La peau étant le premier tissu exposé aux diverses agressions de l’environnement extérieur, les cellules qui la composent doivent disposer de mécanismes de protection vis-à-vis de ces agressions, afin d’assurer le maintien de l’homéostasie tissulaire. Les cellules souches de l’épiderme assurant le renouvellement du compartiment épithélial pendant toute la vie de l’individu, la préservation de l’intégrité de leur génome est essentielle à la fonctionnalité pérenne de la peau. Mon doctorat avait pour objectif d’explorer les mécanismes mis en œuvre par les cellules souches de l’épiderme interfolliculaire afin de se protéger de deux stress génotoxiques radiatifs, à savoir : les rayonnements gamma et les rayonnements ultraviolets B (UVB). Durant mon doctorat, j’ai tout d’abord participé à la démonstration des mécanismes de protection mis en œuvre par les cellules souches des kératinocytes après irradiation ionisante. En effet, il a été montré que ces cellules sont capables de réparer très rapidement l’ensemble des dommages de l’ADN radio-induits, et que cette réparation était activée par le facteur de croissance FGF2 (Fibroblast Growth Factor 2). Afin de savoir si ce mécanisme de protection était aussi opérant dans les cellules souches de carcinome cutané, nous l’avons recherché dans la sous-population de cellules souches qui peut être isolée d’une lignée de carcinome cutané humain. Comme dans le cas des cellules souches normales, nous avons montré que les cellules souches de cancer présentent une réparation très rapide des dommages de l’ADN radio-induits. De plus, le facteur de croissance FGF2 participe à cette réparation, notamment par la présence d’isoformes de ce facteur dans le noyau cellulaire. Le second projet de mon doctorat avait pour objectif l’étude de la réponse des cellules souches et des progéniteurs de l’épiderme humain aux rayonnements UVB. Une fois mises en place les conditions de tri en cytométrie de flux et d’irradiation par les UVB, la toxicité de ces rayonnements a été évaluée dans un modèle cellulaire primaire. Nous avons caractérisé les effets des photons UVB sur la viabilité et la prolifération cellulaire et étudié la réparation des dommages de l’ADN. Cette étude nous a permis de mettre en évidence des réponses aux UVB différentes entre les cellules souches et leur descendance immédiate, les kératinocytes progéniteurs, notamment au niveau de l’activité de réparation des dommages de l’ADN. Par ailleurs, une étude du transcriptome des cellules irradiées a été réalisée, qui permet d’analyser les mécanismes globaux communs et spécifiques de réponse au stress dans les deux populations. L’ensemble des données obtenues nous permet de proposer plusieurs mécanismes de protection, communs et spécifiques, mis en œuvre par les cellules souches de l’épiderme en réponse aux stress radiatifs UVB et gamma. / Human skin is the first organ exposed to various environmental stresses, which requires the development by skin stem cells of specific mechanisms to protect themselves and to ensure tissue homeostasis. As stem cells are responsible for the maintenance of epidermis during individual lifetime, the preservation of genomic integrity in these cells is essential. My PhD aimed at exploring the mechanisms set up by epidermal stem cells in order to protect themselves from two genotoxic stresses, ionizing radiation ( Gamma Rays) and ultraviolet radiation (UVB). To begin my PhD, I have taken part of the demonstration of protective mechanisms used by keratinocyte stem cells after ionizing radiation. It has been shown that these cells are able to rapidly repair most types of radiation-induced DNA damage. Furthermore, we demonstrated that this repair is activated by the fibroblast growth factor 2 (FGF2). In order to know if this protective mechanism is also operating in cutaneous carcinoma stem cells, we investigated the response to gamma Rays of carcinoma stem cells isolated from a human carcinoma cell line. As in normal keratinocyte stem cells, we demonstrated that cancer stem cells could rapidly repair radio-induced DNA damage. Furthermore, fibroblast growth factor 2 also mediates this repair, notably thanks to its nuclear isoforms. The second project of my PhD was to study human epidermal stem cells and progenitors responses to UVB radiation. Once cytometry and irradiation conditions were set up, the toxicity of UVB radiation has been evaluate in the primary cell model. We then characterized UVB photons effects on cell viability, proliferation and repair of DNA damage. This study allowed us to bring out that responses of stem cells and their progeny to UVB are different, notably at the level of part of their repair activity of DNA damage. Moreover, progenitors and stem cells transcriptomic responses after UVB irradiation have been study in order to analyze the global mechanisms of stress response in the two cell populations. Taken together, data obtained during my PhD allowed us to show that stem cells respond differently than keratinocyte progenitors to radiation stress, and that they developed both intrinsic and radiation-induced strategies allowing a better protection. When comparing gamma Rays and UVB, we found that, although their toxic effects on skin share many similarities, the mechanisms set up by human epidermal stem cells to protect themselves vary according to the type of radiation stress.

