Dissertations / Theses on the topic 'Micronuclei'

Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Nuclear Science and Engineering, 2008.
Includes bibliographical references (p. 42-44).
The bystander effect describes radiation-induced biological effects in nonirradiated cells that have received signals from irradiated cells. In a co-culture experiment, the bystander signaling is proposed to occur via the medium. Using a co-culture setup, the work in this thesis investigates the effects of temperature as an experimental parameter and linear energy transfer (LET) dependence on the bystander effect. Using the micronucleus assay and primary human AG01522 fibroblast cells co-cultured as both the target and bystander cells, the incidence of micronuclei in both X-ray irradiated and alpha particle irradiated bystander experiments were ~2 fold over control averages. In the temperature experiment, there were no significant differences between bystander cells co-cultured with cold (4°C) target cells and those co-cultured with warm control target cells. These results have shown, for AG01522 fibroblasts, that the bystander effect is independent of temperature and LET.
by Carrie C. Buchanan.
S.B.

Currently there is no in vitro testing of glioblastoma biopsy material to assess tumour sensitivity to radiation, which could form a basis for personalised treatment plans for patients. In this work, a model to predict sensitivity to radiation, via the micronucleus assay, is set out and a proof of concept is presented where numbers of micronuclei in a glioblastoma cell line, LN18 is predicted. One key requirement for the model is that any in vitro testing needs to yield results within a few days, as the timeline for glioblastoma patients from diagnosis to treatment is short. In order to achieve this, a flow cytometry technique is assessed against traditional fluorescence microscopy for detection of micronuclei. Flow cytometry was completed using an in vitro Microflow kit from Litron Laboratories. There was no previous experience using this kit in cancerous cell lines and limited experience in cell lines that adhere to their flask’s surface as the kit is used mostly in peripheral blood lymphocytes. The flow cytometry technique can be completed within the required time frame and is much less labour intensive that fluorescence microscopy. However, there is a significant amount of variance between samples which makes the microscopy results more useful for fitting the modelling work to. It is expected with further experience and use of the supplied template, as this was incompatible at the time of the experiments, will play a role in reducing some of the variance. The model, in its current state of development, is able to predict numbers of micronuclei in a cohort of cells following doses of radiation between 1 and 3 Gy. The numerical solution is based on a decision tree structure where each double strand break that would be caused by radiation is run through the tree. The tree is traversed populated using probabilities for each decision, such as the success of a repair pathway, and Monte Carlo methods for predicting the cohort response to radiation. These probabilities are fitted to experimental data. The prediction of micronuclei is the first step for the MiNiMUS model. Future work should prioritise incorporating cell death into the model and further assessing the suitability of flow cytometry for rapid micronuclei detection.

The advent of high-throughput sequencing has brought about the creation of an unprecedented amount of research data. Analytical methodology has not been able to keep pace with the plethora of data being produced. Two assays, ImmunoSEQ and the cytokinesisblock micronucleus (CBMN), that both produce count data and have few methods available to analyze them are considered. ImmunoSEQ is a sequencing assay that measures the beta T-cell receptor (TCR) repertoire. The ImmunoSEQ assay was used to describe the TCR repertoires of patients that have undergone hematopoietic stem cell transplantation (HSCT). Several different methods for spectratype analysis were extended to the TCR sequencing setting then applied to these data to demonstrate different ways the data set can be analyzed. The different methods include CDR3 distribution perturbation, Oligoscores, Simpson's diversity, Shannon diversity, Kullback-Liebler divergence, a non-parametric method and a proportion logit transformation method. Herein we also demonstrate adapting compositional data analysis methods to the TCR sequencing setting. The various methods were compared when analyzing a set of 13 subjects who underwent hematopoietic stem cell transplantation. The eight subjects who developed graft versus host disease were compared to the five who did not. There was no little overlap in the results of the different methods showing that researchers must choose the appropriate method for their research question of interest. The CBMN assay measures the rate of micronuclei (MN) formation in a sample of cells and can be paired with gene expression or methylation assays to determine association between MN formation and other genetic markers. Herein we extended the generalized monotone incremental forward stagewise (GMIFS) method to the situation where the response is count data and there are more independent variables than there are samples. Our Poisson GMIFS method was compared to a popular alternative, glmpath, by using simulations and applying both to real data. Simulations showed that both methods perform similarly in accurately choosing truly significant variables. However, glmpath appears to overfit compared to our GMIFS method. Finally, when both methods were applied to two data sets GMIFS appeared to be more stable than glmpath.

Topoisomerase I (Top1) poisons are among the most clinically-effective drugs used for colon, ovary and lung cancers. Unpublished data from our lab have recently revealed that the structurally-unrelated Top1 poisons, Camptothecin (CPT) and Indimitecan (LMP776), induce the formation of micronuclei (MNi) in human cancer cells. In addition, MNi trigger an innate immune gene response by stimulating the cGAS/STING pathway. As the mechanisms of MNi formation are not fully determined, our aim is here to establish how MNi form after Top1 poisoning. Using immunofluorescence assays and EdU labelling of nascent DNAs, our results show that, after 24 hours of recovery, a short treatment with sub-cytotoxic doses of Top1 poisons induces the formation of MNi that do not contain newly synthetized (EdU+) DNA. We also saw that Top1 poisons delay replication machinery reducing EdU incorporation and produce significant levels of the damage markers γH2AX and p53BP1 in S-phase cells but not in G1 and G2/M cells. The results also show that MNi formation is dependent on R-loops, as RNaseH1 overexpression markedly reduces Top1 induced MNi. Genome-wide mapping of R-loops by DRIP-seq technique revealed that R-loop levels are both decreased and increased by CPT. In particular, increased R-loops are mainly found at active genes and always overlapped with Top1cc sites. We also found that increased R-loops overlap with lamina-associated chromatin domains while decreased R-loops correlate with replication origin sites. Overall, our data are consistent with the formation of MNi due to R-loop increase and under-replication at specific regions caused by Top1 poisons. These results will eventually help in developing new strategies for effective personalized interventions by using Top1-targeted compounds as immuno-modulators in cancer patients.

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.

In the investigation of radiation accidents, it is of great importance to estimate the dose absorbed by exposed persons in order to plan their therapy. Although occasionally in these situations physical dose measurements are possible, most often biological methods are required for dose estimation. The aim of this investigation was to assess the suitability of the cytokinesis blocked (CB) micronucleus assay as a biodosimetric method using lymphocytes irradiated in vivo. The approach adopted to achieve this was to estimate whole body doses by relating micronuclei yields in patients undergoing radiotherapy treatment with an in vitro radiation dose-response curve. These biologically derived estimates were then compared with the corresponding doses obtained by physical measurement and calculation. As a first approach a study was performed of the in vitro dose-response of gamma-ray induced micronuclei following cytokinesis-block in the lymphocytes of peripheral blood samples obtained from 4 healthy donors. The results indicated that the distribution of the induced micronuclei were overdispersed. Furthermore, a linear dose-response relationship was established when a curve was fitted to the data by an iteratively reweighted least squares method. By means of an analysis of covariance it was demonstrated that this result is in agreement with the dose-response relationships found by various other workers (Fenech et al., 1985; Fenech et al., 1986; Fenech et al., 1989; Balasem et al., 1992, and Slabbert, 1993). To assess the suitability and accuracy of dose assessment using the CB micronucleus assay for in vivo exposure of lymphocytes, blood samples obtained from 8 patients undergoing radiotherapy before, during and after treatment were examined. The physical doses of these patients were determined according to conventional radiation treatment plans and cumulative dose-volume histograms. The dose-volume histograms permitted calculation of integral doses and subsequently the estimate of equivalent whole-body doses. The results of the CB micronucleus assay applied to peripheral blood lymphocytes of 6 patients undergoing fractionated partial-body irradiation showed a dose-related increase in micronucleus frequency in each of the patients studied. This demonstrated that micronuclei analysis may serve as a quantitative biological measure of such exposures. The pooled data of these patients compared to the pooled data of the healthy donors show that there was no statistically significant difference between in vitro and in vivo results, however a slightly lower induced micronuclei frequency was observed after in vivo exposure. When the biological dose estimates for equivalent whole-body doses obtained from the in vitro dose response curve were compared with calculated physical doses, it was found that: biologically estimated dose = 0.936 physical dose. However, there was inadequate statistical evidence to discard the hypothesis that the gradient of the equation was equal to one. Therefore, the analysis of micronuclei induced in lymphocytes in vivo yields highly quantitative information on the equivalent whole-body dose. The negative binomial method was used for analysing the micronucleus data from two patients who received single, relatively larger tumour doses of 10 Gy each, with the objective to obtain estimates of the exposed body fraction and the dose to this fraction. The dose estimates to the irradiated volume were found to be within 30% of the physical tumour dose. The irradiated volume estimates seemed to be higher than the physically calculated volumes but by discarding the correction for the loss of cells due to interphase death the agreement was good between the physically and biologically determined integral doses. This study has revealed that the CB micronucleus assay appears to offer a reliable, consistent and relatively rapid biological method of whole body dose estimation. It is recognised that further corroborative work using the techniques described in this thesis is required for estimating localized exposure.

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.

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2016-07-11T18:08:29Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação- JOELMA PESSOA GONÇALVES.pdf: 1383537 bytes, checksum: 125450bea68880058f29778e9d89ead0 (MD5)
Made available in DSpace on 2016-07-11T18:08:29Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação- JOELMA PESSOA GONÇALVES.pdf: 1383537 bytes, checksum: 125450bea68880058f29778e9d89ead0 (MD5) Previous issue date: 2015-02-25
Capes
Os metabólitos secundários dos liquens são responsáveis pela maioria das suas atividades biológicas. Muitos destes compostos apresentam relevante atividade antineoplásica. O objetivo deste trabalho foi verificar as atividades citotóxica e genotóxica in vitro dos extratos orgânicos e do ácido barbático (BAR) purificado de Cladonia salzmannii Nyl. Os extratos orgânicos foram obtidos a partir do talo liquênico (22 g) previamente limpo e seco, com os solventes éter dietílico, clorofórmio e acetona, através do método de esgotamento a quente em aparelho de Soxhlet. O ácido barbático foi purificado a partir do extrato etéreo (1,3 g). A análise química dos extratos orgânicos e do BAR purificado foi realizada através de Cromatografia em Camada Delgada (CCD). A pureza do BAR purificado foi observada através de Cromatografia Líquida de Alta Eficiência (CLAE). A atividade citotóxica dos extratos orgânicos e do BAR purificado foi determinada através do Método do MTT [brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio] e do IPBC (Índice de Proliferação com Bloqueio da Citocinese). O potencial genotóxico dos extratos orgânicos e do BAR purificado foram determinados através do teste do micronúcleo e do ensaio cometa. O dimetilsulfoxido (DMSO) foi utilizado como solvente de diluição das amostras em todos os testes de atividade biológica. Os resultados referentes a CI50 demonstraram relevante potencial citotóxico para o extrato etéreo (Ext E) (50 μg/mL) frente as linhagens celulares NCI-H292 (CI50: 29,91 μg/mL), HEp-2 (CI50: 26,75 μg/mL) e HL-60 (CI50: 3,59 μg/mL), e para o BAR purificado (25 μg/mL) contra as linhagens HEp-2 (CI50: 15,79 μg/mL) e MCF-7 (CI50: 18,28 μg/mL). Porém, a avaliação da citotoxicidade considerando o Índice de Proliferação com Bloqueio de Citocinese (IPBC) demonstrou atividade citotóxica para o BAR purificado em todas as concentrações testadas (5, 10, 20 e 40 μg/mL) e para todos os extratos orgânicos (50 μg/mL) frente as células do Carcinoma de Ehrlich. Entretanto, para o Sarcoma 180 apenas o BAR purificado na concentração de 40 μg/mL e os extratos etéreo e clorofórmico (50 μg/mL) foram considerados citotóxicos. O teste do micronúcleo (MN) demonstrou que o BAR purificado na concentração de 5 μg/mL não apresentou potencial genotóxico em ambas as linhagens celulares tumorais. Além disso, o extrato clorofórmico e BAR purificado na concentração de 10 μg/mL não foram considerados genotóxicos para o Sarcoma 180. No ensaio cometa, todos os compostos testados induziram danos ao DNA em ambas as linhagens tumorais. Com base nos resultados, considera-se que os extratos orgânicos e o BAR purificado de C. salzmannii (Nyl). apresentam atividade citotóxica e genotóxica frente as linhagens celulares tumorais testadas.
The secondary metabolites of lichens are responsible for most of their biological activities. Many of these compounds exhibit significant antineoplastic activity. The objective of this study was to evaluate the in vitro cytotoxic and genotoxic activities of organic extracts and purified barbatic acid from Cladonia salzmannii Nyl. The organic extracts were obtained from liquenic thallus (22 g) previously cleaned and dried with the solvents diethyl ether, chloroform and acetone, through hot exhausted method in a Soxhlet apparatus. The barbatic acid was purified from the ether extract (1.3 g). Chemical analysis of the organic extracts and purified BAR was performed by Thin Layer Chromatography (TLC). The purity of purified BAR was observed by High Performance Liquid Chromatography (HPLC). The cytotoxic activity of the organic extracts and purified BAR was determined by the MTT method [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] and IPBC (Cytokinesis-Block Proliferation Index). The genotoxic potential of the organic extracts and purified BAR was determined by the micronucleus test and comet assay. Dimethyl sulfoxide (DMSO) was used as diluting solvent of the samples in all biological tests. The results for IC50 demonstrated significant cytotoxic potential to the ether extract (Ext E) (50 μg/mL) against the cell lines NCI-H292 (IC50: 29,91 μg/mL), HEp-2 (IC50: 26,75 μg/mL) and HL-60 (IC50: 3,59 μg/mL) and to the purified BAR (25 μg/mL) against the cell lines HEp-2 (IC50: 15,79 μg/mL) and MCF-7 (IC50: 18,28 μg/mL). However, the assessment of cytotoxicity considering the Cytokinesis-Block Proliferation Index (IPBC) showed cytotoxic activity for purified BAR at all concentrations tested (5, 10, 20 and 40 μg/mL) and for all organic extracts (50 μg/mL) against Ehrlich carcinoma cells. However, for Sarcoma 180 only BAR purified at a concentration of 40 μg/mL and ether and chloroform extracts (50 μg/mL) were considered cytotoxic. The micronucleus test (MN) showed that the purified BAR at a concentration of 5 μg/mL showed no genotoxic potential in both tumor cell lines. Furthermore, the chloroform extract and purified BAR at a concentration of 10 μg/mL were not considered genotoxic for Sarcoma 180. In the comet assay, all compounds tested induced DNA damage in both tumor lines. Based on the results, it is considered that the organic extracts and the BAR purified from C. salzmannii (Nyl). exhibit cytotoxic and genotoxic activity front of the tested tumor cell lines.

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.

Thesis: S.B., Massachusetts Institute of Technology, Department of Nuclear Science and Engineering, 2016.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (pages 25-27).
Aside from directly causing DNA damage, the traversal of radiation through cells also induces the bystander effect, which is the biological response of unirradiated cells that are neighboring or sharing medium with the irradiated cells. Although the mechanisms through which irradiated cells send signals to the bystander cells are not well understood, the bystander effect could potentially have clinical relevance or play a significant role in low dose radiation environments. The research in this thesis focuses on the ability of the bystander cells to influence the behavior of cells that share medium with them, which can be separated into three categories: unirradiated cells, irradiated cells, and the original irradiated cells that caused the bystander effect. These can be considered the "secondary bystanders." Human AG01522 fibroblasts were irradiated with 250 kVp X-rays and co-cultured with unirradiated fibroblasts to generate bystander cells, which were then cocultured with one of the three types of secondary bystander cells. The micronucleus assay was used to analyze the amount of chromosome aberrations present. In the unirradiated secondary bystander population, an increase in percentage of binucleated cells with micronuclei from the background level to approximately the level of the primary bystander cells was observed, indicating that bystander cells can send damaging signals. The data also showed that there was a lower frequency of micronuclei formation in the irradiated population with bystander inserts in comparison to irradiated populations without bystanders. However, there were no conclusive data on the effect of the bystander cells on other irradiated cells. Overall, the results suggest that bystander fibroblasts are capable of sending both detrimental and beneficial signals and can induce a range of behaviors in other cells.
by Chang Liu.
S.B.

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\".

