Browsing by Author "Antunes, Susana"
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- Are standard genotoxicity tests useful for the safety evaluation of nanomaterials?Publication . Louro, Henriqueta; Tavares, Ana; Vital, Nádia; Antunes, Susana; Costa, Pedro; Alverca, Elsa; Lavinha, João; Silva, Maria JoãoNanomaterials (NMs) are widely used in a diversity of consumer products, despite uncertainties surrounding the potential health risks that they pose to humans and the environment. One of the major concerns is the potential to induce cancer. To analyze in a short term the carcinogenic properties of a compound, genotoxicity assays in mammalian cell lines or animal models are frequently used. In the context of an EU Joint Action, in the present work we have used standard genotoxicity assays (comet, micronucleus and mutation assays) to investigate the effects associated with the exposure to titanium dioxide nanomaterials (TiO2), following standardized dispersion and assay procedures, in three types of human cells and in a mouse model. The results showed slight but significant increases in the frequencies of micronuclei after exposure to some of the NMs, as compared to controls. No clear dose-response relationships could be disclosed. One of the tested TiO2 yielded equivocal results in vitro micronucleus assay and was positive in the comet assay in pulmonary cells. In view of the inconclusive results,it was further analyzed in vivo, using the lacZ transgenic mouse model. It did not induce genotoxic effects in mice, 28 days after injection, despite the accumulation of the NM observed in the mouse liver. Regarding safety assessment, the different genotoxicity observed for closely related NMs, but that differ in some physicochemical characteristics, highlights the importance of investigating the toxic potential of each NM individually, instead of assuming a common mechanism and equal genotoxic effects for a set of similar NMs. The equivocal genotoxicity of the nanosized TiO2 in human cells in vitro was not confirmed in vivo, and therefore the predictive value of these in vitro tests for identifying genotoxic (and potentially carcinogenic) NMs in vivo should be clarified, before extrapolating the conclusions for human health.
- Assessment of the genotoxicity of a titanium dioxide nanomaterial using a combination of in vitro and in vivo assaysPublication . Tavares, Ana; Louro, Henriqueta; Vital, Nádia; Antunes, Susana; Lavinha, João; Silva, Maria JoãoHuman exposure to manufactured nanomaterials such as titanium dioxide (TiO2), often used in sunscreens and cosmetics, has increased worldwide. Their specific properties, such as size and high surface area/mass, render them attractive for many applications, but may also be associated to higher toxicity in biological systems and adverse effects in humans. In the context of EU Joint Action NANOGENOTOX (www.nanogenotox.com), the present work aimed to analyse the potential genotoxic effects of a well-characterized TiO2 nanomaterial, correlating in vitro and in vivo effects. TiO2 dispersions were prepared according to a standardized protocol and were used for exposure of human cells (in vitro) or mice (in vivo). The cytokinesis-block micronucleus assay (OECD guideline 487) was performed in human bronchial epithelial cells (BEAS-2B) and primary cultures of human lymphocytes. Additionally, Comet assay was conducted in BEAS-2B cells. In vivo testing was carried out on a mouse model after exposure of groups of mice intravenously. The mammalian erythrocyte micronucleus test in mouse blood (OECD guideline 474) and comet assay in mouse organs were performed. Concurrent positive chemical controls and a nanoparticle control (ZnO) were included. While the results obtained in BEAS-2B cells showed no induction of micronucleated cells, a significant increase was observed in human lymphocytes at the dose of 125 μg/ml. Exposure of BEAS-2B to TiO2 caused an increase in DNA damage detected by comet assay (3-fold increase, p< 0.006) although no dose-response effect was seen. In mice, there was no genotoxicity in both assays. In summary, using a standardized preparation of nanomaterials, results obtained were mostly negative after TiO2 exposure, in both in vitro and in vivo assays. However, somewhat different genotoxicity outcomes may reflect tissue-specific effects affecting, e.g., cellular uptake of the nanomaterial.
- Biomonitoring the genetic effects of environmental tobacco smoke exposure in restaurant workersPublication . Vital, Nádia; Louro, Henriqueta; Antunes, Susana; Penque, Deborah; Simões, Tânia; Silva, Maria JoãoEnvironmental 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.
- Caracterização da genotoxicidade de nanomateriais manufaturados e potencial impacto na saúde humanaPublication . Louro, Henriqueta; Tavares, Ana; Antunes, Susana; Vital, Nádia; Lavinha, João; Silva, Maria JoãoOs nanomateriais manufaturados (NMs), isto é, materiais fabricados que contêm partículas em que uma ou mais dimensões externas se situam na gama de tamanhos compreendidos entre 1 nanómetro e 100 nanómetros1 apresentam propriedades físico-químicas únicas (e.g., dimensão, área superficial, funcionalização) que lhes conferem caraterísticas mecânicas, óticas, elétricas e magnéticas muito vantajosas, relativamente aos mesmos materiais na forma não nanométrica. Assim, tem-se assistido a um incremento significativo no desenvolvimento, produção e utilização de nanomateriais manufaturados a nível mundial e a uma rápida progressão das nanotecnologias como promotoras de inovação em termos de aplicações e produtos, nomeadamente, nas áreas da ciência, biomedicina e produtos de consumo. O desenvolvimento exponencial das nanotecnologias contrasta com a avaliação ainda insuficiente dos eventuais perigos associados aos nanomateriais, designadamente ao nível dos potenciais efeitos lesivos do genoma e suas consequências a longo termo na saúde humana e no ambiente. Neste contexto, a nanotoxicologia poderá dar um contributo inestimável, em particular, no que se refere aos efeitos genotóxicos e potencialmente tumorigénicos dos NMs.