Submitted by admin tede (tede@pucgoias.edu.br) on 2018-04-18T19:00:37Z No. of bitstreams: 1 RODRIGO ANSALONI DE OLIVEIRA.pdf: 564136 bytes, checksum: 846f6a16985a9180c2d4d7056354c90d (MD5) / Made available in DSpace on 2018-04-18T19:00:37Z (GMT). No. of bitstreams: 1 RODRIGO ANSALONI DE OLIVEIRA.pdf: 564136 bytes, checksum: 846f6a16985a9180c2d4d7056354c90d (MD5) Previous issue date: 2017-03-08 / The use of medicinal plants, including extracts, of popular use as a therapeutic resource is a widespread trend in the Brazilian population. Euphorbia tirucalli is widely used by Brazilian popular medicine in the treatment of lesions, infectious diseases, tumors and inflammatory diseases. Through this work. Objective: To evaluate the genotoxic and antigenotoxic activity of Euphorbia tirucalli (aveloz). Methodology: Latex was obtained from a plant specimen, in the city of Goiânia, and diluted to the concentration of 1mg / ml. To evaluate the genotoxic and antigenotoxic activity of the aqueous solution of Euphorbia tirucalli latex were performed by the micronucleus test in the hematopoietic bone marrow of mice. The cytogenetic analysis of the slides was performed in order to detect possible chromosomal changes and / or losses (micronuclei) in the polychromatic erythrocytes of the bone marrow of the animals submitted to the different treatments. Results: In the treatments concomitant with the aqueous solutions of latex of Euphorbia tirucalli and Mitomycin C, there was a decrease in micronuclei numbers in 2000 EPC. The results allowed to conclude that the aqueous solutions of Euphorbia Tirucalli latex presented genotoxic, antigenotoxic, cytotoxic and anticitotoxic activity and the concomitant use of the aqueous solutions of the latex of Euphorbia tirucalli and Mitomycin C was not indicated, as the genotoxic and cytotoxic effects of MMC were decreased And thus not achieving the expected effect of this highly genotoxic agent. / A utilização de plantas medicinais, incluindo os extratos, de uso popular como recurso terapêutico é uma tendência generalizada na população brasileira. A Euphorbia tirucalli é amplamente utilizada pela medicina popular brasileira no tratamento de lesões, doenças infecciosas, tumores e doenças inflamatórias. Por meio deste trabalho. Objetivo: Avaliar a atividade genotóxica e antigenotóxica de Euphorbia tirucalli (aveloz). Metodologia: O látex foi obtido de um exemplar da planta, na cidade de Goiânia, e diluído para a concentração de 1mg/ml. Para a avaliação da atividade genotóxica e antigenotóxica da solução aquosa do látex da Euphorbia tirucalli foram realizadas pelo teste de micronúcleo na medula óssea hematopoiética de camundongos. A análise citogenética das lâminas foi realizada com a finalidade de se detectar possíveis alterações e/ou perdas cromossômicas (micronúcleos) nos eritrócitos policromáticos (EPC) da medula óssea dos animais submetidos aos diferentes tratamentos. Resultados: Nos tratamentos concomitantes com as soluções aquosas do látex da Euphorbia tirucalli e Mitomicina C, houve diminuição dos números de micronúcleos em 2.000 EPC. Os resultados permitiram concluir que as soluções aquosas do látex da Euphorbia Tirucalli apresentaram atividade genotóxica, antigenotóxica, citotóxica e anticitotóxica e não indicado o uso concomitante das soluções aquosas do látex da Euphorbia tirucalli e Mitomicina C, pois os efeitos genotóxicos e citotóxicos da MMC foram diminuídos e assim não obtendo o efeito esperado deste agente altamente genotóxico.

Asf1 est une protéine chaperon d’histone, qui participe à l’assemblage et au désassemblage des histones H3/H4 sur l’ADN. Asf1 n’est pas essentiel pour la viabilité cellulaire chez S. cerevisiae, mais les voies de surveillance des dommages à l’ADN sont activées de façon constitutive dans les cellules dépourvues d’Asf1 et celles-ci sont hypersensibles à plusieurs types de stress génotoxiques. Chez S. cerevisiae, Asf1 forme un complexe stable avec Rad53 en absence de stress génotoxique. Nos résultats suggèrent qu’au moins trois surfaces d’interaction sont impliquées dans le complexe Asf1-Rad53. Le domaine FHA1 de Rad53 fixe Asf1 phosphorylé sur T270, l’extrémité C-terminale de Rad53 fixe la même surface d’Asf1 impliquée dans la fixation des co-chaperones HirA/CAF-1, et un troisième site putative est constituée de la surface d’Asf1 impliquée dans la fixation de l’histone H3 avec le domaine kinase de Rad53. Lors des stress génotoxiques, Rad53 est phosphorylée et activée. Mes résultats montrent une dissociation totale du complexe Rad53-Asf1 après traitement HU, mais la préservation du complexe après traitement des cellules avec une gamme de concentration de MMS. Nous pensons que la régulation du complexe traduisent des réponses cellulaires distinctes adaptées à des stress génotoxiques spécifiques. Par ailleurs, grâce à la structure du complexe formé par un peptide C-terminal de Rad53 et le domaine N-terminal d’Asf1, nous avons isolé une mutation rad53_A806R-L808R. Nous avons constaté que cette mutation déstabilise l’interaction entre Asf1 et Rad53 et augmente la viabilité des mutants rad9 et rad24 aux stress génotoxiquex. Ce mutant rad53_A806R-L808R semble retourne plus vite dans le cycle cellulaire et/ou traverse plus vite la phase S par rapport à Rad53-WT, et augmente la réparation de l’ADN ou l’adaptation aux dommages du simple mutant rad24Δ. / Asf1 is a histone chaperone, which participates in the assembly and disassembly of histones H3/H4 on DNA. Asf1 is not essential for cell viability in yeast, but the DNA damage checkpoints are constitutively activated in cells lacking Asf1 and they are hypersensitive to several types of genotoxic stress. In yeast, Asf1 forms a stable complex with Rad53 in the absence of genotoxic stress. Our results suggest that this complex involves at Ieast three interaction surfaces. One site involves the H3-binding surface of Asf1 with an as yet undefined surface of Rad53, probably reside in the kinase domain of Rad53. A second site is formed by the Rad53-FHA1 domain binding to Asf1-T270. The third site involves the C-terminal 21 aa of Rad53 bound to the conserved Asf1 N-terminal domain, where Rad53 competes with histone H3/H4 and co-chaperones HirA/CAF-1 for binding to the same surface of Asf1. Rad53 is phosphorylated and activated upon genotoxic stress. The Asf1-Rad53 complex dissociated when cells were treated with hydroxyurea but not methyl methane sulfonate, suggesting a regulation of the complex as a function of the stress.In addition to these results, we also found that the rad53-A806R+L808R mutation at the C-terminus of Rad53 destabilized the Asf1-Rad53 interaction and increased the viability of rad9 and rad24 mutants to genotoxic stress. The rad53-ALRR mutant also appeared to re-enter the cell cycle and/or traverse S-phase more rapidly than wild type and increased repair or adaptation when combined with the rad24 mutant.

