Browsing by Author "Loureiro, Susana"
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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.
- Assessing Contaminant Uptake and Elimination in Insects: Implications for Sustainable Food Production and Waste ManagementPublication . Cardoso, Diogo; Silva, Ana; Duarte, Regina; Brandão, Pedro; Epifânio, Joana; Azevedo, Abraão; Prodana, Maria; Mostafaie, Amid; Pinto, José; Silva, Patrícia; Coelho, Inês; Rego, Andreia; Alvito, Paula; Brooks, Bryan; Loureiro, SusanaConsidering the global food crisis and the pressing issue of waste generation, sustainable solutions for food demand are crucial. Insects have emerged as a promising protein source for food and feed due to their efficient bioconversion capacity and ability to utilize nonvaluable products. However, ensuring the safety of insects for food and feed is essential. This study aims to comprehensively evaluate the uptake and elimination of contaminants by insects, going beyond conventional measurements of contamination levels. Methodology: The study employed two-phase bioaccumulation studies involving an uptake phase with a contaminated substrate followed by an elimination phase in a clean substrate. Data from these studies were used in toxicokinetic modelling. The objective was to assess the bioaccumulation capacity of mercury (Hg), cadmium (Cd), lead (Pb), arsenic (As), and various polycyclic aromatic compounds (benzo[a] pyrene [B[a]P], benz[a]anthracene [B[a]A], benzo[b]fluoranthene [B[a] F], and crysene [Cry]) in black soldier fly (BSF) larvae (Hermetia illucens). Although high metal bioaccumulation potential (e.g., for Hg, Cd, Pb, As) in edible insects has already been identified as risk, there is a gap regarding hazard assessment of the organic contaminants (e.g., B[a]P, B[a]A, B[a]F, Cry). The findings indicate that when insects were exposed to the maximum allowed levels of metals according to European Union feed regulations, they accumulated contaminant levels unsuitable for feeding other animals. However, after a depuration period, the insects could reduce the contaminant concentrations to comply with the existing legislation. For instance, a 4 to 5 days depuration period was found sufficient for safer consumption of insects exposed to Hg, while a longer period was required for Cd elimination. Interestingly, no significant accumulation of polycyclic aromatic compounds was observed when insects were fed at the maximum allowed levels. Conclusion: These findings have significant implications, particularly when insects are employed as waste converters, where concerns arise regarding contaminants. This study supports the implementation of a depuration period after exposure to waste that may vary depending on the classes of contaminants involved. Toxicokinetics is an adequate tool for the safety assessment of insects as feed and food, but it also defines the conditions that will assure their sustainable use in waste management.
- Combined cytotoxic and genotoxic effects of ochratoxin A and fumonisin B1 in human kidney and liver cell modelsPublication . Pinhão, Mariana; Tavares, Ana Maria; Loureiro, Susana; Louro, Henriqueta; Alvito, Paula; Silva, Maria JoãoFood products can be contaminated by several fungi species and each species may produce different mycotoxins, leading to human combined exposure. Although predictions about the joint toxic effects of mycotoxins can be made from their individual toxicities, experimental data is still limited to allow a reliable hazard assessment. Thus, this study aimed to characterize the combined cytotoxic and genotoxic effects of ochratoxin A (OTA) and fumonisin B1 (FB1) in cell lines representative of their target organs, kidney and liver. Interactions were ascertained using mathematical extensions to the reference models of concentration addition and independent action. Cytotoxicity (MTT assay) data modeling revealed a synergistic pattern for low doses of both FB1 and OTA shifting to antagonism at higher concentration levels, irrespectively of the reference model applied. Concerning genotoxicity assessment, neither OTA nor FB1, individually or in combination, induced a prominent increase in DNA damage (comet assay) or oxidative DNA damage (FPG-comet assay). In conclusion, this study revealed a synergistic cytotoxic effect for OTA and FB1 at low concentration levels. Given that human co-exposure to these two mycotoxins is probable to occur at low doses, these results raise concerns regarding their potential health outcomes that seem to differ from those predicted by an additive model.
