The irradiation of cells in culture is an essential part of many radiation biology experiments. Since these experiments necessarily involve the irradiation of cell culture vessels and nutrient medium, the possibility of effects due to the interactions of irradiated material with growing cells needed to be investigated. In the present study the micronucleus frequency in Chinese hamster ovary (CHO) cells as a function of such parameters as type of radiation, type of cell substrate, changes in cell environment, and time course of the effect were characterized. Observations of the persistence of micronucleus formation in irradiated CHO cells reveal that the number of cells containing micronuclei reaches its maximum within nine hours after irradiation and remain elevated for at least five days. The influence of the cell environment on micronucleus formation in CHO cells was examined by plating cells in preirradiated nutrient medium or on preirradiated cell culture vessels. In all experiments, pre-irradiation of the cell substrate (the culture dish or culture dish filled with medium) led to a significantly higher micronucleus frequency than when cells were plated on un-irradiated substrate. The difference is most pronounced at the lowest doses examined. These results suggest that methods of cell culture vessel sterilization and the composition of cell attachment surfaces could be confounding factors, particularly in the experiments which are intended to examine the response of cells exposed to low doses of ionizing radiation.

Η δοξορουβικίνη αποτελεί μια ευρέως χρησιμοποιούμενη αντικαρκινική ένωση σε διάφορους τύπους νεοπλασιών και ανήκει στην οικογένεια των ανθρακυκλινών. Για την κυτταροστατική της δράση έχουν προταθεί διάφοροι μηχανισμοί, μεταξύ των οποίων επαγωγή απόπτωσης και γενετικές αλλαγές. Η απόπτωση αποτελεί μια φυσιολογική διαδικασία, η οποία ελέγχει τον αριθμό των κυττάρων σ’ έναν οργανισμό. Λειτουργεί ως αμυντικός μηχανισμός σε περιπτώσεις κυττάρων που έχουν υποστεί βλάβη και παρεμποδίζει την εξάπλωση μεταλλάξεων συμπεριλαμβανομένων και εκείνων που οδηγούν σε καρκινογένεση. Η απόπτωση μπορεί να ενεργοποιηθεί κυρίως μέσω της εξωγενούς και της ενδογενούς οδούς. Στην παρούσα ερευνητική εργασία μελετήθηκε η σχέση επαγωγής απόπτωσης και γενετικών βλαβών που προκαλούνται από τη δοξορουβικίνη σε κυτταρική σειρά ποντικού C2C12. Για τη διερεύνηση των γενετικών βλαβών μελετήθηκε η συχνότητα των μικροπυρήνων (ΜΝ) με χρώση DAPI. H επαγωγή απόπτωσης εξετάστηκε με τη μέθοδο διπλής χρώσης αννεξίνη V/ιωδιούχο προπίδιο. Η μελέτη του μηχανισμού προέλευσης των μικροπυρήνων πραγματοποιήθηκε με σήμανση πρωτεϊνών του κινητοχώρου, την ανοσοχημική μέθοδο CREST σε συνδυασμό με ανοσοσήμανση της α-τουμπουλίνης για την εμφάνιση του κυτταροπλάσματος. Παρατηρήθηκε ότι η δοξορουβικίνη στις μελετηθείσες συγκεντρώσεις (0.05 μg/ml, 0.10 μg/ml, 0.20 μg/ml, 1.00 μg/ml, 2.50 μg/ml και 5.00 μg/ml,) προκαλεί αυξημένες συχνότητες αποπτωτικών κυττάρων και κυττάρων που περιέχουν μικροπυρήνες. Διαπιστώθηκε ότι η αύξηση της απόπτωσης σχετίζεται με μείωση της συχνότητας των μικροπυρήνων, υποδεικνύοντας πιθανή συμμετοχή της απόπτωσης στον περιορισμό των κυττάρων που φέρουν γενετική βλάβη. Η μελέτη του μηχανισμού προέλευσης των μικροπυρήνων έδειξε ότι η δοξορουβικίνη επάγει τη χρωμοσωματική θραύση και σε μικρότερο βαθμό τη χρωμοσωματική καθυστέρηση. Η τελευταία παρατήρηση μας οδήγησε στη μελέτη της οργάνωσης της μιτωτικής συσκευής μετά την επίδραση με δοξορουβικίνη. Η μιτωτική συσκευή αποτελεί στόχο χημικών ενώσεων που επηρεάζουν το χρωμοσωματικό αποχωρισμό και η μελέτη της πραγματοποιήθηκε με διπλή ανοσοσήμανση για τους μικροσωληνίσκους (β-τουμπουλίνη) και το κεντρόσωμα (γ-τουμπουλίνη). Παρατηρήθηκαν επίσης, ανώμαλες μεταφάσεις και τροποποιημένος αριθμός