Made available in DSpace on 2017-07-24T19:22:10Z (GMT). No. of bitstreams: 1 Ricardo_Kern.pdf: 242426 bytes, checksum: a174b7b70f1d6dd81756070f120c5387 (MD5) Previous issue date: 2006-02-23
O constante aperfeiçoamento dos testes genéticos, como o Teste de Micronúcleos (MN), os tornaram importantes auxiliares na prevenção do câncer. Com o objetivo de melhor compreender a etiologia do desenvolvimento de neoplasias bucais, o presente trabalho se propôs a estudar a influência do fumo, álcool, trauma mecânico e de substância contidas em colutórios sobre a Freqüência de Micronúcleos (FMN) em células epiteliais bucais de alunos do curso de Odontologia. Para tanto, 40 alunos foram divididos em 4 grupos assim caracterizados G1 – Abstêmios (controle); G2 Alcoolistas; G3 – Usuários de aparelho ortodôntico; G4 – Fumantes alcoolistas, e submetidos ao TMN. Posteriormente estes mesmos alunos receberam de forma aleatória, 4 diferentes tratamentos, por 10 dias, a base de colutórios: T1 (óleos essenciais), T2 (álcool 11%), T3 (álcool, 11% + clorexidina 0,12%), T4 (clorexidina 0,12%), sendo então novamente submetidos ao TMN. Os resultados da FMN demonstraram diferenças significativas entre os 4 grupos G (p = 0,043 Kruskal-Wallis), sendo indicado diferença entre G1 e G3 (Mann-Whitney p ? 0,01) e entre G1 e G4 (Mann-Whitney p ? 0,05), contudo, em relação aos tratamentos com colutórios, todos os tratamentos T não alcançaram resultados significativos (Wilcoxon p > 0,05). Os resultados sugerem que o trauma mecânico causado por aparelho ortodôntico assim como a associação de fumo com bebidas alcoólicas favorecem um aumento na prevalência de MN quando comparados ao controle. A utilização de colutórios bucais, em curto prazo, não foi capaz de causar aumento na freqüência de Micronúcleos

With the influx of high-dimensional data there is an immediate need for statistical methods that are able to handle situations when the number of predictors greatly exceeds the number of samples. One such area of growth is in examining how environmental exposures to toxins impact the body long term. The cytokinesis-block micronucleus assay can measure the genotoxic effect of exposure as a count outcome. To investigate potential biomarkers, high-throughput assays that assess gene expression and methylation have been developed. It is of interest to identify biomarkers or molecular features that are associated with elevated micronuclei (MN) or nuclear bud (Nbud) frequency, measures of exposure to environmental toxins. Given our desire to model a count outcome (MN and Nbud frequency) using high-throughput genomic features as predictors, novel methods that can handle over-parameterized models need development. Overdispersion, when the variance of a count outcome is larger than its mean, is frequently observed with count response data. For situations where overdispersion is present, the negative binomial distribution is more appropriate. Furthermore, we expand the method to the longitudinal Poisson and longitudinal negative binomial settings for modeling a longitudinal or clustered outcome both when there is equidispersion and overdispersion. The method we have chosen to expand is the Generalized Monotone Incremental Forward Stagewise (GMIFS) method. We extend the GMIFS to the negative binomial distribution so it may be used to analyze a count outcome when both a high-dimensional predictor space and overdispersion are present. Our methods were compared to glmpath. We also extend the GMIFS to the longitudinal Poisson and longitudinal negative binomial distribution for analyzing a longitudinal outcome. Our methods were compared to glmmLasso and GLMMLasso. The developed methods were used to analyze two datasets, one from the Norwegian Mother and Child Cohort study and one from the breast cancer epigenomic study conducted by researchers at Virginia Commonwealth University. In both studies a count outcome measured exposure to potential genotoxins and either gene expression or high-throughput methylation data formed a high dimensional predictor space. Further, the breast cancer study was longitudinal such that outcomes and high-dimensional genomic features were collected at multiple time points during the study for each patient. Our goal is to identify biomarkers that are associated with elevated MN or NBud frequency. From the development of these methods, we hope to make available more comprehensive statistical models for analyzing count outcomes with high dimensional predictor spaces and either cross-sectional or longitudinal study designs.

Trois bioindicateurs, l'ethoxyresorufine-o-deethylase (erod), les adduits a l'adn et les micronuclei (mn) ont ete etudies pour une application en biosurveillance des cotes francaises. L'erod a ete developpe a titre experimental sur les sites pilotes de la baie de seine et de fos sur mer-marseille. Quatre especes sentinelles, la limande limanda limanda et le callionyme callionymus lyra, le rouget barbet de vase mullus barbatus ainsi que le serran serranus hepatus ont ete selectionnees. Des inductions de l'activite erod dans le foie des poissons ont ete observees dans la zone subestuarienne de la baie de seine et sur les stations les plus proches des sources de polluants a cortiou et fos sur mer. La determination du nombre de poissons necessaire pour detecter une difference significative entre deux activites erod en fonction de la variance d'echantillonnage et pour une puissance donnee ont ete realise. Une etude preliminaire sur la culture d'hepatocytes de callionyme a permis d'evaluer l'effet toxique des pcb presente dans le milieu marin. Des profiles d'adduits d'hap a l'adn ont ete realises en complementarite de mesures d'activites erod de foies de mullus barbatus peches en mediterranee et de dosages chimiques d'hap sur le sediment. La mesure d'adduits hap a l'adn constitue un outil interessant en surveillance future des zones tres polluees. La mesure d'effets mutagenes par les micronuclei a ete testee in vitro et in vivo chez la moule et l'huitre. Un manque de sensibilite du test mn a ete demontre pour des crassostrea gigas collectees sur un gradient de pollution en gironde. Les resultats obtenus par contamination des cellules de cur en culture primaire par du bap, du sulfate de cuivre et un effluent de pate a papier semblent plus prometteurs. Des variations de la frequence des mn ont ete mesurees chez mytilus galloprovincialis pendant un suivi de quatre mois sur les sites les plus contamines. L'obstacle majeur de l'application du test mn en surveillance est une observation microscopique laborieuse et parfois subjective

Made available in DSpace on 2016-08-10T10:39:07Z (GMT). No. of bitstreams: 1 Douglas Dantas Rodrigues.pdf: 1094258 bytes, checksum: be59e50a44653cbac4abda0ac7ca028f (MD5) Previous issue date: 2015-03-30
The agent to combat epidemic (ACEs) are the more exposed classes to effect of pesticides, since their expositions is given to preparation to application of pesticides. The uses of PPE are essential to protection of these workers, yet they often dispense their uses. Factors such as lack of knowledge about the correct use of pesticides or no respect for the PPE uses may be related to the risk of intoxication. It is well known that pesticides may cause DNA damage in people who are occupationally exposed. Although there are many studies on the damage to farmers, few studies have been performed with ACEs. In this context, this study aimed to evaluate the genomic damage in ACEs of the city of Aparecida de Goiânia (GO) and to correlate the damage with intoxication reports and use of PPE and styles of life of the agents. We selected for research 39 ACEs and 39 people from the local community who were not occupationally exposed to pesticides. Based on a structured questionnaire was collected lifestyle information of the participants, such as age, gender, alcohol consumption, tobacco use, and data on the use of PPE and signs and symptoms of intoxication of ACEs. The evaluation of genomic damage was made by micronucleus technique, and the cholinesterase test was performed in ACEs to observe the presence of chronic intoxication. The exposed group consisted of 21 men (53.8%) and 18 women (46.2%). Of agents 39, 10 (25.6%) smoked and 29 (74.4%) did not smoke; 23 (58.9%) drank and 16 (41.1%) did not drink. The evaluation of cholinesterase was normal in all agents, but when comparing the frequency of micronuclei in the case group with control, a statistical difference (p> 0.05) was observed. By correlating the use of PPE with genomic damage, was not found a significant difference. Based on the findings of this research, it can be states that the use of pesticides by ACEs is a risk to it in genomic damage, which can lead to serious healths problems for the same. Worth pointing out that there are a need for education about the risk of using pesticides, particularly for occupationally exposed group.
Os agentes de combate a endemias (ACEs) são as categorias mais exposta aos efeitos de pesticidas, pois sua exposição se dá deste o preparo da calda dos pesticidas até a aplicação do mesmo. O uso de EPIs é essencial para a proteção desses trabalhadores, todavia eles muitas vezes os dispensam. Fatores como a falta de conhecimento acerca do uso correto dos pesticidas ou não respeito aos usos das EPIs podem estar relacionados com o risco de intoxicação. Sabe-se que os pesticidas podem causar danos no DNA em pessoas que são ocupacionalmente expostas. Apesar de existir muitos estudos sobre o dano em agricultores, poucos estudos foram realizados com ACEs. Neste contexto, o presente trabalho teve como objetivo avaliar o dano genômico em ACEs da cidade de Aparecida de Goiânia (GO) e correlacionar o dano com os quadros de intoxicação e com uso de EPIs e aos estilos de vidas dos agentes. Foram selecionados para pesquisa 39 ACEs e 39 pessoas da comunidade local que não foram ocupacionalmente expostos a pesticidas. Através de um questionário estruturado foram coletados dados sobre estilo de vida dos participantes, tais como idade, sexo, etilismo, tabagismo, além de dados acerca do uso de EPIs e sinais e sintomas de intoxicação dos ACEs. A avaliação do dano genômico foi realizada pela da técnica de micronúcleo, e o teste de colinesterase foi realizado nos ACEs para observar a presença de intoxicação crônica. O grupo exposto foi composto por 21 homens (53,8%) e 18 mulheres (46,2%). Dos 39 agentes, 10 (25,6%) fumavam e 29 (74,4%) não fumavam e 23 (58,9%) bebiam e 16 (41,1%) não bebiam. A avaliação da colinesterase foi normal em todos os agentes, porém, ao comparar a frequência dos micronúcleos do grupo caso com controle, foi observada uma diferença estatística (p<0,05). Ao correlacionar o uso de EPIs com dano genômico, não foi encontrada uma diferença significativa. Com base nos achados dessa pesquisa, pode-se afirmar que o uso de pesticidas por ACEs representa um risco de dano genômico, o que pode trazer sérios problemas de saúde para os mesmos. Vale salientar que há uma necessidade de educação sobre o risco do uso de pesticidas, principalmente para os grupos ocupacionalmente exposto.

O teste de frequência de micronúcleos (FMN) in vitro é uma das metodologias de escolha no desenvolvimento de testes de segurança toxicológica. Para o seu desenvolvimento no trabalho foram realizadas modificações da técnica convencional, relativas ao substrato de cultivo das células e à sua coloração. As culturas celulares foram desenvolvidas diretamente nas lâminas e a coloração foi realizada com laranja de acridina (AO) ao invés da coloração segundo Giemsa. Foram utilizados controles positivos com potenciais clastogênico (mitomicina C, benzo[a]pireno) e aneugênico (colchicina), recomendados pela OCDE (Organização para Cooperação e Desenvolvimento Econômico). Como moléculas-teste, foram utilizados compostos cuja associação a isótopos radioativos compõem radiofármacos produzidos pelo IPEN. DOTATATO e Ubiquicidina foram testados em diferentes concentrações proporcionais às concentrações máximas utilizadas em pacientes adultos. Para tanto, foram realizadas diluições correspondentes às concentrações 0,1X, 1X e 10X e culturas de células CHO-KI foram expostas a estas concentrações para ensaios de citotoxicidade e FMN. Nenhuma das concentrações induziu citotoxicidade significativa. Para análise de FMN, foram contabilizadas todas as células mononucleadas e multinucleadas até atingir a contagem de 1000 células binucleadas, com ou sem micronúcleos. Desta maneira foi possível analisar a frequência de micronúcleos e o índice de proliferação (CBPI). As concentrações dos fármacos em teste (0,1X, 1X e 10X) não induziram agressão às células. Nenhuma das concentrações revelou citotoxicidade ou genotoxicidade, ou ainda qualquer alteração no ciclo celular em comparação aos controles, comprovando sua segurança conforme os parâmetros exigidos pelas normas internacionais. Os resultados mostraram também uma boa concordância entre a comparação das leituras realizadas por analistas independentes, com pequenas discrepâncias discutíveis, e boa correlação com resultados obtidos com a coloração convencional. Desta maneira as modificações realizadas na técnica de FMN mostraram potencial para cumprir todos os quesitos como teste pré-clínico.
The in vitro micronucleus frequency test (FMN) is one method of choice in the development of toxicological safety tests. For its development, this work carried out modifications of the conventional technique regarding the cultivation substrate of the cells and staining for microscopy evaluation. The cell cultures were grown directly on slides, and staining was performed with acridine orange (AO) instead of the classical Giemsa staining. Positive controls were used for potential clastogenic (mitomycin C, benzo [a] pyrene) and aneugenic (colchicine) effects, recommended by the OECD (Organization for Economic Cooperation and Development). As test molecules, compounds were used whose association with radioactive isotopes make up radiopharmaceuticals produced by IPEN. DOTATATE and Ubiquicidine were tested at different concentrations proportional to the maximum concentrations used in adult patients. Therefore, corresponding to the concentrations dilutions were performed 0.1X, 1X and 10X cultures and CHO-KI cells were exposed to these concentrations for cytotoxicity assays and FMN. None of the concentrations induced significant cytotoxicity. For FMN analysis, it was recorded every mononuclear cells and multinucleated up to 1000 counts binucleated cells with or without micronuclei. In this way it was possible to analyze the frequency of micronuclei and the proliferation index (CBPI). The concentrations of the test drug (0.1X, 1X and 10X) did not induce aggression to cells. None of the concentrations showed cytotoxicity and genotoxicity, or any changes in cell cycle compared to controls, demonstrating their safety according to the parameters required by international standards. The results also showed good agreement between the comparison of readings by independent analysts, with minor discrepancies debatable, and good correlation comparing to classical staining technique. Thus the changes made in FMN technique showed potential to fulfill all requirements as preclinical test.

Orientador: Carmem Silvia Fontanetti Christofoletti
Banca: Maria Aparecida Marin Morales
Banca: Silvia Tamie Matsumoto
Resumo: A degradação dos recursos hídricos, como os ambientes lênticos, dentre eles, os lagos, é uma das maiores preocupações atualmente, visto que esta pode causar danos diretos ou indiretos à saúde e à sobrevivência dos organismos expostos. Um dos fatores que contribui para a alteração da qualidade das águas de ambientes lênticos é o despejo de efluentes, principalmente àqueles de origem doméstica, portadores de substâncias que chegam a ser tóxicas para o meio aquático. Por meio dos testes citogenéticos, utilizando os mais diversificados organismos-teste, é possível biomonitorar a extensão da poluição e avaliar os efeitos dessas substâncias presentes no ambiente natural. Com esse intuito, o presente trabalho tomou por modelo de estudo, um lago urbano artificial (Lago Azul, Rio Claro-SP) e objetivou avaliar o potencial citotóxico, genotóxico e mutagênico das águas desse ambiente, por meio dos testes de aberrações cromossômicas e micronúcleos, em células meristemáticas de Allium cepa (cebola), em dois tratamentos: o contínuo e o período de recuperação, em água ultra pura; e, pelo teste do micronúcleo associado às anormalidades nucleares e do ensaio do cometa, aplicados em eritrócitos de Oreochromis niloticus (tilápia). Coletas de águas sazonais foram realizadas na estação seca (agosto/2006 e agosto/2007) e na estação chuvosa (março/2007 e fevereiro/2008). Análises físico-químicas foram feitas para uma coleta de cada estação. A partir dos dados obtidos, pode-se inferir que as águas desse ambiente lêntico apresentam potencial citotóxico, genotóxico e mutagênico, nas duas estações de coletas, para os dois organismos-teste empregados. As análises de metais revelaram concentrações acima do permitido pela legislação de Ag, Cd2+, Cu e Fe3+, em ambas as estações. Embora os... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The degradation of water resources, as the lentics environments, among them, the lakes, is a major concern now, because it can cause direct or indirect damages to health and to the survival of the exposed organisms. One factor that can contribute to change the water quality of lentics environments is the dumping of effluents, mainly those of domestic origin, carriers of substances that come to be toxic to the aquatic environment. Through the cytogenetic tests, using the most diverse systems-test, it is possible monitoring the extent of pollution and assess the effects of substances on the natural environment. To that end, this work has taken a model of study, an urban artificial lake (Lago Azul, Rio Claro-SP) and aimed to evaluate the cytotoxic, genotoxic and mutagenic potentials of waters that environment, through tests of chromosome aberrations and micronuclei in meristematic cells of Allium cepa (onion) in two treatments: the continued and the period of recovery, in ultra pure water, and by the micronucleus and nuclear abnormalities test and of the comet assay, applied in erythrocytes of Oreochromis niloticus (tilapia). Seasonal collections of waters were held in the dry season (august/2006 and august/2007) and the rainy season (march/2007 and february/2008). Physical and chemical analyses were made for a collection of each season. From the data obtained, it can be infered that the waters of this lentic environment had cytotoxic, genotoxic and mutagenic potentials in two seasons of collections for the two systems-test employed. Analyses of metals detected high concentrations of Ag, Cd2+, Cu, Fe3+, whose values are higher than permitted by law, in both seasons. Although the cytotoxic, genotoxic and mutagenic potentials have been detected in two seasons, the dry season is that presented the highest risk... (Complete abstract click electronic access below)
Mestre

Nelle matrici ambientali sono spesso presenti xenoestrogeni, molecole sintetiche o di origine naturale ad attività estrogenica in grado di alterare il normale equilibrio ormonale di organismi esposti, incidendo negativamente su alcune funzioni vitali come la riproduzione ed il metabolismo. Diverse sostanze chimiche presenti in ambiente, tra cui alcune molecole ad attività estrogenica, sono anche potenziali composti genotossici, in grado, cioè, di interagire con il DNA ed esercitare effetti anche a lungo termine come l’insorgenza di tumori nei vertebrati, uomo compreso. L’obiettivo del presente lavoro di tesi è stato quello di mettere a punto ed utilizzare due saggi biologici, il saggio E-screen ed il test dei micronuclei, per valutare la presenza di xenoestrogeni e composti genotossici in campioni di acque prelevate prima e dopo i trattamenti di potabilizzazione, utilizzando cellule MCF-7 di adenocarcinoma mammario come modello sperimentale in vitro. Le indagini biologiche sono state condotte sulla base di una convenzione di ricerca con la Società acquedottistica Romagna Acque- Società delle fonti e hanno previsto tre campagne di monitoraggio. I campioni di acqua sperimentale, raccolti prima e dopo i trattamenti presso diversi impianti di potabilizzazione, sono stati preventivamente filtrati, estratti in fase solida, fatti evaporare sotto leggero flusso di azoto, ed infine, saggiati sulle cellule. Il test E-screen, di cui abbiamo dimostrato un elevato livello di sensibilità, ha permesso di escludere la presenza di composti ad attività estrogenica nei campioni esaminati. Allo stesso modo, i risultati del test dei micronuclei hanno dimostrato l’assenza di effetti genotossici, confermando la buona qualità delle acque analizzate. Nell’ambito delle attività di monitoraggio, le indagini biologiche risultano essenziali per la valutazione di una potenziale contaminazione ambientale, in quanto forniscono informazioni anche quando non sono state condotte analisi chimiche. Inoltre, anche quando le analisi chimiche siano state condotte, i test biologici informano della potenzialità tossica di una matrice causata eventualmente da sostanze non oggetto del saggio chimico. Infine, i test biologici permettono di identificare eventuali sinergie tra più contaminanti presenti nelle acque, affermandosi come test da condurre in maniera complementare ai saggi chimici. I test biologici come quelli impiegati nel lavoro di tesi sono molto sensibili ed informativi, ma necessitano della definizione di protocolli standardizzati per garantirne un’uniforme applicazione alle acque ad uso potabile, almeno a livello nazionale.

