Intestinal and hepatic toxicity of nanomaterials used in food and packaging : comparison of their absorption and mechanisms involved

L’incorporation croissante des (nanomatériaux) NMx dans les aliments et les emballages a contribué à une demande sociétale majeure au regard de l’évaluation des risques des NMx sur la santé. Toutefois, en raison des nombreux paramètres des NMx (taille, forme, structure cristalline, solubilité…), ainsi que des processus physiologiques (comme la digestion) pouvant impacter sur l’absorption et la réponse toxique des NMx, l’évaluation des risques des NMx est compliquée. De plus, leur évaluation in vitro est délicate en raison d’interférences (optiques, catalytiques…) lors de la réalisation des tests. Notre projet de recherche visait à déterminer, l’impact des paramètres d’hydrophobicité des NMx de TiO2 de structure cristalline rutile et l’impact de la solubilité des NMx d’Al0 et Al2O3 sur leur toxicité/génotoxicité au niveau intestinal (organe primo-exposé) et hépatique (organe d’accumulation). Les effets de ces NMx ont été étudiés par une combinaison de méthodes complémentaires in vivo par gavage sur rat, et in vitro sur les lignées humaines Caco-2 et HepaRG différenciées, tout en tenant compte des interférences in vitro. Aucune réponse toxique et génotoxique n’a été observée in vitro malgré la différence de revêtement hydrophobe/hydrophile des NMx de TiO2. Seuls les NMx d’Al2O3 ont induit des lésions oxydatives de l’ADN mais uniquement dans les cellules Caco-2, tandis que de fortes interférences ont empêché de conclure avec les NMx d’Al0. Aucun dommage chromosomique n’a été observé pour ces NMx. In vivo aucun effet génotoxique n’a été observé dans l’intestin, le colon et le foie mais des dommages à l’ADN ont été décelés avec les NMx d’Al2O3 dans la moelle osseuse. La comparaison des résultats avec ceux de la forme ionique AlCl3 suppose un mécanisme de génotoxcité indépendant de la solubilité des NMx d’Al0 et d’Al2O3 dans les milieux biologiques. Malgré les nombreux progrès en nanotoxicologie, l’ensemble de nos résultats a souligné la difficulté d’obtenir des conclusions fiables avec des tests classiques utilisés pour les produits chimiques in vitro, la difficulté d’extrapolation in vitro/in vivo des effets des NMx et le besoin de poursuivre les recherches pour disposer de méthodes et d’outils permettant d’évaluer les effets des NMx de manière fiable.The growing incorporation of nanomaterials (NMs) into food and packaging has contributed to the increasing demand for the assessment of the health hazards of these particles. However, this task is rendered difficult since the numerous intrinsic parameters (size, shape, crystalline structure, solubility ...), as well as physiological processes (such as digestion) can have an impact on the absorption and toxicological responses of NMs. Moreover, their evaluation in vitro is complicated by various sources of interference (optical, catalytic …) with toxicity tests. Our research project aimed to evaluate, the impact of hydrophobicity of rutile TiO2 NMs and solubility of AlO and Al2O3 NMs on their intestinal (first-exposed organ) and hepatic (accumulation organ) toxicity/genotoxicity. The effects of these NMs were investigated by a combination of complementary methods, in the rat in vivo by gavage, and in vitro using differentiated human Caco-2 and HepaRG cell lines, while taking into account potential interference with in vitro tests. No toxic/genotoxic response was observed in vitro despite the difference of hydrophobic/hydrophilic surface coating for TiO2 NMs. Only Al2O3 induced oxidative DNA damage solely in Caco-2 cells, while significant interference led to inconclusive results for AlO NMx. No chromosomal damage was observed for Al0 or Al2O3 NMx. In vivo no genotoxic effect was observed in the intestine, colon and liver but DNA damage was detected with Al2O3NMx in the bone marrow. Comparison of results with those for the ionic form AlCl3 demonstrated that effects observed were not related to the solubility of Al0 and d’ Al2O3 NMs in the biological environment. Despite considerable progress in nanotoxicology, our results have highlighted the difficulty to obtain reliable re sults with the traditional toxicity tests used for chemical compounds in vitro, the difficulty associated with the in vitro/in vivo extrapolation of the effects of NMs, as well as the need to continue research aimed at developing robust and reliable methods and tools for the evaluation of the effects of NMs