κεντροσωμάτων, φαινόμενα που σχετίζονται με λανθασμένο χρωμοσωματικό αποχωρισμό. Τέλος σε προκαταρκτικά πειράματα κατά τα οποία πραγματοποιήθηκε αναστολή της απόπτωσης που προκαλείται από τη δοξορουβικίνη με το γενικό αναστολέα Z-VAD-fmk, παρατηρήθηκε αύξηση της συχνότητας των κυττάρων με μικροπυρήνες που προκαλούνται από την ίδια ένωση. Η παρατήρηση αυτή αποτελεί μια ακόμη ένδειξη για την πιθανή συμμετοχή της απόπτωσης στον περιορισμό των κυττάρων με γενετικές αλλαγές. Επίσης παρατηρήθηκε ότι η αναστολή της απόπτωσης τροποποιεί τη συχνότητα των κυττάρων με ανώμαλο αριθμό κεντροσωμάτων που επάγονται από τη δράση της δοξορουβικίνης, αυξάνοντας τα κύτταρα με ένα κεντρόσωμα και μειώνοντας τα κύτταρα με πολλαπλά κεντροσώματα, υποδεικνύοντας πιθανή εμπλοκή της απόπτωσης στο σχηματισμό ασύμμετρων κυτταρικών διαιρέσεων. Οι τελευταίες παρατηρήσεις χρειάζονται περαιτέρω διερεύνηση. / Doxorubicin is one of the most widely used anticancer drugs due to its broad spectrum of antitumor activity and belongs to the family of anthracyclines. Various mechanisms have been proposed for its cytostatic activity, including induction of apoptosis and genetic alterations. Apoptosis is a normal procedure which provides a cell control mechanism. It is a defense mechanism for the elimination of mutated cells including those that might cause carcinogenesis. Apoptosis is carried out by intrinsic as well as extrinsic pathways. In the present thesis we investigated the relationship between apoptosis and genetic damage induced by doxorubicin in C2C12 mouse cell line. Genetic damage was evaluated by studying the micronucleus (MN) frequency by DAPI staining.The induction of apoptosis was examined by double fluorescence staining of ANNEXIN V/PI. The analysis of the mechanism by which the micronuclei are induced was further investigated by kinetochore labeling using the immunocytochemical method CREST with simultaneous labeling of α- tubulin, for the detection of cytoplasm. It was observed that doxorubicin at the studied concentrations (0.05 μg/ml, 0.10 μg/ml, 0.20 μg/ml, 1.00 μg/ml, 2.50 μg/ml and 5.00 μg/ml) increased frequencies of apoptotic and micronucleated cells. It was shown that the induction of apoptosis was related with decreased micronucleus frequency, indicating a possible involvement of apoptosis in the elimination of the micronucleated cells. The analysis of the mechanism of micronucleus formation showed that doxorubicin induced chromosome breakage and in a lesser extent chromosome loss. The latter ability was further investigated by analyzing the integrity of mitotic apparatus after exposure to doxorubicin. The mitotic apparatus consists one of the main targets of chemical compounds which induce chromosome missegregation. The analysis was achieved by double immunofluorescence staining for microtubules (β-tubulin) and for centrosome (γ- tubulin). It was shown that doxorubicin provoked high abnormal metaphase figures and abnormal centrosome numbers, phenomena that are related with abnormal chromosome segregation. Finally, preliminary experiments, where doxorubicin-induced apoptosis was inhibited by the pancaspase inhibitor Z-VAD-fmk, resulted to an increase of the frequency of micronucleated cells generated by doxorubicin. This observation is an additional evidence for the involvement of apoptosis in the elimination of cells with genetic damage. Moreover, it was observed that inhibition of apoptosis altered the frequency of cells with abnormal centrosome number, which are formed after doxorubicin treatment, by increasing cells with only one centrosome and decreasing cells with multiple centrosomes, indicating a possible participation of apoptosis in the formation of asymmetric divisions. Further investigation is warranted to clarify the last observations.

