Iron oxide particles modulate the ovalbumin-induced Th2 immune response in mice.

This study was designed to investigate the modulatory effects of submicron and nanosized iron oxide (Fe(2)O(3)) particles on the ovalbumin (OVA)-induced immune Th2 response in BALB/c mice. Particles were intratracheally administered four times to mice before and during the OVA sensitization period. For each particle type, three different doses, namely 4×100, 4×250 or 4×500 µg/mouse, were used and for each dose, four groups of mice, i.e. group saline solution (1), OVA (2), particles (3), and OVA plus particles (4), were constituted. Mice exposed to OVA alone exhibited an allergic Th2-dominated response with a consistent increase in inflammatory scores, eosinophil numbers, specific IgE levels and IL-4 production. When the mice were exposed to OVA and to high and intermediate doses of iron oxide submicron- or nanoparticles, the OVA-induced allergic response was significantly inhibited, as evidenced by the decrease in eosinophil cell influx and specific IgE levels. However, the low dose (4×100 µg) of submicron particles had no significant effect on the OVA allergic response while the same dose of nanoparticles had an adjuvant effect on the Th2 response to OVA. In conclusion, these data demonstrate that the pulmonary immune response to OVA is a sensitive target for intratracheally instilled particles. Depending on the particle dose and size, the allergic response was suppressed or enhanced.

Cytotoxicity and genotoxicity of nanosized and microsized titanium dioxide and iron oxide particles in Syrian hamster embryo cells.

Potential differences in the toxicological properties of nanosized and non-nanosized particles have been notably pointed out for titanium dioxide (TiO(2)) particles, which are currently widely produced and used in many industrial areas. Nanoparticles of the iron oxides magnetite (Fe(3)O(4)) and hematite (Fe(2)O(3)) also have many industrial applications but their toxicological properties are less documented than those of TiO(2). In the present study, the in vitro cytotoxicity and genotoxicity of commercially available nanosized and microsized anatase TiO(2), rutile TiO(2), Fe(3)O(4), and Fe(2)O(3) particles were compared in Syrian hamster embryo (SHE) cells. Samples were characterized for chemical composition, primary particle size, crystal phase, shape, and specific surface area. In acellular assays, TiO(2) and iron oxide particles were able to generate reactive oxygen species (ROS). At the same mass dose, all nanoparticles produced higher levels of ROS than their microsized counterparts. Measurement of particle size in the SHE culture medium showed that primary nanoparticles and microparticles are present in the form of micrometric agglomerates of highly poly-dispersed size. Uptake of primary particles and agglomerates by SHE exposed for 24 h was observed for all samples. TiO(2) samples were found to be more cytotoxic than iron oxide samples. Concerning primary size effects, anatase TiO(2), rutile TiO(2), and Fe(2)O(3) nanoparticles induced higher cytotoxicity than their microsized counterparts after 72 h of exposure. Over this treatment time, anatase TiO(2) and Fe(2)O(3) nanoparticles also produced more intracellular ROS compared to the microsized particles. However, similar levels of DNA damage were observed in the comet assay after 24 h of exposure to anatase nanoparticles and microparticles. Rutile microparticles were found to induce more DNA damage than the nanosized particles. However, no significant increase in DNA damage was detected from nanosized and microsized iron oxides. None of the samples tested showed significant induction of micronuclei formation after 24 h of exposure. In agreement with previous size-comparison studies, we suggest that in vitro cytotoxicity and genotoxicity induced by metal oxide nanoparticles are not always higher than those induced by their bulk counterparts.

Identification of contact and respiratory sensitizers according to IL-4 receptor α expression and IL-2 production.