Les organismes vivants sont en intime relation avec leur environnement et sont constamment influencés par de nombreux facteurs chimiques et physiques. Parmi les facteurs physiques présents dans notre environnement, les forces mécaniques, y compris la gravité, les radiations, dont les ultraviolets, et les champs électromagnétiques constituent les trois pôles principaux de nos travaux de doctorat. Des outils biologiques, cellulaires et moléculaires ont été développés afin dévaluer le rôle des RhoGTPases dans les altérations morphologiques, prolifératives et phénotypiques induites par la perte du vecteur gravité au cours de vols spatiaux. Au cours du vol de la capsule spatiale inhabitée FOTON-M3, nous avons pu mettre en évidence que la suppression de Rac1 par ARN interférentiel permettait de contrecarrer les effets délétères de la microgravité sur larchitecture du cytosquelette suggérant que cette molécule de signalisation participe à la réception et à la réactivité à la gravité. RhoA et Cdc42 ne semblent pas impliqués. Nous avons également développé un modèle expérimental dinduction de flux calcique par des peptides mimétiques de la matrice extracellulaire activant les intégrines destiné à être expérimenté au cours de vols paraboliques. Au cours de nos travaux visant à évaluer les effets biologiques des champs électromagnétiques, nous avons observé que les EMF de très basse fréquence (450µT-50Hz) naffectent ni les signaux calciques induits par des concentrations élevées de sérum ou des peptides mimétiques de la matrice extracellulaire, ni lexpression des gènes régulés par les UV-B. Ils sont cependant capables de soutenir les oscillations calciques induites par une concentration sub-optimale de sérum, sans toutefois réguler de manière évidente les voies de signalisation contrôlant la prolifération. Lirradiation par les UV-B dun grand nombre de cellules, primaires, immortalisées et tumorales induit, par épissage alternatif du préARNm du VEGF-A, lexpression dun nouveau variant, le VEGF111. Celui-ci est constitué de la combinaison des exons 1-4 et de lexon 8. Cette nouvelle forme de VEGF-A contient donc les sites de fixation aux VEGF-R1 et R-2 et est pro-angiogène in vitro sur les cellules endothéliales et les cellules souche embryonnaires et in vivo chez la souris. Labsence des exons 6 et 7 codant pour la liaison aux protéines de la matrice extracellulaire lui confère une diffusibilité tissulaire. Une de ses caractéristiques remarquable est sa résistance à la dégradation en raison de labsence du site de clivage par la plasmine et les MMPs. Ce nouveau variant est également induit par diverses substances génotoxiques dont les agents chimiothérapeutiques. Les mécanismes régulant lexpression du VEGF111 dépendent des voies de signalisation ATM/ATR, p53 et MAPKinases. La double personnalité de ce nouveau facteur pro-angiogène, néfaste par son induction potentielle au cours de traitements anti-cancéreux mais bénéfique par son utilisation dans le traitement de pathologies ischémiques particulièrement pertinente en cas dactivités protéolytiques élevées, ouvre un champ considérable dinvestigations. ----------------------------------------------------------------------------------------------------- Living organisms interact with their environment and are constantly influenced by various chemical and physical factors. Among the physical factors present in our environment, mechanical forces, including gravity, radiations, among which ultraviolet radiations, and electromagnetic fields constitute the three main poles of our research. Biological, cellular and molecular tools have been developed with the aim to evaluate the role of RhoGTPases in the morphological, proliferative and phenotypic alterations induced by the loss of gravitational field experienced during space flight. During the flight of the unmanned FOTON-M3 capsule, we have demonstrated that the suppression of Rac1 by small interference RNA was able to counteract the deleterious effects of microgravity on the cytoskeleton architecture. This suggests that this signaling molecule participates to the reception and reaction to gravity. RhoA and Cdc42 do not seem to be implicated. We have also developed an experimental model of induction of intracellular calcium ions fluxes by mimetic peptides of the extracellular matrix activating integrins to be used in parabolic flights. During our investigations aimed at evaluating the biological effects of electromagnetic fields, we observed that EMF of very low-frequency (450µT-50Hz) do not affect neither the calcium signals induced by high concentrations of serum or extracellular matrix mimetic peptides, nor the expression of genes regulated by UV-B. They are however able to sustain calcium oscillations induced by a sub-optimal concentration of serum but without disturbing the cellular proliferation rate. Irradiation by UV-B of a large number of cells, primary, immortalized and tumoral, induces, by alternative splicing of the VEGF-A pre-mRNA, the expression of a new variant, the VEGF111. This isoform is made of a combination of exons 1-4 and exon 8. This new VEGF-A variant contains therefore the binding sites to VEGF-R1 and R-2 and proved to be proangiogenic in vitro for endothelial and ES cells and in vivo in mice. The absence of exons 6 and 7 coding for the heparin binding sites confers it with tissue diffusibility. One of its striking characteristics is its resistance to degradation due to the absence of the cleavage site by plasmin and MMPs. This new variant is also induced by a series of genotoxic agents, including chemotherapeutic drugs. The mechanisms controlling the VEGF111 expression depend on the ATM/ATR, p53 and MAPKinases signaling pathways. The dual faces of VEGF111, detrimental by its potential induction during anti-cancer therapy but beneficial by its use for managing ischemic pathologies, mostly relevant when associated with high proteolytic activities, opens a considerable field of investigations.