There is much debate on the carcinogenic potential of disinfection by-products (DBP) in chlorinated water supplies. Until recently, epidemiological studies have been limited in their ability to examine accurately the risk of cancer with exposure to environmental carcinogens. This has largely been due to the long latency periods associated with cancer development, and the difficulties in accurately estimating chronic exposure. Although there is evidence, from predominantly case-control studies, of increased bladder cancer with exposure to chlorinated water supplies, the evidence is inconclusive. ¶ In an attempt to determine the carcinogenic potential of trihalomethanes (THMs) in chlorinated water, this study utilises DNA damage to bladder cells, evident as micronuclei, as a pre-clinical outcome measure. Using a pre-clinical marker helps overcome some of the limitations associated with long latency periods. The study improves on previous studies by estimating exposure to DBP at an individual level, and takes into consideration ingestion, inhalation and dermal exposure. ¶ A cohort study was undertaken in three Australian communities. The Bungendore (NSW) water supply was not chlorinated thereby providing a community unexposed to DBPs from chlorinated water. Canberra (ACT) and Adelaide (SA) had intermediate and relatively higher (but still within NHMRC guideline levels) of DBPs in the reticulation system. Trihalomethane levels in reticulated water (external dose) and in urine (internal dose) were used as exposure indices. As well, intake dose was computed by adjusting external dose for individual variations in ingestion and bathing. The primary outcome measure was the prevalence of micronuclei in bladder epithelial cells. A DNA index derived from flow cytometry was also used to estimate DNA damage in bladder cells. Associations between exposure and outcome were estimated using Poisson regression models, having identified and adjusted for interaction effects and confounders. ¶ A total of 529 participants were eligible to participate, of which 348 (65.8%) completed all aspects of the study. Analysis was limited to the 228 participants (65.53% of those who completed the study) who had slides suitable for micronuclei scoring. One hundred and forty three (63%) of the 228 participants were from the exposed communities, while 85 (37%) were from the unexposed community. This sample exceeded the estimated 50 per group required to detect a relative risk of 1.4, with a significance level of 0.05 and 80% power. ¶ External dose for total THM for the two chlorinated (exposed) communities ranged from 37.75 to 157.25 mg/l. Intake dose estimated by fluid intake diary ranged from 2.9 to 469.5 mg/l, while a retrospective questionnaire estimated intake dose to range from 0 to 409.4 mg/l. Internal dose (urine levels) of total THM for the same two communities ranged from 0 to 6.82 mg/l. Adjusted risk estimate for DNA damage to bladder cells (using the micronuclei assay) when total THM was assessed by available dose was 1.0002 (0.997 to 1.003), by intake dose estimated by fluid intake diary was 1.0001 (0.998 to 1.002), by intake dose estimated by questionnaire was 1.001 (0.999 to 1.003), and by internal dose was 1.05 (0.89 to 1.24). Using DNA index from flow cytometry as the outcome measure also did not identify significant associations, except when exposure was assessed as available dose of total THM (RR=1.0042; 1.0003 to 1.0081). ¶ The results suggest that THM levels are not significantly associated with DNA damage to bladder cell. This supports suggestions of THMs being non-genotoxic. Further work is required to assess the relationship between THM and the more mutagenic compounds, and to assess the carcinogenicity of the more mutagenic compounds at concentrations occurring in drinking water.

Thesis (doctoral)--Lunds Universitet. / Description based on print version record.

O ensaio de frequência de micronúcleos é uma técnica importante utilizada para avaliar danos genotóxicos de agentes químicos ou físicos (como radiação ionizante) em células, baseado na quantificação de células contendo micronúcleos, que são fragmentos derivados de DNA danificado com coloração semelhante ao dos núcleos principais, mas com 5 a 30% do seu tamanho. Tradicionalmente, este ensaio é realizado usando microscopia de coloração convencional de acordo com Giemsa, mas atualmente esta técnica está sendo atualizada para uma abordagem automatizada que se baseia na dissolução da membrana plasmática por detergentes levando em conta apenas núcleos e micronúcleos com seu DNA corados por corantes fluorescentes, que pode discriminar núcleos de células viáveis dos das células inviáveis e permite a contagem por citometria de fluxo. Este trabalho avaliou a extensão de dano genotóxico radioinduzido por radiação gama (60Co) em doses entre 0,5 e 16Gy, e o potencial genotóxico de três peptídeos utilizados em medicina nuclear (PSMA-617, PSMA-11 e Ubiquicidina 29-41). As membranas celulares foram lisadas na presença dos corantes SYTOX® Green e EMA, e núcleos marcados com os dois fluorocromos foram considerados provenientes de células inviáveis e, portanto, removidos da análise. As quantidades de micronúcleos (porcentagem de eventos) nas amostras, foram proporcionais às doses de radiação, e puderam ser ajustadas a um modelo linear-quadrático (R2 = 0,993). Somente doses mais altas (8 e 16 Gy) e controles positivos induziram aumentos relevantes nas quantidades de micronúcleos. Os radiofármacos foram testados com concentrações de 0,1x, x, 10x e 100x a concentração utilizada em adultos, e não induziram dano em concentrações praticáveis. / The micronucleus frequency assay is an important technique used to evaluate genotoxic damage of chemical or physical agents (such as ionizing radiation) in cells, based on the quantification of micronucleus-containing cells, which are fragments derived from damaged DNA with similar coloration to of the main nuclei, but with 5 to 30% of its size. Traditionally, this assay is performed using conventional staining microscopy according to Giemsa, but currently this technique is being updated to an automated approach that relies on the dissolution of the plasma membrane by detergents taking into account only nuclei and micronuclei with their DNA stained by dyes fluorescence, which can discriminate nuclei from unviable or living cells and allows counting by flow cytometry. The genotoxic potential of three peptides used in nuclear medicine (PSMA-617, PSMA-11 and Ubiquicidine 29-41) was evaluated by gamma radiation (60Co) at doses in the range among 0.5 and 16Gy. Cell membranes were lysed in the presence of the SYTOX® Green and EMA dyes, and cores labeled with the two fluorochromes were considered to be from non-viable cells and therefore removed from the analysis. The amount of micronucleus (percentage of events) in the samples was proportional to the radiation doses, and could be adjusted to a linear-quadratic model (R2 = 0.993). Only higher doses (8 and 16 Gy) and positive control induced significant increases in micronucleus amounts. Radiopharmaceuticals were tested at concentrations of 0.1x, 1x, 10x, and 100x the concentration used in adults, and did not induce damage at feasible concentrations.

