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- Insights of enteropathogenic effects of mycotoxins on the human intestinal gut mucosaPublication . Assunção, Ricardo; Alvito, Paula; Kleiveland, C.; Lea, T.Intestinal mucosa is the first biological barrier encountered by natural toxins and it could be exposed to high amounts of dietary mycotoxins1,2. Trichothecenes, ochratoxin A and patulin (PAT) are the best known enteropathogenic mycotoxins able to alter functions of the intestine3. OBJECTIVES: This study aimed to evaluate the effects of PAT, a mycotoxin produced by Penicillium spp. during fruit spoilage, on barrier properties and function of the gut mucosa. METHODOLOGIES: Viability (MTT), proliferation (3H-thymidine incorporation assay), transepithelial electrical resistance (TER), SDS-PAGE and immunoblotting and flow cytometry methodologies were applied in order to characterize the effects of PAT on intestinal cell model (Caco-2), human peripheral blood lymphocytes (PBL) and human blood monocytederived dendritic cells (DC). RESULTS: PAT exposure reduced Caco-2 cell viability at concentrations above 12μM. The integrity of the Caco-2 monolayer was affected by PAT exposure, as demonstrated by a decrease in TER values, becoming more pronounced at 50μM. No effects were detected on the expression levels of the tight junction proteins occludin, claudin-1 and claudin-3 at 50μM. However, the expression of zonula occludens-1 (ZO-1) and myosin light chain (MLC) declined and levels of phospho-MLC increased, after 24h of exposure to 50μM of PAT. T cell proliferation was highly sensitive to PAT with the major effects for concentrations above 10nM of PAT. The same conditions did not affect the maturation of DC. CONCLUSIONS: PAT causes a reduction in Caco-2 barrier function mainly by perturbation of ZO-1 levels and phosphorylation of MLC. Low doses of PAT strongly inhibited T cell proliferation induced by a polyclonal activator, but had no effect on the maturation of DC. These results provide new information that strengthens the concept that the epithelium and immune cells of the intestinal mucosa are important targets for the toxic effects of food contaminants like mycotoxins.
- 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.
- MYCOMIX - Exploring the toxic effects of mixtures of mycotoxins in infant food and potential health impact – a case studyPublication . Alvito, PaulaMycotoxins are natural contaminants produced by fungi and its common occurrence in food poses a threat to human health, mainly to vulnerable population groups as children. In this presentation, the different questions, teams, methodologies applied in the national project Mycomix concerning the health effects of combined mycotoxins present in foods consumed by Portuguese children will be presented as a case study to assess the health impact of multiple chemicals in foodstuffs. The MycoMix Project (2013-15) funded by the Portuguese Foundation for Science and Technology (FCT, PT), 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 Portuguese children exposed daily to one or several mycotoxins through food? 2) Can this co-exposure affect children´s health? and 3) Are there interactions between mycotoxins? Within this project, Portuguese children (< 3 years old, n=103) food consumption data were obtained using a 3 days food diary in a pilot study performed at a Primary Health Care Unit. The main declared infant foods were purchased at Lisboa market along 2014-15 and analyzed by means of HPLC and LC-MS/MS analytical techniques for multiple mycotoxins co-occurrence. Toxicological studies including bioaccessibility and cyto and genotoxic interactions between mycotoxins detected were also performed using in vitro methodologies.
- Aplicação do seis sigma na avaliação da inexatidão (bias) dos resultados laboratoriais do cortisol sérico (2012-2014)Publication . Gaspar, Ana; Faria, Ana Paula; Requeijo, José; Correia, Helena; Cardoso, Ana; Brito, Cristina; Madureira, DeolindaIntrodução: Na prática laboratorial, é crescente a preocupação com a obtenção de resultados fidedignos, que apoiem corretamente os profissionais de saúde no diagnóstico, tratamento e controlo de patologias nos utentes. Requer especial atenção, a variabilidade dos resultados analíticos entre diferentes laboratórios, para um determinado parâmetro de medição. A trabalhar neste sentido, o Programa Nacional de Avaliação Externa da Qualidade (PNAEQ), tem como missão a promoção, organização e coordenação de programas de Avaliação Externa da Qualidade (AEQ), e o objetivo de avaliar o desempenho interlaboratorial dos laboratórios participantes.
