Rat pulmonary responses to inhaled nano-TiO₂: effect of primary particle size and agglomeration state

Abstract

Background: The exact role of primary nanoparticle (NP) size and their degree of agglomeration in aerosols on the determination of pulmonary effects is still poorly understood. Smaller NP are thought to have greater biological reactivity, but their level of agglomeration in an aerosol may also have an impact on pulmonary response. The aim of this study was to investigate the role of primary NP size and the agglomeration state in aerosols, using well-characterized TiO₂ NP, on their relative pulmonary toxicity, through inflammatory, cytotoxic and oxidative stress effects in Fisher 344 male rats.

Methods: Three different sizes of TiO₂ NP, i.e., 5, 10-30 or 50 nm, were inhaled as small (SA) (< 100 nm) or large agglomerates (LA) (> 100 nm) at 20 mg/m³ for 6 hours.

Results: Compared to the controls, bronchoalveolar lavage fluids (BALF) showed that LA aerosols induced an acute inflammatory response, characterized by a significant increase in the number of neutrophils, while SA aerosols produced significant oxidative stress damages and cytotoxicity. Data also demonstrate that for an agglomeration state smaller than 100 nm, the 5 nm particles caused a significant increase in cytotoxic effects compared to controls (assessed by an increase in LDH activity), while oxidative damage measured by 8-isoprostane concentration was less when compared to 10-30 and 50 nm particles. In both SA and LA aerosols, the 10-30 nm TiO₂ NP size induced the most pronounced pro-inflammatory effects compared to controls.

Conclusions: Overall, this study showed that initial NP size and agglomeration state are key determinants of nano-TiO₂ lung inflammatory reaction, cytotoxic and oxidative stress induced effects.

Figure 1

Cumulative distributions based on number concentration of the NP aerosols. NP aerosols cumulative size distributions based on number concentration measured with the ELPI. For each aerosol 5 thirty-minute samples were collected every hour of the experiment.

Figure 2

NP aerosol agglomerate structure observed by transmission electron microscopy. Air samples were collected on pre-carbon coated Formvar copper grids glued onto 25-mm polycarbonate filters. Characterization (shape, agglomeration degree and structure) of the nano-aerosols was performed by TEM.

Figure 3

Computed fractional deposition of NP agglomerates in the rat respiratory tract. Estimated deposition fraction for the aerosols at 20 mg/m³ composed of SA or LA modeled for multiple diameters. TB = tracheobronchial; P = pulmonary. Functional residual capacity (FRC) volume 4 ml; head volume: 0.42 ml; nasal breathing route; tidal volume 2.1 ml; breathing frequency 110/min and inspiratory fraction 0.1.

Figure 4

BALF cytology analyzed by the cytospin method. Data were expressed as fold increases of exposed groups compared to controls. Bars represent the mean value and the standard error of the mean obtained for 6 rats in each exposure group. Statistical procedures: ANOVA followed by a Tukey’s test. *Mean value is statistically different from control level p < 0.05.

Figure 5

Relative levels of BALF pro-inflammatory cytokines. BALF pro-inflammatory cytokines were expressed as fold increases of exposed groups compared to controls. Samples from all rats of the same exposure group were pooled together. The ratio for each cytokine was calculated as described in materials and methods. Results with ratios ≥1.2 were considered to represent a slight inflammation. (A) Aerosols with a primary NP size of 5 nm. (B) Aerosols with a primary NP size of 10–30 nm. (C) Aerosols with a primary particle size of 50 nm.

Figure 6

BALF cytotoxicity (LDH) and oxidative stress (8-isoprostane) markers. Data were expressed as fold increases of exposed groups compared to controls. Bars represent the mean value and the standard error on the mean obtained for 6 rats in each exposure group. LDH assay for each rat was done in triplicate and 8-isoprostane was in duplicate. Statistical procedures: ANOVA followed by a Tukey’s test. * Mean value is statistically different from control level p < 0.05; ** Mean value is statistically different from control level and 10–30; 50 nm SA aerosols p < 0.05; ŧ Mean value is statistically different from all LA aerosol levels p < 0.05.

Figure 7

Representative cell morphology from BALF cytopreparations of rats. Optical microscopy (magnification 400-x) of cells cytopreparation collected in BALF of sham, controls (exposed to compressed air) and TiO₂ NP exposed rats. For all TiO₂ exposed groups, the images show giant foamy macrophages and the distribution of macrophages containing phagocytized TiO₂ NP (arrows). Scale bar = 50 μm.

Figure 8

Optical microscopy images (100-x) of lung tissue sections. Morphological assessments of lung tissue stained with haematoxylin and eosin of sham (A), control (exposed to compressed air) and TiO₂ NP exposed rats by means of inhalation for 6 hours. Responses to nano-TiO₂(C to G) were different in intensity compared to the controls (B), except for the 50 nm SA group (H). The lungs of rats exposed to 5 and 10–30 nm SA aerosols (D and F) as well as rats exposed to 10–30 and 50 nm LA aerosols (E and G) showed more leukocyte infiltration compared to the control group (B).

Figure 9

Optical microscopy images (400-x) of lung tissue sections. (A) Lung of control rat exposed to compressed air. (B) Lung of a rat exposed to small agglomerates of 5 nm NP. This figure demonstrates TiO₂ NP engulfed by alveolar macrophages (arrows).

Figure 10

Experimental set-up for the generation of small agglomerate aerosols. Experimental set-up using three nebulizers in parallel to produce aerosols essentially composed of small NP agglomerates. ELPI: electrical low pressure impactor; HEPA filter: high-efficiency particulate air filter.