Françoise Rogerieux

Comparative toxicity of 24 manufactured nanoparticles in human alveolar epithelial and macrophage cell lines

BackgroundA critical issue with nanomaterials is the clear understanding of their potential toxicity. We evaluated the toxic effect of 24 nanoparticles of similar equivalent spherical diameter and various elemental compositions on 2 human pulmonary cell lines: A549 and THP-1. A secondary aim was to elaborate a generic experimental set-up that would allow the rapid screening of cytotoxic effect of nanoparticles. We therefore compared 2 cytotoxicity assays (MTT and Neutral Red) and analyzed 2 time points (3 and 24 hours) for each cell type and nanoparticle. When possible, TC50 (Toxic Concentration 50 i.e. nanoparticle concentration inducing 50% cell mortality) was calculated.ResultsThe use of MTT assay on THP-1 cells exposed for 24 hours appears to be the most sensitive experimental design to assess the cytotoxic effect of one nanoparticle. With this experimental set-up, Copper- and Zinc-based nanoparticles appear to be the most toxic. Titania, Alumina, Ceria and Zirconia-based nanoparticles show moderate toxicity, and no toxicity was observed for Tungsten Carbide. No correlation between cytotoxicity and equivalent spherical diameter or specific surface area was found.ConclusionOur study clearly highlights the difference of sensitivity between cell types and cytotoxicity assays that has to be carefully taken into account when assessing nanoparticles toxicity.

Biodistribution and clearance of instilled carbon nanotubes in rat lung

BackgroundConstituted only by carbon atoms, CNT are hydrophobic and hardly detectable in biological tissues. These properties make biokinetics and toxicology studies more complex.MethodsWe propose here a method to investigate the biopersistence of CNT in organism, based on detection of nickel, a metal present in the MWCNT we investigated.Results and conclusionOur results in rats that received MWCNT by intratracheal instillation, reveal that MWCNT can be eliminated and do not significantly cross the pulmonary barrier but are still present in lungs 6 months after a unique instillation. MWCNT structure was also showed to be chemically modified and cleaved in the lung. These results provide the first data of CNT biopersistence and clearance at 6 months after respiratory administration.

Induction of apoptosis and absence of inflammation in rat lung after intratracheal instillation of multiwalled carbon nanotubes.

Several studies performed by intratracheal instillation showed that carbon nanotubes (CNT) induced pulmonary fibrosis, granulomas or inflammation. But, recently, two inhalation studies did not observed such pathological phenomena and suggest that granulomas could be due to the instillation of unbreathable agglomerates. In a previous study, we have described a simple method (using albumin as dispersing agent) which produced solutions containing more than 80% of agglomerate of breathable size. We report here results from intratracheal instillation of rats by 0, 1, 10 or 100 microg of MWCNT dispersed with albumin. After 1, 7, 30, 90, and 180 days, inflammation, apoptosis, fibrosis, respiratory parameters and granuloma formation were assessed. Results obtained by plethysmography, soluble collagen quantification, qRT-PCR and luminex measurement of cytokines expression and histopathological observation showed only evidence of apoptosis of alveolar macrophages. These result underline the importance of controlling MWCNT agglomerate size when exposing animals, through appropriate dispersion methods.