O teste do micronúcleo (MN) vem sendo utilizado omo indicador de exposição genotóxica uma vez que sua ocorrência está associada às aberrações cromossômicas. Comparou-se a frequência de MN de 30 pacientes alcoólicos crônicos portadores de carcinomas orais e orofaríngeos, nos quais o consumo de álcool variou de quatro à 59 anos, com a de 30 indivíduos abstinentes de semelhante nível sócio-econômico. A diferença (14,5 anos) entre a média da idade dos pacientes (52,9 ± 1,6) e a dos controles (38,4 ± 1,5) foi estatisticamente significante (P< 0,0001). A pesquisa consiste na análise de 2000 células de escamação da mucosa oral de três regiões distintas da boca de pacientes e controles: ao redor da lesão (B), na região contra-lateral à lesão (A) e na região fundo de saco gengivo-labial superior (C), previamente considerada controle devido à baixa incidência de tumores. As células foram fixadas, coradas e analisadas em "teste cego" através de técnica modificada e adaptada aos requisitos específicos da pesquisa. O número de MN por 2000 células por indivíduo nos pacientes, assim como nos controles mostrou uma distribuição de Poisson com dispersão assimetricamente positiva, e aumento da variância nos pacientes. A distribuição de anomalias metanucleares também exibiu desvios significantes da dispersão normal. A heterogeneidade da frequência de MN nas três regiões orais dos pacientes, avaliada através do teste de Kruskal-Wallis, mostrou-se extremamente significante (P = 0,005). Enquanto a comparação entre as regiões B vs C foi estatisticamente significante (P < 0,01), as comparações entre as regiões A vs B ou A vs C (P > 0,05) não revelaram diferenças estatisticamente significante através do teste de comparação múltipla de Dunn. As comparações das diferençasnas frequências de MN resultantes do emparelhamento entre as três regiões orais (A-B, A-C, BC), aumentou o nível de significância dos resultados quanto à heterogeneidade regional (P = 0,0003) e tornou a comparação A vs C estatisticamente significante. Entretanto, a análise da variância não paramétrica da distribuição de MN nas três regiões orais dos controles não indicou heterogeneidade (P = 0,943). As frequências de MN e de células metanucleadas nas regiões orais dos pacientes também foram comparadas à dos controles através do teste de Mann-Whitney. A diferença na região tumoral foi extremamente significante (P < 0,001) e, significante na região oposta à lesão (P = 0,03) porém não significante na região fundo de saco gengivo-labial superior (P = 0,44). Esses resultados indicam um aumento de sete vezes na frequência de MN ao redor da lesão, três vezes na região oposta à lesão e duas vezes, embora não estatisticamente significante, na região fundo de saco gengivo-labial superior, revelando um gradiente na frequência de MN no sentido C -> A -> B. Comparações das frequências de células metanucleadas: binucleadas (BI), cariorréxes (CR), cariólises (CL) e broken egg (BE) nas três regiões orais de pacientes e controles, revelaram diferenças extremamente significantes, com exceção apenas de BE, em todas as regiões orais e de CR na região fundo de saco gengivo-labial superior. As comparações dicotômicas das variáveis independentes não paramétricas com a frequência de MN através dos testes de contingência e Mann-Whitney, não foram estatisticamente significantes ao nível de 5% de probabilidade, com exceção de uma única questão do questionário de diagnóstico do alcoolismo CAGE, que confirmou o efeito do álcool. Ao contrário dos resultados esperados, as frequências de MN e anomalias metanucleares não mostraram associações significantes com a idade tanto nos pacientes como nos controles. Ainda mais, a análise de regressão múltipla escalonada da frequência de células com MN ou anomalias metanucleares sobre fatores intervenientes, como idade, fim e tempo de consumo de álcool e tempo de uso de tabaco, nos pacientes, revelou coeficientes de regressão pequenos e negativos, mas significantes. Já os coeficientes de regressão da variável CL sobre o consumo de álcool foi significantemente pequeno, mas positivo, com ou sem transformação das frequências das variáveis dependentes através da raiz quadrada. Entretanto, os resultados da análise de regressão dos pacientes, aparentemente contraditórios, podem ser explicados supondo-se que as frequências de MN, durante a exposição crônica ao álcool, teriam um forte aumento inicial, diminuindo posteriormente até um nível significantemente maior que aquele no início do consumo de álcool; por outro lado, a frequência de CL aumentaria inversamente como resultado da transformação de MN durante o processo de reparo. Pode-se deduzir a partir resultados da presente pesquisa que a análise de células com MN e anomalias metanucleares devem levar em consideração critérios de padronização citológicos rígidos. O número de células a ser