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Determining factors of the toxicity in intestine and liver for two similar sized nanoparticles used in food and packaging: In vitro and in vivo investigation on uptake and mechanisms involved (SolNanoTox)
Project
Project code: BfR-LMS-08-1350-011, LA 1177/9-1
Contract period: 01.01.2014
- 30.06.2018
Purpose of research: Applied research
The application of manufactured nanomaterials (MNMs) in food and packaging industries is expected to increase considerably in the near future, and the evaluation of the safety of MNMs present in foodstuff is thus a major concern in Europe and worldwide. Although some consumer food products contain MNMs (additives or contaminants from packaging), little is known concerning the toxicity of these MNMs following ingestion. Moreover, their size, morphology and state of agglomeration together with physiological modifications (e.g. digestion) are likely to play a considerable role in the uptake and toxicity of these materials to humans. Although numerous in vitro studies have begun to shed light on mechanistic effects, very little data is available concerning the toxic effects of MNMs following oral exposure in vivo. Nevertheless, results from in vivo experiments are the main data useful for risk evaluation. However, due to the vast quantity of different MNMs and the variability of their physic-chemical properties together with the inherent limitations of animal experimentation, the toxic effects in vivo cannot be investigated for each MNM. Therefore it is clearly necessary to establish key guidelines in the classification of MNMs according to their potential adverse effects. Among the properties of MNMs, the solubilisation capacity is likely an important determinant of nanomaterial uptake and the initiation of specific pathways of toxicity. In the SolNanoTox project, representatives of two different classes of MNMs will be investigated: titanium dioxide as an example for insoluble species due to its stability in water and aluminium representing the soluble category. Moreover, several reports in the literature suggest that aluminium and titanium oxide nanomaterials target different organs following oral exposure. It is hypothesized that aluminum nanoparticles form aluminum ions, either before or during the uptake in the intestine, whilst titanium dioxide nanoparticles may cross the intestine as intact nanoparticles. This difference in behaviour could then explain the different target organs and toxicity for the two MNMs (Figure 1). In this project, we plan to test this hypothesis by using an innovative combination of modern analytical methods for nanomaterials in tissues and single cells. The characterization of Al and TiO2 nanomaterials will be performed in solution, as well as in cell and tissue. The interaction of lipids, proteins, cell media and intestinal mucus on the characterization parameters of the MNMs will be also addressed. To explore the different solubility in physiological matrices and its influence on the potential uptake mechanisms, the project combines integrative in vitro and in vivo approaches to compare the fate, cytogenotoxic and toxicogenomic effects of the two selected MNMs. Firstly, the oral uptake and fate of MNMs in intestine and liver will be investigated in vivo after short-term oral treatment of rodents and compared to in vitro data obtained in human intestinal and hepatic cell models. Moreover, various toxic effects (genotoxicity, apoptosis, inflammation, proliferation,...) will be studied in vivo and compared the responses observed in in vitro models. In addition, to gain precise information concerning the molecular mechanisms of response following MNM treatment in vivo and in vitro, this project will employ transcriptomic and proteomic approaches. The integrative and multidisciplinary studies outlined in this project will permit to identify and distinguish the determining factors of MNMs driving their uptake, distribution and mechanisms of action. The combined expertise of accomplished research groups in the areas of MNM characterization and analytical analysis, uptake, in vitro and in vivo cytotoxicity and genotoxicity involved in this ambitious project will solve some critical questions raised for MNMs health impacts.
