shapes the reproduction's strategy and performance of a population, influencing its fitness and a fortiori its survival. Therefore, biomarkers that alter reproductive functions represent a great interest in ecotoxicology. The reduction of male fertility in wildlife can represent a threat to the population's survival. Moreover, fcw methodologies are available for species that are relevant to address this issue on envimnmental biomonitoring programs, especially for crustaceans, despite their representativeness in the wildlife and their essential functions within ecosysterns. The present work aimed to propose methodologies based on the measurement of functionality and integrity spermatic biomarkers on Palaemonidae shrimps. We studied two species, an estuarine, Palaemon longirostris, and a coastal species. Palaemon serratus. due to their complementaiity on the continuum estuary-littoral ecosystem. Howevcr, crustaceans' sperm has many structuraI and functional characteristics, the number of transposable markers is limited. Thereafter, the research effort was refocused on the measurement of the DNA integrity and this inethod was evaluated for its adequacy for a biomonitoring study. We optitrtized and validate the Cornet assay for the Palaemonidae species, and the dynamic of the biological response in ternis of appearances, persistence, and possible effects on fitness was evaluated under controlled conditions in the laboratoiy. Furthermore, ex-vivo and in-vivo exposures were conducted using genotoxic models with different modes of action. On the one hand, results attested to the sensibility, the reproducibility and the integrating character of the response, on the other hand, no correlation between damaged sperm DNA and an altération of the pre-hatch stage of development was established. In paralIel, an in-situ approach was conducted to characterize the baseline level of the measured response, taking into consideration the specific constraints of each species' habitat. We were able to define a common baseline level and a threshold value for both species, highlighting the potential of the tool for inter-species transferability. This method was tested with native populations from the estuary and from the Seine Bay in 2015 and 20] 6. And, the results proved to be consistent with the contamination pressure and the hydro-sedimentary dynamics of the Seine Bay. Ultimately, the DNA integrity of sperm in Palaemonidae seems to be functional for in-situ deploytnents as a biomarker of exposure to genotoxic stress. Nevertheless, future studies should be conducted (1) to botter discern the implications of this spermatic damage on the recruitment of new cohorts and, (2) to test the transferability of the methodology to other shrimp species and on a wider geographical scale.

The occupational exposures of sewage workers are complex and variable, and include a great variety of biological and chemical agents. Previous research has focused mostly on infections and various symptoms among sewage workers, e.g. abdominal and respiratory symptoms. At several sewage plants in Sweden, concern arose about occupational cancer, specifically cancer of the stomach, the kidney, and the lung. The aim of this study was to study the cancer incidence among municipal sewage workers, some exposures that might be connected with cancer risk, and self reported abdominal and respiratory symptoms. In a cohort of municipal sewage workers there was no increase in the overall incidence of cancer when compared with the general population. However, there was a slight increase in the incidence of prostate cancer, but not in the sites of original concern among the workers. Infection by the gastric carcinogen Helicobacter pylori (determined from the presence of IgG antibodies in serum against H pylori) was no more prevalent in sewage workers than in comparable referents. Neither were sewage workers more exposed to genotoxic agents than comparable referents, as measured by the alkaline single cell gel electrophoresis (SCG) assay performed on peripheral lymphocytes. There was no increase in the three-month prevalence of abdominal symptoms when compared with other municipal workers. Specifically, there was no difference in prevalence of the common disorders dyspepsia and irritable bowel syndrome. Sewage workers reported adult bronchial asthma significantly more than the referents.

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

Ionisierende Strahlung verursacht in Abhängigkeit von den strahlenphysikalischen Eigenschaften der Radionuklide Zellschäden unterschiedlicher Komplexität. An humanen Lymphozyten wurde untersucht, ob die biologische Wirksamkeit von Alpha- und Betastrahlung sowie der Einfluss von Doxorubicin der Qualität des Strahlenschadens zugewiesen werden kann. Die DNA-Schäden und deren Reparatur wurden mit zellbiologischen Methoden quantifiziert.