- X-linked adrenal hipoplasia congenita: clinical and follow-up findings of two kindreds, one with a novel NR0B1 mutationPublication . Dias Pereira, Bernardo; Portugal, Jorge Ralha; Pereira, Iris; Gonçalves, João; Raimundo, LuísaX-linked adrenal hypoplasia congenita typically manifests as primary adrenal insufficiency in the newborn age and hypogonadotropic hypogonadism in males, being caused by mutations in NR0B1 gene. We present the clinical and follow-up findings of two kindreds with NR0B1 mutations. The proband of kindred A had a diagnosis of primary adrenal insufficiency when he was a newborn. Family history was relevant for a maternal uncle death at the newborn age. Beyond 2 year-old steroid measurements rendered undetectable and delayed bone age was noticed. Molecular analysis of NR0B1 gene revealed a previously unreported mutation (c.1084A>T), leading to a premature stop codon, p.Lys362*, in exon 1. His mother and sister were asymptomatic carriers. At 14 year-old he had 3 mL of testicular volume and biochemical surveys (LH < 0.1 UI/L, total testosterone < 10 ng/dL) concordant with hypogonadotrophic hypogonadism. Kindred B had two males diagnosed with adrenal insufficiency at the newborn age. By 3 year-old both siblings had undetectable androgen levels and delayed bone age. NR0B1 molecular analysis identified a nonsense mutation in both cases, c.243C>G; p.Tyr81*, in exon 1. Their mother and sister were asymptomatic carriers. At 14 year-old (Tanner stage 1) hypothalamic-pituitary-gonadal axis evaluation in both males (LH < 0.1UI/L, total testosterone < 10 ng/dL) confirmed hypogonadotropic hypogonadism. In conclusion, biochemical profiles, bone age and an X-linked inheritance led to suspicion of NR0B1 mutations. Two nonsense mutations were detected in both kindreds, one previously unreported (c.1084A>T; p.Lys362*). Mutation identification allowed the timely institution of testosterone in patients at puberty and an appropriate genetic counselling for relatives.
- Safety of food packaging plastic materialsPublication . Vilarinho, Fernanda; Santos, Mariana; Calhau, Maria AntóniaPackaging has become an indispensible element in the food manufacturing process, and different types of additives, such as antioxidants, stabilizers, lubricants, anti-static and anti-blocking agents, have also been developed to improve the performance of polymeric packaging materials. Recently the packaging has been found to represent a source of contamination itself through the migration of substances from the packaging into food. Various analytical methods have been developed to analyze the migrants in the foodstuff. Overall migration corresponds to the total components, identified or not, that migrate from the packaging material. The objective of the present work is the optimization and validation of the methodology to determine the overall migration from plastic materials to foodstuffs aiming the accreditation according ISO/IEC 17025 (1). The chosen method consists in the total immersion of plastic materials intended to come in contact with foodstuffs into aqueous based food simulants. The selection of the simulating conditions was made in agreement with National Legislation and NP EN 1186 (2). The overall migration from plastic samples was determined as the mass of non-volatile residue after evaporation of the food simulant following immersion. The validation of the analytical procedure consisted on the evaluation of the parameters: limit of detection (LOD), limit of quantification (LOQ), repeatability (ri) and intermediate precision (Pi). LOD and LOQ were determined through the analyses of a series of blank samples. Repeatability (ri) and Intermediate Precision (Pi) were evaluated using real samples of different kinds of plastic with different levels of overall migration, according ISO 5725 (3). The uncertainty estimation was based on intralaboratory validation data. We concluded the laboratory performance complies with the requirements of EN 1186 (4) and is able to evaluate the requirements laid down in Commission Regulation (EU) No 10/2011(5).
- Interleukine-6 promoter polymorphism -174G/C is not associated with acute rejection episodes after kidney transplantation: a meta-analysisPublication . Lima, Bruno; Mendes, Miguel; Alves, HelenaProinflammatory and anti-inflammatory cytokines play key roles in immunologic phenomena leading to Acute Rejection Episodes (ARE) after kidney transplantation which are responsible for kidney graft loss. In particular, interleukin-6 (IL-6), regulates the immune response by acting on various cells including differentiation and maturation of B and T cells or macrophages. This investigation seek to summarize current knowledge on the clinical impact on ARE of IL-6 -174G/C polymorphisms. Relevant published data was retrieved through Medline pertaining to kidney transplant outcome and IL-6 polymorphisms. Odds ratios (OR) with 95 % confidence intervals (CI) were used to assess the strength of the association. Z test was used to determine the significance of the pooled OR. Statistical heterogeneity was measured using the Q statistic. The effect of heterogeneity was quantified using the I2-statistic. A total of 16 studies, including 672 ARE transplanted cases and 1290 transplanted controls without rejection episodes, were collected in this meta-analysis. For high vs. low IL-6 genotypes, no heterogeneity (Q =12.07, p=0.67, I2 =0.0%) was observed among individual estimates, and original data was combined using the fixed-effects model. For the total population, we found no association between G/G and G/C IL-6 genotypes with ARE, we obtained an effect summary OR=1.14, with a 95 % CI=0.84-1.55, and p=0.4. In conclusion, in recipients with a high producer (G/G and G/C) genotype of IL-6, the -174G/C polymorphism is not associated with acute rejection of renal allograft.