- A contribution to hazard assessment of combined exposure to mycotoxins using in vitro toxicity testingPublication . Tavares, Ana; Pinhão, Mariana; Mendonça, Inês; Louro, Henriqueta; Loureiro, Susana; Alvito, Paula; Silva, Maria JoãoHumans may be exposed through diet to a complex and variable combination of mycotoxins co-occurring in food. These toxins may impact on their health, particularly on the long-term, because most of them are mutagenic and carcinogenic (e.g., aflatoxin B1). Moreover, the overall hazard of a particular mycotoxin may be further modified by its interaction with other mycotoxins and, in this context, many data gaps still remain. To overcome this issue, EFSA has recommended the use of concentration addition (CA) as a default assumption for the assessment of combined toxicity of chemicals, provided they produce a common adverse outcome. The present study aimed at identifying potential interactive toxic effects of two binary mixtures of mycotoxins – ochratoxin A (OTA)/aflatoxin M1 (AFM1) and OTA/fumonisin B1 (FB1) – in human cell lines. Cytotoxic effects were measured by the neutral red or the MTT assays while genotoxicity was assessed by the comet assay. Comparison of the viability results obtained for OTA in intestinal (Caco-2), liver (HepG2) and kidney (HK-2) cell lines showed that HK-2 cells were the most sensitive ones. On the other hand, the highest doses of AFM1 exerted a significant toxicity in Caco-2 cells, while FB1 displayed a lower toxic effect in HepG2 and HK-2 cells. The interactive toxic effects of several combinations of AFM1 and OTA were inferred from modelling the experimental data obtained for mixtures, comparatively to that expected from the individual effects, using a CA conceptual model. The results indicated that both mycotoxins exerted antagonistic effects in Caco-2 cells. In contrast, in HK-2 cells the combination of OTA and FB1 was synergistic at low dose levels of both mycotoxins and changed to antagonism at higher dose levels. Genotoxicity analysis of the same combinations is underway and data will be presented and discussed. Overall, this study emphasizes the relevance of assessing the combined toxicity of mycotoxins, thereby contributing to fill the data gaps on mixtures effects and allowing the development of qualitative/semi-quantitative or probabilistic models for the hazard assessment of combined exposure to these food contaminants.
- ENTOSAFE - Edible insects: From a sustainable food production to a food safety concern.Publication . Cardoso, Diogo; Mostafaie, Amid; Silva, Ana Rita; Motta, Carla; Murta, Daniel; Pinto, Glória; Coelho, Inês; Prodana, Marija; Silva, Patricia; Alvito, Paula; Calisto, Vania; Loureiro, SusanaThe use of insects as a food and feed source is nowadays considered a solution for the increasing food demands in the following years. With that in mind, the recently funded project “ENTOSAFE - Edible insects: From a sustainable food production to a food safety concern” aims to integrate the food safety and the environmental sustainability aspects of edible insect farming. ENTOSAFE will take advantage of a symbiotic multidisciplinary team with members from industry (EntoGreen® - Ingredient Odyssey, Portugal) and academia (Univ. of Aveiro and National Health Institute Doutor Ricardo Jorge, Portugal). The project is committed to provide knowledge on the accumulation and transfer of potential contaminants (e.g., metals, mycotoxins, PAHs, pharmaceuticals) in insects farmed in rearing facilities, contributing directly with data sets to the new legislation and regulatory limits, concerning the presence of these compounds in the substrate. ENTOSAFE brings new approaches, focusing on full bioaccumulation studies through a complete uptake phase, assessing the contaminant into insects in different times during this phase. Considering the lack of information on insects’ elimination capacity to excrete contaminants from their body, an elimination phase will be studied in a non-contaminated substrate. ENTOSAFE is also promoting environmentally sustainable strategies while avoiding any wasted components from the process. The application of frass, as an organic fertilizer - produced by the digestion of the agricultural wastes or other residues by insects - on crops is presented as a promising solution to a zero-waste policy. Despite promising, the available information on the benefits that this by-product has on crop productivity is scarce. In this context, ENTOSAFE seeks evidence on the impact of this frass on agricultural soil’s main functions and the key processes behind. The project will evaluate the effects in soil quality and functions, soil-biota interactions in soils enriched with organic amendments and plant responses to stressors, by performing a series of experiments from laboratory to greenhouse scale. In summary, ENTOSAFE will contribute to an upscaling on the use of edible insects as feed and food, covering not only safety concerns but also promoting the sustainability of insect rearing processes.