Agriculture is a valuable economic resource in western Canada, but for decades farmers have focused on intensive production practices while ignoring the long-term health and maintenance of the land. In recent years, the use of conservation agricultural techniques has been encouraged in an effort to conserve prairie landscape while sustaining cropland productivity. Sustainable agricultural practices that promote soil and water conservation and benefit wildlife and prairie biodiversity include conservation tillage and planting of winter cereal crops. Many species of wild birds nest in the ground cover provided by minimum tillage and fall seeded cropland in the spring. Although habitat quality in conservation areas is superior for birds, there is potential for eggs of ground nesting birds to be exposed to herbicides during spring weed control operations. Herbicides commonly used on the prairies to control weed growth in conservational systems include 2,4-D and Buctril-M®. Since the subtlethal effects of exposure to these herbicides may include DNA damage and immunomodulation, the overall goal of this study was to assess whether <i>in ovo</i> exposure to the herbicides 2,4-D and Buctril-M® adversely affects genetic material and/or immune system function in newly hatched domestic chickens (<i>Gallus gallus</i>) and ducks (<i>Anas platyrhynchos</i>), as surrogates for wild bird species.<p>Study design attempted to reproduce actual field exposures by use of an agricultural field spray simulator to apply formulated herbicides (as opposed to pure active ingredients) at recommended crop application rates. In three separate experiments, fertile chicken eggs were sprayed with 2,4-D ester formulation or with Buctril-M® formulation, and fertile duck eggs were sprayed with 2,4-D ester formulation, during either an early (embryonic day 6) or late (embryonic day 15 for chickens or embryonic day 21 for ducks) stage of incubation. Genotoxicity and immune system function were evaluated in the hatchlings as the main toxicological endpoints to assess potential subtle effects from herbicide exposure, but additional measures of general health and development were also evaluated. Two endpoints were used to assess subtle changes to genetic integrity. The comet assay was used to detect structural damage (strand breaks) in avian lymphocyte DNA, as an index of acute genotoxic effects. Flow cytometry was used to examine potential clastogenic effects of the herbicides, by determining if chromosomal changes resulted in variability in the DNA content of avian erythrocytes. Several endpoints were examined to evaluate potential exposure-induced effects on the immune system. Immunopathological assessment of chicks and ducklings included differential lymphocyte counts, as well as immune organ weights and histopathology. The cell-mediated and humoral immune responses in hatchlings were assessed using the delayed-type hypersensitivity test and measurement of systemic antibody production in response to immunization, respectively. Exposure of fertile chicken and duck eggs to Buctril-M® or 2,4-D had no effects on the biomarkers of genetic integrity in this study. Differences in herbicide treatment (high and low concentrations) and times of exposure (early and late incubation stages) did not translate into noticeable factor effects in final model analyses for any of the genotoxicity assay variables evaluated in newly hatched chickens exposed in ovo to 2,4-D. Similarly, comet assay outcomes in chicks exposed to Buctril-M® were not significantly associated with either herbicide treatment or time of exposure as fixed effect factors. Results of the comet assay using peripheral lymphocytes from ducklings provided evidence of potential primary genetic damage associated with the time of spray exposure in ovo. Comet tail DNA content was significantly associated (P = 0.0269) with exposure times, suggesting that ducks may be increasingly sensitive to spray exposure conditions at an early stage of embryological development. Effects of exposure timing were not attributable to herbicide treatment. Although 2,4-D exposure time was associated with DNA strand breakage in ducklings, there was no evidence of chromosomal damage. However, an association between the HPCV values (a measure of DNA content variability) and time of spray exposure was observed in the experiment where 21-day-old chickens were treated in ovo with Buctril-M®. The mean HPCV value for the early exposure group (E6) was significantly greater (P = 0.0210) than that of the group treated later in incubation (E15). However, Buctril-M® the concentration of herbicide did not have any influence on this outcome, and the reason for the difference between exposure times is uncertain, but may be attributed to stress associated with manipulations during spraying. An increase in HPCV, reflecting greater intercellular DNA variability, is indicative of increased incidence of chromosomal damage, which may be an effect of disturbance during early periods of incubation as a result of exposure conditions.<p>Among the panel of immunotoxicity tests conducted to evaluate the effects of <i>in ovo</i> exposure to 2,4-D and Buctril-M® on the developing avian immune system, only heterophil/ lymphocyte (H/L) ratios and relative immune organ weights were significantly associated with either herbicide treatment or time of spray exposure in all three experiments. In 21-day-old chicks exposed in ovo to 2,4-D, relative bursa weight was associated with the different herbicide treatments (P = 0.0006). Relative bursa weights were significantly lower in chicks in the low dose group, while the opposite effect was observed in the high dose chicks, compared with the controls. It is unlikely that the observed decrease in bursa weight in the low dose group is causally related to herbicide exposure because a consistent dose-response effect was not observed, but this outcome may be explained by a compensatory immune response. The relative spleen weights of newly hatched chickens exposed in ovo to Buctril-M® exhibited a significant association with herbicide treatment (P = 0.0137). Relative spleen weights for birds in the low dose treatment groups were significantly different than both the control (P = 0.0179) and high dose groups (P = 0.0125). However, there was no significant difference between high dose and control groups, and this outcome reduces the likelihood of a causal relationship between spleen weight and herbicide exposure. In the parallel experiment involving in ovo exposure to 2,4-D to ducklings, relative bursa weight was associated with time of spray exposure (P = 0.0434). Ducklings that hatched from eggs exposed to spray on day 6 of incubation exhibited greater mean relative bursa weights than the birds exposed to spray at a later incubation stage (E21). This result implies that spray exposure during earlier stages of development may result in conditions which affect the humoral immune response, if increased bursal weight is associated with increased B lymphocyte and antibody production. In the same experiment, mean H/L ratios in peripheral blood samples from 21-day-old ducklings were significantly different between the groups treated with the high concentration of 2,4-D and water (control) (P = 0.0395). Although ratios from the birds in the low dose groups were not significantly different from the control groups, changes in H/L ratio values demonstrate a dose dependent relationship with increasing herbicide exposure.<p>Residue analysis of chicken and duck eggs in this study measured transfer of herbicide through the shell and into the embryo 24 hours and up to 5 days (chickens only) after spraying. Mean 2,4-D residue concentrations were higher in both chicken and duck eggs from the high dose (10X) groups than in eggs exposed to the recommended field rate of herbicide application (1X). Embryo residue concentrations in the chicken eggs increased from the day following exposure to 5 days after spraying, in both low and high dose groups. This observation indicates that the risk of contaminant-induced adverse effects may continue to increase for at least several days after exposure, thereby influencing the concentration of herbicide to which the developing embryo is exposed.<p>On the Canadian prairies, wild bird eggs are potentially to be exposed to 2,4-D and Buctril-M® during various stages of embryonic development. The present study examined effects of herbicide exposure at two distinct times during incubation, and demonstrated the potential for subtle impacts on genetic integrity and the immune system. Results indicate that spray exposure during earlier stages of organogenesis may cause more significant adverse effects. Given the possible harmful consequences of the observed changes on the long-term health of wild birds, further research is needed in order to better characterize the risks of in ovo agrochemical exposure in prairie ecosystems.