Comparatively little is known about the toxicity and distribution of nanomaterials (NM) after oral exposure. Different properties of the particles seem to be important for this. Since it is impossible to test all of them, a classification according to different characteristics of the NM is an approach to hazard assessment. Solubility is an important factor for oral uptake and further distribution in the organism. The aim of the project is to investigate two NM with different solubility: aluminum as a representative of NM with a possible solubility, e.g. with strong pH changes, and titanium dioxide as an example of rather insoluble NM. Comprehensive particle characterization NM as well as in vitro and in vivo studies should provide information on the uptake, distribution and toxicity of NM. Characterization of NM in stock solutions A thorough characterization of TiO2 NM (NM103 and NM104) is available in the JRC report. Al and Al2O3 NMs morphology and agglomeration were determined by transmission electron microscopy (TEM). Size distribution was also determined by Small-Angle X-Ray scattering (SAXS). Comparable results for the particle sizes were obtained and the results of the manufacturers verified. Further techniques for determining the core diameter of the NM are Single Particle Inductively Coupled Plasma Mass Spectrometry (SP-ICP-MS) and X-ray powder diffraction (XRD), which confirmed the results of the TEM and SAXS investigations. Furthermore, TEM with electron dispersive X-Ray detection (EDX) and Electron Energy Loss Spectroscopy (EELS) were applied for a discrimination of aluminum and aluminum oxide NM in relation to their composition and structure. For aluminum NM, a core-shell-structure with aluminum as core and oxygen as shell, was identified. Aluminum oxide on the other hand is fully oxidized. XRD verified the different composition of Al and Al2O3 NM via surface investigations. The hydrodynamic diameter, size distribution and zeta potential of NM was analyzed using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Since these two techniques differ significantly from those mentioned above, much larger diameters have been obtained here, since not only the particle core, but also the shell with proteins and other adsorbents is analyzed. By using ion beam microscopy (IBM) it was possible to detect elementary and molecular impurities. These resulted for TiO2 NM in an aluminum content of 4%. The adsorption of different elements and proteins after sample preparation could be visualized by confocal Raman microscopy (CRM). For the SolNanoTOX project, the Nanogenotox protocol was applied and validated following the NANoREG “SOP for probe-sonicator calibration of delivered acoustic power and de-agglomeration efficiency for in vitro and in vivo toxicological testing”. For this purpose the deagglomeration efficiency of this procedure on standard SiO2 (NM200) NM was tested and inter-laboratory verified by Dynamic Light Scattering (DLS). These measurements were accomplished by University of Leipzig in cooperation with BfR and ANSES. Interactions with lipids, proteins, cell media and intestinal mucus For the interaction of MNMs with cell media and intestinal mucus the following experiments were performed: suspensions with porcine stomach mucin types 2 and type 3 were characterized by measuring the zeta potential. Even for acidic conditions in the stomach, a negative charge was confirmed. After mixing the suspensions with positively charged NM104, the mucin adsorbed at the surface of the particles. By fluorescence measurements it was determined that 80% of the mucin were adsorbed to the particle surface. Mucin experiments were done at ISCR. Stock dispersions of aluminum oxide and zinc oxide diluted in cell culture medium did not show significant differences from the ones diluted in BSA. This was found by both partners, BfR and University of Leipzig. In case of TiO2, this dilution led to smaller particles and a higher stability. After adding cell culture medium and performing several wash steps, an accumulation of chlorine, potassium, phosphor and calcium at the surface of aluminum oxide and titanium dioxide particles was elucidated by CRM. Experiments were performed to determine the protein corona that emerges around the particles when they get in contact with serum-containing cell culture medium. For all applied nanoparticle species, the specific composition of the highest abundant corona proteins was determined by 2D-SDS-PAGE, tryptic digestion and MALDI-TOF-MS. Internal exposure in vivo: Quantification and characterization of particle uptake in gut and liver A 3-day oral exposure study in male Sprague Dawley rats was performed. Al and Al2O3 NM in three different concentrations as well as AlCl3 in the highest concentration were given to the rats. In the following, the intestine, here duodenum and colon, and the liver, kidney, spleen and blood were taken, and prepared for determination of Al organ burden by microwave assisted acid digestion followed by ICP-MS analysis. Significant differences in uptake of Al2O3 compared to Al NM were detected. Especially in blood, a much longer retention time and higher amounts for Al2O3 NM were found compared to Al NM. Highest amounts in the liver were found for the ionic AlCl3. The 28-day study was