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Browsing by Author "Simões, Tânia"

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  • Biomonitoring the genetic effects of environmental tobacco smoke exposure in restaurant workers
    Publication . Vital, Nádia; Louro, Henriqueta; Antunes, Susana; Penque, Deborah; Simões, Tânia; Silva, Maria João
    Environmental tobacco smoke (ETS) is recognized as one of the most common indoor pollutants worldwide. Portuguese legislation prohibits smoking in most indoor public spaces. However, in some restaurants/bars smoking is still allowed, representing a potential risk factor for the workers health, particularly for chronic respiratory diseases and cancer onset.The aim of this study was to characterize early signs of ETS-associated adverse systemic effects in workers from restaurants with smoking permission in comparison with workers from smoke-free spaces, considering the modulating effects of genetic susceptibility.The ETS-exposed workers did not display differences in the frequencies of SCEs, cells with high frequency of SCEs (HFCs), MN or DNA strand breaks, as assessed by the comet assay, when compared to non-ETS workers. Smoking workers presented a significantly increased level of HFCs as compared to non-smokers. Interestingly, the ex vivo challenge of leukocytes with EMS resulted in a lower level of DNA breaks in ETS-exposed as compared to non-exposed workers (P<0,0001), suggesting an increased DNA repair capacity associated to ETS-exposure. Regarding the genetic polymorphisms studied, GSTM1 null genotype carriers presented increased frequencies of SCEs and HFCs associated with ETS exposure, suggesting an increased susceptibility to this environmental stressor. On the contrary, XRCC1-399 wild type carriers presented a lower level of MN than the variant allele carriers, in response to ETS exposure. Finally, among the ETS-exposed subjects, those carrying the hOGG1 variant alleles presented a lower level of ex vivo EMS-induced DNA damage comparatively to the wild type subjects, suggesting a higher DNA repair capacity.The study of ETS exposure in an occupational setting in Lisbon restaurants revealed that the exposed workers did not display systemic genetic effects detectable by the biomarkers analysed. However, the results of the ex vivo challenge comet assay, suggests that ETS may have a more subtle modulating effect on the DNA repair response of blood cells to a genotoxic insult. In addition, the association between some genetic polymorphisms and increased genotoxic effects in subsets of individuals, highlights the possibility of increased health risks in susceptible individuals exposed to ETS, that should be further investigated.
    2013-11-18Conference object Open access Show more
  • Effects of occupational exposure to tobacco smoke: is there a link between environmental exposure and disease?
    Publication . Pacheco, Solange A.; Torres, Vukosava M.; Louro, Henriqueta; Gomes, Filomena; Lopes, Carlos; Marçal, Nelson; Fragoso, Elsa; Martins, Carla; Oliveira, Cátia L.; Hagenfeldt, Manuela; Bugalho-Almeida, António; Penque, Deborah; Simões, Tânia
    In a previous study, evidence was provided that indoor secondhand tobacco smoke (SHS) air pollution remains high in Lisbon restaurants where smoking is allowed, regardless of the protective measures used. The aim of this study was to determine in these locations the levels of polycyclic aromatic hydrocarbons (PAH) associated with the particulate phase of SHS (PPAH), a fraction that contains recognized carginogens, such as benzo[a]pyrene (BaP). Data showed that restaurant smoking areas might contain PPAH levels as high as 110 ng/m(3), a value significantly higher than that estimated for nonsmoking areas (30 ng/m(3)) or smoke-free restaurants (22 ng/m(3)). The effective exposure to SHS components in restaurant smoking rooms was confirmed as cotinine levels found in workers' urine. Considering that all workers exhibited normal lung function, eventual molecular changes in blood that might be associated with occupational exposure to SHS and SHS-associated PPAH were investigated by measurement of two oxidative markers, total antioxidant status (TAS) and 8-hydroxyguanosine (8-OHdG) in plasma and serum, respectively. SHS-exposed workers exhibited higher mean levels of serum 8-OHdG than nonexposed workers, regardless of smoking status. By using a proteomics approach based on 2D-DIGE-MS, it was possible to identify nine differentially expressed proteins in the plasma of SHS-exposed nonsmoker workers. Two acute-phase inflammation proteins, ceruloplasmin and inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), were predominant. These two proteins presented a high number of isoforms modulated by SHS exposure with the high-molecular-weight (high-MW) isoforms decreased in abundance while low-MW isoforms were increased in abundance. Whether these expression profiles are due to (1) a specific proteolytic cleavage, (2) a change on protein stability, or (3) alterations on post-translational modification pattern of these proteins remains to be investigated. Considering that these events seem to precede the first symptoms of tobacco-related diseases, our findings might contribute to elucidation of early SHS-induced pathogenic mechanisms and constitute a useful tool for monitoring the effects of SHS on occupationally exposed individuals such as those working in the hospitality industry.
