Browsing by Author "Tavares, Ana"
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- Aflatoxins and ochratoxin A in baby foods and analysis of interactive cyto- and genotoxic effects in a human intestinal cell linePublication . Tavares, Ana; Alvito, Paula; Loureiro, Susana; Louro, Henriqueta; Silva, Maria JoãoMycotoxins are natural fungal metabolites and food contaminants with potential to cause severe acute and chronic conditions. Food contamination with mycotoxins such as aflatoxins (AF) and ochratoxin A (OTA) have been causing great concern, especially due to their potential mutagenic and carcinogenic effects. Children are especially vulnerable to the deleterious effects of these mycotoxins due to their physiological immaturity and high metabolic rate. Previous studies showed the co-occurrence of low concentrations of aflatoxins and OTA in baby foods. However, studies addressing potential interactive cyto- and genotoxic effects between these toxins are still scarce. In the present study we aimed to develop and validate a method for detection and quantification of total aflatoxins (B1, B2, G1, G2), AFM1 and OTA, and to evaluate the cytotoxic and genotoxic effects of mixtures of AFM1 and OTA, comparatively to their individual effects, in a human-derived intestinal cell line. A method based on immunoaffinity column cleanup and High Performance Liquid Chromatography with fluorescence detection (HPLC-FD), was applied and validated for total aflatoxins, AFM1 and OTA. The method was adequate for the analysis of these mycotoxins in baby foods and met the requirements of validation and quality control. The application of the method to a small set of baby foods marketed in Portugal showed an absence of quantifiable amounts of these mycotoxins. The individual and combined cytotoxic and genotoxic effects of AFM1 and OTA were characterized in Caco-2 cells using the Neutral Red and the Comet assays, respectively. A dose-dependent cytotoxicity was observed after individual exposure to OTA and AFM1, and the IC50 values were determined. The cytotoxic effect observed for several AFM1 and OTA mixtures was compared to the expected effect predicted by concentration addition (CA) and independent action (IA) conceptual models, using the MIXTOX model. A preliminary approach regarding the total data pool and considering the CA model as the most conservative model, pointed to an antagonistic cytotoxic effect caused by the mixture of both mycotoxins. However, a dose level deviation was observed after IA modelling, reflecting antagonism at low dose levels and synergism at higher dose levels. To better support data modelling, further cytotoxicity results from mixtures will be obtained and analyzed. To which respects the genotoxic effects, no induction of DNA damage was observed for the tested low doses, neither for individual toxins nor for their mixtures. The present study reinforces the relevance of exploring possible interactive adverse effects of mycotoxins that can contaminate foodstuff and thus having impact in human health. Future studies will face the challenge of understanding the mode of action of such mycotoxins when in mixture, in order to try predicting their effects.
- Alpha Amylase Activity Assay for In Vitro Food DigestionPublication . Alvito, Paula; Martins, Carla; Assunção, Ricardo; Tavares, Ana; Cost Action INFOGEST - Improving Health Properties of Food by Sharing our Knowledge on the Digestive Process
- 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.
- Avaliação integrada dos efeitos genotóxicos de nanomateriais manufaturados no ratinho transgénico LacZPublication . Louro, Henriqueta; Tavares, Ana; Vital, Nádia; Costa, Pedro M.; Alverca, Elsa; Lavinha, João; Silva, Maria JoãoNum estudo recente, demonstrámos que um nanomaterial de dióxido de titânio na forma cristalina designada anatase, o NM-102 (do repositório do Joint Research Center; Ispra, Itália), induziu um aumento significativo de quebras cromossómicas - detetáveis na forma de micronúcleos - em linfócitos humanos expostos ex vivo(3) não se tendo, porém, observado um efeito dose-resposta. No sentido de prosseguir e aprofundar a avaliação da genotoxicidade do NM-102, o presente estudo teve por objetivo caracterizar os seus efeitos genotóxicos, num modelo animal, utilizando uma abordagem integrada, a qual abrangeu a análise de vários parâmetros de genotoxicidade no mesmo animal.