also performed, and the results for organ burden are under evaluation. In a 28-day exposure study ToF-SIMS for the highest exposure concentration results showed clearly nanoparticle uptake for both nanoparticle species (Al0-NP and Al2O3-NP) across the microvilli membrane with distinct distribution patterns for each nanoparticle species. Whilst Al0-NP were detected intra-cellular as smaller (ca. 200nm up to 1µm) or larger agglomerates (ca. 3-5µm) in the Lamina propria of the ileum and in crypts (lacteal) in the jejunum, Al2O3-NP were detected as smaller agglomerates (200nm – 1µm) in the Lamina propria of the jejunum. Internal exposure in vitro: Quantification and characterization of particle uptake in gut and liver cells In order to elucidate particle uptake, ToF-SIMS investigations were applied by BfR. It was revealed, that both aluminum particle forms were taken up and distributed in the Caco-2 cells, independent whether they were proliferating or differentiated cells. Different aluminum ions were observed under physiological conditions, for which different distribution patterns were recorded. While Al NMs were shown to form complexes with amino acids, Al2O3 NMs mostly formed polyoxo-complexes out of two or more Al2O3 molecules. Generally, smaller agglomerates of different chemical entities, Al NMs, aluminum-(III)-serine, leucine aluminate, phenylalanine aluminate and polyoxo-aluminum complexes, which do not co-localize in the same area, were observed. In addition to areas where predominantly Al2O3 NMs localize, Al2O3 NMs in DMEM show a similar pattern with areas where all chemical entities co-localize but are clearly separated from each other. A starting mineralization of the larger agglomerates, where different chemical entities co-localize and form mixed agglomerates of Al2O3 NMs, amino acids and aluminum salts, could be indicated by this. Further agglomerate compositions and chemical entities were detected. Furthermore, several ions were identified as biomarkers, suitable for the investigation of changes in the phospholipid composition of the cellular membrane. Hydrophilic NM104 nanoparticles significantly lowered lyso-phosphatidylethanolamine levels in HepaRG liver cells after 48h of exposure, whilst levels of all three lyso-phosphatidylethanolamines were similar to unexposed control cells, when cells were exposed to hydrophobic NM103. In contrast, hydrophobic NM103 increased phosphatidylethanolamine levels and ceramide levels in HepaRG liver cells after 48h of exposure, whilst hydrophilic NM104 had similar levels as unexposed control cells. In differentiated cells nanoparticle specific differences in the cell membrane pattern, which were not observed in ionic AlCl3 treated Caco-2 cells or in unexposed control cells were observed. Two ceramides were significantly enhanced in Al-NP and Al2O3-NP exposed cells, whilst levels were similar to controls in with ionic aluminum (AlCl3) exposed Caco-2 cells. One major difference for differentiated Caco-2 cells was the lower ion yields for Al0-NP in comparison to all other treatment groups of lyso phosphatidylethanolamine and lyso phosphatidylethanolamine and lyso phosphatidyl glycerol in comparison to unexposed control cells, Al2O3- exposed and ionic AlCl3 exposed Caco-2 cells. This indicated Al0-NP specific cell membrane changes in differentiated Caco-2 cells, which are not observable in Al2O3-NP or with ionic AlCl3 exposed Caco-2 cells. Nanoparticle-uptake of Al-containing nanomaterials was determined by Transwell-experiments with differentiated Caco-2 cells and two different cocultures (M-cell-model, mucus-model) following Al-quantification by AAS. In all three cell models, a particle uptake of 1-4% of the particulate species was determined, while ionic AlCl3 was not taken up significantly. For no species, a transport across the cell layer was found. NM quantification and distribution in tissue and cells down to the subcellular level Uptake and distribution of NM down to single cell level in intestine models was addressed by a combination of innovative analytical methods. University of Leipzig has applied µProton-Induced-X-Ray Emission (PIXE) and µRutherford Backscattering Spectrometry which both showed an inhomogeneous distribution of TiO2. At BfR, Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) in the imaging mode showed, that both, aluminum and aluminum oxide particles are taken up by the cells but distribute differently. The results show that Al0-NP exposed Caco-2 cells incorporated besides larger nanoparticle agglomerates of ca. 1 to 2µm diameter also smaller nanoparticle agglomerates probably in the range between 100nm and 1 µm. The results show also that not all cells of the culture have taken up nanoparticles and that there are also cells present in the culture that have taken up only smaller nanoparticle agglomerates. Therefore the overall distribution of nanoparticles within single cells is not homogenous. Al2O3-NP exposed cells showed a more homogenous distribution of smaller nanoparticle agglomerates (ca. 200nm – 2µm) throughout the whole cell culture, but also in the cell culture exposed to Al2O3-NP cells without any nanoparticle agglomerates could be detected. In Vitro Toxicokinetic Studies (Transwell-quantification-experiments) For both TiO2 NMs, NM103 and NM104, the