Identification of allergenic chemicals is an important occupational safety issue. While several methods exist to identify contact sensitizers, there is currently no validated model to predict the potential of chemicals to act as respiratory sensitizers. Previously, we reported that cytometry analysis of the local immune responses induced in mice dermally exposed to the respiratory sensitizer trimellitic anhydride (TMA 10%) and contact sensitizer dinitrochlorobenzene (DNCB 1%) could identify divergent expression of several immune parameters. The present study confirms, first, that IgE-positive B cells, MHC class II molecules, interleukin (IL)-2, IL-4 and IL-4Rα can differentiate the allergic reactions caused by high doses of strong respiratory (TMA, phthalic anhydride and toluene diisocyanate) and contact sensitizers (DNCB, dinitrofluorobenzene and oxazolone). The second part of the study was designed to test the robustness of these markers when classing the weakly immunogenic chemicals most often encountered. Six respiratory allergens, including TMA (2.5%), five contact allergens, including DNCB (0.25%), and two irritants were compared at doses of equivalent immunogenicity. The results indicated that IL-4Rα and IL-2 can be reliably used to discriminate sensitizers. Respiratory sensitizers induced markedly higher IL-4Rα levels than contact allergens, while irritants had no effect on this parameter. Inversely, contact allergens tended to induce higher percentages of IL-2⁺CD8⁺ cells than respiratory allergens. In contrast, the markers MHC-II, IgE and IL-4 were not able to classify chemicals with low immunogenic potential. In conclusion, IL-4Rα and IL-2 have the potential to be used in classifying a variety of chemical allergens.

Effect of submicron and nano-iron oxide particles on pulmonary immunity in mice.

Due to advances in nanotechnology, exposure to particle compounds in the workplace has become unavoidable. Assessment of their toxicity on health is an important occupational safety issue. This study was conducted in mice to investigate the toxicological effects of submicron and nano-iron oxide particles on pulmonary immune defences. In that purpose, we explored for the first time, inflammatory and immune responses in lung-associated lymph nodes. For each particle type, mice received either a single intratracheal instillation at different concentrations (250, 375, or 500µg/mouse) or four repeated instillations at 500µg/mouse each. Cytokine production, inflammatory and innate immune response, and humoral immune response were respectively assessed 1, 2, and 6 days after particle exposures. Both types of particles induced lung inflammation associated with increased cytokine productions in lymph node cell cultures and decreased pulmonary immune responses against sheep erythrocytes. Natural killer activity was not modified by particles. In comparison to single instillation, repeated instillations resulted in a reduction of inflammatory cell numbers in both bronchoalveolar lavages and pulmonary parenchyma. Moreover, the single instillation model demonstrated that, for a same dose, nano-iron oxide particles produced higher levels of inflammation and immunodepression than their submicron-sized counterparts.

Simultaneous analysis of the local and systemic immune responses in mice to study the occupational asthma mechanisms induced by chromium and platinum.

As a result of industrial development, increased exposure to platinum and chromium compounds and the subsequent development of occupational asthma (OA) has been reported. Although specific IgE antibodies, an indicator of allergic asthma, against chromium and platinum have been detected in workers with OA, the immunopathological mechanisms involved in this disease are not fully understood. To better understand these complex mechanisms, the local and systemic immune responses were simultaneously analyzed in mice sensitized and challenged three, four, or five times with sodium hexachloroplatinate (Pt salt) and with potassium dichromate (Cr salt) via the respiratory route. Dinitrochlorobenzene (DNCB) and anhydride trimellitic (TMA) were included in this study as reference compounds that induce Th1 and Th2 responses respectively. All the compounds studied may provoke pulmonary sensitization. In the Pt salt-treated mice with a significant increase in local Th2 cytokine production, the increase in IgE and mucus production and in eosinophil number had a positive correlation with the number of challenges (r=0.942, 0.976, and 0.978 respectively), whereas in the Cr salt-treated mice with no local increase in Th2 cytokines, the increase in IgE production and eosinophil numbers had an inverse correlation with the number of challenges (r=-0.895 and -0.999 respectively). The Th2-dominated response induced by Pt salt was very close to that induced by TMA and may thrive after the fifth challenge, probably due to the constancy of the significant decrease in IFN-γ level in the spleens. The results of the present work may increase our understanding of the immunopathological mechanisms of OA induced by platinum and chromium, and emphasize the advantage of simultaneously analyzing local and systemic immune response when studying respiratory allergy.

In Vitro Study of Mutagenesis Induced by Crocidolite-Exposed Alveolar Macrophages NR8383 in Cocultured Big Blue Rat2 Embryonic Fibroblasts.

