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- Ingested nanomaterials: impact of digestion process in the physicochemical characteristics and biological consequences in intestinal cellsPublication . Vieira, Adriana; Vital, Nádia; Roque, Rossana; Gramacho, Ana Catarina; Rolo, Dora; Gonçalves, Lídia D.; Bettencourt, Ana; Martins, Carla; Assunção, Assunção; Alvito, Paula; Silva, Maria João; Louro, HenriquetaNanomaterials(NMs) provide a basis for key enabling technologies, in view of their potential to improve many products and processes, namely in food and feed industry. That is the case of titanium dioxide NMs(TiO2 NMs), presenting beneficial properties for a broad range of innovative applications such as food additives, toothpaste, pharmaceuticals, food products, etc., that may drive ingestion. The oral exposure can occur directly, by consumption of products/pharmaceuticals or foods containing NMs, or indirectly, through the ingestion of foods contaminated with NMs released from food-contact materials or environmental sources. As such, the gastrointestinal tract is the first site of contact of the ingested NMs, allowing a systemic exposure if the intestinal barriers is surpassed. This work aimed to investigate how the digestion process affects the physicochemical properties of three different TiO2 NMs(NM-102, NM-103 and NM-105) and their toxic effects on intestinal cells. After undergoing digestion through the standardized static INFOGEST 2.0 in vitro digestion method, the cytotoxicity of the TiO2 NMs was determined in Caco-2 and HT29-MTX-E12 intestinal cells, using the MTT assay. Furthermore, the cytokinesis-blocked micronucleus assay was used to investigate their genotoxicity in both cell lines in order to predict their carcinogenic potential. The results showed that, for one TiO2 NM(NM-105), the digestion caused changes in the hydrodynamic size of the NM and a more pronounced toxicity in HT29-MTX-E12 intestinal cells, as compared to the undigested one. The micronucleus assay suggests effects on the chromosomal integrity in the HT29-MTXE12 cells, for all the tested TiO2 NM especially after the in vitro digestion. Overall, we conclude that including the digestion prior to the in vitro bioassays for the safety evaluation of ingested NMs, allows integrating the physiological modifications that the NMs suffer in the organism, contributing to an improved hazard assessment of ingested NMs.
- Evaluation of the cytotoxicity and genotoxicity of ingested titanium dioxide nanomaterials in intestinal cellsPublication . Gramacho, Ana Catarina; Martins, Carla; Assunção, Ricardo; Gonçalves, Lídia; Simão Bettencourt, Ana; Paula, Alvito; Silva, Maria João; Louro, HenriquetaMany products already available, namely in agriculture and food, contain nanomaterials (NMs) and thus human ingestion of these compounds is probable through consumer products or food chain. Although NMs have been extensively investigated in recent years, the studies have generated contradictory results, possibly due to differences in the physicochemical properties of the NMs studied and to other variables in the tested systems. Furthermore, the NMs properties have been recognized as being context-dependent, i.e. can be affected by the surrounding matrix. These secondary features may be potentially more relevant for determining the toxicological outcome. In particular, processes like digestion may modify the NMs characteristics leading to unexpected toxicity in intestine cells. This work aimed to investigate the nano-bio interactions of titanium dioxide NMs (from Joint Research Centre, Ispra) in the context of intestinal tract and digestion processes, to better understand key events that may be linked to an adverse outcome pathway (AOP). In vitro for digestion was simulated and the NMs secondary properties in the intestinal cell moiety were characterized after this process. The cytotoxic and genotoxic effects of digested NMs were determined after the in vitro exposure of human intestinal cells (Caco-2). In addition, the FPG-comet assay was used to analyze oxidative DNA lesions. The digestion products without the NM showed cytotoxic effects above the concentration of 10% in cell culture medium, leading to the need to reduce its concentration below this level. Therefore, initial dose-range studies set a working range of NM concentrations of 0.14 up to 14 µg/ml (0.5-4.5 % of digestion product), which relates also with the predicted levels of exposure of human intestinal cells in real life conditions. Under these conditions, the preliminary results suggest that the tested titanium dioxide NMs do not yield cytotoxic or genotoxic effects upon 24h of exposure of Caco-2 cells, directly after the digestion process. Future studies will investigate the subcellular localization of NMs, integrity of cell junctions, activation of stress signaling pathways and secretion of inflammatory cytokines, to allow an integrated approach to potential adverse effects of the NMs. By elucidating key events elicited by NMs, linking exposure to adverse outcomes, it is expected to contribute to the safety evaluation of NMs within an AOP landscape.