Effect of BSA on carbon nanotube dispersion for in vivo and in vitro studies

Carbon nanotubes (CNT) are one of the most promising nanomaterials because of their intrinsic properties. So, it becomes urgent to assess their toxicity. However, CNT are insoluble in aqueous media required for toxicological studies. Thus, we propose a simple method to disperse CNT for toxicological studies using a biomolecule: The albumin. To evaluate this method, several nanotubes were suspended in saline solution (NaCl 0.9%) without or with albumin at a concentration of 0.5 mg/ml or equal as CNT concentration. These suspensions were visually compared to suspensions obtained with classical dispersing methods using Tween 80 or serum. Homogeneity of the suspensions with or without BSA and CNT structure were analyzed by TEM, agglomerates quantification and total carbon dosage. The effect of coupled albumin-CNT was then tested on A549 and U937 cells in vitro and on rats in vivo. Total carbon dosage, agglomerates quantification and TEM revealed that, in the presence of albumin, the tested nanotubes were better dispersed without any modification of their structure. The CNT suspension was tested in vitro and in vivo in rats. Albumin solution alone induced no modification of the biological responses studied (i.e., cell viability in vitro and inflammatory response and histopathology in vivo) compared to the saline. CNT in NaCl or BSA altered cellular viability in vitro in a similar way but results obtained with CNT suspension in the presence of albumin showed a better reproducibility that can be explained by the better homogeneity of the suspensions. CNT in BSA but not in NaCl significantly increased the cell number in BAL and also the number of apparent CNT-containing cells. Taken together, these results highlight the potential importance of CNT dispersion (and thus of the vehicle) for the toxicological studies.

Two-step purification method of vitellogenin from three teleost fish species : rainbow trout (Oncorhynchus mykiss), gudgeon (Gobio gobio) and chub (Leuciscus cephalus)

A two-step purification protocol was developed to purify rainbow trout (Oncorhynchus mykiss) vitellogenin (Vtg) and was successfully applied to Vtg of chub (Leuciscus cephalus) and gudgeon (Gobio gobio). Capture and intermediate purification were performed by anion-exchange chromatography on a Resource Q column and a polishing step was performed by gel permeation chromatography on Superdex 200 column. This method is a rapid two-step purification procedure that gave a pure solution of Vtg as assessed by silver staining electrophoresis and immunochemical characterisation.

Modifications of Phleum pratense Grass Pollen Allergens following Artificial Exposure to Gaseous Air Pollutants (O3, NO2, SO2)

Background: Air pollution is frequently proposed as a potential cause of the increased incidence of allergy in industrialised countries. Our objective was to investigate the impact of the major gaseous air pollutants on grass pollen allergens. Methods: Timothy grass pollen was exposed to ozone (O3), nitrogen dioxide (NO2) and sulphur dioxide (SO2) alone or in combination. Allergen contents were analysed by 2-dimensional immunoblot using grass pollen-sensitive patient sera. Results: For O3-treated pollen, immunoblotting showed an acidification of allergens Phl p 1b, Phl p 4, Phl p 5 and Phl p 6 and an IgE recognition decrease in Phl p 1, Phl p 2, Phl p 6 and Phl p 13. NO2 exposure induced a decrease in Phl p 2, Phl p 5b and Phl p 6 recognition, and SO2 treatment induced a decrease in Phl p 2, Phl p 6 and Phl p 13 recognition. Moreover, samples treated with a mix of NO2/O3 or NO2/SO2 showed a higher decrease in allergen content, compared with samples treated with only one pollutant. The O3 acidification was also observed with the NO2/O3 mix. Conclusion: Exposure of pollen to gaseous pollutants induced a decrease in allergen detection in pollen extracts. This decrease could be due to a mechanical loss of allergens from the altered pollen grains and/or post-translational modifications affecting allergen recognition by IgE.

Biodistribution and Clearance of TiO2 Nanoparticles in Rats after Intravenous Injection

Titanium dioxide (TiO2) nanoparticles are used in many applications. Due to their small size, easy body penetration and toxicological adverse effects have been suspected. Numerous studies have tried to characterize TiO2 translocation after oral, dermal or respiratory exposure. In this study, we focused on TiO2 nanoparticle biodistribution, clearance and toxicological effects after intravenous injection, considering TiO2 translocation in the blood occurs. Using ICP-OES, transmission electron microscopy, and histological methods, we found TiO2 accumulation in liver, lungs and spleen. We estimated TiO2 nanoparticles’ half life in the body to about 10 days. Clinical biomarkers were also quantified for 56 days to identify potential toxicological impact on lungs, blood, liver, spleen and kidneys. Results showed absence of toxicological effects after TiO2 intravenous injection at concentrations of 7.7 to 9.4 mg/kg.