The objective of this study was to evaluate the effect of thorium (Th) on the metabolism of silver catfish (Rhamdia quelen) through biochemical parameters from the muscle tissue (glycogen, glucose, lactate, protein and ammonia), lipidic peroxidation levels (TBARS), catalase (CAT) and glutathione-S-transferase (GST) in the hepatic and muscular tissues and cytogenetic parameters through the evaluation of nuclear abnormalities in blood cells. Silver catfish juveniles (8.78 ± 0.10cm; 6.41 ± 0.17g) were exposed to different waterborne concentrations of ²³²Th (in μg.L-1): 33.6±8.7; 106.5±37.1; 191.6±19.0 and 758.4±150.4 for 15 days. The levels of muscle glycogen were significantly reduced in fish exposed to 106.5 μg.L-¹ Th, while glucose and protein increased in those exposed to 758.4 μg.L-¹ Th. Lactate levels were higher in fish maintained at 191.6 μg. L-¹ Th and ammonia was higher in those exposed to 33.6, 106.5 and 191.6 μg.L-¹ Th. The lipidic peroxidation levels were diminished in the liver of silver catfish exposed to all tested concentrations of Th. In the muscle lipidic peroxidation was higher in juveniles maintained at 106.5 μg.L-¹ Th and lower in those exposed to 191.6 and 758.4 μg.L-¹ Th. The CAT activity was higher in the hepatic tissue (but not muscle) of fish exposed to all tested concentrations of Th. The GST activity in the liver was lower in fish exposed to 33.6 and 106.5 μg.L-¹ Th, and in the muscular tissue of those maintained at 758.4 μg.L-1 Th. Silver catfish exposed to 106.5 μg.L-¹ presented a significant induction of micronuclei, but no alterations in other erythrocyte abnormalities were observed. These results suggest that exposure to waterborne Th induces changes in the metabolic state, increase of lipidic peroxidation in the liver, some alterations of CAT and GST, and DNA damage.
O objetivo deste estudo foi avaliar o efeito de tório (Th) sobre parâmetros metabólicos (glicogênio, glicose, lactato, proteína e amônia), em tecido muscular de jundiás (Rhandia quelen), níveis de lipoperoxidação (TBARS), catalase (CAT) e glutationa-S-transferase(GST) nos tecidos hepático e muscular e parâmetros citogenéticos através da avaliação de anormalidades nucleares em células sanguíneas. Juvenis de jundiás (8,78±0,10cm; 6,41± 0,17g) foram expostos a diferentes concentrações de ²³²Th 33,6±8,7; 106,5±37,1; 191,6±19,0 e 758,4±150,4 em μg. L-¹ (três repetições por tratamento) por 15 dias. Os níveis de glicogênio muscular diminuíram significativamente a 106,5 μg. L-¹ Th. Glicose e proteína aumentaram na 758,4 μg. L-¹ Th. Os níveis de lactato apresentaram-se elevados em 106,5 μg. L-¹ Th. A amônia aumentou a 33,6; 106,5 e 191,6 μg.L-¹ Th. Os níveis de peroxidação lipídica diminuíram no fígado dos jundiás expostos a todas as concentrações de Th testadas. No músculo esquelético aumentaram a 106,5 μg. L-¹Th e diminuíram a 191,6 e 758,4 μg.L-¹ Th. A atividade da CAT no tecido hepático apresentou aumento em todas as concentrações testadas de Th. Nenhuma alteração foi observada no tecido muscular. A GST diminui no fígado a 33,6 e 106,5 μg.L-¹Th. No tecido muscular diminui a 758,4 μg.L-1 Th. Jundiás expostos a 106,5 μg. L-¹ apresentaram maior indução de micronúcleos. Não foi observadas alterações para outras anormalidades nucleares eritrocíticas. Os resultados obtidos sugerem mudanças nos intermediários metabólitos devido ao estresse provocado pelo Th, aumento da lipoperoxidação no fígado, sendo observado níveis variados no músculo esquelético, alterações das enzimas CAT e GST além de danos no DNA dos peixes expostos ao Th, comparando aos grupos controles.

Improved survival for women receiving chemotherapy for breast cancer (BC) has been accompanied by the development/persistence of psychoneurological symptoms (PNS) that compromise their quality of life. The biological basis for these PNS is unknown, but could reflect the acquisition of soma-wide chromosomal/epigenetic alterations. An important first step in testing this hypothesis is to determine if somatic genetic/epigenetic changes arise and persist following treatment. To answer this question we longitudinally studied 71 women (ages 23-71) with early-stage BC and collected measures before chemotherapy (baseline), and 4 weeks (mid-chemo); six months (during radiation therapy for a subset of women); and one year following the initiation of chemotherapy. Acquired lymphocyte chromosomal instability (scored by micronuclei frequencies [MNF]) showed a significant increase in post-treatment compared to baseline time-points (p<0.0001), with these increases persisting for at least one year following chemotherapy. Significant predictive associations were observed between MNF and tumor characteristics [luminal B (lower MNF; p=0.0182); triple negative (higher MNF; p=0.0446)], radiotherapy (higher MNF; p=0.0004), the type of chemotherapy received (p=0.0463), race (Caucasians > African Americans; p=0.0037), perceived stress levels (positive-association; p=0.0123), and cognitive flexibility domain measures (positive-association; p=0.0238). Genome-wide acquired methylation changes were also measured in peripheral blood cells, with 1265 sites showing significant differential methylation following chemotherapy. These sites were localized to open sea, shores, shelves, and CpG island sequences and included sites within genes involved in cell cycle, DNA repair, transcription regulation, signal transduction pathways, neuronal regeneration, and immunity. To determine if the genetic/epigenetic alterations acquired in peripheral blood cells correlated with those in tumor cells, BC tumors from 10 participants were analyzed using a genome-wide copy number/targeted mutations (CN/M) microarray. While no clear blood-tumor cell correlations were detected, genome-wide CN/M evaluations showed promise for stratifying tumors. Lastly, in an unrelated project studying a rare case of fetuses in fetu, methylation changes acquired in embryogenesis were shown to be influenced by both environmental and genetic cues. In summary, acquired chromosomal/epigenetic alterations do arise following chemotherapy (and in embryogenesis). Further delineation of these acquired changes could increase our understanding of the biological basis for cancer-related side-effects and help to identify “at risk” individuals.

Childhood sexual abuse (CSA) is a stressful life experience with lasting/far-reaching health and psychopathological consequences. Our laboratory recently identified a significantly increased frequency of acquired chromosomal anomalies (assessed using the cytokinesis-blocked micronucleus assay) in adult female twins exposed to CSA when compared to their unexposed co-twin. The primary aim of this study was to evaluate potential mechanism(s) underlying the observed increases in levels of micronuclei in an expanded group of 90 female identical twins (61 CSA+ females and 29 CSA- females [including a total of 27 MZ co-twin pairs]) using fluorescence in situ hybridization (FISH) methodologies, with PNA probes specific for the centromeric and telomeric regions of all chromosomes coupled with the standard CBMN assay, we were able to characterize the chromosomal contents of MN and, thus, gain insight into the mechanisms underlying MN formation. By scoring 100 MN per study participant for the number of centromeric signal(s) and/or telomeric signal(s) present, we categorized the MN as harboring either: (1) terminal fragments (only a telomeric signal); (2) acentric interstitial fragments (no telomeric or centromeric signal); (3) centric interstitial fragments (only a centromeric signal); or (4) an intact chromosome(s) or chromatid(s). We identified elevated frequencies of intact chromosome-derived MN in CSA+ women as compared to CSA- women (P=0.014), implicating chromosome loss as a mechanism potentially underlying the increased frequencies of MN identified in adult females with a history of CSA. MN containing fragmented chromosomes were also observed in all of the study participants evaluated; however MN containing terminal fragments and MN containing acentric interstitial fragments were seen less frequently in CSA+ women compared to CSA- women. This study represents the first time that the chromosomal contents of MN have been evaluated in individuals in the context of a psychosocial factor. As chromosomal loss and breakage contributes to the development of age-related health problems, these observations provide important insight into the biological mechanisms that may underlie the latent morbidity and psychopathology associated with childhood adversity. Future studies aimed at understanding the biological impact of early-life trauma could determine if the observed increase in acquired chromosomal abnormalities results in detectable somatic clonal mosaicism. This knowledge could ultimately be used to develop screening tools to identify individuals “at risk” for negative health outcomes in adulthood.

Submitted by Verena Bastos (verena@uefs.br) on 2015-10-08T12:18:43Z No. of bitstreams: 1 Jeanderson_Disserta??o_vers?o final.pdf: 369468 bytes, checksum: 851a8492d73fc0106bfe7784166fdf98 (MD5)
Made available in DSpace on 2015-10-08T12:18:43Z (GMT). No. of bitstreams: 1 Jeanderson_Disserta??o_vers?o final.pdf: 369468 bytes, checksum: 851a8492d73fc0106bfe7784166fdf98 (MD5) Previous issue date: 2013-03-25
Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES
Exposure to genotoxic agents induces changes in the DNA molecule to commit that genes involved with the repair mechanisms and the control of cell proliferation and differentiation pathways or genes associated with apoptosis can lead to cancer development. Among the many chemicals that have been identified as mutagenic action, include the Androgenic Steroids (AAS), hormones widely used in the search for improved physical performance and increased muscle mass. Objective: In this context, the aim of the development of this study was to evaluate the potential of androgenic anabolic steroid nandrolone decanoate, testosterone propionate and testosterone cypionate to induce chromosome damage, apoptosis and necrosis, using the micronucleus test in exfoliated cells from the oral mucosa of users of AAS with a view to its application as a tool in cancer prevention. Method: The study sample consisted of 55 volunteers, male, divided into two (02) groups, matched for age: 25 subjects (G1) users of nandrolone decanoate, testosterone propionate and testosterone cypionate (alone or simultaneously ) and 30 subjects in the control group (G2). The collection methodology and cytological analysis followed the protocol of Tolbert et al. (1992) and Thomas et al. (2009), which includes, in addition to micronuclei, the computation of degenerative nuclear changes indicative of apoptosis (cariorr?xis, condensed chromatin and pyknosis) and necrosis (karyolysis, cariorr?xis, condensed chromatin and pyknosis). Statistical analysis of the endpoints analyzed (micronucleus, cariorr?xis, condensed chromatin, karyolysis, pyknosis and broken eggs) was performed using the conditional test to compare proportions in situations of rare events. Results: Statistical analysis revealed no significant difference in the occurrence of micronuclei, karyolysis and broken eggs between groups. The occurrence of apoptosis was significantly greater in cells from control individuals. Conclusion: The results show inhibition of apoptosis induced by EAA, suggesting that the described association between the use of these substances and the carcinogenic process can be permeated by this mechanism.
A exposi??o a agentes genot?xicos induz altera??es na mol?cula do DNA que ao comprometerem genes envolvidos com os mecanismos de reparo e com o controle da prolifera??o e diferencia??o celular ou genes associados ?s vias de apoptose, podem levar ao desenvolvimento de c?ncer. Entre os muitos agentes qu?micos que t?m sido identificados como de a??o mutag?nica, incluem-se os Ester?ides Anabolizantes Androg?nicos (EAA), horm?nios amplamente utilizados na busca da melhoria do desempenho f?sico e aumento da massa muscular. Objetivo: Neste contexto, objetivou-se com o desenvolvimento do presente estudo, avaliar o potencial dos ester?ides anabolizantes androg?nicos decanoato de nandrolona, propionato de testosterona e cipionato de testosterona em induzir danos cromoss?micos, apoptose e necrose, atrav?s do uso do Teste de Micron?cleo em c?lulas esfoliadas da mucosa oral de usu?rios de EAA com vista ? sua aplica??o como ferramenta na preven??o do c?ncer. M?todo: A amostra do estudo foi composta por 55 volunt?rios, do sexo masculino, distribu?dos em dois (02) grupos, pareados por idade: 25 indiv?duos (G1) usu?rios de decanoato de nandrolona, propionato de testosterona e cipionato de testosterona (isoladamente ou simultaneamente) e 30 indiv?duos no grupo controle (G2). A metodologia de coleta e an?lise citol?gica seguiu o protocolo de Tolbert et al. (1992) e Thomas et al. (2009), que inclui, al?m de micron?cleos, o computo de altera??es nucleares degenerativas indicadoras de apoptose (cariorr?xis, cromatina condensada e picnose) e necrose (cari?lise, cariorr?xis, cromatina condensada e picnose). A an?lise estat?stica dos endpoints analisados (micron?cleo, cariorr?xis, cromatina condensada, cari?lise, picnose e broken eggs) foi realizada com o uso do teste condicional para compara??o de propor??es em situa??es de eventos raros. Resultados: A an?lise estat?stica revelou que n?o houve diferen?a significativa na ocorr?ncia de micron?cleo, cari?lise e broken eggs entre os grupos. A ocorr?ncia de apoptose foi, significativamente, maior em c?lulas dos indiv?duos do grupo controle. Conclus?o: Os resultados obtidos mostram inibi??o da apoptose induzida pelo uso de EAA, sugerindo que a associa??o descrita entre uso destas subst?ncias e o processo carcinog?nico possa ser permeada por este mecanismo.

Made available in DSpace on 2014-06-11T19:22:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-08-04Bitstream added on 2014-06-13T20:49:39Z : No. of bitstreams: 1 christofoletti_ca_me_rcla.pdf: 3763933 bytes, checksum: d9284f200972936e72e67bb3de745be4 (MD5)
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
A degradação dos recursos hídricos, como os ambientes lênticos, dentre eles, os lagos, é uma das maiores preocupações atualmente, visto que esta pode causar danos diretos ou indiretos à saúde e à sobrevivência dos organismos expostos. Um dos fatores que contribui para a alteração da qualidade das águas de ambientes lênticos é o despejo de efluentes, principalmente àqueles de origem doméstica, portadores de substâncias que chegam a ser tóxicas para o meio aquático. Por meio dos testes citogenéticos, utilizando os mais diversificados organismos-teste, é possível biomonitorar a extensão da poluição e avaliar os efeitos dessas substâncias presentes no ambiente natural. Com esse intuito, o presente trabalho tomou por modelo de estudo, um lago urbano artificial (Lago Azul, Rio Claro-SP) e objetivou avaliar o potencial citotóxico, genotóxico e mutagênico das águas desse ambiente, por meio dos testes de aberrações cromossômicas e micronúcleos, em células meristemáticas de Allium cepa (cebola), em dois tratamentos: o contínuo e o período de recuperação, em água ultra pura; e, pelo teste do micronúcleo associado às anormalidades nucleares e do ensaio do cometa, aplicados em eritrócitos de Oreochromis niloticus (tilápia). Coletas de águas sazonais foram realizadas na estação seca (agosto/2006 e agosto/2007) e na estação chuvosa (março/2007 e fevereiro/2008). Análises físico-químicas foram feitas para uma coleta de cada estação. A partir dos dados obtidos, pode-se inferir que as águas desse ambiente lêntico apresentam potencial citotóxico, genotóxico e mutagênico, nas duas estações de coletas, para os dois organismos-teste empregados. As análises de metais revelaram concentrações acima do permitido pela legislação de Ag, Cd2+, Cu e Fe3+, em ambas as estações. Embora os...
The degradation of water resources, as the lentics environments, among them, the lakes, is a major concern now, because it can cause direct or indirect damages to health and to the survival of the exposed organisms. One factor that can contribute to change the water quality of lentics environments is the dumping of effluents, mainly those of domestic origin, carriers of substances that come to be toxic to the aquatic environment. Through the cytogenetic tests, using the most diverse systems-test, it is possible monitoring the extent of pollution and assess the effects of substances on the natural environment. To that end, this work has taken a model of study, an urban artificial lake (Lago Azul, Rio Claro-SP) and aimed to evaluate the cytotoxic, genotoxic and mutagenic potentials of waters that environment, through tests of chromosome aberrations and micronuclei in meristematic cells of Allium cepa (onion) in two treatments: the continued and the period of recovery, in ultra pure water, and by the micronucleus and nuclear abnormalities test and of the comet assay, applied in erythrocytes of Oreochromis niloticus (tilapia). Seasonal collections of waters were held in the dry season (august/2006 and august/2007) and the rainy season (march/2007 and february/2008). Physical and chemical analyses were made for a collection of each season. From the data obtained, it can be infered that the waters of this lentic environment had cytotoxic, genotoxic and mutagenic potentials in two seasons of collections for the two systems-test employed. Analyses of metals detected high concentrations of Ag, Cd2+, Cu, Fe3+, whose values are higher than permitted by law, in both seasons. Although the cytotoxic, genotoxic and mutagenic potentials have been detected in two seasons, the dry season is that presented the highest risk... (Complete abstract click electronic access below)