contado deve ser fixo e superior a 2000 de modo que inclua a variabilidade normal (espontânea) da distribuição de MN e, ainda, eliminar viéses estatísticos na estimativa de suas frequências. Ainda, o tamanho da amostra deve ser superior à 30 indivíduos, a fim de assegurar a representatividade estatística. A significância das diferenças inter-grupais deve ser estimada através de testes não paramétricos. Além disso, análises intra-individuais (ou inter-regionais) bem como a utilização de controles específicos inter-individuais pareados quanto aos fatores intervenientes (sexo, idade, grupo étnico, nível sócio-econômico, etc.) deveriam ser práticas metodológicas usuais. Como conclusão, o teste do MN deve ser considerado como uma técnica de triagem simples, prática, econômica e não invasiva para a prevenção e monitoramento clínicos de indivíduos sob risco carcinogênico, após exposição à agentes ou situações genotóxicas, como consumo crônico e abusivo de álcool, tabaco e/ou outras drogas mutagênicas, ou ainda, manipulação profissional de derivados de petróleo e outras substâncias tóxicas industriais. No contexto da presente investigação, o teste do MN deve ser particularmente indicado para monitoramento da evolução clínica de indivíduos com tumores ou lesões leucoplásicas curadas ou removidas cirurgicamente ou após tratamentos quimo ou radioterápicos, através de comparações intra e interindividuais. / Micronucleus (MN) test has been used as an indicator of genotoxic exposition since it is associated with the occurrence of chromosomal aberrations. The frequency of MN among 30 subjects with oral and oropharyngeal carcinomas, whose alcohol consumption varied from four to 59 years, was compared to that of 30 healthy control individuals, abstinent for alcohol and matched for social-economic status. Difference (14.5 years)between average age of patients (52.9 ± 1.6) and that of controls (38.4 ± 1.5) was statistically significant (P <0.0001). The investigation includes the examination of 2000 cells per individual from each of three distinct areas in the mouth of patients and controls: around the lesion (B), opposite to the lesion (A) and in the upper gengivo-labial gutter (C) taken as control site because of its low tumour occurrence. The cells were fixed, dried and analyzed under "blind test", according to the technique of Sarto, modified and fitted strictly to the requirements of the research. The number of MN per 2000 cells per individual among patients as well as controls showed a Poisson distribution with a positively asymmetric dispersion and an increased variance among patients. Distribution of metanucleated cells also departed significantly from normal dispersion. The heterogeneity of MN in the three oral regions of patients, evaluated through Kruskal-Wallis test, was highly significant (P = 0.005) and pairwise comparison B vs C was statistically significant (P < 0.01) but not comparisons between A vs B or A vs C (P > 0.05), through Dunn´s multiple comparison test. Comparisons of pairwise inter-regional oral differences of MN frequencies (A-B, A-C, B-C), increased the significance levels of the results for regional heterogeneity (P =0.0003) becoming also the comparison A vs C statistically significant. Otherwise, non parametric analysis of variance of the MN distribution in the three oral regions of the controls indicated great statistical homogeneity (P = 0.943). Frequencies of MN and metanucleated cells in the oral regions of patients were also compared to those of controls, through Mann-Whitney test. Differences were highly significant (P< 0.001) for tumoral region and significant for the region opposite to the lesion (P = 0.03) but not for the upper gengivo-labial gutter (P = 0.44). These results indicated a seven-time increase in the frequency of MN in the region around the lesion, a three-time increase in the opposite region and a two-time but non significant increase in the upper gengivo-labial gutter, revealing a gradient frequency in the way C -> A -> B. Comparisons of frequencies of metanucleated cells: binucleated (BI), cariorrhexis (CR), cariolisis (CL) and broken egg (BE) in the three oral regions, between patients and controls, showed highly significant differences, except for BE frequencies in all oral regions and for CR frequency in the upper gengivo-labial gutter. Dichotomous comparisons of non parametric independent variables, with MN frequencies, through contingency and Mann-Whitney tests, were not significant at 5% level of probability, except for CAGE diagnosis of alcoholism, which confirmed the alcohol effect. Contrary to the expected results, sistematically frequencies of MN and metanucleated cells were not significantly correlated to age among patients as well as controls. Moreover, stepwise multiple regression analysis of MN and metanucleated cells in the patients revealed