Toxicological effects of zinc oxide nanoparticle exposure: an in vitro comparison between dry aerosol air-liquid interface and submerged exposure systems

Figures & data

Figure 1. (a) Particle number size distributions of the aerosolized ZnO NPs, measured with the SMPS and the APS (left axis), and the extrapolated deposition efficiency in the NACIVT system from Jeannet et al. (2015) (right axis), and (b) calculated corresponding deposited mass dose size distributions.

Figure 2. SEM-images of aerosolized ZnO NP deposited on Si wafers in the NACIVT system. (a) Medium dose (x600 magnification), (b) high dose (x600 magnification), (c) medium dose (x50,000 magnification), and (d) high dose (x50,000 magnification).

Table 1. Description of experiments in the three exposure systems NACIVT, SUB(iso) and SUB(growth).

Exposure system	Seeding	Flipping to ALI (24 h after seeding)	Exposure (48 h after seeding)	Analysis
				1 h	3 h	24 h
NACIVT	100 µl cell suspension on top of the membrane 1 ml basal growth medium	Apical growth medium removed	1 h exposure to ZnO NP aerosol	WST-1 LDH Cytokines: IL-6, IL-8, TNF-α, MCP-1	WST-1 LDH Cytokines: IL-6, IL-8, TNF-α, MCP-1	WST-1 LDH Cytokines: IL-6, IL-8, TNF-α, MCP-1
SUB(iso)	100 µl cell suspension on top of the membrane 1 ml basal growth medium	Apical growth medium removed	24 h exposure in isotonic solution	No analysis	No analysis	WST-1 LDH Cytokines: IL-6, IL-8, TNF-α, MCP-1
SUB(growth)	100 µl cell suspension on top of the membrane 1 ml basal growth medium	No change	24 h exposure in growth medium	No analysis	No analysis	WST-1 LDH Cytokines: IL-6, IL-8, TNF-α, MCP-1

Schematic images show the cells (dark red) grown in growth medium (light red) on 24-well inserts (surface area: 0.33 cm²). The inserts were flipped 24 h after seeding in two of the exposure systems (NACIVT and SUB(iso)), bringing the cells to the air-liquid interface (ALI). 48 h after seeding, the cells were exposed to zinc oxide nanoparticles (ZnO NPs, yellow) as an aerosol or suspended in isotonic NaCl solution (blue) or growth medium. Metabolic activity was evaluated with the WST-1 assay and cytotoxicity was evaluated with the LDH-assay. Release of the cytokines interleukins IL-6 and IL-8, the tumor necrosis factor, TNF-α, and the monocyte chemotactic protein, MCP-1 were analyzed with Multiplexed Luminex.

Table 2. Exposure parameters in the three exposure systems NACIVT, SUB(iso) and SUB(growth).

Exposure system	Low dose	Medium dose	High dose
NACIVT
ZnO NP suspension concentration (µg/ml)	150	150	600
Compressed air pressure (bar)	1	5	5
Deposited mass dose (µg/cm^2)	0.2	1.0	3.0
Deposited surface area dose (cm^2/cm^2)	0.06	0.31	0.92
SUB(iso)
ZnO NP suspension concentration (µg/ml)	10.0	17.6	70.0
Added suspension volume (µl)	20	20	20
Deposited mass dose (µg/cm^2)	0.6	1.1	4.2
Deposited surface area dose (cm^2/cm^2)	0.18	0.34	1.29
SUB(growth)
ZnO NP suspension concentration (µg/ml)	3.3	5.7	29.8
Added suspension volume (µl)	50	50	50
Deposited mass dose (µg/cm^2)	0.5	0.9	4.5
Deposited surface area dose (cm^2/cm^2)	0.15	0.28	1.38

Figure 3. Comparison of toxicological responses after ZnO NP exposures in the three different exposure systems NACIVT, SUB(iso) and SUB(growth) 24 h after exposure. a) Metabolic activity measured with the WST-1 assay, b) IL-8 release measured in basal medium with Multiplexed Luminex, and c) MCP-1 release measured in basal medium with Multiplexed Luminex. The WST-1, IL-8 and MCP-1 results were normalized to the unexposed controls. The symbol (*) indicates significant difference from unexposed control levels at p < 0.05 and (**) at p < 0.01. The symbol (#) indicates significant difference between exposure systems at p < 0.05 and (# #) at p < 0.01. The doses corresponded to 0.5, 0.6 and 0.2 µg/cm² (low), 0.9, 1.1 and 1.0 µg/cm² (medium), and 4.5, 4.2 and 3.0 µg/cm² (high) for SUB(growth), SUB(iso) and NACIVT respectively.

Figure 4. Toxicological responses measured after ZnO NP exposures in the NACIVT system at three different time points after exposure. a) Metabolic activity measured with the WST-1 assay, b) IL-8 release measured in basal medium with Multiplexed Luminex, and c) MCP-1 release measured in basal medium with Multiplexed Luminex. The WST-1, IL-8 and MCP-1 results were normalized to the unexposed controls. The symbol (*) indicates significant difference from unexposed control levels at p < 0.05 and (**) at p < 0.01. The symbol (#) indicates significant difference between different doses at 24 h after exposure at p < 0.05 and (# #) at p < 0.01. The doses corresponded to 0.2 (low), 1.0 (medium), and 3.0 µg/cm² (high).

Jeannet, N., M. Fierz, M. Kalberer, H. Burtscher, and M. Geiser. 2015. “Nano Aerosol Chamber for In-Vitro Toxicity (NACIVT) Studies.” Nanotoxicology 9 (1): 34–42. doi:10.3109/17435390.2014.886739.

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