Agriculture is a valuable economic resource in western Canada, but for decades farmers have focused on intensive production practices while ignoring the long-term health and maintenance of the land. In recent years, the use of conservation agricultural techniques has been encouraged in an effort to conserve prairie landscape while sustaining cropland productivity. Sustainable agricultural practices that promote soil and water conservation and benefit wildlife and prairie biodiversity include conservation tillage and planting of winter cereal crops. Many species of wild birds nest in the ground cover provided by minimum tillage and fall seeded cropland in the spring. Although habitat quality in conservation areas is superior for birds, there is potential for eggs of ground nesting birds to be exposed to herbicides during spring weed control operations. Herbicides commonly used on the prairies to control weed growth in conservational systems include 2,4-D and Buctril-M®. Since the subtlethal effects of exposure to these herbicides may include DNA damage and immunomodulation, the overall goal of this study was to assess whether <i>in ovo</i> exposure to the herbicides 2,4-D and Buctril-M® adversely affects genetic material and/or immune system function in newly hatched domestic chickens (<i>Gallus gallus</i>) and ducks (<i>Anas platyrhynchos</i>), as surrogates for wild bird species.<p>Study design attempted to reproduce actual field exposures by use of an agricultural field spray simulator to apply formulated herbicides (as opposed to pure active ingredients) at recommended crop application rates. In three separate experiments, fertile chicken eggs were sprayed with 2,4-D ester formulation or with Buctril-M® formulation, and fertile duck eggs were sprayed with 2,4-D ester formulation, during either an early (embryonic day 6) or late (embryonic day 15 for chickens or embryonic day 21 for ducks) stage of incubation. Genotoxicity and immune system function were evaluated in the hatchlings as the main toxicological endpoints to assess potential subtle effects from herbicide exposure, but additional measures of general health and development were also evaluated. Two endpoints were used to assess subtle changes to genetic integrity. The comet assay was used to detect structural damage (strand breaks) in avian lymphocyte DNA, as an index of acute genotoxic effects. Flow cytometry was used to examine potential clastogenic effects of the herbicides, by determining if chromosomal changes resulted in variability in the DNA content of avian erythrocytes. Several endpoints were examined to evaluate potential exposure-induced effects on the immune system. Immunopathological assessment of chicks and ducklings included differential lymphocyte counts, as well as immune organ weights and histopathology. The cell-mediated and humoral immune responses in hatchlings were assessed using the delayed-type hypersensitivity test and measurement of systemic antibody production in response to immunization, respectively. Exposure of fertile chicken and duck eggs to Buctril-M® or 2,4-D had no effects on the biomarkers of genetic integrity in this study. Differences in herbicide treatment (high and low concentrations) and times of exposure (early and late incubation stages) did not translate into noticeable factor effects in final model analyses for any of the genotoxicity assay variables evaluated in newly hatched chickens exposed in ovo to 2,4-D. Similarly, comet assay outcomes in chicks exposed to Buctril-M® were not significantly associated with either herbicide treatment or time of exposure as fixed effect factors. Results of the comet assay using peripheral lymphocytes from ducklings provided evidence of potential primary genetic damage associated with the time of spray exposure in ovo. Comet tail DNA content was significantly associated (P = 0.0269) with exposure times, suggesting that ducks may be increasingly sensitive to spray exposure conditions at an early stage of embryological development. Effects of exposure timing were not attributable to herbicide treatment. Although 2,4-D exposure time was associated with DNA strand breakage in ducklings, there was no evidence of chromosomal damage. However, an association between the HPCV values (a measure of DNA content variability) and time of spray exposure was observed in the experiment where 21-day-old chickens were treated in ovo with Buctril-M®. The mean HPCV value for the early exposure group (E6) was significantly greater (P = 0.0210) than that of the group treated later in incubation (E15). However, Buctril-M® the concentration of herbicide did not have any influence on this outcome, and the reason for the difference between exposure times is uncertain, but may be attributed to stress associated with manipulations during spraying. An increase in HPCV, reflecting greater intercellular DNA variability, is indicative of increased incidence of chromosomal damage, which may be an effect of disturbance during early periods of incubation as a result of exposure conditions.<p>Among the panel of immunotoxicity tests conducted to evaluate the effects of <i>in ovo</i> exposure to 2,4-D and Buctril-M® on the developing avian immune system, only heterophil/ lymphocyte (H/L) ratios and relative immune organ weights were significantly associated with either herbicide treatment or time of spray exposure in all three experiments. In 21-day-old chicks exposed in ovo to 2,4-D, relative bursa weight was associated with the different herbicide treatments (P = 0.0006). Relative bursa weights were significantly lower in chicks in the low dose group, while the opposite effect was observed in the high dose chicks, compared with the controls. It is unlikely that the observed decrease in bursa weight in the low dose group is causally related to herbicide exposure because a consistent dose-response effect was not observed, but this outcome may be explained by a compensatory immune response. The relative spleen weights of newly hatched chickens exposed in ovo to Buctril-M® exhibited a significant association with herbicide treatment (P = 0.0137). Relative spleen weights for birds in the low dose treatment groups were significantly different than both the control (P = 0.0179) and high dose groups (P = 0.0125). However, there was no significant difference between high dose and control groups, and this outcome reduces the likelihood of a causal relationship between spleen weight and herbicide exposure. In the parallel experiment involving in ovo exposure to 2,4-D to ducklings, relative bursa weight was associated with time of spray exposure (P = 0.0434). Ducklings that hatched from eggs exposed to spray on day 6 of incubation exhibited greater mean relative bursa weights than the birds exposed to spray at a later incubation stage (E21). This result implies that spray exposure during earlier stages of development may result in conditions which affect the humoral immune response, if increased bursal weight is associated with increased B lymphocyte and antibody production. In the same experiment, mean H/L ratios in peripheral blood samples from 21-day-old ducklings were significantly different between the groups treated with the high concentration of 2,4-D and water (control) (P = 0.0395). Although ratios from the birds in the low dose groups were not significantly different from the control groups, changes in H/L ratio values demonstrate a dose dependent relationship with increasing herbicide exposure.<p>Residue analysis of chicken and duck eggs in this study measured transfer of herbicide through the shell and into the embryo 24 hours and up to 5 days (chickens only) after spraying. Mean 2,4-D residue concentrations were higher in both chicken and duck eggs from the high dose (10X) groups than in eggs exposed to the recommended field rate of herbicide application (1X). Embryo residue concentrations in the chicken eggs increased from the day following exposure to 5 days after spraying, in both low and high dose groups. This observation indicates that the risk of contaminant-induced adverse effects may continue to increase for at least several days after exposure, thereby influencing the concentration of herbicide to which the developing embryo is exposed.<p>On the Canadian prairies, wild bird eggs are potentially to be exposed to 2,4-D and Buctril-M® during various stages of embryonic development. The present study examined effects of herbicide exposure at two distinct times during incubation, and demonstrated the potential for subtle impacts on genetic integrity and the immune system. Results indicate that spray exposure during earlier stages of organogenesis may cause more significant adverse effects. Given the possible harmful consequences of the observed changes on the long-term health of wild birds, further research is needed in order to better characterize the risks of in ovo agrochemical exposure in prairie ecosystems.