- Human biomonitoring in health risk assessment in Europe: current practices and recommendations for the futurePublication . Louro, Henriqueta; Heinälä, Milla; Bessems, Jos; Buekers, Jurgen; Vermeire, Theo; Woutersen, Marjolijn; van Engelen, Jacqueline; Borges, Teresa; Rousselle, Christophe; Ougier, Eva; Alvito, Paula; Martins, Carla; Assunção, Ricardo; Silva, Maria João; Pronk, Anjoeka; Schaddelee-Scholten, Bernice; Del Carmen Gonzalez, Maria; de Alba, Mercedes; Castaño, Argelia; Viegas, Susana; Humar-Juric, Tatjana; Kononenko, Lijana; Lampen, Alfonso; Vinggaard, Anne Marie; Schoeters, Greet; Kolossa-Gehring, Marike; Santonen, TiinaHuman biomonitoring (HBM) is an important tool to survey the internal exposure of humans which represents the real life chemical body burden to chemicals and/or their metabolites. It results from total exposure to chemical substances from different sources and via different routes. These substances may be regulated under different legislative frameworks on chemicals (e.g., environmental, occupational, food safety etc). In occupational health, HBM has long traditions to control the exposures at workplaces. By providing accurate data on internal exposure, HBM data can improve human health risk assessment (RA) for both the general population and workers. Although the past few years have shown good examples on the use of HBM in the RA of chemicals, there is still quite some work to be done to improve its use in a regulatory RA. Under the scope of the European Human Biomonitoring Initiative (project HBM4EU, 2017-2021), the current study reviews the state-of-the-art of HBM use in chemicals RA with a special focus in Europe, and attempts to identify hurdles and challenges faced by regulators. To gather information on the use of HBM, including the availability of guidance on how to use it in RA, the RA schemes applied by different European or international organizations were analysed. Examples of such use were identified for a few selected groups of chemicals of concern for human health. In addition, we present the results of a survey, aimed at collecting information from national regulatory risk assessors on their day-to-day RA practices, the use of HBM data, and the obstacles and challenges related to their use. The results evidenced and explained some of the current obstacles of using HBM data in RA. These included the lack of HBM guidance values or biomonitoring equivalents (BEs), limited toxicokinetic information to support the interpretation of HBM data and, in the occupational health and safety (OSH) field, the lack of legal enforcement. Therefore, to support the integration of HBM in regulatory RA, we recommend, on one hand, the elaboration of a EU level guidance on the use of HBM in RA and, on the other hand, the continuation of research efforts to integrate HBM with new RA approaches using in vitro/in silico data and Adverse Outcome Pathways (AOPs).
