DAN - Apresentações orais em encontros internacionais
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- Aflatoxin exposure through food consumption – are we ready to face the risk associated to climate change?Publication . Assunção, Ricardo; Alvito, Paula; Jakobsen, Lea; Pires, S.Climate change (CC) has been indicated as a driver for food safety issues worldwide, mainly due to the impact on the occurrence of food safety hazards at various stages of food chain. Mycotoxins, natural contaminants produced by fungi, are mentioned to be one of the most important food safety hazards affected by CC. Aflatoxins, which have the highest acute and chronic toxicity of all mycotoxins, assumes particular importance within this context. A recent study predicted aflatoxin contamination in maize and wheat crops in Europe within the next 100 years. The authors concluded that aflatoxin B1 (AFB1) is predicted to become a food safety issue in Europe, especially in the +2 °C scenario, the most probable scenario of CC expected for the next years. In Europe, previous reported studies estimated mean dietary exposures to aflatoxins for the general population from all food sources ranging from 0.93 ng kg bw-1 day-1 to 2.4 ng kg bw-1 day-1. In Portugal, an estimation of a probable daily exposure to aflatoxins of 0.501 ng kg bw-1 day-1 by the Portuguese population was reported. In addition, other authors also estimated aflatoxin exposure of Portuguese children (1-3 years) and reported a potential health concern associated to the exposure, wherein AFB1 was the main contributor. Considering the potential risks associated to Portuguese exposure to aflatoxins through diet, and the potential influence of CC on the temperature, humidity, precipitation and consequently on the mycotoxin contamination of food products, this presentation will discuss the CC impact on public health. To face this objective, burden of disease (BoD) associated to aflatoxin exposure of the Portuguese population will be estimated, for the first time, using a model of three components: an exposure, health-outcome and disability adjusted life years (DALY) modules. BoD of different scenarios will be discussed in light of the current knowledge about recent estimates of CC impact in Europe.
- 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.
- An emergent challenge: mycotoxins, climatic change and human healthPublication . Alvito, PaulaDuring the past 60 years, it has become clear that the world has to deal with mycotoxin exposure. Mycotoxins are a group of naturally occurring toxic chemical substances, produced mainly by filamentous fungi that can produce adverse health effects when consumed by humans and animals. It is known that mycotoxins have nephrotoxic, genotoxic, teratogenic, carcinogenic, and cytotoxic properties and, as a consequence, these toxins may cause liver carcinomas, renal dysfunctions, and also immunosuppressed states. Changes in temperature, precipitation, and atmospheric CO2 concentration are expected to carry along an increased risk of mycotoxin contamination mainly associated with cereal crops in the field, and might have an impact on the geographical distribution of certain cereals, mycotoxigenic fungi and their mycotoxins. The large impacts of global warming projected on crops worldwide will subsequently influence food security, by reducing yields and thus food availability, and food and feed safety. Despite prevention methods and strict regulations, mycotoxins are still present in the feed and food chain, and could produce mycotoxicoses. Mycotoxicosis can occur at every stages of life, and it can affect the individuals differently according to their age. The harmful effects of mycotoxins on cell division can lead to drastic consequences, which are even more severe during intrauterine life. Within this context, a recent ongoing project on early-life exposure to mycotoxins and its impact on health, EarlyMYCO (PTDC/MED-TOX/28762/2017), will be presented and discussed, as a contribute to evaluate mycotoxins exposure of pregnant women and child and associated risks, during first 1000 days of life.
- Are Portuguese children exposed to mycotoxins through infant foods? A preliminary approachPublication . Assunção, Ricardo; Vasco, Elsa; Nunes, Baltazar; Alvito, PaulaMycotoxins are a wide group of fungal secondary metabolites that cause toxic and carcinogenic outcomes in humans exposed to them. The major foodborne mycotoxins of public health interest are the aflatoxins, fumonisins, trichothecene and ochratoxin A (OTA)1,2. Infants have a more restricted diet and they generally consume more food on a body weight basis than adults thus they are a particularly vulnerable population group to food contaminants as mycotoxins. In Portugal, Alvito et al. had reported the presence of aflatoxin B1 (AFB1) and M1 (AFM1) and OTA in baby foods3 and no data are available until now concerning the exposure assessment of Portuguese children to these mycotoxins. Exposure assessment, which evaluates the degree of intake of a certain contaminant, is one of the four steps included in risk assessment. Although several scientific reports have been published in order to propose the best methodologies for the exposure assessment framework, to date harmonization is far from being achieved4. The aim of the present study was to estimate the exposure of Portuguese children to mycotoxins due to infant foods ingestion, based on a probabilistic approach, using a risk analysis software (@Risk 6 for Excel, Palisade). The mycotoxin occurrence3 of AFB1, AFM1 and OTA in infant foods and consumption data for children until 3 years old were modeled. Consumption data were based on preliminary results recently obtained during a pilot study performed at Primary Health Care Unit from Cascais under the Mycomix Project. Different strategies had been considered to treat the left censored data.
