Centre for Environmental and Marine Studies

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A Shared Perspective on in Vitro and in Vivo Models to Assay Intestinal Transepithelial Transport of Food Compounds

Publication . Hevia, Arancha; Ruas-Madiedo, Patricia; Faria, Miguel Angelo; Petit, Valérie; Alves, Bruna; Alvito, Paula; Arranz, Elena; Bastiaan-Net, Shanna; Corredig, Milena; Dijk, Wieneke; Dupont, Didier; Giblin, Linda; Graf, Brigitte Anna; Kondrashina, Alina; Ramos, Helena; Ruiz, Lorena; Santos-Hernández, Marta; Soriano-Romaní, Laura; Tomás-Cobos, Lidia; Vivanco-Maroto, Santiaga María; Recio, Isidra; Miralles, Beatriz

Assessing nutrient bioavailability is complex, as the process involves multiple digestion steps, several cellular environments, and regulatory-metabolic mechanisms. Several in vitro models of different physiological relevance are used to study nutrient absorption, providing significant challenges in data evaluation. However, such in vitro models are needed for mechanistic studies as well as to screen for biological functionality of the food structures designed. This collaborative work aims to put into perspective the wide-range of models to assay the permeability of food compounds considering the particular nature of the different molecules, and, where possible, in vivo data are provided for comparison.

2023-11-30Journal article

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Overview of in vitro approaches to investigate chemical contaminants intestinal transport

Publication . Alves de Sousa, B.; Faria, M.; Miralles, B.; Alvito, Paula

The occurrence of chemical contaminants in food products is a serious threat to public health and can lead from mild gastroenteritis to fatal liver, kidney and neurological syndromes. The main route of exposure to chemical contaminants is ingestion, with the intestine being the first organ to come into contact with these compounds. In vitro intestinal cell assays may constitute a useful tool to predict the impact of chemical contaminants on the intestinal barrier. Differentiated Caco-2 cells cultivated in porous Transwell inserts, as an in vitro model to evaluate the intestinal transport of food chemical contaminants, is recurrent. These toxicants can be transported via transcellular (transport that occurs through the epithelial cell) or paracellular (transport that occurs in between cells) pathway. This study aims to make an overview of in vitro approaches to investigate chemical contaminants intestinal transport and associated pathways. A bibliographical search was carried out in two databases of scientific publications, “PUBMED” and “WEB OF SCIENCE”, using the keywords “food”, “chemical contaminants”, “cell transport”, in scientific publications from 1999 to 2021. Forty publications were used for data extraction. In sum, Caco-2 cells were the most frequently used model for the evaluation of the food contaminants intestinal transport. In a few works, this cell line was combined with the human HT-29 cells. In other studies, specifically evaluating the implication of membrane transporters, Madin-Darby canine kidney (MDCK) cells were used. HTC-8 intestinal organoids were also reported and gastric absorption of food toxicants was described in a single work using NCI-N87 cells. The use intestinal mucosa explants mounted in Ussing chambers, in studies of food contaminants intestinal transport in farmed animals, was also reported, although in much less extension. The transcellular route was the most reported pathway for chemical contaminants, with paracellular being less referred (mycotoxins and chloropropanols). Further studies concerning absorption and transport of chemical contaminants are needed to better characterize its permeability across epithelial membranes which will contribute to a comprehensive understanding of its impact on human health. In addition, their frequent co-occurrence raises an additional number of questions that can only be answered using high throughput in vitro methods.

2023-09Conference object

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Assessment of multiple mycotoxins in maize flour samples from Portugal under SafeGrains project

Publication . Duarte, S.; Magro, A.; Oliveira, C.; Rosim, R.; Alvito, Paula; Carvalho, M.O.