A contaminação do solo é uma das fontes pela qual os contaminantes são escoados para os ecossistemas aquáticos, afetando a qualidade dos sedimentos. Neste compartimento alguns compostos são capazes de bioacumular e interferir nos fluxos de energia e de nutrientes da cadeia biológica, gerando efeitos agudos, crônicos e genotóxicos para as comunidades que vivem ou entram em contato com este local. A região de estudo está localizada em área às margens do rio Taquari, no município de Triunfo, RS, próxima a um sítio com solo contaminado por preservantes de madeira, com passivo ambiental identificado (creosoto, pentaclorofenol e a hidrossal CCA). Esta área foi submetida à primeira fase do processo de intervenção para retirada das principais fontes ativas. Em estudos anteriores foi definido o provável escoamento de contaminantes para o rio a partir do solo contaminado. Para avaliar a qualidade dos sedimentos, nas diferentes fases de intervenção da área, foi utilizado o ensaio Salmonella/microssoma, para avaliar mutagênese, além da dosagem de HPAs nos extratos orgânicos de sedimentos. Foram testados quatro áreas de coleta, identificadas pela distância em Km a partir da foz, abrangendo local a montante do sítio, Ta032, em frente, Ta010, e a jusante, Ta006 e Ta004. As coletas foram nas fases, antes (inverno e verão), durante (verão) e após (verão) o processo de intervenção. A mutagênese foi avaliada através de linhagens que medem erro no quadro de leitura (TA98 e Ta97a) e substituição de pares de bases (TA100), na presença e ausência de S9mix (±S9) em extratos de compostos moderadamente polares de sedimento. Os resultados indicaram presença de pró-mutágenos em todas as amostragens na área Ta010, sendo mais elevada na anterior à intervenção (verão) para TA100+S9 (1672 rev/g) decrescendo durante e após este processo. Foram também observados valores constantes de HPAs totais e presença das espécies com potencial cancerígeno nos diferentes períodos avaliados. O local Ta006, após a intervenção, foi o que mostrou mutagênese mais alta (764 rev/g), sendo que as espécies consideradas cancerígenas foram detectadas em maiores concentrações, em especial o benzo(b+k) fluoranteno, que também esteve elevado, neste período, em Ta004. Presença de mutagênese e HPAs em arroio interno ao sítio, a montante de Ta010, marcaram uma das rotas de dispersão dos contaminantes. A presença de contaminação em frente ao sítio, após a intervenção, pode estar relacionada com a técnica escolhida para retirada das fontes ativas, contribuindo na presença de contaminantes a jusante deste rio, Ta006 e Ta004. / Soil contamination is one fo the sources of contaminant runoff into acquatic system, affecting sediment quality. In this compartment some compounds can bioaccumulate and interfere in the energy and nutrient flux of the biological chain with acute, chronic and genotoxic effects on the communities who live there or have contact with this place. The region studied is located on the area on the banks of Taquari river, in the municipality of Triunfo, RS, close to a site with soil contaminated by wood preservatives and identified environmental liabilities (creosote, pentachlorophenol and hydrosalt CCA). This area was submitted to the first stage of the intervention process to remove the main active sources. Previous studies defined the probable runoff of contaminants into river from contaminated soil. In order to evaluate the sediment quality in the different phases of intervention in the area, the Salmonella/microsome assay was used to evaluate mutagenesis, besides the dosage of PAHs in the sediment organic extracts. Four collection areas were tested, identified by the distance in km from the mouth, covering a location upstream from the site, Ta032, in front, Ta010, and downstream, Ta006 and 004. The collections were performed in the phases, before (winter and summer), during (summer) and after (summer) the intervention process. Mutagenesis was evaluated using frameshift (TA98 and Ta97a) and base pair substitution (TA100) strains in the presence and absence of S9mix (±S9), in extracts of moderately polar sediment compounds. The results indicate the presence of promutagens in all samplings in the Ta010 area, and it is higher before the intervention (summer) for TA100+S9 (1672 rev/g) decreasing during and after this process. Constant values of total PAHs and presence of species with a carcinogenic potential were also observed in the different periods evaluated. Site Ta006, after intervention, showed the highest mutagenesis (764 rev/g), and the species considered carcinogenic were detected at higher concentrations, especially benzo(b+k) fluoranthene, which was also high during this period at Ta004. The presence of mutagenesis and PAHs in a stream inside the site, upstream from Ta010 marks one of the contaminant dispersion routes. The presence of contamination in front of the site after intervention may be related to the technique chosen to remove the active sources, contributing to the presence of contaminants downstream of this river, Ta006 and Ta004.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Carbon nanotubes (CNTs) are rigid molecules, flexible and resistant to tensions formed from sheets of carbon (graphene), which at high temperature, ultimately to curl, forming nanometer diameter tubes. Among the different types of CNTs, the multi-walled carbon nanotubes (MWCNTs) can be noted for comprising a set of concentric nanotubes with perfect structures and defect- free materials. Several applications for this material are suggested, including biological applications such as manufacturing of biosensors, backers of drugs and vaccines and other biomaterials. However, before these materials can be incorporated into the market, is necessary to know their cytotoxic and genotoxic effects. Thus, this study aims to evaluate the mutagenicity of carbon nanotubes in somatic cells of Drosophila melanogaster, using the Somatic Mutation and Recombination Test (SMART). For this purpose we have used 72-hour larvae from standard (ST) and high bioactivation (HB) crosses. The larvae from both crosses were treated with solutions containing different concentrations of multi-walled carbon nanotubes (MWCNTs) functionalized (0,50, 100, 150, 200, 250 m&#956; /mL). For positive control, doxorubicin DXR (0,4 mM) was used, and reverse osmosis water for negative control. The descendants trans-heterozygous (MH) analyzed from both ST and HB crosses, had no significant effect on the frequency of mutant spots when compared with negative control (reverse osmosis water). This way, based on the results and on the experimental conditions mentioned in this study, it can be concluded that MWCNTs were not mutagenic for Drosophila melanosgaster. / Nanotubos de carbono (NTCs) são