Use of transepithelial electrical resistance in the study of pentachlorophenol toxicity.

The toxicity of pentachlorophenol (PCP), a polluting substance believed to exert a narcotic effect, was assayed using the Caco-2 cell line as a model. In order to assess this toxicity as fully as possible, several viability tests, each examining different endpoints, have been used. Neutral red uptake was found to be more sensitive to PCP than MTT and Alamar Blue tests. Transepithelial electrical resistance (TEER) was shown to be the most sensitive to PCP at concentrations and exposure times where the Alamar Blue, LDH leakage and Blue Dextran passage did not evidence any effect. Blue Dextran passage and optical microscopy revealed cellular detachment at concentrations where LDH and Alamar Blue showed little or no cytotoxicity. Thus, PCP seems to affect the integrity of the intestinal barrier at levels where no cytotoxicity is seen. Our results support the notion that TEER can be used as a very sensitive method for evaluating membrane-perturbing toxicants.

Cell cooperation and role of the P2X7 receptor in pulmonary inflammation induced by nanoparticles

Abstract Macrophages and alveolar epithelial cells are the first targets of inhaled nanoparticles (NPs) reaching the alveoli. Mono- or co-cultures of lung epithelial (A549 or NCI-H441) and macrophage (THP-1) cell lines were used to study the cell cooperation and the involvement of the P2X7 cell death receptor during the inflammation caused by SiO2 and TiO2 NPs. Here we show that, secretion of pro-inflammatory cytokines (IL-1β, IL-6 and IL-8) in response to NPs exposure was higher in co-cultures than in mono-cultures. A functional P2X7 receptor was found in all the cell lines studied. Its involvement in IL-1β secretion in co-cultures was demonstrated using a specific antagonist, the brilliant blue G. Furthermore, mono and co-cultures exhibited distinct secretion patterns of pro-inflammatory cytokines in response to NPs exposure, and we provide the first evidence that the P2X7 receptor is involved in the inflammation triggered by SiO2 and TiO2 NPs, by increasing IL-1β secretion, and likely through the inflammasome pathway. Altogether, our data indicate that cell co-cultures used in this study represent valid models to study the inflammatory mechanisms of NPs within the alveoli.

Ability of Pollen Cytoplasmic Granules to Induce Biased Allergic Responses in a Rat Model

Background: Grass pollen is one of the most important aeroallergens in Europe. It highly contributes to respiratory allergic diseases, mainly allergic rhinitis. In contact to water or airborne pollutants, pollen grains can release pollen cytoplasmic granules (PCGs) containing allergens. Because of their size (<5 µm), PCGs may penetrate deeper into the lungs to induce higher allergic responses, such as asthma. They have been associated with thunderstorm-related asthma. The aim of this study was to evaluate, with Brown Norway rats, the allergenic potential of isolated PCGs and to compare it with the allergenicity of whole timothy grass pollen. Methods: Rats were sensitized (day 0) and challenged (day 21), in controlled comparative conditions, with pollen grains (0.5 mg) or PCGs (4.5 × 107 and 0.5 mg). At day 25, blood samples, bronchoalveolar lavage fluid (BALF) and bronchial lymph node were collected. IgE and IgG1 levels in sera were assessed by ELISA. Alveolar cells, protein and cytokine concentrations were quantified in BALF. T cell proliferation, in response to pollen or granules, was performed by lymph node assay. Results: The results showed that proliferative responses of lymph node cells were similar in PCG- and pollen-sensitized rats. IgE and IgG1 levels were higher in pollen- than in PCG-sensitized rats. However, eosinophils, lymphocytes and pro-allergy cytokines in BALF were higher in PCG- than in pollen-sensitized rats. Conclusions: Thus, PCGs, able to deeply penetrate in the respiratory tract, induced local and strong allergic and inflammatory responses more linked with asthma- than rhinitis-related allergic symptoms.