- Estudos da toxicidade de misturas de aflatoxina M1 e ocratoxina A numa linha celular humanaPublication . Tavares, Ana Maria; Alvito, Paula; Loureiro, Susana; Louro, Henriqueta; Silva, Maria João
- Exploring BPA alternatives - Environmental levels and toxicity reviewPublication . Adamovsky, Ondrej; Groh, Ksenia J.; Białk-Bielińska, Anna; Escher, Beate I.; Beaudouin, R.; Mora Lagares, Liadys; Tollefsen, Knut Erik; Fenske, Martina; Mulkiewicz, Ewa; Creusot, Nicolas; Sosnowska, Anita; Loureiro, Susana; Beyer, Jonny; Repetto, Guillermo; Štern, Alja; Lopes, Isabel; Monteiro, Marta; Zikova-Kloas, Andrea; Eleršek, Tina; Vračko, Marjan; Zdybel, Szymon; Puzyn, Tomasz; Koczur, Weronika; Ebsen Morthorst, Jane; Holbech, Henrik; Carlsson, Gunnar; Örn, Stefan; Herrero, Óscar; Siddique, Ayesha; Liess, Matthias; Braun, Georg; Srebny, Vanessa; Žegura, Bojana; Hinfray, Nathalie; Brion, François; Knapen, Dries; Vandeputte, Ellen; Stinckens, Evelyn; Vergauwen, Lucia; Behrendt, Lars; Silva, Maria João; Blaha, Ludek; Kyriakopoulou, KaterinaBisphenol A alternatives are manufactured as potentially less harmful substitutes of bisphenol A (BPA) that offer similar functionality. These alternatives are already in the market, entering the environment and thus raising ecological concerns. However, it can be expected that levels of BPA alternatives will dominate in the future, they are limited information on their environmental safety. The EU PARC project highlights BPA alternatives as priority chemicals and consolidates information on BPA alternatives, with a focus on environmental relevance and on the identification of the research gaps. The review highlighted aspects and future perspectives. In brief, an extension of environmental monitoring is crucial, extending it to cover BPA alternatives to track their levels and facilitate the timely implementation of mitigation measures. The biological activity has been studied for BPA alternatives, but in a non-systematic way and prioritized a limited number of chemicals. For several BPA alternatives, the data has already provided substantial evidence regarding their potential harm to the environment. We stress the importance of conducting more comprehensive assessments that go beyond the traditional reproductive studies and focus on overlooked relevant endpoints. Future research should also consider mixture effects, realistic environmental concentrations, and the long-term consequences on biota and ecosystems.
- Hazard identification and characterization of leachable chemicals from plastic products – a new PARC projectPublication . Dirven, Hubert; Bogusz, Aleksandra; Hans Bouwmeester; Busch, Mathias; Duflos, Guillaume; Eriksen, Gunnar S.; Fardilha, Margarida; Flores-Gomez, Daniela; Franko, Nina; Gaté, Laurent; Guichard, Yves; Silva, Maria Joao; Kamstra, Jorke H.; Kasiotis, Konstantinos M.; Kim, Sunmi; Kim, Young Jun; Kim, Youngsam; van der Koogh, Elise; Loureiro, Susana; Louro, Henriqueta; Machera, Kyriaki; Pieters, Raymond H. H.; Spyropoulou, Anastasia; Tzanetou, Evangelia N.; Malheiro, Catarina; Ravnjak, Tim; Repetto, Guillermo; Rivière, Gilles; Ryu, Chang Seon; Papadopoulou, Evgenia Anna; Aliferis, Konstantinos A.; Solhaug, Anita; Sollner Dolenc, Marija; Štampar, Martina; Tavares, Ana M.; Tollefsen, Knut Erik; Ventura, Célia; Walkowiak, Radoslaw; Zobl, Walter; Žegura, Bojana; Snapkow, Igor; Herzke, DorteA recent study has suggested that plastics may contain more than 16,000 chemicals, including additives, processing aids, starting substances, intermediates and Non-Intentionally Added Substances. Plastic chemicals are released throughout the plastic life cycle, from production, use, disposal and recycling. Most of these chemicals have not been studied for potential hazardous properties for humans and in the environment. To refine the risk assessment of these leachable chemicals, additional hazard data are needed. The PlasticLeach project within the EU co-funded Partnership for the Assessment of Risks from Chemicals (PARC) aims to address this data gap by screening several plastic products in daily use. Leachates will be prepared from a number of these plastic items, and these chemical mixtures will be further tested using several test guideline compliant assays and New Approach Methodologies covering both human health and environmental endpoints. The most toxic leachates will be characterized using a non-targeted analysis pipeline to identify chemicals in the leachate. When single chemicals of concern are identified, these