    2013-03Journal article Restricted access Show more
  • Environmental Tobacco Smoke in Occupational Settings: Effect and Susceptibility Biomarkers in Workers From Lisbon Restaurants and Bars
    Publication . Vital, Nádia; Antunes, Susana; Louro, Henriqueta; Vaz, Fátima; Simões, Tânia; Penque, Deborah; Silva, Maria João
    Environmental tobacco smoke (ETS) has been recognized as a major health hazard by environmental and public health authorities worldwide. In Portugal, smoke-free laws are in force for some years, banning smoking in most indoor public spaces. However, in hospitality venues such as restaurants and bars, owners can still choose between a total smoke-free policy or a partial smoking restriction with designated smoking areas, if adequate reinforced ventilation systems are implemented. Despite that, a previous study showed that workers remained continuously exposed to higher ETS pollution in Lisbon restaurants and bars where smoking was still allowed, comparatively to total smoke-free venues. This was assessed by measurements of indoor PM2.5 and urinary cotinine, a biomarkers of tobacco smoke exposure, demonstrating that partial smoking restrictions do not effectively protect workers from ETS. The aim of the present work was to characterize effect and susceptibility biomarkers in non-smokers from those hospitality venues occupationally exposed to ETS comparatively to non-exposed ones. A group of smokers was also included for comparison. The sister chromatid exchange (SCE), micronucleus (MN) and comet assays in whole peripheral blood lymphocytes (PBLs) and the micronucleus assay in exfoliated buccal cells, were used as biomarkers of genotoxicity. Furthermore, a comet assay after ex vivo challenge of leukocytes with an alkylating agent, ethyl methanesulfonate (EMS), was used to analyze the repair capacity of those cells. Genetic polymorphisms in genes associated with metabolism and DNA repair were also included. The results showed no clear association between occupational exposure to ETS and the induction of genotoxicity. Interestingly, the leukocytes from non-smoking ETS-exposed individuals displayed lower DNA damage levels in response to the ex vivo EMS challenge, in comparison to those from non-exposed workers, suggesting a possible adaptive response. The contribution of individual susceptibility to the effect biomarkers studied was unclear, deserving further investigation.
    2021-06-04Journal article Open access Show more
  • Exposição ao Fumo Passivo no Local de Trabalho: Biomarcadores de Genotoxicidade e Susceptibilidade
    Publication . Antunes, Susana; Vital, Nadia; Louro, Henriqueta; Gomes, Filomena; Penque, Deborah; Simões, Tânia; Silva, Maria João
    Apesar dos limites impostos ao fumo em locais públicos, ainda é permitido fumar nalguns bares/restaurantes, o que constitui um potencial risco para a saúde dos indivíduos expostos. Neste trabalho pretendeu-se analisar uma potencial associação entre exposição ocupacional ao fumo de tabaco ambiental(ETS) e indução de alterações genéticas, atendendo à influência da susceptibilidade genética individual. Estudaram-se 81 empregados de bares/restaurantes, 37 expostos e 44 não expostos a ETS. Caracterizaram-se os efeitos genotóxicos recorrendo ao ensaio do micronúcleo (MN) em linfócitos e em células do epitélio oral e ao ensaio do cometa em leucócitos. Paralelamente, avaliou-se a capacidade de reparação do DNA através da quantificação de lesões induzidas pelo SEM (Ethyl methanesulfonate) no DNA de leucócitos. Analisaram-se os polimorfismos em genes de metabolização e reparação de DNA através de PCR/RFLP. Relativamente aos biomarcadores de genotoxicidade, não se observaram diferenças significativas entre o grupo exposto a ETS e o controlo. Contudo, o estímulo com EMS induziu um número significativamente menor de lesões no DNA de leucócitos dos indivíduos expostos a ETS comparativamente aos não expostos, sugerindo uma melhor capacidade de reparação do DNA - resposta adaptativa. Evidenciou-se também a influência de alguns polimorfismos em genes de metabolização na frequência de MN em células do epitélio oral e de quebras de DNA em leucócitos. Em conclusão, este estudo revelou que apesar da exposição ocupacional a ETS em bares/restaurantes não ter provocado efeitos genotóxicos detectáveis, induziu uma possível resposta adaptativa em células dos indivíduos expostos, cujo potencial impacto na saúde ainda se desconhece.
