Browsing by Author "Loureiro, S."
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- Aflatoxin B1 accumulation in Tenebrio molitor: a preliminary assessmentPublication . Andrade, M.A.; Cardoso, D.N.; Silva, A.R.R.; Prodana, M.; Santos, J.; Loureiro, S.; Alvito, PaulaWith 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.
- Application of mathematical models to mycotoxins children risk assessment: a case study of Portuguese children exposure to co-occurring mycotoxins in processed cereal-based foodsPublication . Assunção, Ricardo; Vasco, Elsa; Nunes, Baltazar; Loureiro, S.; Martins, Carla; Alvito, PaulaPeople, animals and the environment can be exposed to multiple chemicals at once from a variety of sources, but current risk assessment is usually carried out based on one chemical substance at a time. In human health risk assessment, ingestion of food is considered a major route of exposure to many contaminants, namely mycotoxins, a wide group of fungal secondary metabolites that are known to potentially cause toxicity and carcinogenic outcomes. Mycotoxins are commonly found in a variety of foods including those intended for consumption by infants and young children and have been found in processed cereal-based foods available in the Portuguese market. The use of mathematical models, including probabilistic approaches using Monte Carlo simulations, constitutes a prominent issue in human health risk assessment in general and in mycotoxins exposure assessment in particular. The present study aims to characterize, for the first time, the risk associated with the exposure of Portuguese children to single and multiple mycotoxins present in processed cereal-based foods (CBF). Portuguese children (0-3 years old) food consumption data (n=103) were collected using a 3 days food diary. Contamination data concerned the quantification of 12 mycotoxins (aflatoxins, ochratoxin A, fumonisins and trichothecenes) were evaluated in 20 CBF samples marketed in 2014 and 2015 in Lisbon; samples were analyzed by HPLC-FLD, LC-MS/MS and GC-MS. Daily exposure of children to mycotoxins was performed using deterministic and probabilistic approaches. Different strategies were used to treat the left censored data. For aflatoxins, as carcinogenic compounds, the margin of exposure (MoE) was calculated as a ratio of BMDL (benchmark dose lower confidence limit) to the aflatoxin exposure. The magnitude of the MoE gives an indication of the risk level. For the remaining mycotoxins, the output of exposure was compared to the dose reference values (TDI) in order to calculate the hazard quotients (ratio between exposure and a reference dose, HQ). For the cumulative risk assessment of multiple mycotoxins, the concentration addition (CA) concept was used. The combined margin of exposure (MoET) and the hazard index (HI) were calculated for aflatoxins and the remaining mycotoxins, respectively. 71% of CBF analyzed samples were contaminated with mycotoxins (with values below the legal limits) and approximately 56% of the studied children consumed CBF at least once in these 3 days. Preliminary results showed that children exposure to single mycotoxins present in CBF were below the TDI. Aflatoxins MoE and MoET revealed a reduced potential risk by exposure through consumption of CBF (with values around 10000 or more). HQ and HI values for the remaining mycotoxins were below 1. Children are a particularly vulnerable population group to food contaminants and the present results point out an urgent need to establish legal limits and control strategies regarding the presence of multiple mycotoxins in children foods in order to protect their health. The development of packaging materials with antifungal properties is a possible solution to control the growth of moulds and consequently to reduce mycotoxin production, contributing to guarantee the quality and safety of foods intended for children consumption.
- Efeitos tóxicos combinados de misturas de micotoxinas em linhas celulares humanasPublication . Tavares, Ana; Mendonça, I.; Loureiro, S.; Louro, H.; Alvito, Paula; Silva, M.J.
- Effects of mercury and tobacco smoke prenatal exposure in newborns from Aveiro region, PortugalPublication . Alves, A.C.; Monteiro, M.S.; Costa, C.; Soares, A.M.V.M.; Teixeira, João Paulo; Loureiro, S.It is widely known that people are daily exposed to a number of chemicals due to environmental contamination and/or habits or lifestyle. One of those chemicals is mercury (Hg), which is a naturally occurring, nonessential metal and it is ubiquitous in its different forms. Humans can be exposed to Hg through diet (fish and shellfish), industry, and medical applications such as dental amalgams and cosmetics. Nowadays the Ria de Aveiro region is still considered an hotspot for Hg contamination and therefore an important study area. Another potential threat to humans is tobacco smoke either by first or secondhand exposure, despite the effort on tobacco control policies in work and social places. Hg and tobacco smoke have been considered a concern to the public health since they cause irreversible adverse effects on functional and organic systems. During organogenesis, the developing embryo is particularly sensitive to teratogens and therefore the mother’s diet and lifestyle play a crucial role in the risk of fetus exposure to contaminants in this critical window of development. In this context, the main objective of this work was to assess the exposure of mothers and their newborns from the Aveiro district to Hg and tobacco smoke and to assess their relation with neurotoxic and genotoxic biomarkers of effects.