- 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.
- Building capacity in risk-benefit assessment of foods: lessons learned from the RB4EU ProjectPublication . Boué, Géraldine; Assunção, Ricardo; Alvito, Paula; Brazão, R.; Carmona, Paulo; Carvalho, Catarina; Correia, D.; Fernandes, Paulo; Jakobsen, Lea; Lopes, Carla; Martins, Carla; Membré, Jeanne-Marie; Monteiro, Sarogini; Nabais, Pedro; Thomsen, Sofie; Torres, Duarte; Pires, SaraRisk-Benefit Assessment (RBA) of food, food ingredients and diets, has recently emerged to address multidisciplinary public health issues. RBA aims to assess in a global perspective potential risks and benefits related to microbiology, toxicology and nutrition; in order to evaluate different options and to support decision-making process. At this time, significant methodological progress have been made but only few research groups have experience in RBA. To promote knowledge transfer, the RiskBenefit4EU project was funded by the European Food Safety Authority (EFSA). The objective of the presentation will be to introduce the strategy developed to capacitate a new team in RBA and to give an overview on lessons learned from this experience. The new team was comprised of experts in risk assessment in toxicology, microbiology or nutrition, epidemiology, dietary assessment or data analysis to answer to a specific multidisciplinary public health issue on cereal-based foods consumed by young children considering breakfast and infant cereals. Activities of the capacity building experience were designed by experienced researchers in RBA to transfer a harmonized methodology. Two weeks of face-to-face training were organized, one on theoretical concepts used in RBA and one on their application to the cereal case study. This experience has highlighted the necessity to build a common language within the team by harmonizing important concepts including hazard, health effect, adverse health effect, beneficial health effect, risk, benefit, health and health impact. It was also important that all participant learn basis of all area of expertise used in RBA. A harmonized RBA stepwise approach was introduced with key steps illustrated with examples of previous RBA performed. Finally, the RBA case study was initiated to design a shared protocol. The learning by doing process experienced under the RB4EU project enabled to create the first training in RBA. The strategy developed, with materials and method used, can now be re-used to capacitate other new teams in RBA and can be considered as a robust basis to build on.
- Cellular, Molecular and Genotoxic Effects of Digested Titanium Dioxide NanomaterialsPublication . Rolo, Dora; Pereira, Joana F.S; Vieira, Adriana; Roque, Rossana; Gramacho, Ana Catarina; Vital, Nádia; Matos, Paulo; Gonçalves, Lídia; Bettencourt, Ana F.; Silva, Mafalda A.; Martins, Carla; Assunção, Ricardo; Alvito, Paula; Jordan, Peter; Silva, Maria João; Louro, HenriquetaHuman exposure to titanium dioxide nanomaterials(TiO2NMs) occurs particularly by ingestion, due to food/food contact materials and consumer products. However, the possibility of adverse effects in gastrointestinal tract is unclear. Aiming to study the impact of digestion on the NMs’ properties and their cellular/molecular effects, two human intestinal cell lines were used, Caco-2 and HT29-MTX-E12. After exposure to TiO2NMs(NM-102, NM-103, NM-105), undigested or subjected to standardized static in vitro digestion method (mimicking human digestion), the cells were analyzed for toxicity, genotoxicity, reactive oxygen species, NM uptake and intestinal translocation. We showed that in vitro digestion of TiO2NMs may increase their toxicity and DNA-damaging effect, depending on the NM, more relevant for the rutile/anatase NM-105, possibly due to its smaller hydrodynamic size in the cellular medium. Effects on chromosomal integrity were seen in HT29-MTX-E12 cells, for all tested TiO2NMs, especially after digestion. Internalization into early endosomes was confirmed for NM-103 and NM-105, before and after digestion, in monolayers of both cell lines, and at the apical membrane of polarized Caco-2 cells. The internalized NMs accumulated in late endosomes/multivesicular bodies, partially transversing the basolateral membrane of polarized Caco-2 cells without changing transepithelial electrical resistance or epithelial marker abundance. These results suggest that part of the TiO2NMs can be transcytosed through colonic epithelia without disrupting intestinal barrier integrity. Overall, the biological outcomes from TiO2NMs interaction with intestinal cells were more pronounced after digestion, highlighting its relevance in the hazard assessment of ingested NMs.