will be further tested to determine hazardous properties and identify the respective potency factors to better understand their specific hazard profiles. A tiered approach for hazard testing will be followed. The experimental work will be complemented by toxicological profiling, using publicly available toxicity databases and tools, including Artificial Intelligence tools that cover both human and environmental endpoints. A comprehensive array of endpoints, including cytotoxicity, endocrine disruption, genotoxicity, immunotoxicity, reproductive toxicity and effects related to ecotoxicity will be evaluated. In this paper, we outline the plastic products to be tested and the battery of assays that will be used to identify hazards relevant to both human health and the environment. Data generated from approaches will be reported using standardized formats, stored within a centralized repository, and harmonized to adhere to the FAIR data principles (Findable, Accessible, Interoperable, and Reusable). This integrated strategy will not only advance our understanding of the risks associated with plastic-derived chemicals but will also provide critical support for regulatory decision-making and facilitate the development of safer, and more ecofriendly plastic materials in the future.
- Mixtures health effects: mycotoxins in foodPublication . Pinhão, Mariana; Loureiro, Susana; Louro, Henriqueta; Alvito, Paula; Silva, Maria JoãoIn recent years, the risk assessment paradigm has shifted from the single-exposure and single-chemical adverse effect scenario to the one of multiple exposures and combined adverse effects. The present work describes the in vitro combined toxicity of mycotoxins at the renal level, as a case study. Mycotoxins are often present in food and feed, as secondary metabolites of contaminating fungi and human co-exposure mainly occurs though diet. Even though predictions about the toxic effects of mycotoxins mixtures can be based on their individual toxicities, experimental data is still limited to allow a reliable hazard assessment. This study aimed at characterizing the combined cytotoxic and genotoxic effects of ochratoxin A (OTA) and fumonisin B1 (FB1), in a kidney human cell line. The toxicity of several combinations of OTA and FB1 was compared with their individual toxicities (MTT assay) and interactions were ascertained using the reference models of concentration addition and independent action. A synergistic pattern for combinations of FB1 with the lower doses of OTA was detected, shifting to antagonism at higher dose levels, irrespectively of the reference model applied. Neither OTA nor FB1, individually or in combination, were genotoxic. In conclusion, this study revealed that, OTA and FB1 exert a synergistic toxic effect at the lowest dose levels, which are the most realistic ones in terms of human co-exposure. This finding emphasizes the relevance of assessing the combined toxicity of mycotoxins to allow the development of qualitative/semi-quantitative or probabilistic models for the hazard assessment of combined human exposure to these food contaminants.
- A multi-endpoint approach to the combined toxic effects of patulin and ochratoxin A in human intestinal cellsPublication . Assunção, Ricardo; Pinhão, Mariana; Loureiro, Susana; Alvito, Paula; Silva, Maria JoãoHumans can be exposed to a complex and variable combination of mycotoxins. After ingestion, intestinal mucosa constitutes the first biological barrier that can be exposed to high concentrations of these toxins. The present study aimed to characterize the combined cytotoxicity, genotoxicity and impact on the gastrointestinal barrier integrity of patulin (PAT, 0.7 μM to 100 μM) and ochratoxin A (OTA, 1 μM to 200 μM) mixtures in Caco-2 cells. A dose-ratio deviation was verified for cytotoxicity, implying that OTA was mainly responsible for synergism when dominant in the mixture, while this pattern was changed to antagonism for the highest PAT concentrations. Genotoxicity (comet assay) results were compatible with an interactive DNA damaging effect at the highest PAT and OTA concentrations, not clearly mediated by the formation of oxidative DNA breaks. Regarding gastrointestinal barrier integrity, a potential synergism was attained at low levels of both mycotoxins, changing to antagonism at higher doses. The present results indicate that combined mycotoxins effects may arise at the intestinal level and should not be underestimated when evaluating their risk to human health.