- Biofilm-forming bacteria on the pharyngeal tonsil in a pediatric populationPublication . Rodrigues, João; Reis, Lucia; Subtil, Joao; Jordão, LuísaObjective: The main goal of this study is to evaluate the existence of a link between biofilm assembly on pharyngeal tonsil and the incidence of recurrent infections within a paediatric population. Methods: Nasal and pharyngeal tonsils scrubs as well as a biopsies of pharyngeal tonsils tissue were collected (n=70) and processed for bacterial identification using standard microbiologic methods. Bacteria were identified at the species level using a VITEC apparatus. Another pharyngeal tonsil biopsies were processed for electron microscopy. Initially, a screening for the presence of biofilms was performed using scanning electron microscopy (SEM) being the positive samples further processed for transmission electron microscopy (TEM). Biofilm assembly assay ability in vitro was evaluated by the microtiter-plate test. The assay was performed at 37ºC in Muller-Hinton broth for different periods of time. Results: As expected a vast number of bacterial species were identified in the samples. The majority were aerobic and a balance between Gram negative and positive bacteria was observed. A good correlation between the floras of the three samples from the same patient was found. The presence of biofilms pharyngeal tonsils was assessed by SEM. The data collected so far suggest that in at least 30% of the samples biofilms were present. In vitro all the microorganisms evaluated were able to assemble biofilms. Conclusion: Biofilm formation can be part of the mechanism involved both in onset and propagation of infections. Further studies must be performed to validate the thesis that pharyngeal tonsils function as a reservoir for microorganisms.
- Assessment of mixtures of mycotoxins in breakfast cereals available in Portuguese marketPublication . Martins, Carla; Assunção, Ricardo; Cunha, S.; Jager, A.; Alvito, PaulaObjective: Mycotoxins are secondary metabolites of fungi that cause toxic and carcinogenic outcomes in humans exposed to them1. Mycotoxins affect several commodities including cereal grains and their finished products, infant formula and baby foods2. This study aimed to determine the incidence and levels of 20 mycotoxins (AFB1, AFB2, AFG1, AFG2, AFM1, OTA, NIV, NEO, DAS, FUS-X, DON, 15-AC-DON, 3-AC-DON, HT-2, T-2, VER, T-2 TETROL, T-2 TRIOL), in breakfast cereals available in the Portuguese market, and compare the results with the maximum limits established by the EU. Methodology: Twenty six breakfast cereal samples, including corn, wheat, oat, rice and multigrain, were collected from supermarkets in Lisbon region and analyzed by HPLC-FLD, LC-MS/MS and GC-MS. Results: Results showed that 88 % breakfast cereals samples were contaminated with mycotoxins (with values above the detection limit), although all samples presented levels below the maximum limits established by the Commission Regulation 1881/20063. OTA and DON were the most commonly detected mycotoxins, with 88% and 73% of samples revealing values above the LOD, respectively. The co-occurrence of different mycotoxins in the same sample was observed in 92 % of the analyzed samples. From these, 46% include mixtures of 3 or 4 mycotoxins. These results are accordingly to those reported by Juan et al (2014)4 and Iqbal et al (2014)5. Conclusions: These results contribute to the increased knowledge on mycotoxin contents in breakfast cereals marketed in Portugal, and they highlight the deep need of further studies to overcome the absence of legislated limits for mycotoxins in breakfast cereals other than DON and FB1 and the absence of legislated limits for mycotoxin mixtures in food. The last issue is particularly important considering the potential synergistic effects that could occur between mycotoxins and its potential impact on human and, mainly, children health.
- The impact of cooking process in bioacessibility of minerals from chenopodium quinoaPublication . Mota, Carla; Assunção, Ricardo; Martins, Carla; Nascimento, A.; Santos, M.; Torres, D.; Alvito, Paula; Castanheira, I.Quinoa seeds (Chenopodium quinoa) are usually reported as a good source of minerals; however, information about in vitro bioacessibility is scarce. The aim of the present study was to estimate the minerals bioaccessibility of quinoa seeds using different cooking processes: raw, steamed and boiled. The digestion of samples were performed using the harmonized static “in vitro” digestion protocol method (IVD)1, with a minor modification concerning the oral phase enzymatic composition (bacterial α-amylase). The copper (Cu), manganese (Mn), iron (Fe), zinc (Zn), magnesium (Mg), calcium (Ca), phosphorous (P), sodium (Na) and potassium (K) contents were determined by inductively coupled plasma optical emission spectrometer (ICP-OES). Raw quinoa seeds presented the highest bioaccessibility values for Mn, K, P (100%) and the lowest for Ca (19%). After cooking process the highest values of bioaccessibility were observed for Ca (100% in boiled and 74% in steamed) and Cu (90% in boiled and 78% in steamed). Mn showed the highest decrease in bioacessibility when comparing raw and cooked samples (100% and 20% in raw and cooked samples, respectively). Zn presented bioaccessibility values near 40% for raw, boiled and steamed samples. The boiling process was the procedure that allowed to obtain the highest mineral bioaccessibility values for all the assayed minerals. These are the first results that describe the bioaccesisiblity of minerals of quinoa seeds using the harmonized IVD method.