We examined the use of toxicogenomics data to 1) elucidate the underlying mechanisms of pulmonary responses following exposures to titanium dioxide (TiO2) nanoparticles (NPs) of different size and surface charge, and 2) determine if such responses are influenced by embedding particles in complex paint matrix. Adult C57BL/6 mice were exposed via single intratracheal instillation to three doses of free forms of NRCWE-030 (10.5 nm), NRCWE-025 (38 nm), NRCWE-001 (10 nm, neutral charge) NRCWE-002 (10 nm, positive charge) or to sanding dusts of paint consisting of NRCWE-030+NRCWE-025 or NRCWE-025 alone. Controls were exposed to dispersion medium without NPs. TiO2NPs were characterized for size, surface area, and agglomeration status in the exposure medium. Hyperspectral microscopy was performed to detect TiO2NPs in lungs and to determine the in vivo status of matrix-embedded TiO2NPs. Pulmonary gene expression profiles were generated using DNA microarrays on lung tissues collected at 24 h and 28 d post exposure time points. Bioinformatics tools were used to characterize size and surface property-pertinent effects. The data from 360 individual arrays were collapsed into 567 differentially expressed genes (False discovery rate adjusted P ≤ 0.05 and fold change ≥ 1.5 in any one condition). Unsupervised hierarchical clustering of the 567 genes revealed that TiO2NPs clustered mainly based on the post-exposure time points and then by the particle types. A meta analysis using pathway tools showed that the combination of smaller size and positive surface charge contributes to the potency of TiO2NPs. Although embedding in matrix dampens the overall toxicity of TiO2NPs, the smaller size positively influences the toxicity. Paint matrix itself did not induce any response. Finally, the observed differences in response reflected the differences in severity of the response and not the underlying mechanisms. Thus, transcriptional profiling is an effective tool to determine the important properties responsible for eliciting a response.
Overall design
Female C57BL/6 mice of 5-7 weeks old were obtained from Taconic (Ry, Denmark) and were allowed to acclimatise for 1-3 weeks before the exposure. All mice were given food (Altromin no.1324, Christian Petersen, Denmark) and water ad libitum. Mice were grouped in polypropylene cages with sawdust bedding at controlled temperature 21±1°C, humidity 50 ±10 % in a 12 h-light: dark cycle. The experiments were approved by the Danish “Animal Experiments Inspectorate” and carried out following their guidelines for ethical conduct and care when using animals in research. Animals were exposed to 18, 54, or 162 ug of NRCWE-025 and NRCWE-030 dispersed in 2% serum in water. Each exposure group consisted of minimum of 5 animals. Controls were exposed to vehicle only. The data were compared to their respective controls within the group.
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