- Caracterização da genotoxicidade de nanomateriais manufaturados numa linha celular de epitélio brônquico humanoPublication . Tavares, Ana; Louro, Henriqueta; Vital, Nádia; Antunes, Susana; Lavinha, João; Silva, Maria JoãoOs nanomateriais manufaturados (NMs) apresentam propriedades físico-químicas específicas que lhes conferem caraterísticas mecânicas, óticas, elétricas e magnéticas únicas e vantajosas para aplicações industriais e biomédicas. Contudo, o desenvolvimento exponencial das nanotecnologias contrasta com a avaliação ainda insuficiente dos seus eventuais perigos, nomeadamente ao nível dos potenciais efeitos lesivos do genoma e seu impacto na saúde humana e no ambiente. O presente trabalho, desenvolvido no âmbito do Projeto NANOGENOTOX (www.nanogenotox.com), teve como objetivo contribuir para a caracterização dos efeitos genotóxicos representativos de três classes de NMs utilizados em produtos de consumo - sílica sintética amorfa, dióxido de titânio (TiO2) e nanotubos de carbono de parede múltipla (MWCNTs) - numa linha celular derivada do epitélio respiratório humano. As propriedades físico-químicas dos NMs testados, bem como o seu comportamento em meio aquoso, foram previamente determinados por outros participantes do projeto através de vários métodos, incluindo dynamic light scattering e microscopia eletrónica de transmissão. Para a caracterização dos efeitos genotóxicos de cada NM recorreu-se a uma linha celular de epitélio brônquico humano (células BEAS-2B) expostas a várias concentrações de cada um dos NMs dispersos num meio aquoso, de acordo com um protocolo desenvolvido e estandardizado para o efeito. Foram utilizados, em simultâneo, controlos negativos e positivos incluindo, como controlo nanoparticulado, o óxido de zinco (ZnO). Perante a incerteza sobre qual o método mais adequado para testar a genotoxicidade dos NMs, selecionou-se a combinação de dois ensaios complementares: o ensaio do cometa que permite a quantificação de quebras ao nível do DNA e o ensaio do micronúcleo, um dos métodos mais sensíveis para detetar a indução de instabilidade cromossómica. Globalmente, os resultados de ambos os ensaios foram concordantes, sendo que não se identificaram efeitos genotóxicos inequívocos após exposição das células BEAS-2B aos NMs estudados. No caso do NM de TiO2, observou-se um ligeiro aumento de quebras no DNA após 24h de exposição, apesar de não se ter detetado indução de quebras cromossómicas pelo ensaio do micronúcleo, sugerindo a indução de um efeito lesivo transitório ao nível do DNA. Por sua vez, apenas a concentração mais elevada do ZnO nanoparticulado induziu um aumento significativo de quebras no DNA, sendo, no entanto, já citotóxica. Estes resultados serão comparados com os obtidos noutros Laboratórios em condições experimentais similares (comparação inter-laboratorial), para avaliação da sua consistência e, também, da adequação destes ensaios in vitro na avaliação da genotoxicidade dos NMs. Para além disso, espera-se que a integração dos presentes resultados com os obtidos em ensaios in vivo, contribua para uma caracterização mais completa e fidedigna da genotoxicidade destes nanomateriais. (*Ambas as autoras contribuiram igualmente para o trabalho.)