Genotoxicity of Aluminum and Aluminum Oxide Nanomaterials in Rats Following Oral Exposure

Due to several gaps remaining in the toxicological evaluation of nanomaterials (NMs), consumers and public health agencies have shown increasing concern for human health protection. In addition to aluminum (Al) microparticles, Al-containing nanomaterials (Al NMs) have been applied by food industry as additives and contact materials. Due to the limited amount of literature on the toxicity of Al NMs, this study aimed to evaluate the in vivo genotoxic potential of Al0 and Al2O3 NMs after acute oral exposure. Male Sprague-Dawley rats were administered three successive gavages at 6, 12.5 and 25 mg/kg bw. A comparison with AlCl3 was done in order to assess the potential effect of dissolution into Al ions. Both DNA strand breaks and oxidative DNA damage were investigated in six organs/tissues (duodenum, liver, kidney, spleen, blood and bone marrow) with the alkaline and the Fpg-modified comet assays. Concomitantly, chromosomal damage was investigated in bone marrow and colon with the micronucleus assay. The comet assay only showed DNA damage with Al2O3 NMs in bone marrow (BM), while AlCl3 induced slight but non-significant oxidative DNA damage in blood. No increase of chromosomal mutations was observed after treatment with the two Al MNs either in the BM or in the colons of rats

Aluminum and aluminum oxide nanomaterials uptake after oral exposure -a comparative study

International audienceThe knowledge about a potential in vivo uptake and subsequent toxicological effects of aluminum (Al), especially in the nanoparticulate form, is still limited. This paper focuses on a three day oral gavage study with three different Al species in Sprague Dawley rats. The Al amount was investigated in major organs in order to determine the oral bioavailability and distribution. Al-containing nanoparticles (NMs composed of Al 0 and aluminum oxide (Al 2 o 3)) were administered at three different concentrations and soluble aluminum chloride (AlCl 3 ·6H 2 O) was used as a reference control at one concentration. A microwave assisted acid digestion approach followed by inductively coupled plasma mass spectrometry (ICP-MS) analysis was developed to analyse the Al burden of individual organs. Special attention was paid on how the sample matrix affected the calibration procedure. After 3 days exposure, AlCl 3 ·6H 2 o treated animals showed high Al levels in liver and intestine, while upon treatment with Al 0 NMs significant amounts of Al were detected only in the latter. In contrast, following Al 2 o 3 NMs treatment, Al was detected in all investigated organs with particular high concentrations in the spleen. A rapid absorption and systemic distribution of all three Al forms tested were found after 3-day oral exposure. The identified differences between Al 0 and Al 2 o 3 NMs point out that both, particle shape and surface composition could be key factors for Al biodistribution and accumulation

Quantification of ongoing APOBEC3A activity in tumor cells by monitoring RNA editing at hotspots.

APOBEC3A is a cytidine deaminase driving mutagenesis, DNA replication stress and DNA damage in cancer cells. While the APOBEC3A-induced vulnerability of cancers offers an opportunity for therapy, APOBEC3A protein and mRNA are difficult to quantify in tumors due to their low abundance. Here, we describe a quantitative and sensitive assay to measure the ongoing activity of APOBEC3A in tumors. Using hotspot RNA mutations identified from APOBEC3A-positive tumors and droplet digital PCR, we develop an assay to quantify the RNA-editing activity of APOBEC3A. This assay is superior to APOBEC3A protein- and mRNA-based assays in predicting the activity of APOBEC3A on DNA. Importantly, we demonstrate that the RNA mutation-based APOBEC3A assay is applicable to clinical samples from cancer patients. Our study presents a strategy to follow the dysregulation of APOBEC3A in tumors, providing opportunities to investigate the role of APOBEC3A in tumor evolution and to target the APOBEC3A-induced vulnerability in therapy