Made available in DSpace on 2016-08-10T10:38:32Z (GMT). No. of bitstreams: 1 CAROLINNE BORGES KHAYAT.pdf: 2297476 bytes, checksum: 992aa1b9dc7b17cdf96375464ab8d719 (MD5) Previous issue date: 2012-04-13
The term pesticide is used to refer to a wide variety of chemicals used to kill weeds, insects and fungi. In Brazil, the use of pesticides in agriculture has been expanding continuously, and therefore the analysis of the effects of this type of environmental exposure begins to document an epidemiological profile of the distribution of cancer in both populations occupationally exposed to these chemicals as in the general population indirectly affected by contaminated food and water resources. The possible toxic effects of such exposures are unknown and the information related only to the toxicity of active ingredients are not sufficient to assess the risk of adverse effects of pesticides on human health and the environment. Workers exposed to pesticides when compared with the control group showed increased 8 times more micronucleus and 20 times more binucleated cells and increased DNA damage measured by comet assay. Variables such as smoking, alcohol consumption, age, sex, duration of exposure and type of pesticide did not affect the appearance of genetic damage. However, there was an increase of micronucleus in workers who did not use personal protective equipment. Pesticide exposure can cause genotoxicity and mutagenicity to individuals who deal with such agents.
O termo pesticida é usado para denominar uma ampla variedade de produtos químicos utilizados para destruir ervas daninhas, insetos e fungos. No Brasil, o uso de pesticidas na agricultura vem se ampliando de forma contínua e, consequentemente, a análise sobre os efeitos deste tipo de exposição ambiental começa a documentar um perfil epidemiológico da distribuição de câncer tanto em populações ocupacionalmente expostas a estes agentes químicos, como na população geral indiretamente afetada por contaminação alimentar e dos recursos hídricos. Os possíveis efeitos tóxicos de tais exposições ainda são desconhecidos e as informações da toxicidade relacionada apenas aos ingredientes ativos não são suficientes para avaliar o risco dos efeitos adversos dos pesticidas à saúde humana e ambiental. Os trabalhadores expostos a pesticidas, quando comparados ao grupo controle, tiveram aumento de 8 vezes mais micronúcleos e 20 vezes mais células binucleadas e maior dano ao DNA avaliado pelo ensaio cometa, em relação ao grupo controle (p<0,0001), como demonstrado pelo teste t e análises de regressão linear simples. Nesse estudo, variáveis como tabagismo, consumo de álcool, idade, sexo, tempo de exposição e tipo de pesticida não influenciaram o aparecimento de danos genéticos. Entretanto, houve aumento de micronúcleos em trabalhadores que não utilizavam equipamentos de proteção pessoal (p=0,006). Assim, a exposição a pesticidas, independente do tempo e tipo de agente utilizado, pode causar genotoxicidade e mutagenicidade aos indivíduos que os manipulam.

Doses de radiação de baixa energia podem induzir quebras de dupla fita no DNA assim como também produzir perfis alterados de expressão de genes relacionados a estas lesões. Os danos não reparados ou mal reparados levam a uma maior suscetibilidade à transformação oncogênica já que os efeitos biológicos mais importantes causados pela radiação ionizante são mutação e carcinogênese. As lesões no DNA provocadas pela radiação podem também ocasionar o surgimento micronúcleos e as células podem ser induzidas à apoptose. O objetivo deste trabalho é estudar in vitro a presença de micronúcleos e a apoptose ocasionados por Raios-X de baixa energia. Pretende-se analisar estes efeitos biológicos em relação à energia equivalente à utilizada em exames mamográficos usando duas linhagens estabelecias de células de mama: a MCF-7 (tumoral) e a HB-2 (não-tumoral). As células, em crescimento exponencial, foram irradiadas no equipamento de arranjo experimental de mamografia do LCR/UERJ. A dose de 5Gy na energia de 30 kV foi aplicada com taxa de 0,1 Gy/seg utilizando filtro de 0,03 mm de molibdênio. As irradiações foram realizadas duas vezes, após as irradiações, as células foram incubadas por 4, 24 ou 48 horas e posteriormente coradas com o corante Hoechst33258 para análise em microscopia de fluorescência. Para cada análise, 1000 células foram categorizadas pela morfologia do núcleo. Os resultados mostraram que a HB-2, utilizada neste estudo como célula mamária normal, apresentou maior sensibilidade aos efeitos da radiação, com 37 % das células em apoptose após 4 hs de incubação, enquanto a MCF-7 apresentou 6,5 %. Nas análises após 24 hs foi possível confirmar a radioresistência da MCF-7 tendo sido observadas 11% de células em apoptose no grupo irradiado. Houve um aumento crescente de micronúcleos radioinduzidos nas duas linhagens de acordo com os tempos de incubação. Na análise de 4 hs a HB-2 apresentou 3%, em 24 hs, 8,5% e 48 hs, 11,5 %. Diante destes resultados, foi possível concluir que a energia do feixe de raios-X utilizada na mamografia pode ser capaz de ocasionar aumento de ocorrência de apoptose e geração de micronúcleos nas duas linhagens estudadas
Radiation doses of low-energy can induce double-stranded breaks in DNA as well resulting in altered profiles of gene expression related to these injuries. The damage is not repaired or poorly repaired leading to increased susceptibility to oncogenic transformation since most important biological effects caused by ionizing radiation are mutation and carcinogenesis. The DNA lesions caused by radiation can also provoke the presence of micronuclei and as result cells can be induced to apoptosis. The aim of this work is to perform a preliminary study "in vitro" to identify the presence of micronuclei caused by low energy. X-rays. It is intended analyze these biological effects caused by x-rays with energy equivalent to the ones used in mammography in two very well established breast: cell lines the MCF-7 (tumor) and HB-2 (non-tumor). Cells in exponential growth were irradiated using an experimental arrangement with a mammography tube of the LCR / UERJ. The dose of 5Gy was used for 30 kVp x-rays using 0.03 mm filter molybdenum with a fixed dose rate of 0.1 Gy / sec. The irradiations were repeated twice and. the cells were incubated for 4, 24 or 48 hours and stained with dye Hoechst 33258 for fluorescence microscopy analysis. For each analysis 1000 cells were categorized by the morphology of the nucleus. The results revealed that the HB-2, used in this study as normal mammary cell have showed high sensitivity to the effects of radiation, with 37% of cells undergoing apoptosis after incubation of 4hs, while MCF-7 showed 6.5%. In the 24 hour-analysis it was confirmed the radiation resistance of MCF-7 were it was observed 11% of cells in apoptosis in the irradiated group. There was an increasing number of radiation-induced micronuclei in both cell lines, according to the incubation times. After 4 hours, HB-2 presented 3% of micronuclei, in 24 hours, the experiment reveled 8.5%, and after 48 hours it increased to 11.5%. As result of this study we may conclude that the energy used in mammography is capable to cause an increase in incidence of apoptosis and in the production of micronuclei in both cell lines studied

The primary study aim was to evaluate the latent biological effect of childhood sexual abuse (CSA) on adults by quantifying acquired cytogenetic changes and cortisol levels in identical twins who were discordant (N=22) or concordant (N=2) for a history of CSA. Although the difference scores for cortisol values between discordant identical co-twins were not significantly different from zero, a trend was observed for the twins exposed to intercourse, the most severe form of CSA, to have a blunted cortisol awakening response. Acquired cytogenetic changes were assessed by scoring telomere lengths and somatic cell abnormality frequencies via a cytokinesis-block micronucleus (MN) assay. No significant difference in overall telomere intensity values was observed between co-twins, but chromosome-specific telomere differences were observed in the individuals exposed to intercourse compared to their unabused co-twins ([χ2(45)= 62.88; p= 0.040 and χ2(45)= 73.72; p= 0.004). Specifically, shortened telomeres were observed on the short arms of chromosomes 3, 5, & 6, and long arms of chromosomes 11 & 13. A significant increase in MN frequencies was observed in the abused twins compared to unabused twins (t=2.65; df=16; p=0.009). A significant interaction between micronuclei frequencies and age was also observed, suggesting that the biological effects of stress are cumulative (coefficient [SE] = 0.030 [0.009]; p=0.0006). However, the pattern of chromatin present in MN, which was assessed using spectral karyotyping methodologies, was not limited to the subset of chromosomes with telomeric attrition. In summary, this is the first assessment of acquired chromosomal abnormalities, chromosome-specific telomere lengths and cortisol levels in identical adult twins discordant for exposure to CSA. Given that a portion of biological changes were most pronounced in the intercourse discordant twins, these findings support a possible dose-response relationship with CSA severity. Our data also suggest that the MN assay is a superior tool in assessing the latent effects of stress compared to either cortisol profiling or the measurement of telomere lengths. Collectively, application of the information gained from these studies may allow for novel screening techniques to identify individuals who are most at risk for developing stress-associated disease states.

Submitted by Alison Vanceto (alison-vanceto@hotmail.com) on 2017-01-03T13:03:15Z No. of bitstreams: 1 DissPRS.pdf: 2244710 bytes, checksum: d11e0b755d5503e93a47a8a6dc113814 (MD5)
Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2017-01-16T16:23:49Z (GMT) No. of bitstreams: 1 DissPRS.pdf: 2244710 bytes, checksum: d11e0b755d5503e93a47a8a6dc113814 (MD5)
Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2017-01-16T16:23:57Z (GMT) No. of bitstreams: 1 DissPRS.pdf: 2244710 bytes, checksum: d11e0b755d5503e93a47a8a6dc113814 (MD5)
Made available in DSpace on 2017-01-16T16:24:06Z (GMT). No. of bitstreams: 1 DissPRS.pdf: 2244710 bytes, checksum: d11e0b755d5503e93a47a8a6dc113814 (MD5) Previous issue date: 2016-10-27
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Titanium dioxide nanoparticles (TiO2-NP) are commonly used in many industrial activities. Consequently, the daily consumption by humans is estimated in 5.4 mg day, with an input in the environment of 4.2 mg day per person, receiving or not appropriated treatment before disposure. The cytotoxicity, genotoxicity and mutagenicity of TiO2-NP were investigated using the established fish cell line derived from zebrafish (Danio rerio) liver (i. e. ZF-L cells). Prior to the evaluation of nanoparticle’s toxic potential, a careful characterization was realized in culture medium in the presence or not of fetal bovine serum (FBS). Regarding to the characterization in terms of size was accessed using a transmission electron microscope (TEM), the agglomeration potential and surface charge were accessed by diameter light scattering (DLS) and zeta potential measurements, respectively, using a spectrophotometer. TiO2-NP in environmentally relevant concentrations were tested for cytotoxicity, genotoxicity and mutagenicity. Cell viability was accessed by four different tests, the trypan blue assay (membrane integrity), MTT reduction assay (mitochondria), neutral red retention assay (lysosomes) and finally, induction of apoptosis and necrosis. Genotoxicity was determined by observing the fragmentation of DNA by the comet assay, while mutagenicity was determined by Cytokinesis-block micronucleus technique. The characterization showed that the FBS was effective in dispersing the nanoparticles and prevent the formation of large agglomerates allowing robust responses on the real toxicity of NP. After 24 hours of treatment, there was cell membranes rupture, decreasing cell viability to 35.33%, at the highest concentration (1.0 μg mL-1). Mitochondrial metabolic activity remained unchanged, but it was possible to detect the proliferation of lysosomes, which was mainly attributed to the NP endocytosis. The induction of apoptosis was 50.4%, and necrosis was 13.9%, both in the concentration 1.0 μg mL-1 TiO2-NP. In the case of necrosis, a result 10 times greater than that presented by the negative control. Added necrosis and apoptosis indicated a decrease in cell viability to 35.7%. The comet test showed the fragmentation of the DNA, it was also possible to observe the formation of micronuclei, bridges and shoots demonstrated by the micronucleus assay. In general, this study demonstrated that TiO2-NP, after 24 hours of exposure, significantly affect cell viability and cause DNA damage, which may become irreversible. In conclusion, this study showed the cytotoxic, genotoxic and mutagenic potential of TiO2-NP for ZF-L cells. Mitochondrial and lysosome responses require further studies on the effect of TiO2-NP on these organelles.
Nanopartículas de dióxido de titânio (NP-TiO2) são comumente usadas em muitos produtos industriais. Por conseguinte, seu consumo diário por seres humanos é estimado em 5,4 mg dia-1, o que acarreta em um aporte no ambiente de 4,2 mg dia-1 por pessoa, podendo ou não receber um tratamento apropriado antes do seu despejo. Foram avaliadas a citotoxicidade, genotoxicidade e mutagenicidade de NPTiO2 para a linhagem permanente derivada de fígado de peixe-zebra (Danio rerio) (células ZF-L). Antes da avaliação do potencial tóxico das nanopartículas, foi realizada uma caracterização cuidadosa em meio de cultura com e sem a adição de soro fetal de bovino (SFB). A caracterização em termos do tamanho físico da nanopartícula (NP) foi realizada utilizando um microscópio eletrônico de transmissão (TEM); os tamanhos hidrodinâmicos dos aglomerados e as cargas de superfície foram acessados por medidas de DLS (diameter light scattering) e potencial zeta, respectivamente utilizando um espectrofotômetro. A viabilidade celular foi avaliada por três ensaios diferentes, o ensaio de exclusão do corante azul de tripano (integridade de membrana), ensaio de redução do sal MTT (atividade metabólica mitocondrial) e ensaio de retenção do corante vermelho neutro (viabilidade dos lisossomos). A indução de apoptose e necrose foi avaliada por citometria de fluxo. A genotoxicidade foi determinada pela observação da fragmentação do DNA por meio do ensaio do cometa, enquanto a mutagenicidade foi determinada pelo ensaio de micronúcleos com bloqueio da citocinese. A análise DLS mostrou que o SFB foi eficaz quanto à dispersão das nanopartículas e preveniu a formação de grandes aglomerados, o que permitiu a obtenção de respostas mais robustas relativas à toxicidade real das nanopartículas. Após 24 horas de tratamento, houve ruptura das membranas celulares diminuindo a viabilidade celular a 35,33%, na concentração mais elevada (1,0 μg mL-1). A atividade metabólica mitocondrial manteve-se inalterada, mas foi possível detectar a proliferação dos lisossomos, que foi atribuída principalmente à endocitose das NP. A indução de apoptose foi de 50,4%, e de necrose de 13,9%, ambos na concentração 1,0 μg mL-1 NP-TiO2. No caso da necrose, um resultado 10 vezes maior do que o apresentado pelo controle negativo. Necrose e apoptose somadas indicaram a diminuição da viabilidade celular a 35,7%. O teste do cometa mostrou a fragmentação do DNA, também foi possível observar a formação de micronúcleos, pontes e brotos demonstrados pelo ensaio do micronúcleo. Em geral, este estudo demonstrou que as NP-TiO2, após 24 horas de exposição, afetam significativamente a viabilidade celular e causam danos ao DNA, que podem se tornar irreversíveis. Em conclusão, este estudo mostrou o potencial citotóxico, genotóxicos e mutagênico das NP-TiO2 para células ZF-L. Respostas mitocondriais e dos lisossomos requerem novos estudos quanto ao efeito das NP-TiO2 sobre essas organelas.