- Contribution to micro- and nanoplastics hazard assessment: the lessons learnt from nanomaterials toxicity assessmentPublication . Silva, Maria João; Alvito, Paula; Louro, HenriquetaThe last decade has witness growing scientific concerns and public debate over the adverse effects that may result from marine and freshwater organisms’ exposure to plastics and microplastics. Moreover, due to microplastics degradation or direct release from domestic and industrial sources, aquatic organisms are equally exposed to nano-sized plastics (size between 1 and 100 nm, EFSA 2016). These micro- and nanoplastics have the potential to accumulate in edible tissues of the exposed organisms, including those that are part of the human diet, thus entering the human food chain (Toussaint et al., 2019). Apart from this indirect exposure route, humans are additionally exposed to tiny plastic particles through water consumption and consumer products that incorporate the so-called microbeads. In contrast to the wide range of investigation on the impact of plastics/microplastics on the ecosystem, a major question is whether micro- and nanoplastics will have a negative impact on human health. This negative impact may derive from the physicochemical nature of the micro- and nanoplastics, that resemble those of engineered nanomaterials, including their capacity to cross barriers and reach all organs and tissues, inducing deleterious effects e.g., inflammation or genotoxic effects that may lead long-term disease, such as cancer. Moreover, these plastic particles might be associated with a wide range of substances, including chemicals present in the plastic composition, e.g., metals, polychlorinated biphenyls and plasticizers or chemicals adsorbed to their surface, which may also be hazardous to human health. An effective risk assessment of micro- and nanoplastics needs to be supported by a robust hazard assessment. The experimental approach to be applied should rely on previously acquired knowledge from the toxicity assessment of different engineered nanomaterials. Indeed, the potential cytotoxic, immunotoxic and genotoxic effects of these plastic particles have to be assessed using complementary in vitro and in vivo assays and considering their specific physicochemical properties. In this work, we will present the testing strategy that has been recently applied to the genotoxicity characterization of nanomaterials in human cell models, e.g., a co-culture of human intestinal epithelial cells and mucus-producing cells, and in an integrative in vivo model (Louro et al., 2014). Particularly, the approach used to the toxicity assessment of titanium dioxide following a harmonized in vitro digestion process will be presented and its advantages and pitfalls discussed. Furthermore, the results of our previous approaches to the toxicity assessment of nanomaterials have supported the view that a thorough understanding of the relationship between the physicochemical properties, the behaviour of nanoparticles in biological systems and their mechanism of action is of utmost importance to predict their biological activity (Louro et al., 2019). Particularly, the investigation of genotoxic and epigenetic effects will be focused, given their strong association with carcinogenic effects. In summary, we propose the use of a predictive toxicology approach based on defining the key events at cellular and molecular levels, to identify and characterize the hazard of micro- and nanoplastics, reducing the in vivo experimentation to the lowest possible level.
- Harmonized human biomonitoring in European children, teenagers and adults: EU-wide exposure data of 11 chemical substance groups from the HBM4EU Aligned Studies (2014-2021)Publication . Govarts, Eva; Gilles, Liese; Rodriguez Martin, Laura; Santonen, Tiina; Apel, Petra; Alvito, Paula; Anastasi, Elena; Andersen, Helle Raun; Andersson, Anna-Maria; Andryskova, Lenka; ANTIGNAC, Jean-Philippe; Rüther, Maria; Sarigiannis, Denis; Silva, Maria João; Šlejkovec, Zdenka; Snoj Tratnik, Janja; Stajnko, Anja; Szigeti, Tamas; Tarazona, Jose; Thomsen, Cathrine; Tkalec, Žiga; Trnovec, Tomas; Tolonen, Hanna; Uhl, Maria; Van Nieuwenhuyse, An; Vasco, Elsa; Verheyen, Veerle J.; Viegas, Susana; Vinggaard, Anne