Genotoxicity biomarkers micronucleus, nucleoplasmic bridges and nuclear buds

The increase of mortality from cancer brought urgency in identification and validation of predictive markers of risk and therefore early diagnosis. There is evidence that cytogenetic biomarkers are positively correlated with risk of cancer, and this is validated by studies of cohort and case-control. Cytokinesis-blocked micronucleus (CBMN) assay is used extensively in molecular epidemiology, and can be considered as a “cytome” assay covering cell proliferation, apoptosis, necrosis and chromosomal changes. The chromosomal alterations most reported and studied by the CBMN are: micronucleus (MN), nucleoplasmic bridges (NPB) and nuclear buds (NBUDS). The use of the MN assay in biomonitoring studies had a large increase in the last 15 years and international projects such as the HUMN have helped to increase the applicability and reliability of these tests

Evaluation of staining techniques on the results of micronucleus in exfoliated oral mucosa cells

Micronuclei (MN) in exfoliated epithelial cells are widely used as biomarkers of cancer risk in humans. MN are classified as biomarkers of the break age and loss of chromosomes. They are small, extra nuclear bodies that arise in dividing cells from centric chromosome/chromatid fragments or whole chromosomes/chromatids that lag behind in anaphase and are not included in the daughter nuclei in telophase. Buccal mucosa cells have been used in biomonitoring exposed populations because these cells are in the direct route of exposure to ingested pollutant, are capable of metabolizing proximate carcinogens to reactive chemicals, and are easily and rapidly collected by brushing the buccal mucosa. The objective of the present study was to further investigate if, and to what extent, different stains have an effect on the results of micronuclei studies in exfoliated cells. These techniques are: Papanicolaou (PAP), Modified Papanicolaou, May-Grünwald Giemsa (MGG), Giemsa, Harris’s Hematoxylin, Feulgen with Fast Green counterstain and Feulgen without counterstain

Biomarcadores genotóxicos e polimosfismos genéticos em trabalhadores expostos a formaldeído

Tese de mestrado, Biologia (Biologia Molecular Humana), 2009, Universidade de Lisboa, Faculdade de CiênciasO formaldeído é um agente químico, classificado como carcinogénico em humanos e animais experimentais, amplamente utilizado nos laboratórios de Anatomia Patológica. A exposição a esta substância está epidemiologicamente associada a cancro e a alterações nucleares detectáveis através do teste dos micronúcleos por bloqueio da citocinese. Este método é utilizado extensivamente em epidemiologia molecular, uma vez que permite determinar vários biomarcadores de genotoxicidade, tais como: micronúcleos (biomarcadores de quebra ou perda de cromossomas), pontes nucleoplásmicas (biomarcador de re-arranjo cromossómico, má reparação e/ou fusão de telómeros) e protusões nucleares (biomarcador de eliminação de DNA amplificado e/ou complexos de reparação de DNA). O objectivo deste trabalho é investigar a relação entre a frequência de alterações nucleares e factores genéticos e ambientais, em indivíduos expostos e não-expostos ocupacionalmente a formaldeído. Para tal, foram constituídos 2 grupos: 56 indivíduos expostos ocupacionalmente a formaldeído (casos) e 85 indivíduos não expostos (controlos). Foram recolhidos sangue periférico e células epiteliais esfoliadas da mucosa bucal. Realizou-se o estudo dos biomarcadores genotóxicos e extracção de DNA para estudo dos polimorfismos nos genes XRCC3 e ADH3. Os resultados obtidos fornecem evidência inequívoca para associação entre a exposição ocupacional a formaldeído e a presença de alterações nucleares. Os hábitos tabágicos e o consumo de álcool, exercem efeitos significativos na presença de micronúcleos na mucosa bucal, pontes nucleoplásmicas e protusões nucleares. Relativamente aos factores genéticos, verificaram-se associações estatisticamente significativas entre o polimorfismo do gene da ADH3 e a presença de micronúcleos em linfócitos periféricos e do polimorfismo do gene XRCC3 nas protusões nucleares.Formaldehyde is a chemical agent, classified as carcinogenic in humans and experimental animals, widely used in laboratories of Pathology. The exposure to this substance is epidemiologically linked to cancer and nuclear changes detected by the cytokinesis-block micronucleus test. This method is extensively used in molecular epidemiology, since it determines several biomarkers of genotoxicity, such as micronuclei (biomarkers of chromosomes breakage or loss), nucleoplasmic bridges (biomarker of chromosome rearrangement, poor repair and / or telomeres fusion) and nuclear buds (biomarker of elimination of amplified DNA). The aim of this study is to investigate the relationship between the frequency of nuclear changes and genetic and environmental factors in individuals exposed and non-occupationally exposed to formaldehyde. To this end, were made 2 groups: 56 individuals occupationally exposed to formaldehyde (cases) and 85 unexposed individuals (controls). It was collected peripheral blood and exfoliated epithelial cells of oral mucosa and carried out the study of biomarkers of genotoxic and DNA extraction for verification of polymorphisms in XRCC3 and ADH3 genes. The results provide unequivocal evidence for association between occupational exposure to formaldehyde and the presence of nuclear changes. The smoking and alcohol consumption have significant effects in the presence of micronuclei in oral mucosa, nucleoplasmic bridges and nuclear buds. For genetic factors, there were significant associations between gene polymorphism of ADH3 and the presence of micronuclei in peripheral lymphocytes and gene polymorphism in XRCC3 and nuclear buds