One of the most relevant problems in stored grains is the contamination by insects and mycotoxin-producing fungi. Tribolium castaneum is one of the most common insect pests of stored products. Its presence makes cereals more susceptible to the spread of the fungi Aspergillus flavus, which may produce mycotoxins. The aim of this work was to evaluate the mycotoxin profile of maize flour infested by T. castaneum adults, alone or in combination with mycotoxigenic A. flavus strain (MUM-UMinho). Maize collected directly from fields was stored at 4 ºC and then ground and sieved to obtain maize fl our. Multiple mycotoxin profile of maize flour was determined in three different assays: i) controls (n=8), ii) inoculated with T. castaneum adults (insect assay, n = 24), iii) inoculated with A. flavus conidia (fungus assay, n = 8) and iv) inoculated with both organisms (mixed assay, n = 24). Samples were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) according to Franco et al. (2019) for determination of aflatoxins (AFs) B1, B2, G1 and G2, deoxynivalenol (DON), zearalenone(ZEN) and fumonisins (FBs) B1 and B2. The limits of detection (LOD) and quantification (LOQ) of the analytical method were 6.1 and 18.0 μg/ kg for DON, respectively. For AFs, ZEN and FBs, LOD and LOQ varied between 0.2 – 0.9 and 0.6 – 2.5 μg/kg, respectively. DON, ZEA, FB1 and FB2 were detected in control assays at mean levels ranging from 33-50, 2-6, 112-144 and 49-75 μg/kg, respectively. AFs were not detected in controls. Insect, fungi and mixed assays presented the same mycotoxin profile (with contents in the same order of magnitude), except for AFB1, which was detected only in mixed assays. Although the levels of mycotoxins are below the legislated ones (Commission Regulation (EU) 2023/915), they point out to the presence of multiple mycotoxins in raw maize flour and to a potential impact between insects and mycotoxigenic strains in which concerns AFB1. Data presented here agree with previous assays from this team describing significantly higher concentration in the maize flour inoculated with both organisms (Duarte et al, 2021).

2023-09Conference object

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Aflatoxin B1 accumulation in Tenebrio molitor: a preliminary assessment

Publication . Andrade, M.A.; Cardoso, D.N.; Silva, A.R.R.; Prodana, M.; Santos, J.; Loureiro, S.; Alvito, Paula

With the population growth rate, there are some concerns that food production will not be able to keep up with this growth. Edible insects seem to present a sustainable solution. Farming these insects presents an opportunity to drain the production of the by-products by reusing them as bio-feedstocks and reintroducing these components into the food value chain. However, these products can present several contaminations, including mycotoxins, which can be accumulated in insects after exposure to the contaminant, and be detected at the end of the food chain. The ENTOSAFE project aims to address these concerns and evaluate the potential risk for the consumer. The principal aim of this study was to evaluate the mycotoxin aflatoxin B1 (AFB1) accumulation in Tenebrio molitor (yellow Mealworm, YMW) exposed to a spiked AFB1 feed subtract at maximum levels in cereals and products derived from cereals (2 µg/kg) and ten times higher (20 µg/kg). AFB1 contents were quantified in both feed substrate and T. molitor samples, before and after a 14-days of exposure and mycotoxins (aflatoxins and ochratoxin A, OTA), detected by HPLC-FD detection. Results concerning non-contaminated feed substrate revealed absence of AFB1 and presence of OTA, the latter (0,8 µg/kg) presenting values below the legislated value of 3 µg/kg, for cereals and products derived from cereals (European Commission, 2023). AFB1 spiked feed substrates revealed values slightly higher (4 and 23 µg/kg) than the theoretical contamination levels of 2 and 20 µg/kg. OTA values remain close to the previously reported. No changes occurred in contamination levels at beginning and 14-days AFB1 exposure assays. Results concerning T. molitor larvae, revealed absence of OTA along exposure assays and different AFB1 contamination levels. AFB1 contents in low (0.011 µg/kg) and high (0.022 µg/kg) AFB1 contamination levels were close, and below the 2 µg/kg legislated level for cereals and products derived from cereals (European Commission, 2023), after the 14-days exposure. The reported results are preliminary, so several aspects need to be improved as mycotoxin analytical method validation, mycotoxin contamination procedure and a higher number of samples to get representative results on AFB1 accumulation in insect larvae.

2023-09Conference object

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