Marie; Vogel, Nina; Vorkamp, Katrin; Wasowicz, Wojciech; Wimmerova, Sona; Weber, Till; Woutersen, Marjolijn; Zimmermann, Philipp; Zvonar, Martin; Koch, Holger; Kolossa-Gehring, Marike; Esteban López, Marta; Castano, Argelia; Stewart, Lorraine; Sepai, Ovnair; Appenzeller, Brice; Schoeters, Greta; Barbone, Fabio; Barnett-Itzhaki, Zohar; Barouki, Robert; Berman, Tamar; Bil, Wieneke; Borges, Teresa; Buekers, Jurgen; Cañas-Portilla, Ana; Covaci, Adrian; Csako, Zsofia; Den Hond, Elly; Dvorakova, Darina; Fabelova, Lucia; Fletcher, Tony; Frederiksen, Hanne; Gabriel, Catherine; Ganzleben, Catherine; Göen, Thomas; Halldorsson, Thorhallur; Haug, Line Småstuen; Horvat, Milena; Huuskonen, Pasi; Imboden, Medea; Jagodic Hudobivnik, Marta; Janasik, Beata; Janev Holcer, Natasa; Karakitsios, Spyros; Katsonouri, Andromachi; Klanova, Jana; Kokaraki, Venetia; Kold Jensen, Tina; Koponen, Jani; Laeremans, Michelle; Laguzzi, Federica; Lange, Rosa; Lemke, Nora; Lignell, Sanna; Lindroos, Anna Karin; Lobo Vicente, Joana; Luijten, Mirjam; Makris, Konstantinos C.; Mazej, Darja; Melymuk, Lisa; Meslin, Matthieu; Mol, Hans; Montazeri, Parisa; Murawski, Aline; Namorado, Sónia; Niemann, Lars; Nübler, Stefanie; Nunes, Baltazar; Olafsdottir, Kristin; Palkovicova Murinova, Lubica; Papaioannou, Nafsika; Pedraza-Diaz, Susana; Piler, Pavel; Plichta, Veronika; Poteser, Michael; Probst-Hensch, Nicole; Rambaud, Loic; Rauscher-Gabernig, Elke; Rausova, Katarina; Remy, Sylvie; Riou, Margaux; Rosolen, Valentina; Rousselle, ChristopheAbstract: As one of the core elements of the European Human Biomonitoring Initiative (HBM4EU) a human biomonitoring (HBM) survey was conducted in 23 countries to generate EU-wide comparable HBM data. This survey has built on existing HBM capacity in Europe by aligning national or regional HBM studies, referred to as the HBM4EU Aligned Studies. The HBM4EU Aligned Studies included a total of 10,795 participants of three age groups: (i) 3,576 children aged 6–12 years, (ii) 3,117 teenagers aged 12–18 years and (iii) 4,102 young adults aged 20–39 years. The participants were recruited between 2014 and 2021 in 11–12 countries per age group, geographically distributed across Europe. Depending on the age group, internal exposure to phthalates and the substitute DINCH, halogenated and organophosphorus flame retardants, per- and polyfluoroalkyl substances (PFASs), cadmium, bisphenols, polycyclic aromatic hydrocarbons (PAHs), arsenic species, acrylamide, mycotoxins (deoxynivalenol (total DON)), benzophenones and selected pesticides was assessed by measuring substance specific biomarkers subjected to stringent quality control programs for chemical analysis. For substance groups analyzed in different age groups higher average exposure levels were observed in the youngest age group, i.e., phthalates/DINCH in children versus teenagers, acrylamide and pesticides in children versus adults, benzophenones in teenagers versus adults. Many biomarkers in teenagers and adults varied significantly according to educational attainment, with higher exposure levels of bisphenols, phthalates, benzophenones, PAHs and acrylamide in participants (from households) with lower educational attainment, while teenagers from households with higher educational attainment have higher exposure levels for PFASs and arsenic. In children, a social gradient was only observed for the non-specific pyrethroid metabolite 3-PBA and di-isodecyl phthalate (DiDP), with higher levels in children from households with higher educational attainment. Geographical variations were seen for all exposure biomarkers. For 15 biomarkers, the available health-based HBM guidance values were exceeded with highest exceedance rates for toxicologically relevant arsenic in teenagers (40%), 3-PBA in children (36%), and between 11 and 14% for total DON, Σ (PFOA + PFNA + PFHxS + PFOS), bisphenol S and cadmium. The infrastructure and harmonized approach succeeded in obtaining comparable European wide internal exposure data for a prioritized set of 11 chemical groups. These data serve as a reference for comparison at the global level, provide a baseline to compare the efficacy of the European Commission's chemical strategy for sustainability and will give leverage to national policy makers for the implementation of targeted measures.