Orientador: Maria Aparecida Marin-Morales
Banca: Maria Luiza Silveira Mello
Banca: Cláudia Bueno dos Reis Martinez
Banca: Maria Angélica Maciel Martinho Ferreira
Banca: Edson Luis Maístro
Resumo: Os azocorantes são substâncias químicas extremamente utilizadas em indústrias têxteis que podem induzir mudanças no material genético de organismos expostos, mesmo que essas alterações no DNA não se expressem de imediato. Foram avaliadas as citotoxicidade, genotoxicidade e mutagenicidade de diferentes concentrações (1, 10, 100 e 1000 mg/L - na ausência dos microorganismos - e 50 e 200 mg/L - na presença dos microorganismos) do azocorante Black Dye Commercial Product (BDCP), antes e após tratamentos de biodegradação por diversos microrganismos (1. "Pool" de bactérias heterotróficas provenientes de uma estação de tratamento biológico de efluentes, 2. Levedura Candida viswanathii, e 3. Fungo Basidiomiceto Phanerochaete chrysosporium), por meio de diferentes técnicas citogenéticas (coloração convencional, bandamento C, bandamento RON, bandamento por fluorocromos base-específicos CMA/DAPI e hibridação in situ fluorescente - FISH) aplicadas sobre o organismo teste Allium cepa. Pela técnica citogenética de coloração convencional, foi possível verificar que o corante, com e sem ação microbiana, induziu apoptose, necrose, células micronucleadas, aberrações cromossômicas e alterações nucleares. As aberrações cromossômicas e alterações nucleares foram visualizadas em todos os estágios do ciclo celular: na intérfase foram observados brotos nucleares e células poliplóides; na prófase, perdas de material genético; na metáfase, aderências cromossômicas, perdas cromossômicas, C-metáfases e poliploidias; na anáfase e telófase, multipolaridades, pontes e perdas cromossômicas. Os brotos nucleares apareceram com maior freqüência nas células submetidas aos testes do azocorante tratado com microorganismos, sendo que esse tipo de alteração deve estar associado à presença dos metabólitos do corante. As freqüências de micronúcleos... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Azo dyes are chemical substances extremely used by textile industries that may induce changes in the genetic material of exposed organisms, even if these changes in the DNA do not express themselves immediately. Cytotoxicity, genotoxicity and mutagenicity evaluations of the different azo dye (BDCP) concentrations were performed (1, 10, 100 e 1000 mg/L - without microorganisms - and 50 and 200 mg/L - with microorganisms), before and after biodegradation tests, using different microorganisms (1. Heterotrofic bacteria "pool" proceeding from an effluent biological treatment station, 2. Candida viswanathii - Yeast, and 3. Phanerochaete chrysosporium - Basidiomicet Fungi), by means the different cytogenetical assays (conventional staining, C-banding, RON-banding, CMA/DAPI banding and fluorescent in situ fluorescent hybridization), using Allium cepa as test organism. By the conventional staining cytogenetic assay, it was possible to verify that the azo dye induced apoptosis, necrosis, micronuclei, chromosome aberrations and nuclear alterations. The chromosome aberrations and nuclear alterations were visualized in all phases of the cell cycle: in the interphase were observed nuclear buds and polyploidizated cells; in the prophase were observed genetic material losses; in the metaphase were observed chromosome adherences, chromosome losses, C-metaphases and polyploid cells; and in the anaphase and telophase were observed multipolar cells, chromosome bridges and chromosome losses. The frequencies of nuclear buds were the higher when the cells had been submitted to the azodye treated with microorganisms, suggesting that this kind of alteration must be associated to the presence of the azodye metabolites. The micronuclei and chromosome breaks frequencies... (Complete abstract click electronic access below)
Doutor

Made available in DSpace on 2014-06-11T19:30:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-12-17Bitstream added on 2014-06-13T20:01:10Z : No. of bitstreams: 1 ventura_bc_dr_rcla.pdf: 1747414 bytes, checksum: d6fd71edeabed9a63deeb67289ab785e (MD5)
Os azocorantes são substâncias químicas extremamente utilizadas em indústrias têxteis que podem induzir mudanças no material genético de organismos expostos, mesmo que essas alterações no DNA não se expressem de imediato. Foram avaliadas as citotoxicidade, genotoxicidade e mutagenicidade de diferentes concentrações (1, 10, 100 e 1000 mg/L – na ausência dos microorganismos – e 50 e 200 mg/L – na presença dos microorganismos) do azocorante Black Dye Commercial Product (BDCP), antes e após tratamentos de biodegradação por diversos microrganismos (1. “Pool” de bactérias heterotróficas provenientes de uma estação de tratamento biológico de efluentes, 2. Levedura Candida viswanathii, e 3. Fungo Basidiomiceto Phanerochaete chrysosporium), por meio de diferentes técnicas citogenéticas (coloração convencional, bandamento C, bandamento RON, bandamento por fluorocromos base-específicos CMA/DAPI e hibridação in situ fluorescente – FISH) aplicadas sobre o organismo teste Allium cepa. Pela técnica citogenética de coloração convencional, foi possível verificar que o corante, com e sem ação microbiana, induziu apoptose, necrose, células micronucleadas, aberrações cromossômicas e alterações nucleares. As aberrações cromossômicas e alterações nucleares foram visualizadas em todos os estágios do ciclo celular: na intérfase foram observados brotos nucleares e células poliplóides; na prófase, perdas de material genético; na metáfase, aderências cromossômicas, perdas cromossômicas, C-metáfases e poliploidias; na anáfase e telófase, multipolaridades, pontes e perdas cromossômicas. Os brotos nucleares apareceram com maior freqüência nas células submetidas aos testes do azocorante tratado com microorganismos, sendo que esse tipo de alteração deve estar associado à presença dos metabólitos do corante. As freqüências de micronúcleos...
Azo dyes are chemical substances extremely used by textile industries that may induce changes in the genetic material of exposed organisms, even if these changes in the DNA do not express themselves immediately. Cytotoxicity, genotoxicity and mutagenicity evaluations of the different azo dye (BDCP) concentrations were performed (1, 10, 100 e 1000 mg/L – without microorganisms – and 50 and 200 mg/L – with microorganisms), before and after biodegradation tests, using different microorganisms (1. Heterotrofic bacteria ”pool” proceeding from an effluent biological treatment station, 2. Candida viswanathii - Yeast, and 3. Phanerochaete chrysosporium - Basidiomicet Fungi), by means the different cytogenetical assays (conventional staining, C-banding, RON-banding, CMA/DAPI banding and fluorescent in situ fluorescent hybridization), using Allium cepa as test organism. By the conventional staining cytogenetic assay, it was possible to verify that the azo dye induced apoptosis, necrosis, micronuclei, chromosome aberrations and nuclear alterations. The chromosome aberrations and nuclear alterations were visualized in all phases of the cell cycle: in the interphase were observed nuclear buds and polyploidizated cells; in the prophase were observed genetic material losses; in the metaphase were observed chromosome adherences, chromosome losses, C-metaphases and polyploid cells; and in the anaphase and telophase were observed multipolar cells, chromosome bridges and chromosome losses. The frequencies of nuclear buds were the higher when the cells had been submitted to the azodye treated with microorganisms, suggesting that this kind of alteration must be associated to the presence of the azodye metabolites. The micronuclei and chromosome breaks frequencies... (Complete abstract click electronic access below)

Made available in DSpace on 2016-05-02T13:54:44Z (GMT). No. of bitstreams: 1 Dissertacao completa Patricia Scotini Freitas.pdf: 380463 bytes, checksum: 593d35f67c31f8a091c16952ef946b32 (MD5) Previous issue date: 2007-04-27
Coordenacao de Aperfeicoamento de Pessoal de Nïvel Superior
Blueberry Vaccinium corymbosum is a vegetable very rich in anthocyanins which have strong antioxidant capacity and other potential health benefits and because of that is widely consumed in the world as medicinal plant In this work the mutagenic potential of the crude extract from this plant was studied in mice after acute treatment using the comet and micronucleus assay Animals were treated orally with three different concentrations of the extract (1000 1500 and 2000 mg/kg) Peripheral blood cells of Swiss mice were collected 4 and 24 hours after the treatment for the comet assay and 48 and 72 hours for the micronucleus test The results have shown that the extract of Vaccinium corymbosum did not induce statistically significant increases in the average number of damages to desoxyribonucleic acid in peripheral blood cells However a significant increase in the mean of the micronucleated polychromatic erythrocytes was observed at three tested doses It is suggested that its consumption could be moderate until a definitive risk for humans is established
Vaccinium corymbosum é um vegetal muito rico em antocianinas que tem uma grande capacidade antioxidante e outros potenciais benefícios à saúde e por este motivo é altamente utilizado no mundo inteiro como planta medicinal Neste trabalho foi analisado o potencial mutagênico da administração aguda do extrato bruto de frutos desta planta em células de camundongos utilizando o ensaio cometa e o teste do micronúcleo Os animais foram tratados oralmente com três diferentes concentrações do extrato (1000, 1500 e 2000 mg/kg de peso corporal) Células do sangue periférico de camundongos foram coletados 4 e 24 horas após o tratamento para a realização do ensaio cometa e 48 e 72 horas para o teste do micronúcleo Os resultados mostraram que o extrato de Vaccinium corymbosum não induziu aumentos estatisticamente significativos de danos ao ácido desoxirribonucléico nas células de sangue periférico Entretanto o teste do micronúcleo evidenciou um aumento significativo na média de eritrócitos policromáticos micronucleados nas três concentrações testadas Sugere-se que o consumo deste extrato seja moderado até que seu risco definitivo para os seres humanos seja melhor estabelecido

A emissão de uma grande diversidade de substâncias através dos efluentes industriais gasosos, líquidos ou sólidos acarreta que uma gama de compostos seja introduzida no ambiente atmosférico e aquático de meios urbanos. A exposição a substâncias genotóxicas no ambiente oferece freqüentemente um grande risco à saúde humana. Com essa preocupação, o presente trabalho avaliou a exposição das pessoas à qualidade do ar atmosférico e à da água para abastecimento público de duas cidades urbanas do Rio Grande do Sul (Brasil), que recebem diferentes contribuições antrópicas (cidades-alvo), comparando-as com uma cidade menos impactada - cidade referência. Os extratos orgânicos da água tratada para abastecimento público e das amostras de ar foram testados quanto a mutagenicidade através do ensaio Salmonella/microssoma (linhagens TA98 e TA100 para água e ar, com e sem fração de metabolização e, ainda para ar, YG1021 e YG1024) - marcador de mutagenicidade. As amostras de ar ainda foram analisadas por HPLC para a caracterização de HPAs e algumas amostras também por HRGC-HRMS para a composição dos PCDD/Fs. Para o biomonitoramento humano foram avaliados através do ensaio do cometa e do micronúcleo (MN), em linfócitos e células da mucosa bucal, respectivamente, as pessoas que viviam nas três cidades - biomarcador de efeito. Análises de polimorfismos genéticos (CYP1A1, GSTM1 e GSTT1) foram utilizados como biomarcadores de susceptibilidade. As duas cidades-alvo apresentaram resultados significativos quanto a mutagenicidade da água de abastecimento apresentando todos os resultados positivos, principalmente para mutação de substituição de pares de bases (TA100), indicando a presença de compostos como THMs e MX. Valores de PTS atmosféricos apresentaram-se em algumas amostragens acima dos limites da legislação brasileira (CONAMA, 1990). Os HPAs encontrados em maior quantidade foram indeno(1,2,3-c,d)pireno e benzo(ghi)perileno, sendo que em uma das amostras a análise dos PCDD/Fs, mostrou valores que superaram em dez vezes a outra amostra analisada. As duas cidades alvo apresentaram altas respostas mutagênicas nas amostras de ar tanto por mutágenos de ação direta, como indireta e também altas concentrações de nitro-HPAs. Estes resultados sinalizaram para a necessidade de uma revisão da legislação para material particulado atmosférico, já que dentro dos atuais parâmetros, grande quantidade de compostos mutagênicos foram encontrados. Para os indivíduos estudados quanto aos biomarcadores de efeito, tanto os linfócitos analisados pelo ensaio do cometa quanto às células da mucosa bucal pelo ensaio do MN não apresentaram resultados significativos, sendo que apenas uma cidade alvo apresentou quanto ao ensaio do cometa. Como a maioria dos indivíduos CYP1A1, GSTM1 e GSTT1 apresentaram genótipos prevalentes, pode estar ocorrendo uma eficiência no sistema de detoxificação garantidos pela presença desses genótipos. Este trabalho mostrou a importância de estudos ambientais que relacionem marcadores de mutagenicidade com biomarcadores de efeito e de susceptibilidade, para que haja uma maior compreensão dos efeitos da exposição humana diária à agentes genotóxicos ambientais.
The emission of a great substances amount through gaseous, liquid and solid industrial effluents have been introduced many kinds of chemical compounds in aquatic and atmospheric environments. The environmental exposure to genotoxic substances often brings great risk to human health. Therefore, the aim of this work was make an avaliation of people exposition to atmospheric air and water supply quality in two urban cities in Rio Grande do Sul State (Brazil) which were under influence of different anthropogenic sources (target cities), compared to a less intense influenced urban city (reference city). The organic extracts from supply water and airborne samples were tested to mutagenicity using the Salmonella/microsome assay (TA98 and TA100 strains for water and airborne samples, with and without metabolization fraction, and also, YG1021 and YG1024 strains for airborne) - mutagenicity marker. Moreover, the airborne samples were analysed by HPLC to PAHs composition and by HRGC-HRMS to PCDD/Fs composition. Human biomonitoring was evaluated by samples of lymphocytes and bucal mucosa cells from individuals living in this three cities using the comet and the micronuclei (MN) assays, respectively - effect biomarkers. Analysis of gene polymorphisms (CYP1A1, GSTM1 and GSTT1) was taken as susceptibility biomarkers. Both target cities showed significative results for the water mutagenicity with mutagenic responses for all samples, mainly to base-pair substitution mutation (TA100 strain), denoting the presence of THMs and MX compounds. The TSP concentrations were above the Brazilian Legislation and World Health Organization regulation limits few times. The PAHs detected in greater amout were indeno(1,2,3-c,d)pyrene and benzo(ghi)perylene and one of the samples overcame ten times the values of PCDD/Fs compared to the other analysed. The two target cities showed high mutagenicity response in all airborne samples, both by direct and indirect mutagens, besides high concentrations of nitro-PHAs. Actually, these results indicate a law revision necessity, because great amount of mutagenic compounds were assessed in smaller concentration that those present in current legal parameters. The analysed subjects for effect biomarkers, both limphocytes tested by comet assay and buccal mucosa cells tested by MN, did not show significant results, only one city show increased primary DNA damage detected through comet assay. As the majority of subjects CYP1A1, GSTM1 e GSTT1 showed prevalent genotype, it maybe occuring an efficient system detoxification warranted by these genotypes presence. This work has shown the importance of environmental studies that relate mutagenicity markers with effect and susceptibility biomarkers, thus a greater understanding about environmental genotoxic agents, which people are exposed daily, will be done carefully.

Aescin is a mixture of several acidic triterpenoid saponin glycosides found in the extracts of the horse chestnut tree. Horse chestnut, Aesculus Hipoocastanum, is one of the 25 domestic species of Aesculus that are mostly large, ornamental shade trees. Although known to be poisonous, the nuts of the horse chestnut are used by Amerindians, after detoxification. Horse chestnuts are said to have several traditional medicinal usages including even cancer. In this study the biochemical, genotoxic, and cytotoxic effects of aescin was studied using isolated lymphocytes, whole blood lymphocytes and HL-60 promyeloid leukemia cell lines. Cytotoxicity of aescin was examined by trypan blue viability staining of the cells in culture treated with varying aescin concentrations. It was observed that aescin was cytotoxic at all concentrations, for all cell types studied, except whole blood lymphocytes, where it was not cytotoxic at 10-9 and 10-10 M concentrations. Genotoxicity of aescin was examined by sister chromatid exchange and micronucleus. The genotoxic effect of Aescin was observed to be more significant over isolated lymphocytes compared to other cell lines. On the otherhand, aescin at 10-8 M and lower concentrations were observed to be non-genotoxic over whole blood lymphocytes whereas this concentration was considerably toxic for isolated lymphocytes and for HL-60 cell lines. Apoptotic properties of aescin were determined by DNA fragmentation, cytochrome c release and negative NAPO staining. All the Aescin concentrations tested resulted in apoptosis over HL-60 cell lines, whereas necrosis was not observed. However, isolated lymphocytes showed both apoptosis and necrosis upon treatment with 10-6 M to 10-8 M aescin, exhibiting apoptosis only at 10-9 M and 10-10 M. Biochemical effects of aescin were investigated by following GST and NAT enzyme activities. An increase in GST enzyme activity was observed over all cell lines treated with increasing aescin concentrations for 72 hours. Whereas NAT activity was decreased upon treatment with aescin in similar manner.

