Michèle Goutet

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.

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.

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.

Effects of nickel sulfate on pulmonary natural immunity in Wistar rats

This study was designed to investigate the in vivo effect of nickel sulfate on the pulmonary non-specific immune defences. Groups of four male Wistar rats were treated with a single intratracheal instillation of NiSO(4) at different doses: 1, 2, 4 and 8 micromol of NiSO(4) per rat. Control rats received a corresponding instillation of the saline vehicle. The effect of NiSO(4) on the cytotoxic activity of the pulmonary natural killer (NK) cells and alveolar macrophages (AM), as well as the pulmonary production of cytokines such as alpha-tumor necrosis factor (TNF-alpha) and gamma-interferon (IFN-gamma), were examined 1, 2 and 7 days later. Spontaneous NK-cytotoxicity towards mouse-derived tumor cell line, Yac-1 was suppressed 1 day after treatment at doses of 2 micromol/rat and above with only one result significant (P<0.05); 2 days after treatment the suppression was increased with all results significant at the same doses; 1 week after treatment NK activity restoration was observed except for the highest dose, 8 micromol/rat. AM-mediated cytotoxicity towards mouse-derived tumor cell line, 3T12, did not show any significant difference in treated and untreated animals. In contrast, whereas moderate levels of TNF-alpha were detected in the broncho-alveolar lavage (BAL) fluid supernatants of controls, the NiSO(4) treatment highly suppressed TNF-alpha production with a maximum observed after 2 days. TNF-alpha suppression was found to be transient, at least with the lowest NiSO(4) dose, with levels returning to normal after 7 days. A non-significant increase in IFN-gamma was observed in the BAL fluids of treated animals at each time of examination. Taken together, these results indicate that NK cell activity and TNF-alpha secretion are sensitive targets for instilled NiSO(4) in Wistar rats.

TDI inhalation in guinea-pigs involves migration of dendritic cells.

Toluene diisocyanate (TDI) can cause occupational asthma, but the mechanism underlying sensitization to this chemical compound remains controversial. The present study aims to investigate whether tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) liberated in the lungs after TDI inhalation can contribute to the migration of dendritic cells from respiratory airways towards lung associated lymph nodes for presentation of TDI hapten. Exposure was studied in two modes: (1) acute exposure (experiment no. 1, 2 and 3) where animals were exposed to 2.962, 1.060, and 1.076 ppm TDI for 1, 4, and two periods of 4 h, respectively; (2) subacute exposure (experiment no. 4, 5 and 6) where animals were exposed to 0.066, 0.110, and 0.999 ppm TDI for 48 h for the two lower doses and 5 days for the highest dose. Depending on the modes of exposure, two to four post exposure times were selected. After acute exposure to 2.962 ppm TDI for 1 h, the increase in TNF-alpha and IL-6 levels in bronchoalveolar lavage (BAL) fluid was observed immediately at the end of inhalation exposure, whereas the maximum number of dendritic cells and total cells occurred at post exposure times of 48 h and 5 days, respectively. In two other acute exposures, the peak increases in TNF-alpha, IL-6 and total cell numbers were observed at 48 h post exposure time, whereas the peak increase in dendritic cells occurred at 24 h. After subacute exposure to 48 h TDI, where TDI concentrations were relatively low (0.006 or 0.110 ppm), a parallel increase in TNF-alpha and IL-6 levels, dendritic and total cell numbers were observed at 0 h post exposure time. This phenomenon was also apparent at 24 h post exposure time when the animals had been exposed to 1.999 ppm TDI for 5 days. From these results, we can conclude that dendritic cells could play a key role as antigen presenting cells in the development of TDI-induced respiratory sensitization, and that their migration toward lung-associated lymph nodes is probably conditioned by cytokine release in their micro-environment. Future work must delineate whether TNF-alpha and IL-6 are solely responsible for the migration of dendritic cells after TDI inhalation, for example by using antibodies to neutralize these cytokines.

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.

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.

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.

Serum-borne factor(s) of 1,1-dichloroethylene and 1,2-dichlorobenzene-treated mice inhibited in vitro antibody forming cell response and natural killer cell activity

1,1-Dichloroethylene and 1,2-dichlorobenzene administered to mice produced liver and/or kidney damage which was quantified in this study by a histochemical method. The in vitro effect of sera obtained from these mice on antibody forming cell (AFC) response and natural killer (NK) cell activity was investigated in parallel with the assessment of sera tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) levels. 1,1-Dichloroethylene (100, 150 and 200 mg/kg) provoked liver and kidney damage. Peak kidney damage occurred 16 h after the dose was administered and at 24 h in the case of the liver. During the peak level of liver damage, a serum-borne immunosuppressive effect was also at its highest level. With respect to sera cytokine levels, an increase of TNF-alpha and IL-6 was detected earlier, i.e. 6 h after toxic administration, followed by a decrease that tended toward a baseline level. There was a relationship between the tissue damage induced by 1,1-dichloroethylene and the immunosuppressive effect of mice sera on AFC response and NK cell activity. 1,2-Dichlorobenzene (300, 500 and 600 mg/kg) provoked only liver damage. Peak liver damage severity was observed 48 h after toxic administration, whereas the highest serum-borne immunosuppressive effect was observed almost immediately, i.e. 6 h after administration. As regards sera cytokine levels, only TNF-alpha could be detected 6 h after administering 500 and 600 mg/kg doses of 1,2 dichlorobenzene. There was a relationship between the liver damage induced by 1,2-dichlorobenzene and the immunosuppressive effect of mice sera on the AFC response. In view of the above results, this study suggests that the immunosuppressive effect in sera of mice treated with 1,1-dichloroethylene and 1,2-dichlorobenzene may result from tissue damage, and that the increased levels of TNF-alpha and IL-6 in sera may contribute to this effect. Further studies are needed to clarify the factor(s) responsible, including transforming growth factor-beta1 (TGF-beta1) causing immunosuppression.