Bisphenol A induces steatosis in HepaRG cells using a model of perinatal exposure

International audienceHuman exposure to bisphenol A (BPA) could favor obesity and related metabolic disorders such as hepatic steatosis. Investigations in rodents have shown that these deleterious effects are observed not only when BPA is administered during the adult life but also with different protocols of perinatal exposure. Whether perinatal BPA exposure could pose a risk in human is currently unknown, and thus appropriate in vitro models could be important to tackle this major issue. Accordingly, we determined whether long-term BPA treatment could induce steatosis in human HepaRG cells by using a protocol mimicking perinatal exposure. To this end, the kinetics of expression of seven proteins differentially expressed during liver development was determined during a 4-week period of cell culture required for proliferation and differentiation. By analogy with data reported in rodents and humans, our results indicated that the period of cell culture around day 15 and day 18 after seeding could be considered as the "natal" period. Consequently, HepaRG cells were treated for 3 weeks with BPA (from 0.2 to 2000 nM), with a treatment starting during the proliferating period. BPA was able to induce steatosis with a nonmonotonic dose response profile, with significant effects on neutral lipids and triglycerides observed for the 2 nM concentration. However, the expression of many enzymes involved in lipid and carbohydrate homeostasis was unchanged in exposed HepaRG cells. The expression of other potential BPA targets and enzymes involved in BPA biotransformation was also determined, giving answers as well as new questions regarding the mechanisms of action of BPA. Hence, HepaRG cells provide a valuable model that can prove useful for the toxicological assessment of endocrine disruptors on hepatic metabolisms, in particular in the developing liver

Chronic effects of two rutile TiO2 nanomaterials in human intestinal and hepatic cell lines

International audienceBackground TiO2 nanomaterials (NMs) are present in a variety of food and personal hygiene products, and consumers are exposed daily to these NMs through oral exposition. While the bulk of ingested TiO2 NMs are eliminated rapidly in stool, a fraction is able to cross the intestinal epithelial barrier and enter systemic circulation from where NMs can be distributed to tissues, primarily liver and spleen. Daily exposure to TiO2 NMs, in combination with a slow rate of elimination from tissues, results in their accumulation within different tissues. Considerable evidence suggests that following oral exposure to TiO2 NMs, the presence of NMs in tissues is associated with a number of adverse effects, both in intestine and liver. Although numerous studies have been performed in vitro investigating the acute effects of TiO2 NMs in intestinal and hepatic cell models, considerably less is known about the effect of repeated exposure on these models. In this study, we investigated the cytotoxic effects of repeated exposure of relevant models of intestine and liver to two TiO2 NMs differing in hydrophobicity for 24 h, 1 week and 2 weeks at concentrations ranging from 0.3 to 80 mu g/cm(2). To study the persistence of these two NMs in cells, we included a 1-week recovery period following 24 h and 1-week treatments. Cellular uptake by TEM and ToF-SIMS analyses, as well as the viability and pro-inflammatory response were evaluated. Changes in the membrane composition in Caco-2 and HepaRG cells treated with TiO2 NMs for up to 2 weeks were also studied. Results Despite the uptake of NM-103 and NM-104 in cells, no significant cytotoxic effects were observed in either Caco-2 or HepaRG cells treated for up to 2 weeks at NM concentrations up to 80 mu g/cm(.)(2) In addition, no significant effects on IL-8 secretion were observed. However, significant changes in membrane composition were observed in both cell lines. Interestingly, while most of these phospholipid modifications were reversed following a 1-week recovery, others were not affected by the recovery period. Conclusion These findings indicate that although no clear effects on cytotoxicity were observed following repeated exposure of differentiated Caco-2 and HepaRG cells to TiO2 NMs, subtle effects on membrane composition could induce potential adverse effects in the long-term

Quantification of ongoing APOBEC3A activity in tumor cells by monitoring RNA editing at hotspots.