moléculas rígidas, flexíveis e resistentes a tensões formadas a partir de folhas de carbono (grafenos), que em altas temperaturas, acabam por se enrolarem, formando tubos de diâmetro nanométrico. Existem diferentes tipos de NTCs, dentre eles podem-se destacar os nanotubos de carbono de parede múltipla (NTCPM) que compreendem um conjunto de nanotubos concêntricos com uma estrutura livre de defeitos. Várias aplicações para este material são sugeridas, incluindo aplicações biológicas como fabricação de biosensores, veiculadores de drogas e vacinas entre outros biomateriais. Contudo, antes que estes materiais possam ser incorporados ao mercado, há a necessidade de se conhecer seus efeitos citotóxicos e genotóxicos. Sendo assim, o presente estudo tem como objetivo avaliar a mutagenicidade de nanotubos de carbono em células somáticas de Drosophila melanogaster, utilizando o Teste para detecção de mutação e recombinação somática (SMART). Para tanto, foram utilizadas larvas de 72 horas provenientes do cruzamento padrão (ST) e do cruzamento de alta bioativação (HB), que foram tratadas com soluções contendo diferentes concentrações de NTCPM funcionalizados (0,50, 100, 150, 200, 250 m&#956; / mL). Como controle positivo, foi utilizada a doxorrubicina DXR (0,4 mM), e como controle negativo água de osmose reversa. As asas dos descendentes trans-heterozigotos (MH) analisados de ambos os cruzamentos ST e HB, não apresentaram resultado significativo sobre a frequência de manchas mutantes quando comparadas com o controle negativo. Desta forma, com base nos resultados obtidos e nas condições experimentais mencionadas, pode-se concluir que NTCPM não foram mutagênicos para D. melanosgaster. / Mestre em Genética e Bioquímica

A contaminação do solo é uma das fontes pela qual os contaminantes são escoados para os ecossistemas aquáticos, afetando a qualidade dos sedimentos. Neste compartimento alguns compostos são capazes de bioacumular e interferir nos fluxos de energia e de nutrientes da cadeia biológica, gerando efeitos agudos, crônicos e genotóxicos para as comunidades que vivem ou entram em contato com este local. A região de estudo está localizada em área às margens do rio Taquari, no município de Triunfo, RS, próxima a um sítio com solo contaminado por preservantes de madeira, com passivo ambiental identificado (creosoto, pentaclorofenol e a hidrossal CCA). Esta área foi submetida à primeira fase do processo de intervenção para retirada das principais fontes ativas. Em estudos anteriores foi definido o provável escoamento de contaminantes para o rio a partir do solo contaminado. Para avaliar a qualidade dos sedimentos, nas diferentes fases de intervenção da área, foi utilizado o ensaio Salmonella/microssoma, para avaliar mutagênese, além da dosagem de HPAs nos extratos orgânicos de sedimentos. Foram testados quatro áreas de coleta, identificadas pela distância em Km a partir da foz, abrangendo local a montante do sítio, Ta032, em frente, Ta010, e a jusante, Ta006 e Ta004. As coletas foram nas fases, antes (inverno e verão), durante (verão) e após (verão) o processo de intervenção. A mutagênese foi avaliada através de linhagens que medem erro no quadro de leitura (TA98 e Ta97a) e substituição de pares de bases (TA100), na presença e ausência de S9mix (±S9) em extratos de compostos moderadamente polares de sedimento. Os resultados indicaram presença de pró-mutágenos em todas as amostragens na área Ta010, sendo mais elevada na anterior à intervenção (verão) para TA100+S9 (1672 rev/g) decrescendo durante e após este processo. Foram também observados valores constantes de HPAs totais e presença das espécies com potencial cancerígeno nos diferentes períodos avaliados. O local Ta006, após a intervenção, foi o que mostrou mutagênese mais alta (764 rev/g), sendo que as espécies consideradas cancerígenas foram detectadas em maiores concentrações, em especial o benzo(b+k) fluoranteno, que também esteve elevado, neste período, em Ta004. Presença de mutagênese e HPAs em arroio interno ao sítio, a montante de Ta010, marcaram uma das rotas de dispersão dos contaminantes. A presença de contaminação em frente ao sítio, após a intervenção, pode estar relacionada com a técnica escolhida para retirada das fontes ativas, contribuindo na presença de contaminantes a jusante deste rio, Ta006 e Ta004. / Soil contamination is one fo the sources of contaminant runoff into acquatic system, affecting sediment quality. In this compartment some compounds can bioaccumulate and interfere in the energy and nutrient flux of the biological chain with acute, chronic and genotoxic effects on the communities who live there or have contact with this place. The region studied is located on the area on the banks of Taquari river, in the municipality of Triunfo, RS, close to a site with soil contaminated by wood preservatives and identified environmental liabilities (creosote, pentachlorophenol and hydrosalt CCA). This area was submitted to the first stage of the intervention process to remove the main active sources. Previous studies defined the probable runoff of contaminants into river from contaminated soil. In order to evaluate the sediment quality in the different phases of intervention in the area, the Salmonella/microsome assay was used to evaluate mutagenesis, besides the dosage of PAHs in the sediment organic extracts. Four collection areas were tested, identified by the distance in km from the mouth, covering a location upstream from the site, Ta032, in front, Ta010, and downstream, Ta006 and 004. The collections were performed in the phases, before (winter and summer), during (summer) and after (summer) the intervention process. Mutagenesis was evaluated using frameshift (TA98 and Ta97a) and base pair substitution (TA100) strains in the presence and absence of S9mix (±S9), in extracts of moderately polar sediment compounds. The results indicate the presence of promutagens in all samplings in the Ta010 area, and it is higher before the intervention (summer) for TA100+S9 (1672 rev/g) decreasing during and after this process. Constant values of total PAHs and presence of species with a carcinogenic potential were also observed in the different periods evaluated. Site Ta006, after intervention, showed the highest mutagenesis (764 rev/g), and the species considered carcinogenic were detected at higher concentrations, especially benzo(b+k) fluoranthene, which was also high during this period at Ta004. The presence of mutagenesis and PAHs in a stream inside the site, upstream from Ta010 marks one of the contaminant dispersion routes. The presence of contamination in front of the site after intervention may be related to the technique chosen to remove the active sources, contributing to the presence of contaminants downstream of this river, Ta006 and Ta004.