- Evaluation of combined cytotoxic effects of effects of ochratoxin A and fumonisina B1 in human liver and renal cellsPublication . Tavares, A.M.; Mendonça, I.; Alvito, Paula; Loureiro, S.; Louro, Henriqueta; Silva, MariaMycotoxins are fungal food contaminants with potential to cause severe acute and chronic conditions1. Therefore, food contamination with mycotoxins such as ochratoxin A (OTA) and fumonisin B1 (FB1) causes great concern. Previous studies addressed the co-occurrence of these toxins in foods2, however there is little knowledge on their combined cytotoxic effects. In the present study we aimed to evaluate the cytotoxic effects of mixtures of OTA and FB1 in two human-derived cell lines.
- Evaluation of combined cytotoxic effects of ochratoxin a and fumonisin B1 in human liver and renal cellsPublication . Tavares, Ana; Mendonça, I.; Alvito, Paula; Louro, H.; Silva, M.J.; Loureiro, S.Mycotoxins are fungal food contaminants with potential to cause severe acute and chronic conditions1. Therefore, food contamination with mycotoxins such as ochratoxin A (OTA) and fumonisin B1 (FB1) causes great concern. Previous studies addressed the co-occurrence of these toxins in foods2, however there is little knowledge on their combined cytotoxic effects. In the present study we aimed to evaluate the cytotoxic effects of mixtures of OTA and FB1 in two human-derived cell lines. For this purpose, neutral red and MTT assays were performed. In HepG2 cells, OTA caused a significant decrease in cell viability after 24h exposure (above 10 µM; p<0.001), with an IC50 of 27.5 µM. However, no significant cytotoxic effects were observed after 24h exposure with FB1. When in mixture, both mycotoxins caused a non-significant decrease in the viability of HepG2 cells compared to the effects of the FB1 individually. In HK-2, OTA caused a significant decrease in cells viability after 24h exposure (above 5 µM; p<0.001), with an IC50 of 7.5 µM. Also, exposure to FB1 during 24h caused significant cytotoxic effects (above 320 µM; p<0.001), with an IC50 of 1.1 mM. The mixture of both toxins was significantly different from all the respective individual treatments of OTA and FB1 (p< 0.006). After modelling these data with the Concentration Addition conceptual model, there was a significant deviation for the dose level pattern, depicting a synergism at low dose levels of both mycotoxins, but changing to antagonism at higher doses. Therefore, considering the lower doses as the more relevant and potential to occur, this results highlight the importance of studies like this, since an increase in toxicity was observed, being higher than expected. These results agree with those presented by Creppy et al. (2004) with synergistic effects between OTA and FB1 in Vero cells3. Further work must be performed to disclose which genetoxic effects these toxins might cause to these cell lines
- Genotoxic effect of transplacental exposure to tobacco smokePublication . Alves, A.C.; Costa, C.; Tirsina, A.; Monteiro, M.S.; Soares, A.M.; Loureiro, S.; Teixeira, J.P.The effect of transplacental exposure to environmental pollutants in birth outcomes has been a matter of interest to the scientific community for several years. In the past, some studies have been carried out assessing biomarkers of early effect, such as DNA adducts, micronuclei, DNA damage and more recently, epigenetic alterations. Herein, the main goal was to analyse the possible effect of mother tobacco smoke status in the levels of DNA damage evaluated in cord blood samples using the alkaline comet assay.
