Background: Increasing environmental and occupational exposures to nanoparticles (NPs) warrant deeper insight into the toxicological mechanisms induced by these materials. The present study was designed to characterize the cell death induced by carbon black (CB) and titanium dioxide (TiO₂) NPs in bronchial epithelial cells (16HBE14o- cell line and primary cells) and to investigate the implicated molecular pathways. Results: Detailed time course studies revealed that both CB (13 nm) and TiO₂(15 nm) NP exposed cells exhibit typical morphological (decreased cell size, membrane blebbing, peripheral chromatin condensation, apoptotic body formation) and biochemical (caspase activation and DNA fragmentation) features of apoptotic cell death. A decrease in mitochondrial membrane potential, activation of Bax and release of cytochrome c from mitochondria were only observed in case of CB NPs whereas lipid peroxidation, lysosomal membrane destabilization and cathepsin B release were observed during the apoptotic process induced by TiO₂ NPs. Furthermore, ROS production was observed after exposure to CB and TiO₂ but hydrogen peroxide (H₂O₂) production was only involved in apoptosis induction by CB NPs. Conclusions: Both CB and TiO₂ NPs induce apoptotic cell death in bronchial epithelial cells. CB NPs induce apoptosis by a ROS dependent mitochondrial pathway whereas TiO₂ NPs induce cell death through lysosomal membrane destabilization and lipid peroxidation. Although the final outcome is similar (apoptosis), the molecular pathways activated by NPs differ depending upon the chemical nature of the NPs.

The uptake of nanoparticles (NPs) by cells remains to be better characterized in order to understand the mechanisms of potential NP toxicity as well as for a reliable risk assessment. Real NP uptake is still difficult to evaluate because of the adsorption of NPs on the cellular surface. Here we used two approaches to distinguish adsorbed fluorescently labeled NPs from the internalized ones. The extracellular fluorescence was either quenched by Trypan Blue or the uptake was analyzed using imaging flow cytometry. We used this novel technique to define the inside of the cell to accurately study the uptake of fluorescently labeled (SiO₂) and even non fluorescent but light diffracting NPs (TiO₂). Time course, dose-dependence as well as the influence of surface charges on the uptake were shown in the pulmonary epithelial cell line NCI-H292. By setting up an integrative approach combining these flow cytometric analyses with confocal microscopy we deciphered the endocytic pathway involved in SiO₂ NP uptake. Functional studies using energy depletion, pharmacological inhibitors, siRNA-clathrin heavy chain induced gene silencing and colocalization of NPs with proteins specific for different endocytic vesicles allowed us to determine macropinocytosis as the internalization pathway for SiO₂ NPs in NCI-H292 cells. The integrative approach we propose here using the innovative imaging flow cytometry combined with confocal microscopy could be used to identify the physico-chemical characteristics of NPs involved in their uptake in view to redesign safe NPs.

It has been shown that nanomaterials (NMs) are able to translocate to secondary tissues one of the important being the kidneys. Oxidative stress has been implicated as a possible mechanism for NM toxicity, hence effects on the human renal proximal tubule epithelial cells (HK-2) treated with a panel of engineered nanomaterials (NMs) consisting of two zinc oxide particles (ZnO - coated - NM 110 and uncoated - NM 111), two multi walled carbon nanotubes (MWCNT) (NM 400 and NM 402), one silver (NM 300) and five TiO2 NMs (NM 101, NRCWE 001, 002, 003 and 004) were evaluated. In order to assess the toxicological impact of the engineered NMs on HK-2 cells - WST-1 cytotoxicity assay, FACSArray, HE oxidation and the comet assays were utilised. For statistical analysis, the experimental values were compared to their corresponding controls using an ANOVA with Tukey’s multiple comparison. We found the two ZnO NMs (24 hr LC50 – 2.5 μg/cm2) and silver NM (24 hr LC50 – 10 μg/cm2) were highly cytotoxic to the cells. The LC50 was not attained in the presence of any of the other engineered nanomaterials (up to 80 μg/cm2). All nanomaterials significantly increased IL8 and IL6 production. Meanwhile no significant change in TNF-α or MCP-1 was detectable. The most notable increase in ROS was noted following treatment with the Ag and the two ZnO NMs. Finally, genotoxicity was measured at sub-lethal concentrations. We found a small but significant increase in DNA damage following exposure to seven of the ten NMs investigated (NM 111, NRCWE 001 and NRCWE 003 being the exception) with this increase being most visible following exposure to Ag and the positively charged TiO2. While the NMs could be categorised as low and highly cytotoxic, sub-lethal effects such as cytokine production and genotoxicity were observed with some of the low toxicity materials.