A contaminação do solo é uma das fontes pela qual os contaminantes são escoados para os ecossistemas aquáticos, afetando a qualidade dos sedimentos. Neste compartimento alguns compostos são capazes de bioacumular e interferir nos fluxos de energia e de nutrientes da cadeia biológica, gerando efeitos agudos, crônicos e genotóxicos para as comunidades que vivem ou entram em contato com este local. A região de estudo está localizada em área às margens do rio Taquari, no município de Triunfo, RS, próxima a um sítio com solo contaminado por preservantes de madeira, com passivo ambiental identificado (creosoto, pentaclorofenol e a hidrossal CCA). Esta área foi submetida à primeira fase do processo de intervenção para retirada das principais fontes ativas. Em estudos anteriores foi definido o provável escoamento de contaminantes para o rio a partir do solo contaminado. Para avaliar a qualidade dos sedimentos, nas diferentes fases de intervenção da área, foi utilizado o ensaio Salmonella/microssoma, para avaliar mutagênese, além da dosagem de HPAs nos extratos orgânicos de sedimentos. Foram testados quatro áreas de coleta, identificadas pela distância em Km a partir da foz, abrangendo local a montante do sítio, Ta032, em frente, Ta010, e a jusante, Ta006 e Ta004. As coletas foram nas fases, antes (inverno e verão), durante (verão) e após (verão) o processo de intervenção. A mutagênese foi avaliada através de linhagens que medem erro no quadro de leitura (TA98 e Ta97a) e substituição de pares de bases (TA100), na presença e ausência de S9mix (±S9) em extratos de compostos moderadamente polares de sedimento. Os resultados indicaram presença de pró-mutágenos em todas as amostragens na área Ta010, sendo mais elevada na anterior à intervenção (verão) para TA100+S9 (1672 rev/g) decrescendo durante e após este processo. Foram também observados valores constantes de HPAs totais e presença das espécies com potencial cancerígeno nos diferentes períodos avaliados. O local Ta006, após a intervenção, foi o que mostrou mutagênese mais alta (764 rev/g), sendo que as espécies consideradas cancerígenas foram detectadas em maiores concentrações, em especial o benzo(b+k) fluoranteno, que também esteve elevado, neste período, em Ta004. Presença de mutagênese e HPAs em arroio interno ao sítio, a montante de Ta010, marcaram uma das rotas de dispersão dos contaminantes. A presença de contaminação em frente ao sítio, após a intervenção, pode estar relacionada com a técnica escolhida para retirada das fontes ativas, contribuindo na presença de contaminantes a jusante deste rio, Ta006 e Ta004. / Soil contamination is one fo the sources of contaminant runoff into acquatic system, affecting sediment quality. In this compartment some compounds can bioaccumulate and interfere in the energy and nutrient flux of the biological chain with acute, chronic and genotoxic effects on the communities who live there or have contact with this place. The region studied is located on the area on the banks of Taquari river, in the municipality of Triunfo, RS, close to a site with soil contaminated by wood preservatives and identified environmental liabilities (creosote, pentachlorophenol and hydrosalt CCA). This area was submitted to the first stage of the intervention process to remove the main active sources. Previous studies defined the probable runoff of contaminants into river from contaminated soil. In order to evaluate the sediment quality in the different phases of intervention in the area, the Salmonella/microsome assay was used to evaluate mutagenesis, besides the dosage of PAHs in the sediment organic extracts. Four collection areas were tested, identified by the distance in km from the mouth, covering a location upstream from the site, Ta032, in front, Ta010, and downstream, Ta006 and 004. The collections were performed in the phases, before (winter and summer), during (summer) and after (summer) the intervention process. Mutagenesis was evaluated using frameshift (TA98 and Ta97a) and base pair substitution (TA100) strains in the presence and absence of S9mix (±S9), in extracts of moderately polar sediment compounds. The results indicate the presence of promutagens in all samplings in the Ta010 area, and it is higher before the intervention (summer) for TA100+S9 (1672 rev/g) decreasing during and after this process. Constant values of total PAHs and presence of species with a carcinogenic potential were also observed in the different periods evaluated. Site Ta006, after intervention, showed the highest mutagenesis (764 rev/g), and the species considered carcinogenic were detected at higher concentrations, especially benzo(b+k) fluoranthene, which was also high during this period at Ta004. The presence of mutagenesis and PAHs in a stream inside the site, upstream from Ta010 marks one of the contaminant dispersion routes. The presence of contamination in front of the site after intervention may be related to the technique chosen to remove the active sources, contributing to the presence of contaminants downstream of this river, Ta006 and Ta004.