- Investigação e avaliação de risco: contributo do projeto MYCOMIXPublication . Alvito, Paula; Martins, Carla; Assunção, Ricardo; Silva, M.J.; Louro, H.; Borges, T.; Loureiro, S.; Leal, S.; Dupond, D.; Seljak, B.Sumário: 1. Misturas de micotoxinas nos alimentos – uma realidade 2. Avaliação de risco – alteração de paradigma 3. Desafios associados à avaliação de risco de misturas de micotoxinas em alimentos 4. Projeto MYCOMIX – um caso estudo
- Multi-mycotoxin determination in baby foods and in vitro combined cytotoxic effects of aflatoxin M1 and ochratoxin APublication . Tavares, Ana; Alvito, Paula; Loureiro, S.; Louro, H.; Silva, M.J.The co-occurrence of mycotoxins in baby foods, including aflatoxin M1 (AFM1) and ochratoxin A (OTA), has been reported in previous studies, but data on their potential combined toxic effects are still missing. The present work aimed at (1) validating an in-house multi-mycotoxin high performance liquid chromatography with fluorescence detection (HPLC-FLD) method for AFM1, total aflatoxins (aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2)) and OTA in infant formulae (milk powders) and cereal baby foods (flours), and (2) assessing the combined cytotoxic effects of AFM1and OTA in an intestine-derived cell line. The HPLC-FLD method, which included a chloroform extraction, liquid-liquid extraction, immunoaffinity column clean-up and fluorescence detection after post-column derivatisation with electrochemically generated bromine, was adequate for the analysis of baby foods and met the requirements of validation and quality control for the studied working ranges. The limits of quantification for AFM1, AFB1, AFB2, AFG1, AFG2 and OTA were 0.069, 0.032, 0.020, 0.047, 0.020 and 0.244 μg/kg, respectively. The mean recovery values were 96, 114, 112, 107, 101 and 87%, respectively. A dose-dependent cytotoxicity was observed for individual and combined AFM1 and OTA using the Caco-2 cell line, which represents a site of contact of both mycotoxins in the body, after oral exposure. Interactions between both mycotoxins were disclosed by application of the concentration addition (CA) and independent action (IA) models, revealing the predominance of an antagonistic pattern. In conclusion, this study proposes a HPLC-FLD method for multi-mycotoxin monitoring in baby foods and provides evidence for the interaction between AFM1 and OTA, and for the applicability of CA/IA models to predict the effect of mycotoxins mixtures, further contributing to the prevention of mycotoxins-associated adverse health effects.
- MycoMix and risk assessment: a contribute to improve risk analysisPublication . Alvito, Paula; Assunção, Ricardo; Borges, T.; Dupont, D.; Leal, S.; Loureiro, S.; Louro, H.; Martins, Carla; Nunes, Baltazar; Pinhão, M.; Koroušic Seljak, B.; Silva, M.J.; Silva, E.; Vasco, Elsa; Calhau, Maria AntóniaRisk analysis, is a powerful tool for including science-based knowledge in a systematic approach to food safety problems. The use of risk analysis can promote ongoing improvements in public health and provide a basis for expanding international trade in foods. Within risk analysis, the risk assessment results are quantitative or qualitative expressions of the likelihood of harmful effects associated with exposure to a chemical (WHO, 2010). Human risk assessment of combined exposure to multiple chemicals (chemical mixtures) poses several challenges to scientists, risk assessors and risk managers, namely the complexity of the terminology and problem formulation, the diversity of chemical entities, and the toxicological profiles and exposure patterns in test species and humans (EFSA, 2013). Mycotoxins are natural contaminants produced by fungi and its frequent co-occurrence in food poses a threat to human health, mainly to vulnerable population groups as children. MycoMix is an ongoing national project (2013-15) that explores the toxic effects of mixtures of mycotoxins in infant food and its potential health impact. This project aims to study the occurrence of multiple mycotoxins and toxicity interactions in infant foods and cereals consumed by Portuguese children and try to answer 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? Answering these questions will raise novel approaches to: 1) apply new techniques on mycotoxin multiple detection, 2) understand the toxicity responses upon multiple mycotoxin exposures, and 3) implement new methodologies to characterize hazard and risk for children exposure to mycotoxins. A multidisciplinary team has been developing, for the first time in Portugal, i) a liquid chromatography (LC) method coupled with tandem mass-spectrometry (LC-MS/MS) for multimycotoxin detection in infant food developed and applied to study infant food consumed by Portuguese children, ii) cito and genotoxic assays to assess the toxicity of binary mixtures of mycotoxins detected in analyzed infant foods associated with the MIXTOX tool to assess the interactive effects, iii) in vitro methodologies to simulate the digestive and intestinal absorption processes of binary mixtures of mycotoxins, iv) a web-based dietary assessment and diet planning platform, the “OPEN Portugal”, to record infant food consumption data allowing simultaneously the assessment of the nutritional profile of the inquired children, and v) a set of deterministic, probabilistic (@RISK) and cumulative risk assessment approaches that allow the exposure assessment and risk characterization of Portuguese children to multiple mycotoxins in food. An overview of the results obtained within the MycoMix project will be presented, showing the patterns of the exposure of Portuguese infant to multiple mycotoxins as well as the scientific evidence of the toxic effects of mycotoxin mixtures using in vitro models. Hence,MycoMix outputs contribute for hazard identification and characterization as well as to exposure characterization, contributing for risk analysis.