Browsing by Author "Louro, H."
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- Applicability of the harmonized in vitro digestion method to titanium dioxide nanomaterialsPublication . Alvito, Paula; Assunção, Ricardo; Gramacho, A.; Silva, M.J.; Louro, H.; Martins, C.Titanium dioxide nanomaterials (TiO2 NMs) have a high potential for ingestion by human populations, due to their increasing use as food additives, inclusion in dietary supplements and food packaging materials. Whether this oral exposure may lead to adverse local or systemic outcomes, has been the subject of research. In vitro studies have generated contradictory results, possibly due to differences in the physicochemical properties of the TiO2 NMs studied, which can be additionally affected by the surrounding matrix and interactions during digestion. INGESTnano is a national project aimed at investigating the nano-bio interactions of nanomaterials on intestinal cells, at the cellular and molecular levels, after the digestion process, to better understand their potential impacts on human health. Three TiO2 NMs were selected as case-studies to setup a workflow for addressing nanosafety concerns of ingested NMs, while considering the nano-bio interactions under physiological conditions. As an alternative to in vivo testing, this project is focused on the use of the harmonized in vitro digestion method for simulating the human digestion of NMs. This digestion comprises three compartments: a) mouth (pH 7.0, alpha-amylase, salts); b) stomach (pH 3, HCl, pepsin, salts) and c) small intestine (pH 7.5, bile salts, pancreatin, salts). The final product of digestion is tested in bioassays using intestinal cells, to ascertain its toxicity. A high level of toxicity of the final digestion product challenged the applicability of the digestion product in the biological assays. The results revealed that digestion products without the NM showed cytotoxic effects above the concentration of 10% in cell culture medium. Several modifications to the initial protocol were thus performed to overcome this issue. The results suggested that the addition of bile salts accounted for most of the toxicity observed. The applicability of the harmonized in vitro digestion method is discussed in view of its potential use as a tool for addressing the toxicity of ingested NMs or other food contaminants, mimicking the physiological processes, in alternative to animal models.
- Contribute of the MYCOMIX project and future perspectives for Human Biomonitoring and health risk assessment in PortugalPublication . Martins, Carla; Assunção, Ricardo; Silva, M.J.; Louro, H.; Pinhão, M.; Nunes, Baltazar; Vasco, Elsa; Alvito, PaulaThere is a growing concern within public health about mycotoxin involvement in human diseases, namely those related to children. The MycoMix project (2012-2015), funded by the Portuguese Foundation for Science and Technology, gathered a multidisciplinary team aiming at answering 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 interaction effect between mycotoxins? Mycomix results revealed that Portuguese children (< 3 years old, n=103) are exposed to multiple mycotoxins through food consumption. Cumulative risk assessment results revealed a potential health concern for the high percentiles of intake, specially for aflatoxins which are carcinogenic compounds. This fact assumes particular importance considering the interactive effects found in in vitro bioassays. These results highlight the need for a more accurate approach to assess the human exposure to mycotoxins6. Within the Mycomix project the assessment of mycotoxin exposure was based on calculations combining mycotoxin data in food with population data on food consumption. This approach does not consider some aspects as the inter-individual metabolism variation, the exposure through sources other than food and the heterogeneous distribution of mycotoxins in food. Exposure assessment of mycotoxins in Portuguese population through biomarkers is still missing and further studies are urgent to be developed. The European Human Biomonitoring Initiative (EHBMI), a proposal within the European Joint Programme, aims to advance the understanding of the extent of exposure to environmental chemicals across Europe and the impact on human health, by gathering national expertise in human biomonitoring domain. At national level Mycomix project uncovered the potential health risk of exposure of Portuguese children to multiple mycotoxins. The risk assessment expertise acquired within Mycomix, namely in analysis and toxicology of chemical mixtures, will be brought together as a contribute to EHBMI objectives.