small negative,but significant, regression coefficients upon intervinient factors such age, end and time of alcohol consumption and time of tobacco usage, but regression coefficients of CL, upon alcohol consumption, were significantly small, but positive, before or after square root transformation of dependent variables. However, the apparently contradictory results from analysis of regression among patients could be explained by the assumption that frequencies of MN, under alcohol exposure, had a early strong increase, but decreased, afterwards, to a level significantly greater than that before alcohol consumption, while CL frequency conversely increased significantly as a result from MN transformation, during the repair process. It could be stated from the results of the present research that examination of cells with MN and metanucleated anomalies should follow critical and strict cytological criteria of standardization. The number of cell counts should be fixed and above 2000 in order to include normal (spontaneous) variability in the distribution of MN and, therefore, to prevent biases in the estimation of its frequencies. Also, sample size should be above 30 individuals, so that the statistical representativity be assured and significance of intergroup differences, could be estimated through non parametric tests. Moreover, intra-individual (or intra-regional) examinations and specific interindividual controls matched for intervinient factors (sex, age, etnic group, socio-economic level,etc.) should be an usual methodological practice. As a conclusion, it must to be considered that MN test is a simple, practical, non-expensive and non-invasive screening technique of diagnosis for clinical prevention and management of subjects under carcinogenic risks, after exposition to genotoxical agents or situations, such as abusive and chronical consumption of alcohol, tobacco and/or other mutagenic drugs or professional manipulation of derivatives of petroleum and other toxical industrial substances. In the context of the present investigation, the MN test must particularly be indicated for monitoring the clinical evolution of subjects with healed or surgically removed tumours or leukoplasic lesions, after chemio or radiotherapic treatments, by means of intra and inter-individual cellular comparisons.

Micronúcleos são estruturas constituídas por material cromatínico contido por um envoltório nuclear e menores que o núcleo principal. Apesar do amplo uso do teste do micronúcleo na avaliação do potencial genotóxico de diversas substâncias, os trabalhos cujas preocupações sejam elucidar o mecanismo de formação dessas estruturas, seu conteúdo e a fase do ciclo celular em que surgem, são raros. O objetivo do presente trabalho foi estudar a cinética de surgimento de micronúcleos em células A549, provenientes de carcinoma de pulmão humano, submetidas à sincronização. Após duplo bloqueio por ausência de soro fetal bovino e adição de vincristina ao meio, o surgimento de micronúcleos foi acompanhado com o auxílio de um marcador para fase S. Os resultados obtidos indicam que micronúcleos podem surgir tanto durante a mitose quanto na intérfase e possuem capacidade de replicação de DNA, independentemente do núcleo principal. A capacidade de escapar ao duplo bloqueio permite sugerir que o ponto de checagem de ligação ao fuso mitótico em A549 não é funcional. Também foram observadas seqüências amplificadas de EGFR no interior dos micronúcleos. A interpretação do significado dos micronúcleos é importante para a definição de sua relação com a expulsão de oncogenes em células tumorais ou de outras seqüências amplificadas. / Micronuclei are structures composed by chromatin material contained in the nuclear envelope and smaller than the main nucleus. Micronucleus test has been widely used in the genotoxic potential evaluation of different compounds. Although a few report concerning on the mechanism of micronucleus genesis, its DNA sequences content and the cell cycle phase when they arise. The aim of this work was to analyze the kinetic of micronuclei in A549 cells from human lung carcinoma submitted to cell cycle synchronization. After double blocking by fetal bovine serum deprivation and vincristine treatment, micronucleus formation was monitored with a S-phase marker. The results have showed that both in the mitosis and in the interphasic phase, micronuclei may arise and they were able to replicate its DNA. This process seemed to be independent of main nucleus. Cellular ability to escape from double blocking suggests that mitotic spindle checkpoint in A549 is not functional. Moreover, EGFR sequences were detected into the micronucleus. It is important to elucidate the micronucleus meaning to describe precisely its association with elimination of oncogene or other amplified sequences from the tumor cells.

