Agglomeration State of Titanium-Dioxide (TiO2) Nanomaterials Influences the Dose Deposition and Cytotoxic Responses in Human Bronchial Epithelial Cells at the Air-Liquid Interface

Abstract

Extensive production and use of nanomaterials (NMs), such as titanium dioxide (TiO₂), raises concern regarding their potential adverse effects to humans. While considerable efforts have been made to assess the safety of TiO₂ NMs using in vitro and in vivo studies, results obtained to date are unreliable, possibly due to the dynamic agglomeration behavior of TiO₂ NMs. Moreover, agglomerates are of prime importance in occupational exposure scenarios, but their toxicological relevance remains poorly understood. Therefore, the aim of this study was to investigate the potential pulmonary effects induced by TiO₂ agglomerates of different sizes at the air-liquid interface (ALI), which is more realistic in terms of inhalation exposure, and compare it to results previously obtained under submerged conditions. A nano-TiO₂ (17 nm) and a non-nano TiO₂ (117 nm) was selected for this study. Stable stock dispersions of small agglomerates and their respective larger counterparts of each TiO₂ particles were prepared, and human bronchial epithelial (HBE) cells were exposed to different doses of aerosolized TiO₂ agglomerates at the ALI. At the end of 4h exposure, cytotoxicity, glutathione depletion, and DNA damage were evaluated. Our results indicate that dose deposition and the toxic potential in HBE cells are influenced by agglomeration and exposure via the ALI induces different cellular responses than in submerged systems. We conclude that the agglomeration state is crucial in the assessment of pulmonary effects of NMs.

Keywords: agglomerates; air-liquid interface; nanomaterials; pulmonary toxicity; titanium dioxide.

Figure 1

Experimental setup: generation of airborne TiO₂ agglomerates in dry air according to VDI guideline 3491 and exposure of human lung cells in the Air–Liquid-Interface Exposure System with accompanying measurement of particle size distribution using Scanning Mobility Particle Sizer (SMPS) at the dry stage in the reactor as well as for the aerosol inside the exposure system.

Figure 2

Particle size distributions of TiO₂ agglomerates measured by Scanning Mobility Particle Sizer U-SMPS in the range of 8 to 800 nm. Each curve shows the means of number size distributions in dependence of particle type and agglomeration state. Red circles: 17 nm-SA; black squares: 17 nm-LA; blue triangles: 117 nm-SA; and green inverted triangles: 117 nm-LA.

Figure 3

Effect on metabolic activity of HBE cells after 4 h exposure to TiO₂ agglomerates at the ALI. 17 nm-SA (A), 17 nm-LA (B), 117 nm-SA (C), and 117 nm-LA (D). Data are expressed as means ± SD from three independent experiments with six replicates each. p < 0.05 (*) and p < 0.01 (**) represent significant difference compared to control (One-way ANOVA followed by Dunnett’s multiple comparison test).

Figure 4

LDH activity measured in HBE cell supernatants after 4 h exposure to TiO₂ agglomerates at the ALI. 17 nm-SA (A), 17 nm-LA (B), 117 nm-SA (C), and 117 nm-LA (D). Data are expressed as means ± SD from three independent experiments with six replicates each. p < 0.05 (*) represent significant difference compared to control (One-way ANOVA followed by Dunnett’s multiple comparison test).

Figure 5

Glutathione levels measured in HBE cells after 4 h exposure to TiO₂ agglomerates at the ALI. 17 nm-SA (A), 17 nm-LA (B), 117 nm-SA (C), and 117 nm-LA (D). Data are expressed as means ± SD from two independent experiments with six replicates each. p < 0.05 (*) represent significant difference compared to control (One-way ANOVA followed by Dunnett’s multiple comparison test).

Figure 6

DNA damage measured in HBE cells after 4 h exposure to TiO₂ agglomerates at the ALI. 17 nm-SA (A), 17 nm-LA (B), 117 nm-SA (C), and 117 nm-LA (D). Data are expressed as means ± SD from two independent experiments with six replicates each. p < 0.05 (*) represent significant difference compared to control (One-way ANOVA followed by Dunnett’s multiple comparison test).