- Drinking water contaminants: toxicity of halogenated polycyclic aromatic hydrocarbonsPublication . José, S.S.; Pinto, M.; Antunes, A.M.M.; Louro, H.; Jordão, Luísa; Silva, M.J.; Cardoso, A.S.Food may be contaminated with polycyclic aromatic hydrocarbons (PAHs) in the process of smoking or heating. These contaminants or their derivatives can also be present in drinking water when raw water contacts with discharges of untreated industrial/waste water effluents, forest fires or by solubilisation of organic material from contaminated soils. A few studies have shown that water disinfection can lead to halogenated derivatives of PAHs (HPAHs) as chlorinated and brominated derivatives, and there are evidences that these compounds may have greater mutagenicity than the parent PAHs. In this study the cytotoxic and genotoxic effects of chlorinated/brominated derivatives of pyrene (Pyr) and benzo[a]anthracene (BaA), 1-ClPyr, 1-BrPyr and 7-ClBaA, which can be formed as water disinfection by-products, were studied in HepG2 cells to assess their potential hazard to human health. The formation of 1-ClPyr, 1-BrPyr and 7-ClBaA under aqueous disinfection conditions in waters contaminated with Pyr and BaA, was confirmed with an optimized gas chromatography method. Cells exposed (24h) to several concentrations of BaA and 7-ClBaA (1 to 200μM) displayed a dose-related and significant increase of cytotoxicity (neutral red assay) with IC50 values of 3.37 and 12.63µM respectively. For Pyr, 1-ClPyr and 1-BrPyr (10 to 200μM), a lower but significant dose-related cytotoxicity was observed. At non-cytotoxic concentrations (10 and 15µM), 7-ClBaA was able to induce a significantly higher level of oxidative DNA damage in HepG2 cells than its parent compound, as assessed by the FPG-modified comet assay. Under these conditions neither Pyr nor its derivatives were genotoxic. In conclusion, the disinfection process may give rise to genotoxic HPAHs with potential impact on human health and it should be performed in raw waters with minimal content of total organic carbon. In real conditions, humans may be exposed to a mixture of these organic compounds and thus their combined toxic effects should be further evaluated.
- Efeitos tóxicos combinados de misturas de micotoxinas em linhas celulares humanasPublication . Tavares, Ana; Mendonça, I.; Loureiro, S.; Louro, H.; Alvito, Paula; Silva, M.J.
- Effect of Aflatoxin B1 in both Caco-2 and Caco-2/HT29-MTX modelsPublication . Serrenho, I.; Vidal, N.; Rolo, D.; Louro, H.; Pereira, J.; Matos, P.; Jordan, P.; Alvito, P.About effect of Aflatoxin B1 in both Caco-2 and Caco-2/HT29-MTX models within the scope of the intestinal absorption assessment and enabling formulations.
- 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
- Formation of Emerging Disinfection Byproducts in Water and Evaluation of Potential Genotoxic Effects: the Case of Chlorinated Polycyclic Aromatic HydrocarbonsPublication . Rebola, M.; Pinto, M.; Louro, H.; Antunes, A.M.M.; José, S.S.; Rocha, M.R.; Silva, M.J.; Cardoso, A. S.Disinfection byproducts (DBPs) are formed when disinfectants used in water treatment plants (WTPs) react with natural (or anthropogenic) organic matter present in the source water. Many studies have addressed health risks posed by a life-time exposure to DBPs through chlorinated drinking water or through dermal or inhalation exposure routes. Experimental studies have revealed genotoxic and carcinogenic effects of some DBPs and epidemiological studies evidenced potential associations between chlorinated drinking water and bladder or colorectal cancer. In addition, a possible link between chlorinated drinking water and reproductive/developmental effects has been hypothesized. Many DBPs have been identified in treated water, which justifies the growing concern about the potential health effects of emerging unregulated DBPs, some of which appear to be more genotoxic, in some assays, than the regulated DBPs. Polycyclic aromatic hydrocarbons (PAHs) are among the most persistent contaminants detected in environmental samples such as river sediments and tap water. Water chlorination can lead to the formation of chlorinated derivatives of PAHs (Cl-PAHs) and the few available toxicological studies have shown that Cl-PAHs possess greater mutagenicity than the corresponding parent PAHs. The present study had two main objectives: 1) identification of the major chlorinated derivatives of benzo[a]pyrene (BaP) and fluoranthene (Fluo) formed as chlorination by-products and 2) evaluation of their potential hazard to humans, through the characterization of their potential genotoxic effects in a human cell line. To synthesize chlorinated standards of PAHs, a newly two phase (water/n-hexane) method was developed for BaP and Fluo. 6-Cl-BaP was obtained as the major chlorination product of BaP, and 3-Cl-Fluo and 1,3-Cl2-Fluo of Fluo. The formation of these BaP and Fluo chlorinated derivatives was also observed under WTPs chlorination conditions after at 0.5 until 24 h of exposure. The effects of equimolar concentrations of 6-Cl-BaP vs. BaP and of 3-Cl-Fluo/1,3-Cl2-Fluo vs. Fluo on cell viability and DNA integrity were assessed by the neutral red uptake (NR) and the comet assay, respectively. Exposure of HepG2 cells to a dose-range of 6-Cl-BaP and BaP showed that both compounds are cytotoxic above 50 μM and that, at the equimolar doses of 100 and 125 μM, 6-Cl-BaP is able to induce a significantly higher level of DNA damage than BaP. On the other hand, no changes of cell viability were observed after exposure to several concentrations of Fluo and its derivatives. Likewise, none of the compounds was able to significantly induce DNA damage. In conclusion, the present data confirmed that chlorinated derivatives of BaP and Fluo are formed during WTPs chlorination procedures and allowed the identification of their major chlorinated derivatives that should be further analysed in drinking water. On the other hand, the results from the comet assay evidenced a higher DNA damaging effect of Cl-BaP comparatively to its parent compound, suggestive of a more potent genotoxic effect. In spite of the negative results found for Fluo and its chorinated products, further genotoxicity studies are still needed to allow a definite conclusion. Although health risks of DBPs are small compared to health risks of waterborne diseases, the identification of hazardous Cl-PAHs in water emphasizes the need of development of new and safer water disinfection methods.