Tecnologias laboratoriais em anatomia patológica II

Versão original publicada em CDROM. O registo inclui o plano, o cronograma e as aulas teóricas (0. Apresentação; 1. Arquivos e sua importância em anatomia patológica; 2. microscopia; 3. Histotecnologia aplicada; 4. Tissue microarrays; 5. Ictiohistologia; 6. Megacassetes; 7. Culturas celulares).Unidade curricular que fornece, para além dos fundamentos essenciais para o exercício da histopatologia, algumas das técnicas inovadoras utilizadas atualmente.info:eu-repo/semantics/publishedVersio

Human biomonitoring : biomarkers, individual susceptibility, and nutrigenetics

Tese de doutoramento, Biologia (Genética), Universidade de Lisboa, Faculdade de Ciências, 2015Cytokinesis blocked micronucleus (CBMN) assay and comet assay are the most promising short-term genotoxicity assays for human risk assessment and their combination is recommended to monitor populations chronically exposed to genotoxic agents. Nutrition is recognized to be an important lifestyle factor that influences cancer risk, and should be taken into account at an individual level. Laboratories are occupational settings where chemical agents are handled and workers are exposed. Formaldehyde and cytostatic drugs, in particular, are chemical agents handled in laboratories that are considered carcinogenic for humans and special protective measures should thus be adopted against them. The aim of this investigation is to contribute to the development of a biomonitoring programme that includes genotoxicity assessment related with genetic susceptibility biomarkers, and lifestyle factors, namely nutrition. The experimental planning used was a case-control blinded study. Four separated samples were formed comprising two samples of subjects exposed (n = 56 for formaldehyde; n = 46 for cytostatics), and two samples of non-exposed controls (n = 85 and n = 46, respectively). Participants filled-in a personal and a food frequency questionnaires. CBMN and comet assays were used to assess genotoxicity. Individual susceptibility was investigated by Real Time PCR. Measurements of serum vitamins A and E were performed by HPLC, and vitamin D by ELISA. The risk of genotoxicity in those exposed was then compared with the risk in the controls, allowing for the quantitative measurement of association between exposure and genotoxicity. In both occupational settings, the genotoxicity biomarkers were significantly higher in the exposed than in the non-exposed controls (p<0.05). In the formaldehyde occupational context, significant associations were found between XRCC3 genotypes and nuclear buds; but that was not the case either for ADH5 or VDR. Comet assay did not identify significant differences between those exposed to cytostatics and controls, and the same lack of association applies to the OGG1 genotypes. Micronutrients association to the genotoxicity biomarkers was controversial; we have found positive correlations for vitamin A and negative ones for vitamin E. Our findings emphasize the need for the implementation of a regular biomonitoring programme of personnel occupationally exposed to drugs like formaldehyde and cytostatics.Fundação para a Ciência e a Tecnologia, IP (FCT