- How to use human biomonitoring in chemical risk assessment: Methodological aspects, recommendations, and lessons learned from HBM4EUPublication . Santonen, Tiina; Mahiout, Selma; Alvito, Paula; Apel, Petra; Bessems, Jos; Bil, Wieneke; Borges, Teresa; Bose-O'Reilly, Stephan; Buekers, Jurgen; Cañas Portilla, Ana Isabel; Calvo, Argelia Castaño; de Alba González, Mercedes; Domínguez-Morueco, Noelia; López, Marta Esteban; Falnoga, Ingrid; Gerofke, Antje; Caballero, María del Carmen González; Horvat, Milena; Huuskonen, Pasi; Kadikis, Normunds; Kolossa-Gehring, Marike; Lange, Rosa; Louro, Henriqueta; Martins, Carla; Meslin, Matthieu; Niemann, Lars; Díaz, Susana Pedraza; Plichta, Veronika; Porras, Simo P.; Rousselle, Christophe; Scholten, Bernice; Silva, Maria João; Šlejkovec, Zdenka; Tratnik, Janja Snoj; Joksić, Agnes Šömen; Tarazona, Jose V.; Uhl, Maria; Van Nieuwenhuyse, An; Viegas, Susana; Vinggaard, Anne Marie; Woutersen, Marjolijn; Schoeters, GreetThe need for such information is pressing, as previous research has indicated that regulatory risk assessors generally lack knowledge and experience of the use of HBM data in RA. By recognising this gap in expertise, as well as the added value of incorporating HBM data into RA, this paper aims to support the integration of HBM into regulatory RA. Based on the work of the HBM4EU, we provide examples of different approaches to including HBM in RA and in estimations of the environmental burden of disease (EBoD), the benefits and pitfalls involved, information on the important methodological aspects to consider, and recommendations on how to overcome obstacles. The examples are derived from RAs or EBoD estimations made under the HBM4EU for the following HBM4EU priority substances: acrylamide, o-toluidine of the aniline family, aprotic solvents, arsenic, bisphenols, cadmium, diisocyanates, flame retardants, hexavalent chromium [Cr(VI)], lead, mercury, mixture of per-/polyfluorinated compounds, mixture of pesticides, mixture of phthalates, mycotoxins, polycyclic aromatic hydrocarbons (PAHs), and the UV-filter benzophenone-3. Although the RA and EBoD work presented here is not intended to have direct regulatory implications, the results can be useful for raising awareness of possibly needed policy actions, as newly generated HBM data from HBM4EU on the current exposure of the EU population has been used in many RAs and EBoD estimations.
- Avaliação da exposição da população portuguesa a substâncias químicas: resultados de um estudo de âmbito nacionalPublication . Namorado, Sónia; Ogura, Joana; Silva, Susana; Coelho, Inês; Delgado, Inês; Gueifão, Sandra; Ventura, Marta; Martins, Carla; Viegas, Susana; Alvito, Paula; Silva, Maria João; Nunes, Baltazar; Matias Dias, CarlosIntrodução: Tem sido amplamente reconhecido que a exposição humana a determinados químicos tem um impacto negativo na saúde humana. Importa, pois, caracterizar e monitorização a exposição da população por forma a, se necessário, suportar o desenvolvimento de medidas que levem à redução dessa exposição. Objetivo: Caracterizar a exposição da população adulta portuguesa a um conjunto de substâncias químicas consideradas prioritárias no âmbito da Iniciativa Europeia de Biomonitorização Humana (HBM4EU). Metodologia: Em 2019-2020 realizou-se um estudo epidemiológico transversal (INSEF-ExpoQuim) numa subamostra dos participantes do Inquérito Nacional de Saúde com Exame Físico (INSEF 2015). Para tal, selecionaram-se indivíduos de ambos os sexos e entre os 28 e 39 anos que foram recontactados. Cada participante respondeu a um questionário para recolha de dados socio-demográficos e estilos de vida e cedeu uma amostra de primeira urina da manhã. As amostras foram analisadas em laboratórios previamente qualificados para determinação de cádmio (Cd), bisfenóis (BPA, BPF e BPS), hidrocarbonetos aromáticos policíclicos (PAHs), acrilamidas e micotoxinas (desoxinivalenol – DON). Foi realizada uma análise descritiva das concentrações dos biomarcadores e uma avaliação do risco para a saúde utilizando a proporção de participantes com valores acima dos valores de referência disponíveis. Resultados: Observaram-se, na população portuguesa (n=296), valores médios das concentrações urinárias de BPA, BPS, acrilamidas e de alguns PAHs, entre os quais o 1-hidroxipireno, superiores aos valores médios observados para a população europeia, enquanto que a média das concentrações de cádmio era inferior. Comparando as concentrações dos biomarcadores analisados com os valores de referência disponíveis observou-se que 1,4%, 0%, 19,3%, 13,7% e 1,0% das concentrações de Cd, BPA, BPS, DON, e 1-hidroxipireno, respetivamente, se encontravam acima dos valores de referência. Conclusões: O INSEF-ExpoQuim produziu pela primeira vez dados de exposição a várias substâncias químicas na população adulta portuguesa harmonizados e diretamente comparáveis com valores de exposição da população Europeia. Os resultados mostraram que a população portuguesa está exposta aos químicos analisados e considerados prioritários no âmbito do HBM4EU, com uma proporção significativa de indivíduos a apresentar valores de exposição a BPS e DON, que poderão ser preocupantes, em termos de saúde pública. Os presentes resultados poderão contribuir para a definição de prioridades nacionais num futuro programa de monitorização e apoiar o desenvolvimento de políticas que visem a redução da exposição a estes químicos.