    2011-05-20Conference object Open access Show more
  • Exposição ocupacional ao fumo de tabaco ambiental: um estudo em restaurantes na cidade de Lisboa
    Publication . Pacheco, Solange; Aguiar, Fátima; Proença, Carmo; Penque, Deborah; Simões, Tânia
    2012-10Journal article Open access Show more
  • Exposição ocupacional ao fumo de tabaco: alterações subclínicas em proteínas de inflamação aguda
    Publication . Pacheco, Solange; Simões, Tânia; Penque, Deborah
    2013-12-17Journal article Open access Show more
  • Exposure to second-hand smoke in occupational settings: biomarkers of DNA damage and susceptibility
    Publication . Louro, Henriqueta; Carmo, Susana; Vital, Nádia; Gomes, Filomena; Penque, Deborah; Simões, Tânia; Silva, Maria João
    Portuguese legislation prohibits smoking in most indoor public spaces. However, some restaurants/bars remain, in which smoking is still allowed, representing a potential risk for employees, particularly for chronic respiratory diseases and cancer development. The objectives of this project were to compare the indoor air quality of some restaurants with and without smoking permission and, in their workers, to search for associations between respiratory dysfunctions and biomarkers of exposure, biological effects (DNA and proteome alterations) and susceptibility. Herein, we present data on DNA damage and genetic susceptibility in 37 workers occupationally exposed to environmental tobacco smoke (ETS) and from 44 non-exposed workers. DNA damage was assessed by the comet assay in blood leukocytes and by micronucleus (MN) analysis in buccal cells. DNA repair capacity was evaluated by a challenge of blood cells with EMS (32 µM), preceding the comet assay. Polymorphisms in metabolism (GSTP1, GSTM and GSTT) and DNA-repair genes (hOGG1,XRCC1, XRCC3, and NBS1) were analysed by PCR/RFLP. No significant differences in the MN frequency and in level of DNA damage was observed between ETS and non-ETS groups. However, challenge with EMS resulted in a significantly lower level of DNA breaks in ETS-exposed as compared to non-exposed workers (P<0.0001). Concerning polymorphisms analysis, GSTP1 variant allele carriers showed a decreased frequency of MN in buccal cells, which was not influenced by ETS exposure. No association related to the other polymorphisms was detected. In conclusion, the present data show that blood cells from ETS-exposed workers display a lower mutagenic response to EMS challenge, suggestive of an adaptive response elicited by the previous exposure to low levels of ETS.
    2011-03Conference object Open access Show more
  • Fumo de Tabaco em Estabelecimentos Recreativos: Biomarcadores de Genotoxicidade e Susceptibilidade Genética
    Publication . Vital, Nádia; Antunes, Susana; Louro, Henriqueta; Penque, Deborah; Simões, Tânia; Silva, Maria João
    A exposição a fumo de tabaco ambiental (ETS, Environmental Tobacco Smoke), isto é, ao fumo passivo, pode causar uma variedade de efeitos na saúde dos indivíduos expostos, nomeadamente doenças do foro respiratório e, a longo prazo, desenvolvimento de cancro. Desde Janeiro de 2008 que a legislação portuguesa estabeleceu a proibição de fumar na maioria dos espaços públicos fechados. No entanto, alguns bares e restaurantes mantiveram espaços reservados a fumadores, constituindo assim uma fonte de exposição a ETS para os seus frequentadores e, em particular, para os seus trabalhadores. O presente trabalho teve como objectivo principal analisar a existência de uma potencial associação entre exposição ocupacional ao fumo do tabaco e a indução de alterações genéticas em células somáticas dos indivíduos expostos, tendo em consideração a influência da susceptibilidade genética de cada indivíduo. Foram seleccionados para o estudo 33 empregados de bares e restaurantes expostos a ETS no local de trabalho e 30 trabalhadores da mesma área profissional não expostos. Os efeitos genotóxicos foram analisados através do ensaio do micronúcleo (MN) quer em linfócitos com bloqueio da citocinese (CBMN) quer em células do epitélio oral. Como biomarcadores de susceptibilidade genética caracterizaram-se polimorfismos em genes que codificam para enzimas de metabolização de xenobióticos (GSTP1, CYP1A1, GSTM e GSTT) através de PCR/RFLP. Não se observaram diferenças significativas na frequência de MN entre os grupos expostos e não expostos a ETS, tanto ao nível dos linfócitos como ao nível das células do epitélio oral. A análise dos polimorfismos mostrou uma associação entre o alelo variante do gene GSTP1 e uma menor frequência de MN nas celulas do epitélio oral, independentemente da exposição. Em conclusão, o facto de não ter sido detectado um efeito genotóxico inequívoco, nos trabalhadores não fumadores dos espaços com fumo, não poderá ser interpretado como uma total ausência de efeitos genotóxicos nesses fumadores passivos. Para além disso, não se pode excluir a possibilidade de que um efeito genotóxico possa estar a ocorrer em tecidos-alvo, nomeadamente ao nível das células pulmonares. A continuação deste estudo bem como a utilização de outras abordagens que contribuam para a elucidação desta problemática tão importante para a saúde pública, poderá trazer nova luz à compreensão do efeito genotóxico e potencialmente carcinogénico associado ao fumo passivo.