- Children exposure assessment to food additives: an exploratory studyPublication . Calmeiro, Ana; Alvito, Paula; Vasco, ElsaThe increase of processed products in children´s diet has been followed by an increase in the consumption of food additives. The present exploratory study aimed to: i) evaluate the exposure of a Portuguese group of children, from 0 to 3 years of age, to food additives; ii) study its association with age, gender and Body Mass Index (BMI); iii) identify the main food categories that contribute to food additives intake.
- Combined exposure to multiple mycotoxins in food: are we ready for risk assessment and risk management?Publication . Alvito, Paula; Assunção, Ricardo; Silva, M.J.It is nowadays recognized that the risk of human co-exposure to multiple mycotoxins is real. In the last years, a number of studies have approached the issue of co-exposure and the best way to develop a more precise and realistic assessment. Likewise, the growing concern about the combined effects of mycotoxins and their potential impact on human health has been reflected by the increasing number of toxicological studies on the combined toxicity of these compounds. Nevertheless, risk assessment of these toxins, still follows the conventional paradigm of single exposure and single effects, incorporating only the possibility of additivity but not taking into account the complex dynamics associated to interactions between different mycotoxins or between mycotoxins and other food contaminants. Considering that risk assessment is intimately related to the establishment of regulatory guidelines, once the risk assessment is completed, an effort to reduce or manage the risk should be followed to protect public health. Risk assessment of combined human exposure to multiple mycotoxins thus poses several challenges to scientists, risk assessors and risk managers and opens new avenues for research. This presentation aims to give an overview of the different challenges posed by the likelihood of human co-exposure to mycotoxins and the possibility of interactive effects occurring after absorption, towards knowledge generation to support a more accurate human risk assessment and risk management. For this purpose, a physiologically-based framework that includes knowledge on the bioaccessibility, toxicokinetics and toxicodynamics of multiple toxins is proposed. Regarding exposure assessment, the need of harmonized food consumption data, availability of multianalyte methods for mycotoxin quantification, management of left-censored data and use of probabilistic models will be highlight, in order to develop a more precise and realistic exposure assessment. On the other hand, the application of predictive mathematical models to estimate mycotoxins’ combined effects from in vitro toxicity studies will be also discussed. Results from a recent Portuguese project aimed at exploring the toxic effects of mixtures of mycotoxins in infant foods and their potential health impact will be presented as a case study, illustrating the different aspects of risk assessment highlighted in this presentation. Further studies on hazard and exposure assessment of multiple mycotoxins, using harmonized approaches and methodologies, will be crucial towards an improvement in data quality and contributing to holistic risk assessment and risk management strategies for multiple mycotoxins in foodstuffs.
- Exploring the toxic effects of MYCOtoxins MIXtures in infant food and potential health impactPublication . Alvito, Paula; Arruda, Noélia; Assunção, Ricardo; Barreira, M.J.; Vasco, Elsa; Tavares, Ana; Calhau, Maria AntóniaThere is growing concern within public health about mycotoxin involvement in human diseases particularly related to children´s exposure through contaminated food1 . The natural co-occurrence of mycotoxins is an increasing concern which could be expected to exert greater toxicity and carcinogenicity than exposure to single mycotoxins2. The present project aimed to study the occurrence of multiple mycotoxins and toxicity interactions in baby foods and cereals consumed by Portuguese children. Scarce data are available in the literature concerning the co-occurrence of mycotoxins in infant food and their combined toxicity and no data exists in Portugal concerning this issue. This project gathered a multidisciplinary team in order to answer to several questions: 1) Are children exposed daily to mycotoxins through food? 2) What are the quality and quantity that characterize this exposure? 3) Can this exposure bring harm to children? Will it put them on risk? The co-occurrence of several mycotoxins were evaluated in different infant foods (cereal based) marketed at Portugal (Lisboa). These results, combined with the consumption data from a food consumption survey performed at the Primary Health Care Unit from Cidadela, Cascais (children aged until 3 years old) were used to estimate the intake of mycotoxins in Portugal, through a probabilistic approach using @Risk software. Toxicological studies including bioavailability and absorption of mycotoxins3 and its interactive effects4 were also performed.