O presente estudo foi desenvolvido para verificar se o bioensaio Trad-MCN, desenvolvido com inflorescências de Tradescantia pallida cv. Purpurea pode discriminar riscos clastogênicos em diferentes locais e épocas na cidade de Santo André-SP, contaminada por diferentes tipos de poluentes, determinar se as variações da freqüência de micronúcleos podem ser explicadas por fatores ambientais característicos da região e verificar se o potencial acumulador de elementos químicos de T. pallida pode ser usado para mapeamento de fontes emissoras de poluentes contendo metais e outros compostos tóxicos. Vasos com a planta foram expostos em locais com alta contaminação por ozônio (Capuava e Escola), em locais com maior emissão veicular (Centro e Parque Celso Daniel) e em uma área supostamente pouco contaminada (Parque do Pedroso). Durante o período de setembro de 2003 a setembro de 2004, vinte inflorescências jovens foram colhidas quinzenalmente e a freqüência de micronúcleos (MCN) foi estimada. Durante o período de maio a junho de 2004, folhas em diferentes posições nas inflorescências da planta foram colhidas para determinação da concentração de elementos químicos, entre os quais metais pesados, pelo método da ativação de nêutrons. As condições ambientais observadas foram suficientemente estressantes para promover o aumento da freqüência de micronúcleos. O bioensaio Trad-MCN identificou forte risco clastogênico em áreas com maior emissão veicular. Entretanto, a freqüência de micronúcleos em Capuava e no Centro não foram preditas somente por poluentes atmosféricos da região. Condições climáticas extremas, como temperaturas mínima e máxima, baixa umidade relativa do ar e baixa precipitação contribuíram para a intensificação da formação de MCN. Para um sistema eficiente de biomonitoramento é recomendável minimizar os efeitos dos fatores climáticos. A análise por ativação de nêutrons identificou um evidente acúmulo foliar de elementos importantes para biomonitoramento da poluição aérea, tais como: Ba, Ce, Co, Cr, Cs, La, Rb, Sb, Sc e Zn. Verificou-se que a concentração dos metais nas folhas inseridas nas inflorescências não teve relação com a formação de micronúcleos, porém, eles foram marcadores de locais específicos, auxiliando no mapeamento das fontes poluidoras de cada região estudada. A concentração de Ba foi mais elevada nas folhas provenientes das áreas centrais, podendo ser considerado marcador de emissão veicular e La e Zn destacaram-se na área industrial da cidade, sendo considerados marcadores da emissão do pólo petroquímico. Considerando as condições em que foi desenvolvido o presente estudo, a análise das concentrações foliares de elementos tóxicos, foi mais adequada para mapear fontes de emissão de poluentes na atmosfera de Santo André.
The present study aimed at verifying if the Trad-MCN assay, developed with inflorescences of Tradescantia pallida cv. Purpurea might discriminate clastogenic risks among sites and periods at the Santo André city, contaminated by different air pollutants, determining if the variations in the frequency of micronuclei can be explained by environmental factors that characterize the stress situation in each site and verify if the accumulator potential of chemical elements of T. pallida may be used to indicate pollutant sources containing metals or other toxic compounds. Potted plants were exposed in sites characterized by high air contamination by ozone (Capuava and School) and in sites reached by high vehicular emissions (Downtown and C. Daniel Park). From September/2003 to September/2004, twenty young inflorescences were be-weekly collected from each site and the frequencies of micronuclei (MCN) were estimated. From May/2004 to June/2004, leaves of T. pallida inserted in different positions on the inflorecences were collected to determine the concentrations of chemical elements, by instrumental neutron activation analysis, among them heavy metals. The environmental conditions were stressing enough to promote an increase of chromosomal breakages in pollen mother cells of inflorescences of T. Pallida. The Trad-MCN assay identified strong clastogenic risks at the sites reached by vehicular emissions. However, the frequency of micronuclei at Capuava and Downtown could not be only predicted by pollutants that characterized the air contamination in both sites. More extreme climatic conditions, mainly low and high temperatures, low relative humidity and low rainfall, intensified the formation of MCN. Therefore, the biomonitoring system should be improved in order to minimize this negative influence of climatic factors. The neutron activation analysis identified an evident leaf accumulation of important elements for biomonitoring air pollution, such as: Ba, Ce, Co, Cr, Cs, La, Rb, Sb, Sc and Zn. There was no relation between the concentrations of metals in the leaves inserted in the inflorescences and the frequency of micronuclei, however, the metal accumulation could discriminate specific sites, contributing to the mapping of polluted sources in each region studied. Leaf concentrations of Ba were higher in central areas, so that they can be considered markers of vehicular emissions. La and Zn were evidently accumulated in leaves from the industrial areas of the city, being considered indicators of emissions from the petrochemical pole. Taking in account the conditions in which the present study was developed, the analysis of leaf concentrations leaves of toxic elements was more adequate to map emission sources of air pollutants in Santo André.

DA CASTANHA DE CAJU Apesar da importância social e econômica do beneficiamento da castanha de caju para o Rio Grande do Norte, a produção ainda é realizada de forma artesanal. Para a coleta da amêndoa da castanha é necessário assá-la. A fumaça gerada durante a queima da castanha possui altas concentrações de Material Particulado (MP) e esse MP produzido é inalado diariamente por grupos familiares por um período que pode exceder a 10 horas diárias. Em geral, os poluentes atmosféricos oriundos da queima de biomassa são potencialmente nocivos a saúde, relacionando-se com eventos de genotoxicidade, aumento no número de internações hospitalares e ambulatoriais, e mortalidade por doenças cardiovasculares e respiratórias. O presente trabalho teve três objetivos principais: 1° Realizar medições na concentração de O3, NO2, MP, Black Carbon (BC) e composição elementar do MP 2,5 provenientes da queima da castanha. 2° Identificar o potencial mutagênico associado ao beneficiamento artesanal da castanha de caju durante a estação seca, chuvosa e intermediária no RN (Brasil) com o auxílio do teste de micronúcleo (MN) em Tradescantia pallida. 3° Verificar o efeito mutagênico da atividade em MN de células esfoliadas de mucosa oral de trabalhadores envolvidos no beneficiamento. Para isso, três locais distintos foram definidos como pontos testes: Ponto 1. Comunidade do Amarelão, situada no perímetro rural do município de João Câmara-RN (local onde ocorre a queima da castanha de caju); Ponto 2. Fazenda Santa Luzia, situada próxima à região de queima da castanha de caju (local com as mesma condições ambientais do Ponto 1, porém sem a influência da atividade); Ponto 3. Universidade Federal do Rio Grande do Norte (UFRN), zona urbana de Natal-RN. Os resultados obtidos para o O3 e NO2 não excederam os limites estabelecidos pela legislação brasileira. No entanto, os resultados da medição de MP obtidos com o medidor portátil \"DUSTTRAK (TM)Aerossol Monitor \" indicou que entre as 8 medições de MP realizadas no Ponto 1, 7 excederam o nível de exposição definido como \"estado de emergência \"descrito na legislação brasileira (500µg/m 3 ), diferindo significativamente dos resultados obtidos nos Pontos 2 e 3 (6µg/m 3 ). As avaliações realizadas nos meses de Janeiro, Maio e Setembro de 2009, com o Mini-sampler confirmaram os resultados previamente obtidos com o \"DUSTTRAK (TM)Aerossol Monitor \".O valor médio de MP 2,5 (Jan - 548,412 µg/m 3 ; Mai - 1022,232 µg/m 3 ; Set - 1291,946 µg/m 3 ) e BC (Jan - 46,798 µg/m 3 ; Mai 70,068- µg/m 3 ; Set - 69,432 µg/m 3 ) obtido nas três campanhas para o Ponto 1 foram significativamente maiores que o Ponto 2 e 3. Para o Ponto 1 os elementos Si, S, Cl, K, Ni, Cu e Zn quando presentes estiveram em concentrações superiores aos Pontos 2 e 3. Os testes de genotoxicidade com T. pallida indicou aumento significativo no número de MN em todas as campanhas. Os resultados com células de mucosa oral humana corroboraram com o biomonitor vegetal, sendo verificado aumento significativo na freqüência de MN. Os resultados obtidos caracterizaram um dos piores níveis de exposição humana ao MP já relatado na literatura, excedendo amplamente os limites da legislação brasileira assim como os da OMS. Os resultados obtidos apresentaram um problema ocupacional grave, sendo necessária intervenção imediata dos gestores públicos na tentativa de minimizar os efeitos lesivos da atividade.
x Despite the social and economic importance of the processing of cashew nuts to Rio Grande do Norte, the production is still carried out artisanally. To collect the almond nut is necessary to roasting. The smoke generated during the burning of the nut has high concentrations of particulate matter (PM) and the PM produced is inhaled daily by families for a period which can exceed 10 hours a day. In general, air pollutants come from burning biomass are potentially harmful to health, relating to genotoxicity events, increase in the number of hospitalizations and outpatient, and mortality from cardiovascular and respiratory diseases. This project had three main objectives: 1 Make measurements in the concentration of O3, NO2, MP, Black Carbon (BC) and elemental composition of the PM 2.5 from the combustion of the cashew nut. 2 Identify the mutagenic potential associated with artisanally processing of cashew nut during the dry season, wet and intermediate in RN (Brazil) by using a micronucleus (MN) bioassay of T. pallida tetrads. 3 To investigate the effect of mutagenic activity in exfoliated cells of oral mucosa of workers involved in processing. For this, three test sites were chosen for this purpose: Site 1. the Amarelão community - where the roasting occurs, Site 2. the Santa Luzia farm - an area near the roasting site, though without direct influence on the process and Site 3. the Universidade Federal do Rio Grande do Norte (UFRN) - an urban area of Natal, Brazil. The results obtained for the O3 and NO2 do not exceed the limits established by Brazilian legislation. However, the results of the measurement of PM obtained with the portable meter \"DUSTTRAK (TM) Aerosol Monitor\" has indicated that between 8 PM measurements made at Site 1, 7 exceeded the level of exposure defined as \"emergency rule\" described in the Brazilian legislation (500 ? g/m 3 ), differing significantly from the results obtained in Sites 2 and 3 (6 ? g/m 3 ). Evaluations in January, May and September 2009, with the Mini-sampler confirmed the results previously obtained with the \"DUSTTRAK (TM)Aerosol Monitor.The average value of 2.5 MP (Jan - 548.412 mg/m 3 ; May - 1022.232 mg/m 3 ; Set - 1291.946 mg/m 3 ) and BC (Jan - 46.798 mg/m 3 ; May 70.068 - mg/m 3 ; Set - 69.432 mg/m 3 ) obtained in the three campaigns for Site 1 were significantly higher than the Site 2 and 3. To the Site 1 the elements Si, S, Cl, K, Ni, Cu and Zn when present were at concentrations higher than the Sites 2 and 3. Genotoxicity tests with T. pallida showed a significant increase in the number of MN in all campaigns. The results with cells of human oral mucosa have confirmed the biomonitoring, and found significant increase in the frequency of MN. The results marked one of the worst levels of human exposure to PM has been reported in the literature and greatly exceeded the limits of the Brazilian legislation as well as the WHO. The results showed a serious occupational problem, requiring immediate intervention of public officials in an attempt to minimize the harmful effects of the activity.

Made available in DSpace on 2014-12-17T15:55:00Z (GMT). No. of bitstreams: 1 AlineRPC_DISSERT.pdf: 3018551 bytes, checksum: 17363a397c49ea615dc8b9fbdbe31f5c (MD5) Previous issue date: 2012-05-31
Environmental pollution causes the loss of the quality of aquatic resources and also affects the health of human beings. The Golandim River is located in S?o Gon?alo do Amarante (RN Brazil) and had its water recovered seven years ago by measurements of parameters of the water s quality analyses physicochemistry, microbiological and heavy metals. However, it is not well established if this river is truly recovered, so this study provides a wide knowledge about the quality of these waters. Therefore, this investigation was accomplished by assays of ecotoxicology utilizing Ceriodaphnia dubia and of genotoxicity of the river s water using a biomarker Tradescantia Pallida (Trad-MCN). In set, it carried through a study of environmental perception through questionnaires that approached questions related to the profile of the interviewed one, knowledge on the environment and of the river Golandim with the community that lives in the neighborhoods of the river to diagnosis as they perceive the environment where live and its problems. The assays of the water had been carried out by collecting samples in three different sites of the Golandim River. They were collected between the periods of December 2010 (dry season in the northeast of Brazil) and July 2011 (rainy season in the northeast of). The analysis of the data allowed observing that the majority of the inhabitants are adult and presents a global vision of what it is part of the environment, the majority mentions the pollution of the river as one of the problems of the city, considering it serious. The ecotoxicology assay showed that there was not acute toxicity in all three samples collected. Meanwhile, all these three samples demonstrated significant chronic toxicity. The results from the Trad-MCN assay presented an increase in the frequency of micronucleus in one of the sites analyzed (S3) (p<0.01), in both seasons collected. On the other hand, the sites S1 and S2 did not presented a significant increase of micronucleus using this bioassay. The analyses of chemicals detected an increase in the levels of some metals, in different seasons and samples, which can be associates with some compounds found in urban and industrial areas. On the other hand, the physicochemistries parameters demonstrated that the Golandim River is recouped, when compared with the values presented at the CONAMA s legislation. However, these results indicate the presence of compounds capable of inducing chromosomal mutation in plants. On the other hand, the parameters physicistchemistries demonstrate that the river Golandim is if recouping, since when compares the values observed with the legislation of the CONAMA. All these results point to the fact that the Trad-MCN assay was sensitive and efficient biomarker for chromosomal instability and the C. dubia ecotoxicology assay was as though an efficient biomarker of toxicity of water s quality. The results from Trad-MCN associated with the ecotoxicology demonstrates that these analyses are important for environmental monitoring, once the first bioassay described above indicates alterations at the standards of cells and the other one indicates alterations at the standards of organisms. This study alerts for the necessity to carry out biological assays for the analyses of the water s quality
A ?gua ? essencial para a manuten??o da vida, entretanto, a polui??o ambiental vem causando a perda da qualidade dos recursos h?dricos, afetando assim a sa?de humana. Este estudo avaliou a qualidade da ?gua do Rio Golandim no munic?pio de S?o Gon?alo do Amarante/RN/Brasil ap?s a realiza??o do plano de recupera??o atrav?s de medi??es de par?metros de qualidade de ?gua - an?lises f?sico-qu?micas, de metais pesados e microbiol?gicos al?m de incluir ensaio de ecotoxicidade com Daphnia dubia e teste que detecta a presen?a de compostos mutag?nicos em Tradescantia pallida (Trad-MCN). Em conjunto, foi realizado um estudo de percep??o ambiental atrav?s de question?rios que abordou quest?es relacionadas ao perfil do entrevistado, conhecimentos sobre o meio ambiente e do rio Golandim, com a comunidade que vive nas proximidades deste rio para diagnosticar como eles percebem o ambiente em que vivem e seus problemas. Os experimentos foram realizados a partir de amostras de ?gua coletadas em dezembro de 2010 (esta??o do ano seca) e em julho de 2011 (esta??o das chuvas) em tr?s pontos diferentes do rio Golandim. A an?lise dos dados de percep??o ambiental permitiu observar que a maioria dos moradores s?o adultos e apresentam uma vis?o global do que ? meio ambiente e a maioria menciona a polui??o do rio como um dos problemas do munic?pio, considerando-o grave. Os dados resultantes do ensaio ecotoxicol?gico n?o indicou efeito de toxicidade aguda, entretanto demonstrou toxicidade cr?nica para a m?dia de reprodu??o dos clad?ceros nas esta??es do ano seca e chuvosa, em todos os pontos amostrados, os resultados provenientes do ensaio Trad-MCN para a ?gua bruta mostrou resposta positiva, representada pelo aumento na frequ?ncia de micron?cleos, para um dos pontos analisados (P3) em ambas as esta??es de coleta. As an?lises qu?micas detectaram um aumento nos n?veis de alguns metais nos diferentes per?odos e amostras, que podem estar associados ainda ? presen?a de compostos de origem urbana e industrial. Contudo, os par?metros f?sico-qu?micos demonstram que o rio Golandim est? se recuperando, j? que quando se compara os valores observados com a legisla??o brasileira do CONAMA. Nossos resultados apontam para a presen?a na ?gua de compostos capazes de induzir muta??es, podendo gerar s?rios agraves ? sa?de da popula??o que utiliza e reside nas proximidades do Golandim, a qual ainda se sente insegura quanto a utiliza??o do mesmo. Desta forma, este estudo alerta para a necessidade de realizar ensaios biol?gicos al?m de empregar apenas as an?lises provenientes das medi??es f?sico-qu?micas para atestar a qualidade de um corpo d ?gua, como tamb?m da realiza??o de trabalhos de educa??o ambiental com a comunidade ribeirinha

Ionizing radiation (IR) can cause damage to DNA molecules, both through direct ionization and induction of oxidative stress. Exposure to IR may induce single strand breaks (SSB), double strand breaks (DSB) in the genomic DNA as well as trigger oxidative stress (increasing production of reactive free oxygen species, ROS) that ultimately mediates DNA damage. Previous studies have shown that human topoisomerase IB (hTop IB) is proposed to be involved in the SSB and DSB repair as a response to various DNA damages but its role still remains unclear. To study the role of topoisomerases (Top IB) in repair of DNA damage induced by both IR and ROS, we used three model system such as Saccharomyces cerevisiae and human epithelium cell lines (both normal and cancer). In this context, we evaluated the effect of different doses of X-rays and different concentrations of H2O2 on the enzyme. For all cases, it was confirmed that following IR exposure and ROS, Top IB activity is increased as well as the activity of Top IB to bind with genomic DNA is also increased. On the other hand, Top IB has shown no effect on the survival rate of S. cerevisiae after radiation exposure. Whereas, from H2O2 treatment, cancer cell lines showed increased survivability than normal one. Following exposure to IR S. cerevisiae cells bearing Top IB showed a higher initial damage comparing to defective cells, but repair was more efficient and, 20 h from radiation exposure, DNA damage was higher in the defective cells. Whereas, for both normal and cancer cells, number of DNA damage and micronuclei formation shown to be more following IR but cancer cells showed faster repair rate in respect to normal cell. Even for Top IB activity assay normal and cancer cells behaved in a different way that demands for further study for cancer cells. Another fact is that, for both IR and H2O2 stress condition Top IB expression level changes very little but not noticibly, opposite to its activity that drives us to find about post translational modification of this enzyme. Based on all these outcomes, in future, we hope to carry on with different cancer cells and to focus more on posttranslational modifications of this enzyme and also to find out correlation of Top IB with other repair proteins such as tyrosyl DNA phosphodiesterase (TDP1) and poly (ADP-ribose) polymerase 1 (PARP 1) to make our data pharmacologically more significant. We hope that the results of our research could be used to improve the use of camptotecin in radiotherapy or to better understand the early phases of DNA breaks repair and also the role of oxidative stress in reducing repair which ultimately may provide a new insight into the mechanisms underlying the maintenance of genomic integrity and treatment of cancer.

