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.

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.

Use of the caco-2 model in the screening of polluting substance toxicity.

The aim of this work was to investigate the oral toxicity of representative chemicals chosen from each class of the list of 132 substances present in industrial effluents after the EEC Directive 76-464. Owing to its characterization as a model of the intestinal epithelium, the CaCo-2 cell line model was chosen. Cytotoxicity was assayed using the tetrazolium blue (MTT) test. For most of the substances, a linear correlation was observed between the octanol/water partition coefficient (log Kw) and the median inhibition concentration (IC(50)). This relationship between lipophilicity and toxicity is the hallmark of a narcotic mechanism of action. However, diethylamine appeared more toxic than the correlation would predict. Other amines were then tested (tert-butylamine, n-butylamine and benzylamine). All of these did not fit into the baseline correlation. The IC(50) were corrected by taking into account only the non-ionized, lipid insoluble, concentration at pH7.3. The amines still did not fit into the correlation, reinforcing the idea of a non-narcotic mechanism. The toxicity of a large number of substances can thus be predicted from their physico-chemical properties only when the substances exert a direct and non-specific effect. The amines appeared more toxic than substances with the same partition coefficient, showing that knowledge of the only lipophilicity is too restrictive to predict toxicity.

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 microg 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.

Modifications of Phleum pratense grass pollen allergens following artificial exposure to gaseous air pollutants (O(3), NO(2), SO(2)).

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 (O(3)), nitrogen dioxide (NO(2)) and sulphur dioxide (SO(2)) alone or in combination. Allergen contents were analysed by 2-dimensional immunoblot using grass pollen-sensitive patient sera. RESULTS: For O(3)-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. NO(2) exposure induced a decrease in Phl p 2, Phl p 5b and Phl p 6 recognition, and SO(2) treatment induced a decrease in Phl p 2, Phl p 6 and Phl p 13 recognition. Moreover, samples treated with a mix of NO(2)/O(3) or NO(2)/SO(2) showed a higher decrease in allergen content, compared with samples treated with only one pollutant. The O(3) acidification was also observed with the NO(2)/O(3) 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.

The trypsin-induced leucostasis which leads to emphysema in the hamster is not due to contaminating endotoxins.

Intravenous injection of trypsin in the rat induces early lung leucostasis and emphysema of delayed onset. This report confirms that this emphysema is not rat-specific and that the leucostasis is not related to the presence of contaminating endotoxin in the trypsin. In hamsters (n = 37), leucostasis did not occur when they were injected with heat-treated trypsin, but numerous granulocytes were sequestered in the vessels of hamsters receiving a fresh solution of trypsin. In these hamsters, the number of granulocytes harvested by lavage increased significantly (1.87 x 10(6) per ml, P < 0.001) compared with hamsters injected with either heat-denatured trypsin (0.89) or saline (0.86), or compared with controls (0.86). Emphysema was inconstantly observed in hamsters 6 or 12 weeks after injection with trypsin for 1 h. It was frequently (17/20) present and always (20/20) well developed (intercept + 180 per cent) in the 2-h perfused hamsters whose lungs were abnormally heterogeneous (index + 100 per cent) relative to the seven controls and to the nine saline-injected hamsters. The efficiency of trypsin in triggering emphysema (percentage of hamsters having abnormal values of intercept) was dependent on the time of perfusion. This form of experimental emphysema is thus considered to be due to an endotoxin-independent leucostasis.

Determination of the sialic acid linkage specificity of sialidases using lectins in a solid phase assay.

A procedure for the determination of activity and linkage specificity of sialidases is described. The sialoglycoprotein fetuin is coated onto a microtiter plate and incubated with sialidases from different sources. Enzymatic activities and linkage specificities are then determined by a sandwich method which measured the binding of different lectins to fetuin. The lectins used were peanut agglutinin (PNA) from Arachis hypogaea, which binds specifically the galactose beta-1-3-N-acetylgalactosamine structures that are unmasked following sialidase treatment of fetuin, the lectins from Sambucus nigra (SNA) and Maackia amurensis (MAA) that are specific for alpha-2-6 and alpha-2-3 bound sialic acids, respectively, and the slug agglutinin from Limax flavus (LFA) that is specific for N-acetyl and N-glycolyl neuraminic acids. Increased PNA and decreased LFA, SNA, and MAA lectin binding correlated with sialidase-induced desialylation of the substrate. In this report, the assay was used to determine the activities and specificities of influenza, Vibrio cholerae, and Arthrobacter ureafaciens sialidases.

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.