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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.
- Microbiological monitoring of water for control of LegionellaPublication . Coelho, Carla; Pizarro, Cristina; Reinas, Alcina; Rebelo, HelenaLegionellosis is a collection of infections that are caused by Legionella pneumophila and related Legionella bacteria. The severity of legionellosis varies from mild febrile illness (Pontiac fever) to a potentially fatal form of pneumonia (Legionnaires’ disease) that can affect anyone, but principally affects those who are susceptible due to age, illness, immunosuppression or other risk factors, such as smoking. Legionella spp. are heterotrophic bacteria found in a wide range of water environments and proliferate at temperatures between 25°C and 45°C. These bacteria are members of the natural flora of many freshwater environments, such as rivers, streams and impoundments, where they occur in relatively low numbers. Bacteria of the genus Legionella are ubiquitous, they can proliferate in certain human-made water environments, such as water cooling devices associated with air conditioning systems, hot water, spas, distribution systems, on the inside surfaces of shower heads especially in warm waters. Legionellae can be ingested by certain amoebae which play an important role in their persistence in water environments. Devices that support multiplication of Legionella have been associated with outbreaks of Legionnaires’disease. Nosocomial cases usually make up a small proportion of reported cases of legionellosis. However, the proportion of cases that are fatal tends to be much higher with nosocomial infections than with community-acquired infections. Health-care facilities may include environments that support the proliferation and dissemination of Legionella and must be monitored. In Portugal only exists legislation for air conditioning systems and thermal waters. It is important to be aware of the need to develop specific rules for the detection of Legionella in water intended for human consumption in order to act more for the prevention of the disease than to act only in the monitoring of an outbreak. In recent years there has been an increasing incidence of disease caused by this bacterium, atypical pneumonia, and a large variety of water systems are different sources of contamination.
- 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.