Background: Nowadays, effects of fine particulate matter (PM_2.5) are well-documented and related to oxidative stress and pro-inflammatory response. Nevertheless, epidemiological studies show that PM_2.5exposure is correlated with an increase of pulmonary cancers and the remodeling of the airway epithelium involving the regulation of cell death processes. Here, we investigated the components of Parisian PM_2.5involved in either the induction or the inhibition of cell death quantified by different parameters of apoptosis and delineated the mechanism underlying this effect.Results: In this study, we showed that low levels of Parisian PM_2.5are not cytotoxic for three different cell lines and primary cultures of human bronchial epithelial cells. Conversely, a 4 hour-pretreatment with PM_2.5prevent mitochondria-driven apoptosis triggered by broad spectrum inducers (A23187, staurosporine and oligomycin) by reducing the mitochondrial transmembrane potential loss, the subsequent ROS production, phosphatidylserine externalization, plasma membrane permeabilization and typical morphological outcomes (cell size decrease, massive chromatin and nuclear condensation, formation of apoptotic bodies). The use of recombinant EGF and specific inhibitor led us to rule out the involvement of the classical EGFR signaling pathway as well as the proinflammatory cytokines secretion. Experiments performed with different compounds of PM_2.5suggest that endotoxins as well as carbon black do not participate to the antiapoptotic effect of PM_2.5. Instead, the water-soluble fraction, washed particles and organic compounds such as polycyclic aromatic hydrocarbons (PAH) could mimic this antiapoptotic activity. Finally, the activation or silencing of the aryl hydrocarbon receptor (AhR) showed that it is involved into the molecular mechanism of the antiapoptotic effect of PM_2.5at the mitochondrial checkpoint of apoptosis.Conclusions: The PM_2.5-antiapoptotic effect in addition to the well-documented inflammatory response might explain the maintenance of a prolonged inflammation state induced after pollution exposure and might delay repair processes of injured tissues.

Background: The contribution of air particles in human cardio-respiratory diseases has been enlightened by several epidemiological studies. However the respective involvement of coarse, fine and ultrafine particles in health effects is still unclear. The aim of the present study is to determine which size fraction from a chemically characterized background aerosol has the most important short term biological effect and to decipher the determinants of such a behaviour. Results: Ambient aerosols were collected at an urban background site in Paris using four 13-stage low pressure cascade impactors running in parallel (winter and summer 2005) in order to separate four size-classes (PM_0.03–0.17 (defined here as ultrafine particles), PM_0.17–1 (fine), PM_1–2.5(intermediate) and PM_2.5–10 (coarse)). Accordingly, their chemical composition and their pro-inflammatory potential on human airway epithelial cells were investigated. Considering isomass exposures (same particle concentrations for each size fractions) the pro-inflammatory response characterized by Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) release was found to decrease with aerosol size with no seasonal dependency. When cells were exposed to isovolume of particle suspensions in order to respect the particle proportions observed in ambient air, the GM-CSF release was maximal with the fine fraction. In presence of a recombinant endotoxin neutralizing protein, the GM-CSF release induced by particles is reduced for all size-fractions, with exception of the ultra-fine fraction which response is not modified. The different aerosol size-fractions were found to display important chemical differences related to the various contributing primary and secondary sources and aerosol age. The GM-CSF release was correlated to the organic component of the aerosols and especially its water soluble fraction. Finally, Cytochrome P450 1A1 activity that reflects PAH bioavailability varied as a function of the season: it was maximal for the fine fraction in winter and for the ultrafine fraction in summer. Conclusion: In the frame of future regulations, a particular attention should thus be paid to the ultrafine/fine (here referred to as PM1) fraction due to their overwhelming anthropogenic origin and predominance in the urban aerosol and their pro-inflammatory potential.