ability to penetrate a membrane mimicking the gastrointestinal barrier was tested by application of test substances in different concentrations to the apical compartment of transwell membranes simulating the barrier function and subsequent material quantification in both, apical and basal compartments of the simulated barrier system. Prior to analysis with ICP-MS, a digestion with hydrofluoric acid digestion method was performed at BfR. Impact of the digestion process: In Vitro Digestion An artificial digestion following to a normative approach was performed by the Department for Food Safety of BfR in cooperation with the Federal Institute for Materials Research and testing (BAM). With TEM it could be shown, that during the digestion process both nanoparticle species appeared to agglomerate and to be surrounded by organic material, while their size ranges had not substantially changed. However, it has to be kept in mind that preparation for TEM analysis may cause agglomeration due to the necessary drying step. Deagglomeration was observed in intestinal fluid. The results from TEM analysis were verified by SAXS measurements. For aluminum ions, nanoparticle-like structures with different densities were observed in intestinal fluid. These were not detectable in undigested samples, saliva, or gastric fluid. In the following, characterization of the digestion fluid with DLS and NTA revealed a polydisperse suspension of large, agglomerated particles. This finding is in accordance for measurements at BfR and University of Leipzig. Furthermore, Ion Beam Microscopy (IBM) identified mainly phosphor and calcium on the surface of the investigated aluminum and aluminum oxide particles. With SP-ICP-MS applied at BfR, comparable results to DLS and NTA measurements were achieved with broad size distributions and heavy agglomerated particles, especially in the stomach. Ion release of particles in artificial digestion fluids was determined by ICP-MS after microwave assisted acid digestion. Both particle species displayed a very low ion release below 0.03% in stock solution. Similar values were obtained for the ionic content in saliva. In gastric media, Al NM released slightly more ions, whereas Al2O3 NM appeared to be more inert. In intestinal fluid, almost no free ions were detectable. Ion controls showed almost 100% free ions in stocks, saliva, and gastric fluid, while there was a significant decrease of free ions in the intestinal fluid. With ToF-SIMS agglomeration behavior of NM in the digestion fluids were investigated. In the saliva, no agglomeration was observed. Strong agglomeration could be visualized in the gastric fluid. For Al2O3 NM, these intense spots disappeared in the intestine, while strong agglomerated spots were still present for the metallic Al NM. No aluminum-containing spots in the AlCl3 samples in saliva and stomach fluid were detected, while there were some measurable accumulation spots of aluminum in the intestine fluid visible which, however, appeared much weaker than in the nanoparticle samples. In vitro results A variety of cellular endpoints were investigated with in vitro experiments. For cell culture, proliferating and differentiated Caco-2 cells, HepG2 cells and HepaRG cells were used. The experimental setting included cell viability measurements (CTB, MTT, NRU instead of LDH), Apoptosis/Necrosis (AxV/7AAD, cell cycle (PI), Glutathione levels (monochlorobimane assay instead of DCF), Mitochondrial membrane potential (JC1), ATP-levels and measurements of cellular impedance by xCELLigence systems. On intestinal cells, neither Al nor TiO2-species were found out to be cytotoxic. On hepatic cells, high concentrations of AlCl3 (100 µg Al/mL) induced cytotoxic effects and affected other cellular endpoints as well. Particle species did not induce these effects, for which reason no nano-specific toxicity was determined, even though cellular uptake of these species was higher. Artificially digested Al-species were also tested on cellular endpoints. The effects that were visible (cytotoxicity, cellular impedance) were found out to derive rather from the digestion fluids than from the Al-species. These effects did not differ from the intestinal fluid control. An adequate, non-cytotoxic concentration of digestion fluid was determined for following experiments. Digested and undigested Al-species were used for a transcriptome analysis. After incubation on differentiated Caco-2 cells in different concentrations for 24 h, cells were harvested, RNA was isolated and microarray analysis was performed with the HTA 2.0 array. Deregulated genes were identified and evaluated in silico. Fold changes of selected genes were verified in vitro by qPCR. The predicted effects were impact on metal interactions, inflammation, xenobiotic metabolism and oxidative stress. Predicted effects were stronger for ionic AlCl3 than for the particlulate Al species. Additionally, effects of digested species were stronger than of the undigested samples. Summarized, cellular effects of ionic Al-species were much higher, although cellular uptake appears to be preferred in particle form. These effects were stronger on liver cells compared with intestinal cells. No nano-specific toxicity could be determined.
Section overview
Subjects
- Food Chemistry
- Toxicology