    2011-03-19Conference object Open access Show more
  • Glycation potentiates α-synuclein-associated neurodegeneration in synucleinopathies
    Publication . Vicente Miranda, Hugo; Szego, Éva M.; Oliveira, Luís M.A.; Breda, Carlo; Darendelioglu, Ekrem; de Oliveira, Rita M.; Ferreira, Diana G.; Gomes, Marcos A.; Rott, Ruth; Oliveira, Márcia; Munari, Francesca; Enguita, Francisco J.; Simões, Tânia; Rodrigues, Eva F.; Heinrich, Michael; Martins, Ivo C.; Zamolo, Irina; Riess, Olaf; Cordeiro, Carlos; Ponces-Freire, Ana; Lashuel, Hilal A.; Santos, Nuno C.; Lopes, Luisa V.; Xiang, Wei; Jovin, Thomas M.; Penque, Deborah; Engelender, Simone; Zweckstetter, Markus; Klucken, Jochen; Giorgini, Flaviano; Quintas, Alexandre; Outeiro, Tiago F.
    α-Synuclein misfolding and aggregation is a hallmark in Parkinson's disease and in several other neurodegenerative diseases known as synucleinopathies. The toxic properties of α-synuclein are conserved from yeast to man, but the precise underpinnings of the cellular pathologies associated are still elusive, complicating the development of effective therapeutic strategies. Combining molecular genetics with target-based approaches, we established that glycation, an unavoidable age-associated post-translational modification, enhanced α-synuclein toxicity in vitro and in vivo, in Drosophila and in mice. Glycation affected primarily the N-terminal region of α-synuclein, reducing membrane binding, impaired the clearance of α-synuclein, and promoted the accumulation of toxic oligomers that impaired neuronal synaptic transmission. Strikingly, using glycation inhibitors, we demonstrated that normal clearance of α-synuclein was re-established, aggregation was reduced, and motor phenotypes in Drosophila were alleviated. Altogether, our study demonstrates glycation constitutes a novel drug target that can be explored in synucleinopathies as well as in other neurodegenerative conditions.
    2017-05-01Journal article Restricted access Show more
  • Heat-mediated enrichment of α-synuclein from cells and tissue for assessing post-translational modifications
    Publication . Vicente Miranda, Hugo; Xiang, Wei; Oliveira, Rita M. de; Simões, Tânia; Pimentel, José; Klucken, Jochen; Penque, Deborah; Outeiro, Tiago
    α-synuclein (α-syn) is the major component of Lewy bodies, a pathological hallmark of Parkinson's disease and other synucleinopathies. The characterization of α-syn post-translational modifications (PTMs), thought to interfere with its aggregation propensity and cellular signaling, has been limited by the availability of extraction methods of endogenous protein from cells and tissues, and by the availability of antibodies toward α-syn PTMs. Here, by taking advantage of α-syn thermostability, we applied a method to achieve high enrichment of soluble α-syn both from cultured cells and brain tissues followed by proteomics analysis. Using this approach, we obtained 98% α-syn sequence coverage in a variety of model systems, including a transgenic mouse model of PD, and validated the strategy by identifying previously described PTMs such as phosphorylation and N-terminal acetylation. Our findings demonstrate that this procedure overcomes existing technical limitations and can be used to facilitate the systematic study of α-syn PTMs, thereby enabling the clarification of their role under physiological and pathological conditions. Ultimately, this approach may enable the development of novel biomarkers and strategies for therapeutic intervention in synucleinopathies. In this study, we describe a method for enriching alpha-synuclein (α-syn) from a variety of biological samples, from cultured cells to brain tissues. Enrichment of α-syn was achieved by heating samples, further facilitating the identification of specific post-translational modifications by immunoblot, or mass spectrometry-based techniques. This approach will contribute to the clarification of the role of α-syn PTMs in Parkinson's disease.
    2013-09Journal article Restricted access Show more