- Formation of emerging disinfection byproducts in water and evaluation of potential genotoxic effects: the case of halogenated polycyclic aromatic hydrocarbonsPublication . Pinto, M.; Antunes, A.M.M.; José, S.S.; Alves, A.C.; Louro, H.; Silva, M.J.; Cardoso, A.S.Disinfection byproducts (DBPs) are formed when disinfectants used in water treatment plants (WTPs) react with natural (or anthropogenic) organic matter present in the source water. Many studies have addressed health risks posed by a life-time exposure to DBPs through chlorinated drinking water or through dermal or inhalation exposure routes. Experimental studies have revealed genotoxic and carcinogenic effects of some DBPs and epidemiological studies evidenced potential associations between chlorinated drinking water and bladder or colorectal cancer. In addition, a possible link between chlorinated drinking water and reproductive/developmental effects has been hypothesized. Many DBPs have been identified in chlorinated water, which justifies the growing concern about the potential health effects of emerging unregulated DBPs, some of which appear to be more genotoxic, in some assays, than the regulated DBPs. Polycyclic aromatic hydrocarbons (PAHs) are among the most persistent contaminants detected in environmental samples such as river sediments and tap water. A few studies have already proven that water disinfection can lead to the formation of halogenated derivatives of PAHs, such as chlorinated (Cl-PAHs) and brominated PAHs (Br-PAHs). The available toxicological studies have shown that these compounds possess, in general, greater mutagenicity than the corresponding parent PAHs. Our investigation group have also showed that exposure of HepG2 cells to a dose-range of 6-Cl-benzo[a]pyrene (6-Cl-BaP) and BaP resulted in cytotoxicity above 50 µM and that, at the equimolar doses of 100 and 125 µM, 6-Cl-BaP was able to induce a significantly higher level of DNA damage than BaP. The present study had two main objectives: 1) identification of the major chlorinated and brominated derivatives of benzo[a]anthracene (BaA) and pyrene (Pyr) formed as disinfection by-products and 2) evaluation of their potential hazard to humans, through the characterization of their potential cytotoxic and genotoxic effects in a human cell line. To synthesize Cl-PAHs and Br-PAHs the method of Mitchell was developed for BaA and Pyr. 1-Cl-Pyr and 1-Br-Pyr were obtained as the major chlorinated and brominated derivatives of Pyr, and 7-Cl-BaA and 7-Br-BaA as the reaction products of BaA. Cell viability and DNA integrity of those derivatives were assessed by the neutral red uptake (NR) and the comet assay, respectively, allowing the comparison of their genotoxic potential. Although health risks of DBPs are small compared to the health risks of waterborne diseases, the formation of hazardous halogenated-PAHs in chlorinated water water emphasizes the need of development of new and safer water disinfection methods.
- Halogenated polycyclic aromatic hydrocarbons associated with drinking water disinfectionPublication . José, S. S.; Pinto, M.; Antunes, A.M.M.; Louro, H.; Silva, M.J.; Cardoso, A.S.Introduction: Disinfection by-products (DBPs) have been identified in chlorinated water. This fact justifies the growing concern about the potential health effects of emerging unregulated DBPs, some of which appear to be more genotoxic than the regulated DBPs[1]. Polycyclic aromatic hydrocarbons (PAHs) are among the most persistent contaminants detected in environmental samples such as river sediments and tap water. A few studies have already proven that water disinfection can lead to the formation of halogenated derivatives of PAHs, such as chlorinated and brominated PAHs[2] . The available toxicological studies have shown that these compounds possess, in general, greater mutagenicity than the corresponding parent PAHs. Our research group has also shown that exposure of HepG2 cells to a dose-range of 6-Cl-benzo[a]pyrene (6-ClBaP) and BaP resulted in cytotoxicity above 50 µM and that, at the equimolar doses of 100 and 125 µM, 6-ClBaP was able to induce a significantly higher level of DNA damage than BaP[3] . The present study had two main objectives: 1) identification of the major chlorinated and brominated derivatives of benzo[a]anthracene (BaA) and pyrene (Pyr) formed as disinfection by-products and 2) evaluation of their potential hazard to humans, through the characterization of their potential cytotoxic and genotoxic effects in a human cell line.
- Halogenated polycyclic aromatic hydrocarbons associated with drinking water disinfectionPublication . Cardoso, A.S.; José, S.S.; Pinto, Miguel; Antunes, A.M.M.; Louro, H.; Silva, M.J.