- Different genotoxic effects of multi-walled carbon nanotubes in A549 cells: implications for nanomaterials safety investigationPublication . Louro, Henriqueta; Tavares, Ana; Antunes, Susana; Vicente, Ana; Silva, Maria JoãoHuman exposure to nanomaterials (NM) has been increasing worldwide, either due to the growing of environmental sources or from increased deliberated production for application in consumer products and nanomedicine. In particular, single- and multi-walled carbon nanotubes (MWCNT) have been developed for industrial purposes, and their safety must be assured. The same properties that render MWCNT-based materials so attractive may also cause higher toxicity. In particular, the similarity, in size and shape, between MWCNTs and asbestos fibres has raised concerns about their potential effects in human health. Moreover, contradictory results concerning their genotoxicity and carcinogenicity have been reported and further safety assessment is urgent. The objective of the present work was to characterize the potential cyto- and genotoxic effects of two MWCNTs (NM402 and NM403) in a human type-II alveolar epithelial cell line (A549). Dispersions of each NM were freshly prepared and cultures were exposed to NMs concentrations ranging from 0.52-52.08 μg/cm2. The clonogenic assay was used to determine in situ cell survival (8-days exposure) and the cytokinesis-block micronucleus assay was carried out (48h-exposure) to evaluate genotoxicity. Concurrent control cultures were also analysed: vehicle control, positive control (mitomycin C, MMC) and reference NM (ZnO-NM110). The results of the clonogenic assay showed that both NMs induced a concentration-dependent reduction of the cell survival with IC50 of 25.15 and 27.63 μg/cm2 for NM402 and NM403, respectively. The highest concentrations of NM402, 26.04 and 52.08 μg/cm2, induced a 2-fold significant increase in micronucleated binucleate cells (MNBCs) compared with the vehicle controls (P=0.006 and 0.019, respectively). Regression analysis indicated a concentration-response relationship that was best fitted to a linear-quadratic model (R2= 0.861). However, no concentration-response relationship in MNBCs was observed for NM403. The cytokinesis-block proliferation index (CBPI) remained unaltered following A549 cells exposure to NM402 or NM403. The positive controls, MMC and ZnO significantly increased MNBCs frequency and concomitantly decreased CBPI. In summary, while both NMs were cytotoxic for A549 cells, their ability to cause DNA damage was distinct. NM403 was not genotoxic while NM402 caused a dose-dependent genotoxic effect, which may be related to a potential carcinogenic activity. The differences observed may be explained by structural differences between the two MWCNTs. Although both present low diameter, they differ in length, being NM402 the longest. Thus, the result of lower genotoxicity of NM403 is in line with the fibre paradigm of CNT toxicity, whereby the length would be critical to their toxic potential. However, the NMs also differ in the types and contents of impurities, being NM402 the less pure (>90%), which may contribute to the observed genotoxicity. Regarding safety assessment, the different genotoxicity observed for these two closely related NMs highlights the importance of investigating the toxic potential of each NM individually, instead of considering a common mechanism responsible for CNT toxicity, since physical-chemical characteristics are recognized as important toxicity determinants. Co-funded by EU Grant Agreement 2009 21 01 (NANOGENOTOX), in the framework of the Health Programme and INSA.
- Environmental Tobacco Smoke in Occupational Settings: Effect and Susceptibility Biomarkers in Workers From Lisbon Restaurants and BarsPublication . Vital, Nádia; Antunes, Susana; Louro, Henriqueta; Vaz, Fátima; Simões, Tânia; Penque, Deborah; Silva, Maria JoãoEnvironmental 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.
- Exposição ao Fumo Passivo no Local de Trabalho: Biomarcadores de Genotoxicidade e SusceptibilidadePublication . Antunes, Susana; Vital, Nadia; Louro, Henriqueta; Gomes, Filomena; Penque, Deborah; Simões, Tânia; Silva, Maria JoãoApesar 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.
- Fumo de Tabaco em Estabelecimentos Recreativos: Biomarcadores de Genotoxicidade e Susceptibilidade GenéticaPublication . Vital, Nádia; Antunes, Susana; Louro, Henriqueta; Penque, Deborah; Simões, Tânia; Silva, Maria JoãoA 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.
- Genotoxicity evaluation of nanosized titanium dioxide, synthetic amorphous silica and multi-walled carbon nanotubes in human lymphocytesPublication . Tavares, Ana Maria; Louro, Henriqueta; Antunes, Susana; Quarré, S.; Simar, S.; Temmerman, P.D.; Verleysen, E; Mast, J.; Jensen, K.A.; Norppa, H.; Nesslany, F.; Silva, Maria JoãoToxicological characterization of manufactured nanomaterials (NMs) is essential for safety assessment, while keeping pace with innovation from their development and application in consumer products. The specific physicochemical properties of NMs, including size and morphology, might influence their toxicity and have impact on human health. The present work aimed to evaluate the genotoxicity of nanosized titanium dioxide (TiO2), synthetic amorphous silica (SAS) and multiwalled carbon nanotubes (MWCNT), in human lymphocytes. The morphology and size of those NMs were characterized by transmission electron microscopy, while the hydrodynamic particle size-distributions were determined by dynamic light scattering. Using a standardized procedure to ensure the dispersion of the NMs and the cytokinesis-block micronucleus assay (without metabolic activation), we observed significant increases in the frequencies of micronucleated binucleated cells (MNBC) for some TiO2 NMs and for two MWCNTs, although no clear dose–response relationships could be disclosed. In contrast, all forms of SAS analyzed in this study were unable to induce micronuclei. The present findings increase the weight of evidence towards a genotoxic effect of some forms of TiO2 and some MWCNT. Regarding safety assessment, the differential genotoxicity observed for closely related NMs highlights the importance of investigating the toxic potential of each NM individually, instead of assuming a common mechanism and equal genotoxic effects for a set of similar NMs.