Os girinos de rã-touro, Lithobates catesbeianus, podem ser bons bioindicadores de condições ambientais. O objetivo desse trabalho foi determinar o potencial de toxicidade para L. catesbeianus de alguns dos principais agrotóxicos utilizados no cultivo de arroz irrigado. Foram realizados testes de toxicidade aguda para a determinação da CL50-96h do bentazon, penoxsulam, óleo vegetal, permetrina e carbofuran, separadamente, e da mistura desses agrotóxicos. Com esses resultados foram estimados os índices de segurança dos produtos. Girinos em fase pré-metamorfose foram expostos aos agrotóxicos na própria lavoura de arroz e em laboratório por 21 dias, para avaliar os possíveis efeitos crônicos destas substâncias, separadamente e da mistura, sobre o quadro hematológico, metamorfose (regulada pelo eixo tiroideano), e também o possível potencial mutagênico através do teste do micronúcleo. A CL50-96h para girinos foi de 4530 mg/L para o bentazon; 7,52 mg/L para o penoxsulam + 145,66 mg/L do óleo vegetal; 81,57 mg/L para o óleo vegetal, 0,10 mg/L para a permetrina, 29,90 mg/L para o carbofuran (ingredientes ativos) e, 38,79 vezes a dose utilizada no campo para a mistura desses produtos. Foi determinado risco ambiental apenas para o inseticida permetrina. Nos testes in situ, as águas de irrigação não apresentaram toxicidade aguda para os girinos. A taxa de metamorfose não diferiu entre os tratamentos, demonstrando que os agrotóxicos utilizados nas doses indicadas não tem ação desreguladora do eixo tiroideano. As análises do micronúcleo mostraram aumento significativo de eritrócitos micronúcleoados para os testes in situ e, no laboratório, para o herbicida bentazon e para a mistura dos agrotóxicos. As análises hematológias mostraram diminuição da hemoglobina e número de eritrócitos no teste de campo, retornado aos padrões normais na semana seguinte. No laboratório houve queda na contagem de eritrócitos para o bentazon, aumento do VCM e HCM para o bentazon e penoxsulam; aumento do CHCM para o penoxsulam e para a mistura dos agrotóxicos. Para a série branca não houve diferenças no teste in situ, mas obtivemos aumento dos números de neutrófilos dos girinos tratados com o bentazon. / American bullfrogs, Lithobates catesbeianus could be good environmental indicators. The aim of this study was evaluate the potential toxicity of some principal pesticides used in irrigated rice crops to L. catesbeianus tadpoles. The pesticides Bentazon, Penoxsulam, Vegetable oil, Permetrina, Carbofuran and the mixture of them were assessed. Pre-metamorphose tadpoles were exposed to all of these agrochemicals in the laboratory to determinate de LC50-96h and so estimate the index of security by each product. Animals in the same phase were exposed to these pesticides on the rice crops, in situ and in laboratory per 21 days to evaluate the possible chronic effects of the substances, separated and in the mixture of them. The hematological results, red and white series, the mutagenic potential (micronucleous test), and the metamorphose rate (regulated by thyroid axis) were evaluated. The LC50-96h to tadpoles was 4530 mg/L to Bentazon; 7.52 + 145.66 mg/L to Penoxsulam + vegetable oil; 81.57 mg/L to vegetable oil; 0.10 mg/L to Permetrina; 29.90 mg/L to Carbofuran (active ingredients) and 38.79 times to the dose used in the field to the mixture of the products. Only to Permetrina insecticide was observed environmental risk. The metamorphose rate showed no difference between the treatments suggesting that these pesticides, used on indicated doses did not promote deregulated action on the thyroid axis. In situ tests the irrigated waters showed low mortality to the animals. The red series showed in situ, a decrease in the haemoglobin tax and in the counting of erytrocyte\'s number however return to the normal values in the follow week. In laboratory tests showed a decrease in the counting of erytrocyte\'s number to the animals exposed to Bentazon, an increase in the MCV and MCH to the animals exposed to Bentazon and Penoxsulam, an increase in the MCHC to those exposed to Penoxsulam and to the \"mixture\". The white series showed no difference in situ test however an increase in the neutrophils number was observed to the animals exposed to Bentazon in laboratory. The micronucleous analyze showed significant increase in the erytrocyte\'s micronucleated number in situ and in laboratorial tests to animals exposed to Bentazon and to the \"mixture\".