O lodo de esgoto gerado em estações de tratamento de esgoto pode conter substancias tóxicas ainda não regulamentadas nas legislações nacionais e internacionais. Dentre elas as genotóxicas tem recebido especial atenção. Este trabalho teve como objetivos avaliar a genotoxicidade de amostras de lodo de esgoto tratado de diferentes Estações de Tratamento de Esgoto (ETE) do Estado de São Paulo com o teste de micronúcleo em Tradescantia e ampliar os conhecimentos sobre o potencial de utilização da Tradescantia pallida em substituição ao clone 4430. Todas as ETEs estudadas apresentaram pelo menos uma amostra positiva para o teste de micronúcleo em Tradescantia sendo necessários mais estudos para elucidar quais os compostos são responsáveis pelo efeito observado. Os resultados sugerem que a Tradescantia pallida pode substituir o clone 4430 no teste de micronúcleo, porém esse ensaio tem aplicabilidade limitada em programas de monitoramento.
The sludge produced in sewage treatment plants can contain toxic substances which are not yet regulated by national and international legislation. Among these, the genotoxic substances are of great concern. The present paper aimed at evaluating the genotoxicity of treated sludge samples collected in different Sewage Treatment Plants (STP) located in the State of São Paul, Brazil. The micronucleus assay in Tradescantia was the test chosen for this evaluation. Another objective of the study was to verify it Tradescantia pallida could replace the clone 4430 in the Trad-MN assay. All the STPs studied have presented at least one positive sample for the micronucleus assay in the Tradescantia. Further studies are required in order to determine which compounds are responsible for the observed effect. The results obtained suggest that T. pallida can replace clone 4430 in the micronucleus assay, however, this assay presents limited applicability in monitoring programs.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnologico
Made available in DSpace on 2016-01-08T19:35:11Z (GMT). No. of bitstreams: 1 99814.pdf: 1817538 bytes, checksum: c2e26aa7b140f767a75b21a06468f3c1 (MD5) Previous issue date: 1995
Projeto e implementação de um sistema operacional com micronúcleo distribuído para multicomputador, com interface de programação compatível com o sistema UNIX, cujos serviços são suportados por um conjunto de processos servidores. Construção de um simulador do multicomputador com rede de interconexão dinâmica, concebido com ambiente multiprogramado para servir como base de desenvolvimento para o sistema operacional proposto.

Importance and effects of aneuploidy has long been debated. Aneuploidy is now considered sufficiently important to be included in the routine testing of chemicals and radiations. Aneuploidy may arise by at least two mechanisms, chromosome loss and non-disjunction. Over the past few years, the Cytokinesis Blocked Micronucleus (CBMN) technique has evolved into a robust assay for the detection of aneuploidy induction. At present, it is the only assay, which can detect both chromosome loss and non-disjunction reliably when coupled with appropriate molecular probing techniques. The present study aimed at the assessment of genotoxic potential of three major groups of chemicals using CBMN assay, applied in conjunction with kinetochore labelling and Fluorescence in situ Hybridization. In first part, the aneuploidy induction by ethanol and acetaldehyde was studied. The results focussed on the ability of ethanol and acetaldehyde to produce genotoxic effects produced by aneugenic and clastogenic mechanisms respectively. The results from the present study suggest that ethanol is an aneugen itself and the ability of ethanol to produce mutagenic effects depends on the ability of specific cells or tissue to absorb and metabolise ethanol efficiently. In second part, the aneuploidy induction by three groups of hormones was studied using CBMN assay coupled with Fluorescence in situ Hybridization. The results from the present study suggest that 17-β Oestradiol, Diethylstilboesterol, Progesterone and Testosterone are genotoxic and induce aneuploidy by non-disjunctional mechanism. In the third part of this thesis, experiments were carried out to study the toxic consequence of xenobiotic-diacylglycerols, which have been ignored in the past many years. The results from this study, for the first time, have shown that xenobiotic diacylglycerols can induce aneuploidy and this may provide a link between environmental exposure and hyperproliferative diseases in which disruption of cell cycle is a critical factor. At least one xenobiotic-DG (Ibuprofen-DG) was found to induce aneuploidy in vitro in mammalian cultures. Finally, the results from this present study shows the usefulness, reliability and sensitivity of CBMN assay which can now be routinely used for the assessment of genotoxic potential of different factors.

Made available in DSpace on 2014-12-17T14:03:38Z (GMT). No. of bitstreams: 1 MarcosFOG_DISSERT.pdf: 3390000 bytes, checksum: ba6308b2ffb8a1c987acbb7748b9335f (MD5) Previous issue date: 2011-10-14
The Brazil is the third largest producer of cashew nuts in the world. Despite the social and economic importance of the cashew nut, its production is still carried out artisanally. One of the main problems encountered in the cashew production chain are the conditions under which the roasting of the nut occurs to obtain the kernel from the shell. In the present study was conducted a biomonitoring of the genotoxic and cytotoxicity effects associated with the elements from the cashew nut roasting in Jo?o C?mara - RN, semi-arid region of Brazil. To assess the genotoxic was used the bioassay of micronucleus (MN) in Tradescantia pallida. In addition, it was performed a comparative between the Tradescantia pallida and KU-20 and other biomarkers of DNA damage, such as the nucleoplasmic bridges (NBP) and nuclear fragments (NF) were quantified. The levels of particulate matter (PM1.0, PM2.5, PM10) and black carbon (BC) were also measured and the inorganic chemical composition of the PM2.5 collected was determined using X-ray fluorescence spectrometry analysis and the assessment of the cytotoxicity by MTT assay and exclusion method by trypan blue. . For this purpose, were chosen: the Amarel?o community where the roasting occurs and the Santa Luzia farm an area without influence of this process. The mean value of PM2.5 (Jan 2124.2 μg/m3; May 1022.2 μg/m3; Sep 1291.9 μg/m3) and BC (Jan 363.6 μg/m3; May 70.0 μg/m3; Sep 69.4 μg/m3) as well as the concentration of the elements Al, Si, P, S, Cl, K, Ca, Ti, Cr, Mn, Fe, Ni, Cu, Zn, Se, Br and Pb obtained at Amarel?o was significantly higher than at Santa Luzia farm. The genotoxicity tests with T. pallida indicated a significant increase in the number of MN, NBP and NF and it was found a negative correlation between the frequency of these biomarkers and the rainfall. The concentrations of 200 μg/mL and 400 μg/mL of PM2.5 were cytotoxic to MRC-5 cells. All together, the results indicated genotoxicity and citotoxicity for the community of Amarel?o, and the high rates of PM2.5 considered a potential contributor to this effect, mainly by the high presence of transition metals, especially Fe, Ni, Cu, Cr and Zn, these elements have the potential to cause DNA damage. Other nuclear alterations, such as the NPBs and NFs may be used as effective biomarkers of DNA damage in tetrads of Tradescantia pallida. The results of this study enabled the identification of a serious occupational problem. Accordingly, preventative measures and better practices should be adopted to improve both the activity and the quality of life of the population. These measures are of fundamental importance for the sustainable development of this activity.
O Brasil ? o terceiro maior produtor mundial de castanha de caju e apesar da import?ncia social e econ?mica, sua produ??o ainda ? realizada de forma artesanal. Um dos maiores problemas da cadeia produtiva do caju s?o as condi??es nas quais ocorre a queima artesanal da castanha para se obter a am?ndoa. No presente estudo foi realizado um biomonitoramento do potencial genot?xico e avalia??o da citotoxicidade associada aos elementos oriundos da queima artesanal da castanha de caju no munic?pio de Jo?o C?mara - RN, semi-?rido brasileiro. Para a avalia??o genot?xica foi utilizado o bioensaio de micron?cleo (MN) em Tradescantia pallida. Al?m disso, foi realizado um comparativo quanto a sensibilidade da T. pallida frente ao clone KU-20 e outros biomarcadores de danos no DNA, tais como as pontes nucleoplasm?ticas (PNP) e fragmentos nucleares (FN) foram quantificados. A avalia??o citot?xica se deu pelo ensaio MTT e m?todo de exclus?o por tripan blue. As concentra??es de material particulado (MP1,0, MP2,5, MP10) e black carbon (BC) foram determinadas e a composi??o inorg?nica do MP2.5 definida pela t?cnica de fluoresc?ncia de raios-X. Foram definidos dois pontos testes: Comunidade do Amarel?o (local de queima da castanha de caju) e Fazenda Santa Luzia (sem influ?ncia da atividade). Os valores m?dios obtidos para o MP2,5 (Jan - 2124,2 μg/m3; Mai 1022,2 μg/m3; Set 1291,9 μg/m3) e BC (Jan 363,6 μg/m3; Mai 70 μg/m3; Set 69,4 μg/m3), bem como a concentra??o dos elementos Al, Si, P, S, Cl, K, Ca, Ti, Cr, Mn, Fe, Ni, Cu, Zn, Se, Br e Pb obtidos no Amarel?o foram significativamente maiores que na Fazenda Sta. Luzia. Os testes de genotoxicidade com T. pallida indicaram um aumento de 2-7 vezes maior na frequ?ncia de MN para o Amarel?o. Os outros biomarcadores tamb?m apresentaram sua frequ?ncia aumentada. Al?m disso, verificou-se uma correla??o negativa entre a freq??ncia de MN, PNP e FN com a precipita??o pluviom?trica. As concentra??es de 200 μg/mL e 400 μg/mL do MP2,5 em suspens?o foram citot?xicas para as c?lulas MRC-5. O conjunto dos resultados indicaram genotoxicidade e citotoxicidade para a comunidade do Amarel?o, sendo as altas concentra??es de MP2,5 um dos prov?veis contribuintes para esse efeito, principalmente pela elevada presen?a de metais de transi??o, sobretudo Fe, Ni, Cu, Cr e Zn, que potencialmente causam les?es no DNA. Outras altera??es nucleares, como PNP e FN podem ser utilizadas como biomarcadores efetivos de danos no DNA em t?trades de T. pallida. Os conjunto dos resultados possibilitaram a identifica??o de um problema ocupacional grave, com s?rios riscos aos trabalhadores que exercem a atividade. Diante disto, a ado??o de medidas preventivas e de melhores pr?ticas, s?o de fundamental import?ncia para o desenvolvimento sustent?vel da atividade e melhoria na qualidade de vida dos trabalhadores.

O objetivo do presente deste trabalho foi avaliar a toxicidade aguda, crônica e a genotoxicidade sobre E. andrei causadas por solo recém-contaminado com óleo lubrificante usado e após biorremediação por diferentes estratégias, após 22 meses, e paralelamente ao estudo de ecotoxicidade, foi conduzida uma investigação comparativa de três métodos de extração de HTP e HPA de solos para análise cromatográfica. A comparação das técnicas de extração evidenciou que para HTP, a técnica de extração acelerada por solvente-ASE foi a que melhor recuperou n-alcanos; já para as frações HRP e MCNR as técnicas soxhlet e micro-ondas-MARS não apresentaram diferenças significativas e foram melhores que ASE. Para HPA, a técnica de extração por soxhlet foi a que apresentou melhor recuperação em todos os solos. O teste de mortalidade apresentou, aos 14 dias, taxas crescentes de mortalidade de 10 6%, 20 0%, 73 25%, 93 12% e 100 0% para amostras de CONT (solo controle, sem contaminação artificial), BIOS (solo contaminado com 5% de OLU e biorremediado por bioestimulo), BIOA1 (solo contaminado com 5% de OLU e biorremediado por bioestimulo + bioaumento com adição de 10% de RSU maturado), e BIOA2 (solo contaminado com 5% de OLU e biorremediado por bioestimulo + bioaumento com adição de 10% de RSU semi-maturado) e OLU (solo contaminado com 5% de OLU), respectivamente. Aos 28 dias, entretanto, BIOS e OLU apresentaram taxas de mortalidade de 97 % 6 % e de 100 % 0 % respectivamente, valores estes significativamente superiores ao CONT. Foram observadas deformações anatômicas nos indivíduos mantidos em BIOS e OLU, assim como diminuição da biomassa em todas as amostras, evidenciando efeitos crônicos. O teste de reprodução, aos 28 dias, foram observadas grandes quantidades de indivíduos jovens nos solos biorremediados e recém-contaminado. No entanto, aos 56 dias houve uma diminuição dessas formas e o controle (CONT) exibiu uma quantidade maior de formas juvenis. O teste de densidade e viabilidade celular mostrou ser indicador sensível para toxicidade crônica apresentando queda nos solos BIOS e OLU em relação ao CONT com diferenças significativas (p <0.05). Não foram observados micronúcleos nos solos em estudo. Tal observação reforça a necessidade de testes de ecotoxicidade para avaliar a real eficácia de tecnologias de tratamento.
The aim of this study was to evaluate this acute toxicity, chronic and genotoxicity on E. andrei caused by freshly contaminated soil with used lubricating oil and after bioremediation by different strategies, after 22 months, and alongside ecotoxicity study, we conducted a comparative study of three methods of extraction and HTP HPA soil for chromatographic analysis. Comparison of extraction techniques for HTP showed that the technique of accelerated solvent extraction-ASE had the best recovery of n-alkanes, whereas for fractions and HRP MCNR soxhlet techniques and microwave-MARS and no significant differences were better than ASE. For HPA, the soxhlet extraction technique showed the best recovery in all soils. The mortality test showed , at the 14th day, growing mortality rates of 10 6% 20 0%, 73 25% 93 12% and 100 0% for samples CONT (ground control without contamination artificial ), BIOS (soil contaminated with 5% of OLU and bioremediated for biostimulation) BIOA1 (soil contaminated with 5% of OLU and bioremediated for biostimulation + bioaugmentation by adding 10% of MSW matured) and BIOA2 (soil contaminated with 5% bioremediated of OLU and for biostimulation + bioaugmentation by adding 10% of MSW semi-matured) and OLU (soil contaminated with 5% OLU), respectively. At the 28th day, however, OLU BIOS and mortality rates were 97% 6% and 100% 0%, respectively, values which are significantly higher than CONT. Anatomical deformations have been observed in individuals kept in BIOS and OLU, as well as decreased biomass in all samples, suggesting chronic effects. The reproduction test, at the 28th day, were found large quantities of juveniles in bioremediated soils and freshly contaminated soil. However, at the 56th day there was a decrease of these forms and control (CONT) exhibited a greater amount of juveniles. The >test> density and cell viability test showed to be a sensitive indicator for chronic toxicity in soils having decrease BIOS and OLU compared to CONT with significant differences (p <0.05). No micronuclei were observed in soils under study. This observation reinforces the need for ecotoxicity tests to assess the true efficacy of treatment technologies.

Made available in DSpace on 2014-12-17T14:13:58Z (GMT). No. of bitstreams: 1 LucilaCME.pdf: 777399 bytes, checksum: 0872bb60e8e228e6574e4fe1b87a20f8 (MD5) Previous issue date: 2006-12-21
Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico
The Pitimbu River is located at the oriental portion of the State of Rio Grande do Norte, including three importants cities named Maca?ba, Parnamirim e Natal. Although its high importance as a water source, which supplys great part of the South Zone of the Natal city, this river receives a large quantity of domestic and industrial waste water without treatment. The Pitimbu River headhas its river-head located in the city of Macaiba, goes through Parnamirim, then it flowing into at the Jiqui Lake in Natal. The aim of this study was to evaluate, qualitatively and quantitatively, the environmental quality of the Pitimbu River by genotoxicity bio-assays, which are important tools for genetics toxicological evaluation. In this work, five samples sites, distributed along the river, were used to collect water samples. Another point site, located near Jiqui lake, was used to collect drinkable water, which was treated by CAERN, the water treatment entreprise of Rio Grande do Norte. The following assays where used to evaluate the quality of these samples: Allium cepa assay; Comet assay; Micronuclei (MN) assay; and Ames test. For the Allium cepa assay, sixteen specimes where used for each water sample from the sample sites. In this assay both microscopic, like cytogenetic damage, and macroscopic aspects, as morphological variation were evaluated. Red blood cells from periferical blood of the Crenicichla menezesi native specie were used not only for the MN assay, but also for the Comet assay. These fishes were collected at different points on the Pitimbu River and the negative control was developed using fishes of the same species that were bring to the laboratory and maintained for 100 days in the optimal experimental conditions. For the Ames test, TA100, YG1042, TA98 and YG1041 strains were used in the directed method without metabolic activation. The results found by the Allium cepa assay showed that two water sample sites induced increase of mitotic index (IM). Additionally, compared to the control, all the water samples increased the chromossomal aberrations frequency and/or micronucleus. Among the sample sites, two also showed an abnormal growth rate in its root and two samples induced morphological alteration. With the MN test in red blood cells, a high frequence of MN was observed in tree sample. By comparing all the results obtained on the water sample points and with the negative control, a significant variation on the MN frequency was observed. Positive results were also observed for the same sample to water test by the Comet assay. These results allow concluding that the proposed specie Crenicichla menezesi has a good profile as a bio-indicator for the evaluation of environmental water quality and the MN and comet test can be usuful for in situ evaluation. By the Ames test, it was possible to detect the mutagenic activity on the waters from the Pitimbu River in different levels of mutagenicity. This result suggests that this river has several substances that induced changes directly to the DNA. The mechanisms involved to this phenomenon could be by both processes, by changing of the reading frame and by nucleotide substitution. These data set indicate the presence of mutagenic agents, which can represent in risk to biot and human beens
O rio Pitimbu localiza-se no litoral oriental do Estado do Rio Grande do Norte, mais precisamente na grande Natal, nos munic?pios de Maca?ba, Parnamirim e Natal. Embora se trate de um importante manancial, abastecendo parte da zona sul da capital do Estado, suas ?guas t?m sido alvo de dejetos industriais e dom?sticos, os quais n?o recebem tratamento adequado. O rio Pitimbu tem sua nascente situada no munic?pio de Maca?ba e des?gua na lagoa do Jiqu?, e parte em dire??o a praia de Pirangi, lan?ando suas ?guas no mar. O presente estudo teve como objetivo avaliar quantitativamente e qualitativamente a qualidade das ?guas do rio Pitimb?, atrav?s de bioensaios de genotoxicidade que s?o ferramentas de grande import?ncia nos estudos de gen?tica toxicol?gica. Neste trabalho foram coletadas amostras de ?gua de cinco pontos distribu?dos ao longo do rio e amostras da ?gua da lagoa do Jiqui ap?s tratamento feito pela CAERN (Companhia de ?guas e Esgotos do Rio Grande do Norte). Essas amostras foram analisadas atrav?s dos seguintes ensaios: teste de Allium cepa; o ensaio Cometa; o teste de Micron?cleo (MN); e o teste de Ames. Para realiza??o do teste em Allium cepa foram utilizados 16 indiv?duos para cada amostra testada, foram feitas an?lises dos aspectos microsc?picos (danos citogen?ticos) e macrosc?picos (varia??es morfol?gicas). Eritr?citos de sangue perif?rico da esp?cie nativa Crenicichla menezesi, foram utilizados tanto para execu??o do teste de MN como para o ensaio Cometa, os exemplares desta esp?cie foram coletados em diferentes pontos do rio Pitimbu e o controle negativo foi realizado com peixes da mesma esp?cie que foram trazidos para o laborat?rio e mantidos por 100 dias em condi??es ideais. Para execu??o do teste de Ames foram utilizadas as linhagens TA100, YG1042, TA98 e YG1041, sem ativa??o metab?lica, o qual foi realizado pelo m?todo direto. Os resultados obtidos no teste de Alium cepa monstraram que dois pontos de coleta de ?gua induziram altera??es no ?ndice mit?tico (?ndice de prolifera??o celular); al?m disso, todas as amostras de ?gua do rio induziram aumento na freq??ncia de aberra??es cromoss?micas e/ou de MN, em rela??o ao grupo controle. Dois dos pontos de coleta tamb?m apresentaram altera??o no crescimento da raiz e dois pontos produziram modifica??es morfol?gicas. Com o teste MN em eritr?citos uma alta freq??ncia de MN foi observada para tr?s amostras coletadas. Resultados positivos nas mesmas amostras teste tamb?m foram obtidos com o ensaio cometa. Estes resultados sugerem que a esp?cie Crenicichla menezesi pode ser considerado como um bom bioindicador para an?lise de qualidade ambiental e que os testes de MN e cometa s?o vi?veis para an?lise in situ. No teste de Ames, foi poss?vel detectar a presen?a de atividade mutag?nica nas ?guas do rio Pitimbu em diferentes n?veis de mutagenicidade, sugerindo a presen?a de compostos que agem de forma direta no DNA tanto por mudan?a do quadro de leitura quanto por substitui??o de pares de bases. Em conjunto os dados indicam a presen?a de agentes mutag?nicos, o que pode representar risco para a biota e os seres humanos

