Franck Robidel

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.

Longitudinal 3He and proton imaging of magnetite biodistribution in a rat model of instilled nanoparticles

Epidemiological and toxicological studies have provided evidence that accidentally inhaled nanosize ultrafine particles can induce chronic or acute health damage. MRI, being noninvasive, is able to assess the biodistribution and clearance of magnetically labeled nanoparticles induced by instillation or inhalation. We report 3He and proton MRI follow‐up of lung, liver, spleen, and kidney distribution of USPIO (ultrasmall superparamagnetic iron oxide) in a rat model. The sensitivity of the imaging technique to various concentrations of instilled magnetite suspension was first assessed in vivo (n = 12). A 2‐week longitudinal imaging study was then performed on animals (n = 7) instilled with a 0.5 mg magnetite solution. Hypointense and void signal regions associated with intrapulmonary USPIO were observed in the 3He ventilation images throughout the study, whereas no USPIO‐related proton signal intensity changes were found. Intrapulmonary magnetite nanoparticle confinement was confirmed by ex vivo iron assay and histological analysis. This study demonstrates that combined 3He and proton MRI enables noninvasive assessment of the distribution and clearance of magnetically labeled instilled nanoparticles. Magn Reson Med 59:1298–1303, 2008.

Interference of a short-term exposure to nitrogen dioxide with allergic airways responses to allergenic challenges in BALB/c mice

Nitrogen dioxide (NO(2)) is a common indoor and outdoor air pollutant whose role in the induction of asthma is unclear. We investigated the effects of NO(2) on the development of asthma-like responses to allergenic challenge in BALB/c mice. Ovalbumin (OVA)-immunized mice were intranasally challenged with OVA or saline solution just before starting a 3 h exposure to 5 or 20 ppm NO(2) or air. Twenty parts per million of NO(2) induced a significant increase of bronchopulmonary hyperreactivity in OVA-challenged mice and of permeability according to the fibronectin content of the bronchoalveolar lavage fluid (BALF) 24 h after exposure, as compared with air or 5 ppm NO(2). Eosinophilia (cell counts in the BALF and eosinophil peroxidase of lung tissue) was detected at 24 and 72 h with similar levels for air and 20 ppm NO(2), whereas a marked reduction was unexpectedly observed for 5 ppm NO(2). At 24 h, interleukin-5 in the BALF was markedly reduced at 5 ppm compared with 20 ppm NO(2) and was also more intense for 20 ppm NO(2) than for the air group. In contrast to specific IgG1 titers, anti-OVA IgE titers and interleukin-4 in the BALF were not affected by NO(2) exposure. Irrespective of the concentration of NO(2), OVA-challenged mice did not develop late mucosal metaplasia compared with those exposed to OVA-air. These results indicate that a short exposure to NO(2) can exacerbate or inhibit some features of the development of allergic disease in mice and may depend on the concentration of pollutant.

Study of TiO2 P25 Nanoparticles Genotoxicity on Lung, Blood, and Liver Cells in Lung Overload and Non-Overload Conditions After Repeated Respiratory Exposure in Rats

Inhaled titanium dioxide (TiO2) nanoparticles (NPs) can have negative health effects, and have been shown to cause respiratory tract cancer in rats. Inflammation has been linked to oxidative stress, and both have been described as possible mechanisms for genotoxicity of NPs, but rarely examined side-by-side in animal studies. In the present study, a wide range of complementary endpoints have been performed to study TiO2 P25 NP-induced genotoxicity in lung overload and non-overload conditions. Additionally, lung burden, inflammation, cytotoxicity and oxidative stress have also been evaluated in order to link genotoxicity with these responses. To assess quick and delayed responses after recovery, endpoints were evaluated at two time points: 2 h and 35 days after three repeated instillations. This study confirmed the previously described lung overload threshold at approximately 200-300 cm2 of lung burden for total particle surface area lung deposition or 4.2 µl/kg for volume-based cumulative lung exposure dose, above which lung clearance is impaired and inflammation is induced. Our results went on to show that these overload doses induced delayed genotoxicity in lung, associated with persistent inflammation only at the highest dose. The lowest tested doses had no toxicity or genotoxicity effects in the lung. In blood, no lymphocyte DNA damage, erythrocytes chromosomal damage or gene mutation could be detected. Our data also demonstrated that only overload doses induced liver DNA lesions irrespective of the recovery time. Tested doses of TiO2 P25 NPs did not induce glutathione changes in lung, blood or liver at both recovery times.

Effet of Combined Nitrogen Dioxide and Carbon Nanoparticle Exposure on Lung Function During Ovalbumin Sensitization in Brown Norway Rat

The interaction of particulate and gaseous pollutants in their effects on the severity of allergic inflammation and airway responsiveness are not well understood. We assessed the effect of exposure to NO2 in the presence or absence of repetitive treatment with carbon nanoparticle (CNP) during allergen sensitization and challenges in Borwn-Norway (BN) rat, in order to assess their interactions on lung function and airway responses (AR) to allergen and methacholine (MCH), end-expiratory lung volume (EELV), bronchoalveolar lavage fluid (BALF) cellular content, serum and BALF cytokine levels and histological changes. Animals were divided into the following groups (n = 6): Control; CNP (Degussa-FW2): 13 nm, 0.5 mg/kg instilled intratracheally ×3 at 7-day intervals; OVA: ovalbumin-sensitised; OVA+CNP: both sensitized and exposed to CNP. Rats were divided into equal groups exposed either to air or to NO2, 10 ppm, 6 h/d, 5d/wk for 4 weeks. Exposure to NO2, significantly enhanced lung inflammation and airway reactivity, with a significantly larger effect in animals sensitized to allergen, which was related to a higher expression of TH1 and TH2-type cytokines. Conversely, exposure to NO2 in animals undergoing repeated tracheal instillation of CNP alone, increased BALF neutrophilia and enhanced the expression of TH1 cytokines: TNF-α and IFN-γ, but did not show an additive effect on airway reactivity in comparison to NO2 alone. The exposure to NO2 combined with CNP treatment and allergen sensitization however, unexpectedly resulted in a significant decrease in both airway reactivity to allergen and to methacholine, and a reduction in TH2-type cytokines compared to allergen sensitization alone. EELV was significantly reduced with sensitization, CNP treatment or both. These data suggest an immunomodulatory effect of repeated tracheal instillation of CNP on the proinflammatory effects of NO2 exposure in sensitized BN rat. Furthermore, our findings suggest that NO2, CNP and OVA sensitization may significantly slow overall lung growth in parenchymally mature animals.