APOBEC3A is a cytidine deaminase driving mutagenesis, DNA replication stress and DNA damage in cancer cells. While the APOBEC3A-induced vulnerability of cancers offers an opportunity for therapy, APOBEC3A protein and mRNA are difficult to quantify in tumors due to their low abundance. Here, we describe a quantitative and sensitive assay to measure the ongoing activity of APOBEC3A in tumors. Using hotspot RNA mutations identified from APOBEC3A-positive tumors and droplet digital PCR, we develop an assay to quantify the RNA-editing activity of APOBEC3A. This assay is superior to APOBEC3A protein- and mRNA-based assays in predicting the activity of APOBEC3A on DNA. Importantly, we demonstrate that the RNA mutation-based APOBEC3A assay is applicable to clinical samples from cancer patients. Our study presents a strategy to follow the dysregulation of APOBEC3A in tumors, providing opportunities to investigate the role of APOBEC3A in tumor evolution and to target the APOBEC3A-induced vulnerability in therapy

Aluminum in liver cells – the element species matters

International audienceAluminum (Al) can be ingested from food and released from packaging and can reach key organs involved in human metabolism, including the liver via systemic distribution. Recent studies discuss the occurrence of chemically distinct Al-species and their interconversion by contact with biological fluids. These Al species can vary with regard to their intestinal uptake, systemic transport, and therefore could have species-specific effects on different organs and tissues. This work aims to assess the in vitro hepatotoxic hazard potential of three different relevant Al species: soluble AlCl3 and two nanoparticulate Al species were applied, representing for the first time an investigation of metallic nanoparticles besides to mineral bound gamma-Al2O3 on hepatic cell lines. To investigate the uptake and toxicological properties of the Al species, we used two different human hepatic cell lines: HepG2 and differentiated HepaRG cells. Cellular uptake was determined by different methods including light microscopy, transmission electron microscopy, side-scatter analysis, and elemental analysis. Oxidative stress, mitochondrial dysfunction, cell death mechanisms, and DNA damage were monitored as cellular parameters. While cellular uptake into hepatic cell lines occurred predominantly in the particle form, only ionic AlCl3 caused cellular effects. Since it is known, that Al species can convert one into another, and mechanisms including 'trojan-horse'-like uptake can lead to an Al accumulation in the cells. This could result in the slow release of Al ions, for which reason further hazard cannot be excluded. Therefore, individual investigation of the different Al species is necessary to assess the toxicological potential of Al particles

Genotoxic impact of aluminum-containing nanomaterials in human intestinal and hepatic cells

International audienceExposure of consumers to aluminum-containing nanomaterials (Al NMs) is an area of concern for public health agencies. As the available data on the genotoxicity of AlO and Al NMs are inconclusive or rare, the present study investigated their in vitro genotoxic potential in intestinal and liver cell models, and compared with the ionic form AlCl. Intestinal Caco-2 and hepatic HepaRG cells were exposed to Al and AlO NMs (0.03 to 80 μg/cm). Cytotoxicity, oxidative stress and apoptosis were measured using High Content Analysis. Genotoxicity was investigated through γH2AX labelling, the alkaline comet and micronucleus assays. Moreover, oxidative DNA damage and carcinogenic properties were assessed using the Fpg-modified comet assay and the cell transforming assay in Bhas 42 cells respectively. The three forms of Al did not induce chromosomal damage. However, although no production of oxidative stress was detected, AlO NMs induced oxidative DNA damage in Caco-2 cells but not likely related to ion release in the cell media. Considerable DNA damage was observed with Al NMs in both cell lines in the comet assay, likely due to interference with these NMs. No genotoxic effects were observed with AlCl. None of the Al compounds induced cytotoxicity, apoptosis, γH2AX or cell transformation