Made available in DSpace on 2014-08-20T13:32:51Z (GMT). No. of bitstreams: 1 dissertacao_rutilene_roll.pdf: 2016815 bytes, checksum: e7c5da43dfa30476ca94e16d9b7c9f57 (MD5) Previous issue date: 2005-07-29
Among the vast number of types of polysaccharides produced in nature, by plants, algae, and bacteria, xanthan is the one of the few that has functional properties leading to a broad spectrum of uses as well as commercial production in large quantities. It is the second microbial polysaccharide to be developed for an economically process, and it has continued to be the single most important biopolymer in food applications because of this useful properties. The chemical and physical properties of commercial xanthan mainly viscosity and stability with relation to temperature and pH variations, it makes that polysaccharide to be utilised a lot of for food industries, like as thickener and stabilizer of suspensions and emulsions. Because xanthan by patovar pruni is a new material never before introduced into foods, tests should be conduct in conformance with Agência de Vigilância Sanitária (ANVISA) for stablish its safety. And due to a few studies about the genotoxical mighty of xanthan, has been important to make a work about it. In this study, were experimentaly used xanthans produced by 06 and 24 strain from Xanthomonas campestris pv pruni and commercial xanthan from the same bacteria, but by pv campestris (Jungbunzlauer), like a additional component of mice diet, with aim of genotoxical evaluation in this animal model, through comet assay and micronuclei test. The comet assay and micronuclei test data were evaluated and, then, were able to comply no increase DNA damage induced by xanthans evaluated, through of utilised tests. Furthermore, was observed a tendency to decrease cholesterol and glucose levels, in the treatment with xanthans, although its values doesn´t have been significantly inferior to control group.
Entre o vasto número de tipos de polissacarídeos produzidos naturalmente, por plantas, algas e bactérias, xantana é um dos poucos que tem propriedades funcionais com amplo espectro de uso bem como produção industrial em grande escala. É o segundo biopolímero a ser desenvolvido por um processo economicamente viável, e continua a ser o mais importante biopolímero na aplicação em alimentos devido às suas propriedades funcionais. As propriedades químicas e físicas da xantana comercial, principalmente a viscosidade e a estabilidade em relação a variações de pH e temperatura, fazem com que este polissacarídeo seja amplamente utilizado na indústria alimentícia, dentre outras, como espessante e estabilizante de suspensões e emulsões. Devido a xantana do patovar pruni ser um material novo e ainda não utilizado em alimentos, testes devem ser conduzidos de acordo com a Agência de Vigilância Sanitária (ANVISA) para estabelecer a sua segurança. Com base no aumento da utilização destes polímeros na indústria alimentícia e, aos poucos dados referentes à genotoxicidade e aos efeitos sobre os índices de colesterol e glicose dos mesmos, torna-se importante a realização de um estudo sobre os danos ou benefícios que estes polímeros podem proporcionar. No presente estudo, foram usadas experimentalmente as xantanas produzidas pelas cepas 06 e 24 da bactéria Xanthomonas campestris pv pruni e a xantana comercial produzida pela mesma espécie, porém pelo patovar campestris (Jungbunzlauer), como componente adicional na dieta de camundongos, com o objetivo de avaliar o potencial genotóxico destes biopolímeros, neste modelo animal, através do ensaio cometa e do teste de micronúcleos; além de verificar os índices de colesterol e glicose. Os dados do ensaio cometa e do teste de micronúcleos foram avaliados e, então, observou-se que as xantanas produzidas pelas cepas 06 e 24, assim como a xantana comercial, não apresentaram valores significativamente superiores aos controles negativos, na indução de danos no DNA através dos testes utilizados. Além disso, foi observada uma tendência a diminuir os índices de colesterol e glicose, nos tratamentos com as xantanas, embora estes valores não tenham sido significativamente inferiores ao do grupo controle.

Conselho Nacional de Desenvolvimento Científico e Tecnológico
Sorbitol é um poliol encontrado em produtos para fins especiais, como diet e light, Dentro deste contexto, incluem-se as lactantes como grandes consumidoras, almejando o retorno mais rápido ao peso pré-gestacional. Devido à grande carência em dados referentes às consequências metabólicas do consumo excessivo destes tipos de produtos, o objetivo deste trabalho foi avaliar os possíveis efeitos da ingestão materna de sorbitol na lactação sobre os perfis nutricional, bioquímico e toxicológico nas proles amamentadas. O teste da Salmonella/microssoma foi utilizado, inicialmente, na avaliação mutagênica e citotóxica com linhagens de S. enterica sorovar Typhimurium (TA97, TA98, TA100, TA102, TA104 e TA1535). Verificamos a capacidade de reversão da mutação (I.M.) e sobrevivência (%), em diferentes concentrações (0,4; 4; 40; 400; 4000 e 5000 μg/placa). Os resultados foram considerados positivos para valores de I.M. ≥ 2,0. Ratas wistar lactantes (6 por grupo experimental), cada uma com 6 filhotes, receberam sorbitol (0,00015 mg/g/dia; 0,0015 mg/g/dia e 0,15 mg/g/dia), nos primeiros 14 dias da lactação. Neste período, avaliamos a biometria das mães e proles, consumo de ração e ingestão hídrica das mães. Após a lactação, as ratas mães foram ordenhadas, e, junto com as proles, sacrificadas por punção cardíaca, para coleta de sangue total. Os fígados das proles foram submetidos ao método de perfusão, para obtenção de hepatócitos em cultura primária, ao final dos 14 dias de lactação. Os fêmures das proles foram retirados, para obtenção da medula óssea. A bioquímica de sangue das proles (glicose, triglicerídeo, colesterol total, LDL, proteínas totais, albumina, ALT, AST, cálcio total e ionizado) foi analisada, assim como a bioquímica do leite ordenhado (triglicerídeos). Os testes do micronúcleo em medual óssea e hepatócitos, assim como o teste Cometa em sangue total, foram utilizados para avaliação genotóxica e citotóxica, de acordo com as diretrizes da OECD. Os resultados mostraram que, em concentrações mais baixas (0,00015 e 0,0015 mg/g), o sorbitol induziu o ganho de peso, principalmente na menor concentração (0,00015 mg/g), e alterações no perfil lipídico do sangue em todas as concentrações. As quantidades de triglicerídeo no leite variaram em função da dose ingerida, reduzida na maior concentração (0,15 mg/g) e aumentada na menor (0,00015 mg/g). A maior concentração (0,15 mg/g) resultou em perda de peso das mães e proles, diminuição de proteínas viscerais totais, albumina e aumento de enzimas hepáticas (ALT e AST) nas proles. Os resultados do teste da Salmonella/microssoma não indicaram mutagenicidade, entretanto, uma relação de dependência entre dose e Índice de Mutagenicidade (I.M.). Ambos os testes, micronúcleos de medula óssea e hepatócitos, apresentaram uma citotoxicidade dose dependente, estatisticamente significativa em relação ao grupo controle, corroborando com a genotoxicidade do teste Cometa e dependência de dose encontrada no teste da Salmonella/microssoma. Em concentrações mais baixas, parece existir modulação das vias lipogênicas, em contrapartida, nas mais altas a toxicidade parece dificultar tais alterações, induzindo o efeito contrário. Concluimos que o consumo excessivo de sorbitol resulta em alterações metabólicas e toxicológicas em lactentes, mesmo sendo considerado seguro pelo FDA e ANVISA.
Sorbitol is a polyol found in products for special purposes such as diet and light, Within this context, include lactating women as major consumers, aiming for a faster return to pre-pregnancy weight. Due to the great need for data on metabolic consequences of excessive consumption of these types of products, the objective of this study was to evaluate the possible effects of maternal intake of sorbitol in lactation on profiles nutritional, biochemical and toxicological in breastfed offspring. The test Salmonella/microsome was used initially in evaluating cytotoxic and mutagenic to Salmonella enterica sorovar Typhimurium strains deficient in the synthesis of the amino acid histidine. Lactating female Wistar rats (6 per experimental group), each with six puppies received sorbitol (0.00015 mg/g/day, 0.0015 mg/g/day and 0.15 mg/g/day) the first 14 days of lactation. In this period, we evaluate the biometrics of mothers and offspring, feed intake and water intake of the mothers. After lactation, the mother rats were milked, and, along with the proles, sacrificed by cardiac puncture for blood collection total. The livers of offspring were subjected to perfusion method for obtaining hepatocytes in primary culture, the end of the 14 days of lactation. The fêmurs of offspring were removed to obtain bone marrow. The biochemistry of blood offspring (glucose, triglycerides, total cholesterol, LDL, total protein, albumin, ALT, AST, total and ionized calcium) was analyzed as well as the biochemistry of milk milked (triglycerides). Micronucleus tests in bone medual and hepatocytes, as well as the Comet assay in whole blood, were used to evaluate cytotoxic and genotoxic, according to the OECD guidelines. The results showed that at lower concentrations (0.00015 and 0.0015 mg/g), sorbitol induced weight gain, especially at the lower concentration (0.00015 mg/g), and changes in blood lipid profiles in all concentrations. The amounts of triglycerides in milk varied according to the dose ingested reduced at the higher concentration (0.15 mg/g) and increased in the lower (0.00015 mg/g). The highest concentration (0.15 mg/g) resulted in weight loss of mothers and offspring, decreased visceral proteins, albumin and increased liver enzymes (ALT and AST) in the offspring. The test results of the Salmonella/microsome mutagenicity had not, however, a dependency relationship between dose and Mutagenicity Index (MI). Both tests, and bone marrow micronucleus hepatocyte cytotoxicity showed a dose dependent, statistically significant compared to the control group, supporting the Comet genotoxicity testing and dose dependency of the test found Salmonella / microsome. At lower concentrations, there appears to be modulation of lipogenic pathways in return, the higher the toxicity seems to hinder such changes, inducing the opposite effect. We conclude that excessive consumption of sorbitol could result in metabolic and toxicological disorders in infants and lactating rats, even though considered safe by the FDA and ANVISA.

Os problemas da poluição atmosférica atingem todos os grandes centros urbanos, em particular as megacidades com população maior do que 10 milhões de habitantes. Emissões veiculares e industriais destacam-se entre as principais responsáveis pelas altas concentrações de poluentes do ar nesses centros urbanos. Os diferentes componentes da poluição atmosférica, a dose e o tempo de exposição, podem levar a diversos impactos na saúde humana. Esse estudo teve por objetivo avaliar a genotoxicidade da poluição atmosférica (PM2,5 e NO2) e sua correlação com modificações no perfil de metilação das citocinas IL-10 e TNF-alfa, em trabalhadores da cidade de São Paulo/SP ocupacionalmente expostos. Participaram deste estudo 57 indivíduos do gênero masculino, com idades variando entre 28 e 66 anos de idade, trabalhadores em turnos diários de atividades externas na cidade de São Paulo e, portanto, ocupacionalmente expostos à poluição atmosférica. Foram recrutadas 3 categorias de profissionais: 1) controladores de tráfego (n=18); 2) taxistas (n=21) e profissionais do Instituto Florestal (n=18). Esses trabalhadores foram divididos em dois grupos em função dos locais de trabalho e exposição: 1) grupo área urbana (AU), composto pelos controladores de tráfego e taxistas e 2) grupo área periurbana (APU), composto pelos profissionais do Instituto Florestal. Amostradores individuais de poluição atmosférica foram utilizados para a coleta dos poluentes PM2,5 e NO2. A análise da genotoxicidade foi realizada pelo teste de micronúcleos nas células epiteliais da mucosa oral e em linfócitos do sangue periférico. O perfil de metilação das citocinas IL-10 e TNFalfa foi realizado por sequenciamento de nova geração. Nossos resultados mostraram uma diferença na concentração do PM2,5 entre os grupos (AU=32,92?g.m-3, APU=25,77ug.m-3; p=0,0311). Não foi encontrada diferença na concentração de NO2 entre os grupos. Foram encontradas diferenças nas frequências de micronúcleos, tanto em mucosa oral (AU=2,78%, APU=1,16%; p < 0,0001) quanto em linfócitos periféricos (AU=1,51%, APU=0,73%; p < 0,0001). Também foi encontrada diferença na metilação média do gene IL-10 entre os grupos (AU=25%, APU=30%; p=0,0120). Não foi encontrada diferença na metilação média do gene TNF-alfa entre os grupos. Concluímos que os trabalhadores da área urbana da cidade estão expostos a maiores concentrações de PM2,5, possuem maiores frequências de micronúcleos tanto em células da mucosa oral quanto em linfócitos periféricos e apresentam um perfil de hipometilação do gene IL-10 em comparação com os trabalhadores da área periurbana da cidade
The problems of air pollution affect all major urban centers, particularly megacities with populations greater than 10 million. Vehicular and industrial emissions stand out among the main responsible for the high concentrations of air pollutants in these urban centers. The different components of air pollution, the dose and time of exposure, can lead to different impacts on human health. This study aimed to evaluate the genotoxicity of air pollution (PM2,5 and NO2) and its correlation with changes in the methylation profile of the cytokines IL-10 and TNF-alpha in workers of São Paulo/SP occupationally exposed. The study included 57 male individuals, with age range between 28 and 66 years old, workers in daily shifts of outdoor activities in the São Paulo city and therefore occupationally exposed to air pollution. Were recruited professional of three categories: 1) traffic controllers (n=18); 2) taxi drivers (n=21) and professionals from the Forestry Institute (n=18). These workers were divided into two groups according to workplaces and exposure: 1) urban area group (UA), composed of traffic controllers and taxi drivers and 2) peri-urban area group (PUA), composed of professionals from the Forestry Institute. Individual samplers of air pollution were used for the collection of PM2,5 and NO2 pollutants. The analysis was performed by genotoxicity micronucleus test in the buccal mucosa epithelial cells and in peripheral blood lymphocytes. The methylation profile of the cytokines IL-10 and TNF-alpha was done by next generation sequencing. Our results showed a difference in PM2,5 concentration between the groups (UA=32,92ug.m-3, PUA=25,77ug.m-3; p=0,0311). No difference was found in NO2 concentrations between groups. Differences were found in the frequency of micronuclei in both buccal mucosa (UA=2,78%, PUA=1,16%; p < 0,0001) and in peripheral lymphocytes (UA=1,51%, PUA=0,73%; p < 0.0001). Difference was also found in average methylation of the IL-10 gene between the groups (UA=25%, PUA=30%; p=0,0120). There was no difference in the average methylation of TNF-alpha gene between the groups. We conclude that the workers of the urban area of the city are exposed to higher concentrations of PM2,5, have higher frequencies of micronuclei in both the buccal mucosa cells and in peripheral lymphocytes and have a hypomethylation profile of IL-10 gene in comparison with workers the peri-urban area of the city