Tetraploid cells, which typically arise from errors in mitosis, are genomically unstable and promote tumorigenesis. Recent evidence suggests that ~40% of tumors undergo a tetraploid intermediate during their evolution, with ~20% of all solid tumors maintaining a tetraploid karyotype. Consequently, tumor suppression mechanisms have evolved to limit the proliferation of tetraploid cells. However, it remains unclear how tetraploid cells are able to overcome these tumor suppression mechanisms to initiate tumorigenesis. To address this unresolved question, we developed and validated a genome-wide screening assay to comprehensively identify miRNAs whose overexpression promotes tetraploid cell proliferation. We then profiled those miRNAs to mechanistically define how each miRNA functions to overcome tetraploid induced arrest. Our results demonstrate that miRNAs can promote proliferation via multiple mechanisms, including inhibition of the p53 tumor suppressor pathway, hyperactivation of growth factor signaling, and inactivation of the Hippo tumor suppressor pathway. Additionally, we investigated mechanisms that facilitate tumorigenesis from proliferating tetraploid cells. It is well established that tetraploid cell proliferation promotes both numerical and structural chromosome abnormalities, although the precise mechanisms underlying these phenomena remain incompletely understood. Chromosome missegregation can lead to the formation of micronuclei separate from the primary nucleus, a result of either lagging or polar chromosomes. Micronuclei have been shown to rupture during interphase, leading to massive amounts of DNA damage and chromothripsis, resulting in extensive DNA breaks and rearrangements. We followed micronuclei formed from both lagging and polar chromosomes to determine whether all micronuclei are equally prone to nuclear envelope rupture. Our results show that polar micronuclei have nuclear envelopes that are significantly more stable than the nuclear envelopes of micronuclei formed from lagging chromosomes. Furthermore, micronuclei have been shown to be deficient at nuclear import of proteins. Kinetochore assembly, vital for proper chromosome segregation, is dependent upon the nuclear import of many proteins. We sought to establish whether micronuclei have defects in kinetochore assembly since without functional kinetochores, chromosomes cannot bind to the mitotic spindle. We found that chromosomes in micronuclei fail to assemble kinetochores efficiently, and thus promote additional chromosome missegregation. Overall, this dissertation identifies multiple mechanisms that facilitate tumorigenesis from tetraploid intermediates.

Micronúcleos são estruturas constituídas por material cromatínico contido por um envoltório nuclear e menores que o núcleo principal. Apesar do amplo uso do teste do micronúcleo na avaliação do potencial genotóxico de diversas substâncias, os trabalhos cujas preocupações sejam elucidar o mecanismo de formação dessas estruturas, seu conteúdo e a fase do ciclo celular em que surgem, são raros. O objetivo do presente trabalho foi estudar a cinética de surgimento de micronúcleos em células A549, provenientes de carcinoma de pulmão humano, submetidas à sincronização. Após duplo bloqueio por ausência de soro fetal bovino e adição de vincristina ao meio, o surgimento de micronúcleos foi acompanhado com o auxílio de um marcador para fase S. Os resultados obtidos indicam que micronúcleos podem surgir tanto durante a mitose quanto na intérfase e possuem capacidade de replicação de DNA, independentemente do núcleo principal. A capacidade de escapar ao duplo bloqueio permite sugerir que o ponto de checagem de ligação ao fuso mitótico em A549 não é funcional. Também foram observadas seqüências amplificadas de EGFR no interior dos micronúcleos. A interpretação do significado dos micronúcleos é importante para a definição de sua relação com a expulsão de oncogenes em células tumorais ou de outras seqüências amplificadas. / Micronuclei are structures composed by chromatin material contained in the nuclear envelope and smaller than the main nucleus. Micronucleus test has been widely used in the genotoxic potential evaluation of different compounds. Although a few report concerning on the mechanism of micronucleus genesis, its DNA sequences content and the cell cycle phase when they arise. The aim of this work was to analyze the kinetic of micronuclei in A549 cells from human lung carcinoma submitted to cell cycle synchronization. After double blocking by fetal bovine serum deprivation and vincristine treatment, micronucleus formation was monitored with a S-phase marker. The results have showed that both in the mitosis and in the interphasic phase, micronuclei may arise and they were able to replicate its DNA. This process seemed to be independent of main nucleus. Cellular ability to escape from double blocking suggests that mitotic spindle checkpoint in A549 is not functional. Moreover, EGFR sequences were detected into the micronucleus. It is important to elucidate the micronucleus meaning to describe precisely its association with elimination of oncogene or other amplified sequences from the tumor cells.