- Bioaccessibility of aflatoxin M1 in three artificially contaminated infant formula using a standardised static in vitro digestion methodPublication . Tavares, Ana; Egger, C.; Portmann, R.; Martins, Carla; Assunção, Ricardo; Alvito, PaulaMycotoxins are fungal natural contaminants that commonly occur in a great variety of foods, and can form complexes with the food matrix with a significant impact on their bioaccessibility1. To our knowledge, until now no studies were performed to disclose the possible role of milk proteins in the bioaccessibility of aflatoxin M1 (AFM1), a mycotoxin commonly found in milk products, and no data are available on the use of a standardized in vitro digestion method2, to study mycotoxins bioaccessibility. This study aimed to evaluate the bioaccessibility of aflatoxin M1 in three artificially contaminated infant formula containing different casein/soluble protein ratios (A=40/60, B=60/40, C=80/20). Sample A showed mean bioaccessibility values of 105.6% ± 5.7 (n=8), sample B showed 88.7% ± 3.3 (n=7) and sample C showed 93.9% ± 4.4 (n=7). The bioaccessibility values of sample A were also significantly higher than those of samples B (p= 0.002, Mann-Whitney) and C (p= 0.024, Tukey HSD). The high bioaccessibility values observed agree well with those reported by Kabak and Ozbey (2012), for AFM1 in UHT milk (80.5-83.8% for naturally and 81.7-86.3% for artificially contaminated samples)3. More samples need to be analysed in the future in order to confirm a possible implication of caseins in the bioaccessibility of AFM1.
- Bioaccessibility of mycotoxins in baby foods using the harmonized in vitro digestion modelPublication . Martins, Carla; Tavares, Ana; Assunção, Ricardo; Alvito, PaulaObjectives: Food products provide essential nutrients, but also contaminants that affect human health. Mycotoxins are fungal natural contaminants commonly found in a great variety of foods including baby foods. Patulin (PAT) is a mycotoxin found in fruits and fruit based products1 and aflatoxin M1 (AFM1), the hydroxilated metabolite of AFB1, is a potent carcinogen, mainly found in milk and milk based products2. Mycotoxins can form complexes with the food matrix that may cause a significant impact on their bioaccessibility - the proportion of the ingested contaminant in food that reaches the systemic circulation3. This study aimed to evaluate the bioaccessibility of the mycotoxins PAT and AFM1 in powdered baby foods. Methodology: A standardized static in vitro digestion method4 was used to assess the bioaccessibility of PAT and AFM1 in two different powdered baby foods: 3 cereal and fruit based baby food and 3 infant formulae artificially contaminated to 20 µg/kg of PAT and 500 µg/kg of AFM1, respectively. Mycotoxins quantification was performed by HPLC-UV1 for PAT and HPLC-FLD for AFM12. Results: Patulin bioaccessibility in cereal and fruit based baby foods ranged between 49 % to 61 %. These results agree well with those reported by Brandon et al (2006)5 (84-100%) and are higher than those reported by Assunção et al (2014)6 (28 %), both in apple juices. AFM1 bioaccessibility in infant formulae ranged between 86 % and 104 % which agree with results from Kabak et al (2014)7. Both methodologies had a RSD below 15 %. Conclusions: These are the first results on mycotoxins bioaccessibility using the standardized static in vitro digestion method developed by the COST action INFOGEST. Future work must be focused on analyzing a broader number of samples in order to assess the influence of different food matrix in mycotoxin bioaccessibility.
- Biomonitorização humana e genotoxicidade ambiental: quatro décadas de investigação translacional sobre interação gene-ambiente e seu impacto em saúde públicaPublication . Louro, Henriqueta; Ventura, Célia; Vital, Nádia; Tavares, Ana; Silva, Maria João
- 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.)