Métodos e técnicas em histoquímica

Versão original publicada em CDROM. O registo inclui o plano e as aulas teóricas (0. Apresentação; 1. introdução à histoquímica; 2. Hidratos de carbono; 3. Tecido conjuntivo; 4. Microorganismos; 5. Pigmentos; 6. Tecido ósseo; 7. Lípidos; 8. Sistema nervoso central.Unidade curricular que fornece os fundamentos teóricos e a aplicação prática de técnicas complementares ao diagnóstico histopatológico da anatomia patológica.info:eu-repo/semantics/publishedVersio

Técnica laboratorial em anatomia patológica I (Vol. 1)

Versão original publicada em CDROM. Este registo inclui exclusivamente as aulas teóricas (0. Apresentação; 1. Laboratório de anatomia patológica; 2. Segurança no laboratório de AP; 3. Elaboração de soluções; 4. Receção e registo macroscópico; 5. Fixação; 6. Descalcificação; 7. Processamento histológico; 8. Inclusão; 9. Microtomia), os cronogramas e o planeamento.Técnica laboratorial em anatomia patológica I - Unidade curricular que fornece os fundamentos essenciais para o exercício da Histopatologia, pilar basilar da Anatomia Patológica.info:eu-repo/semantics/publishedVersio

EDCs mixture effects in human cell lines

Endocrine disrupting chemicals (EDCs) are exogenous agents commonly employed in a wide variety of consumer products and indoor/outdoor applications. Human exposure to these compounds either by environmental and/or occupational sources is omnipresent, persistent and occurs in complex mixtures which, therefore, can origin a mixed “body burden” of contaminants. Extensive research in human cell lines, particularly from sex hormone-responsive tissues, show that EDCs can simultaneously and differentially trigger specific signaling pathways, predominantly associated to estrogen receptors. Evidence demonstrates that EDCs endorse distinct cellular responses in different cell lines, however, most of the studies have focused exclusively in the independent action of the tested compounds whereas information regarding EDCs mixtures effects is still scared. We performed a revision of the state of the art in PubMed focused on studies that evaluate EDCs mixture effects. Based on published data a review of the utilized EDCs, tested doses, studied cell lines and observed effects of co-exposure studies are presented. Although the independent action of chemicals is still considered the main principle of EDCs mixture toxicity, the evidence demonstrates that some effects cannot be predicted when analyzing single compounds independently and thus it is undoubtedly that the daily doses of each compound are not the only one that can be taken into consideration. There is an emergent concern that the current toxicological assessment based in a chemical-by-chemical approach may not be adequately protected in the context of EDCs mixtures and that recognizing potential aggregate and cumulative exposures are crucial for an adequate risk assessment.info:eu-repo/semantics/publishedVersio

EDCs mixtures: a stealthy hazard for human health ?

ReviewEndocrine disrupting chemicals (EDCs) are exogenous chemicals that may occur naturally (e.g., phytoestrogens), while others are industrial substances and plasticizers commonly utilized worldwide to which human exposure, particularly at low-doses, is omnipresent, persistent and occurs in complex mixtures. EDCs can interfere with/or mimic estrogenic hormones and, consequently, can simultaneously trigger diverse signaling pathways which result in diverse and divergent biological responses. Additionally, EDCs can also bioaccumulate in lipid compartments of the organism forming a mixed “body burden” of contaminants. Although the independent action of chemicals has been considered the main principle in EDCs mixture toxicity, recent studies have demonstrated that numerous effects cannot be predicted when analyzing single compounds independently. Co-exposure to these agents, particularly in critical windows of exposure, may induce hazardous health effects potentially associated with a complex “body burden” of different origins. Here, we performed an exhaustive review of the available literature regarding EDCs mixtures exposure, toxicity mechanisms and effects, particularly at the most vulnerable human life stages. Although the assessment of potential risks to human health due to exposure to EDCs mixtures is a major topic for consumer safety, information regarding effective mixtures effects is still scarceinfo:eu-repo/semantics/publishedVersio