Decrease in ovalbumin-induced pulmonary allergic response by benzaldehyde but not acetaldehyde exposure in a Guinea pig model.

The pulmonary effects of two environmentally relevant aldehydes were investigated in nonsensitized or ovalbumin (OA)-sensitized guineapigs (GPs). Four-week-old male Hartley GPs, weighing about 400 g, were intraperitoneally injected with 1 ml of an NaCl solution containing 100 µ g OA and 100 mg Al(OH) 3 . They were then exposed to either acetaldehyde (200 ppb) or benzaldehyde (500 ppb) for 4 wk (6 h/d, 5 d/wk). At the end of exposure, GPs were challenged with an OA aerosol (0.1% in NaCl) and pulmonary functions were measured. The day after, guinea pigs were anesthetized and several endpoints related to inflammatory and allergic responses were assessed in blood, whole-lung histology, and bronchoalveolar lavage (BAL). Sensitized nonexposed GPs showed bronchial hyperresponsiveness to OA and an increased number of eosinophils in blood and BAL, together with a rise in total protein and leukotrienes (LTB 4 and LTC 4 /D 4 /E 4 ) in BAL. In nonsensitized GPs, exposure to acetaldehyde or benzaldehyde did not induce any change in the tested parameters, with the exception of irritation of the respiratory tract as detected by histology and an increased number of alveolar macrophages in animals exposed to acetaldehyde. In sensitized GPs, exposure to acetaldehyde induced a moderate irritation of the respiratory tract but no change in biological parameters linked to the inflammatory and allergic responses. In contrast, exposure to benzaldehyde induced a decrease both in OA-induced bronchoconstriction and in eosinophil and neutrophil numbers in BAL, an increase in the bronchodilatator mediator prostaglandin E 2 (PGE 2 ), and a decrease in the bronchoconstrictor mediators LTC 4 /D 4 /E 4 . Further investigations are needed to determine if the attenuated response observed in sensitized GPs exposed to benzaldehyde is due to an alteration of the mechanism of sensitization or to a more direct effect on various mechanisms of the allergic response.

Cerebral radiofrequency exposures during adolescence: Impact on astrocytes and brain functions in healthy and pathologic rat models

The widespread use of mobile phones by adolescents raises concerns about possible health effects of radiofrequency electromagnetic fields (RF EMF 900 MHz) on the immature brain. Neuro-development is a period of particular sensitivity to repeated environmental challenges such as pro-inflammatory insults. Here, we used rats to assess whether astrocyte reactivity, perception, and emotionality were affected by RF EMF exposures during adolescence. We also investigated if adolescent brains were more sensitive to RF EMF exposures after neurodevelopmental inflammation. To do so, we either performed 80 μg/kg intra-peritoneal injections of lipopolysaccharides during gestation or 1.25 μg/h intra-cerebro-ventricular infusions during adolescence. From postnatal day (P)32 to 62, rats were subjected to 45 min RF EMF exposures to the brain (specific absorption rates: 0, 1.5, or 6 W/kg, 5 days/week). From P56, they were tested for perception of novelty, anxiety-like behaviors, and emotional memory. To assess astrocytic reactivity, Glial Fibrillary Acidic Protein was measured at P64. Our results did not show any neurobiological impairment in healthy and vulnerable RF EMF-exposed rats compared to their sham-exposed controls. These data did not support the hypothesis of a specific cerebral sensitivity to RF EMF of adolescents, even after a neurodevelopmental inflammation. Bioelectromagnetics. 37:338-350, 2016.

Propylene glycol monomethyl ether. A three-generation study of isomer β effects on reproductive and developmental parameters in rats

Propylene glycol monomethyl ether (PGME) is widely used as a solvent in numerous commercial products. Its chemical synthesis leads to the formation of two isomers: a and b, the latter being usually present in the range of 0.5-1.5%. Isomer α has been shown to be of low toxicity. Isomer β raises concerns as to its reproductive and developmental effects. We evaluated the reproductive and developmental toxicity of two different commercial mixes of PGME (Mix A: 99% isomer α and 0.5% isomer β, Mix B: 98.5% isomer α and 1.5% isomer β) on Sprague-Dawley rats. The use of two mixes allowed us to differentiate between isomer α and isomer β effects. Male and female rats were exposed through drinking water to mixes A or B during a gametogenesis cycle (64 days for males and 15 days for females) to 0, 2, 5, 10 and 15% (v/v) of each mix. These animals (F0) and the three following generations (F1, F2 and F3) were followed. We observed a statistically significant decrease in the number of pups in isomer α-treated animals of generation F1 and a nondose-related variation of the sex ratio in F1 and F2 generations after PGME mix B treatment. The most important effect observed was a decrease in testicular and epididymal sperm counts in relation to PGME isomer β in acute daily exposure, on the first parental generation. The effect evidenced on sex ratio needs further work in order to assay the potential persistent effects of PGME exposure.