Asbestos-induced mutagenicity in the lung may involve reactive oxygen/nitrogen species (ROS/RNS) released by alveolar macrophages. With the aim of proposing an alternative in vitro mutagenesis test, a coculture system of rat alveolar macrophages (NR8383) and transgenic Big Blue Rat2 embryonic fibroblasts was developed and tested with a crocidolite sample. Crocidolite exposure induced no detectable increase in ROS production from NR8383, contrasting with the oxidative burst that occurred following a brief exposure (1 hour) to zymosan, a known macrophage activator. In separated cocultures, crocidolite and zymosan induced different changes in the gene expressions involved in cellular inflammation in NR8383 and Big Blue. In particular, both particles induced up-regulation of iNOS expression in Big Blue, suggesting the formation of potentially genotoxic nitrogen species. However, crocidolite exposure in separated or mixed cocultures induced no mutagenic effects whereas an increase in Big Blue mutants was detected after exposure to zymosan in mixed cocultures. NR8383 activation by crocidolite is probably insufficient to induce in vitro mutagenic events. The mutagenesis assay based on the coculture of NR8383 and Big Blue cannot be used as an alternative in vitro method to assess the mutagenic properties of asbestos fibres.

Murine bone marrow-derived dendritic cells as a potential in vitro model for predictive identification of chemical sensitizers.

The identification of potential sensitizing chemicals is a key step in the safety assessment process. To this end, predictive tests that require no or few animals and that are reliable, inexpensive and easy to perform are needed. The aim of this study was to evaluate the performance of murine bone marrow-derived dendritic cells (BMDCs) in an in vitro skin sensitization model. BMDCs were exposed to six well-known allergens (dinitrochlorobenzene, DNCB; dinitrofluorobenzene, DNFB; Bandrowski's base, BB; paraphenylenediamine, PPD; nickel sulfate, NiSO(4); cinnamaldehyde, Cinn). Surface expression of MHC class II, CD40, CD54, and CD86 was measured by flow cytometry after 48h exposure to these chemicals. All the allergens tested induced a significant increase in marker expression, with an augmentation in the percentage of mature cells ranging from 2.3- to 10.5-fold change over control. The level of up-regulation was dependent on the concentration and the strength of the allergens. In contrast, the irritants (sodium dodecyl sulfate, SDS and 4-aminobenzoic acid, pABA) and the negative control (zinc sulfate, ZnSO(4)) tested induced either no modification or a down-regulation of membrane marker expression. Taken together, our data suggest that murine BMDCs may represent a new and valuable in vitro model to predict the sensitizing properties of chemicals.

Identification of contact and respiratory sensitizers using flow cytometry.

Identification of the chemicals responsible for respiratory and contact allergies in the industrial area is an important occupational safety issue. This study was conducted in mice to determine whether flow cytometry is an appropriate method to analyze and differentiate the specific immune responses to the respiratory sensitizer trimellitic anhydride (TMA) and to the contact sensitizer dinitrochlorobenzene (DNCB) used at concentrations with comparable immunogenic potential. Mice were exposed twice on the flanks (days 0, 5) to 10% TMA or 1% DNCB and challenged three times on the ears (days 10, 11, 12) with 2.5% TMA or 0.25% DNCB. Flow cytometry analyses were conducted on draining lymph node cells harvested on days 13 and 18. Comparing TMA and DNCB immune responses on day 13, we found obvious differences that persisted for most of them on day 18. An increased proportion of IgE+ cells correlated to total serum IgE level and an enhancement of MHC II molecule expression were observed in the lymph node B lymphocytes from TMA-treated mice. The percentage of IL-4-producing CD4+ lymphocytes and the IL-4 receptor expression were clearly higher following TMA exposure. In contrast, higher proportions of IL-2-producing cells were detected in CD4+ and CD8+ cells from DNCB-treated mice. Both chemicals induced a significant increase in the percentage of IFN-gamma-producing cells among CD8+ lymphocytes but to a greater proportion following TMA treatment. In conclusion, this study encourages the use of flow cytometry to discriminate between contact and respiratory sensitizers by identifying divergent expression of immune response parameters.