- earlyMYCO: A Pilot Mother-Child Cohort Study to Assess Early-Life Exposure to Mycotoxins - Challenges and Lessons LearnedPublication . Martins, Carla; Assunção, Ricardo; Costa, Ana; Serrano, Débora; Visintin, Lia; De Boevre, Marthe; Lachat, Carl; Vidal, Arnau; De Saeger, Sarah; Namorado, Sónia; Vidigal, Cristina; Almeida, Elisabete; Alvito, Paula; Nunes, CarlaEarly-life exposure occurs during gestation through transfer to the fetus and later, during lactation. Recent monitoring data revealed that the Portuguese population is exposed to mycotoxins, including young children. This study aimed to develop a pilot study to assess the early-life exposure to mycotoxins through a mother-child cohort, and to identify the associated challenges. Participants were recruited during pregnancy (1st trimester) and followed-up in three moments of observation: 2nd trimester of pregnancy (mother), and 1st and 6th month of the child's life (mother and child), with the collection of biological samples and sociodemographic and food consumption data. The earlyMYCO pilot study enrolled 19 mother-child pairs. The analysis of biological samples from participants revealed the presence of 4 out of 15 and 5 out of 18 mycotoxins' biomarkers of exposure in urine and breast milk samples, respectively. The main aspects identified as contributors for the successful development of the cohort were the multidisciplinary and dedicated team members in healthcare units, reduced burden of participation, and the availability of healthcare units for the implementation of the fieldwork. Challenges faced, lessons learned, and suggestions were discussed as a contribution for the development of further studies in this area.
- Challenges of the Application of In Vitro Digestion for Nanomaterials Safety AssessmentPublication . Vital, Nádia; Gramacho, Ana Catarina; Silva, Mafalda; Cardoso, Maria; Alvito, Paula; Kranendonk, Michel; Silva, Maria Joao; Louro, HenriquetaConsidering the increase in the production and use of nanomaterials (NM) in food/feed and food contact materials, novel strategies for efficient and sustainable hazard characterization, especially in the early stages of NM development, have been proposed. Some of these strategies encompass the utilization of in vitro simulated digestion prior to cytotoxic and genotoxic assessment. This entails exposing NM to fluids that replicate the three successive phases of digestion: oral, gastric, and intestinal. Subsequently, the resulting digestion products are added to models of intestinal cells to conduct toxicological assays, analyzing multiple endpoints. Nonetheless, exposure of intestinal cells to the digested products may induce cytotoxicity effects, thereby posing a challenge to this strategy. The aim of this work was to describe the challenges encountered with the in vitro digestion INFOGEST 2.0 protocol when using the digestion product in toxicological studies of NM, and the adjustments implemented to enable its use in subsequent in vitro biological assays with intestinal cell models. The adaptation of the digestion fluids, in particular the reduction of the final bile concentration, resulted in a reduced toxic impact of digestion products.