Cerium Oxide Enhances the Toxicity of Zinc Oxide Nanoparticles in Human Lung Epithelial Cell Cultures

Abstract

Recently, many approaches have been developed to improve the performance of nanomaterials. Combining more than one nanomaterial is one such approach that achieves superior results. However, during the fabrication of nanomaterials or formulation of end products, materials can be released into the ambient air and be inhaled by workers. The adverse health outcomes of inhaling such compounds are unknown. In this study, we examined such effects in combining two of the most utilized nanomaterials in several industrial sectors: zinc oxide (ZnO) and cerium oxide (CeO₂). These materials can be found together in sunscreens, polyvinyl alcohol (PVA) films, and construction products. The aim of this study was to assess the adverse biological outcomes of CeO₂-ZnO nano-mixtures in human lung epithelial cells. A549 human lung epithelial cells were treated with increasing concentrations of ZnO or CeO₂ NPs alone, or as a mixture of both, under submerged conditions for 24 h. After treatment, cell viability, reactive oxygen species (ROS) formation, cell membrane integrity, and cytokine production were examined. ZnO NPs showed a dose-dependent trend for all endpoints. CeO₂ NPs did not exhibit any toxic effect in any individual concentrations. When higher doses of ZnO were combined with increasing doses of CeO₂, loss of cell viability and an elevation in cell membrane leakage were observed. Interleukin 8 (IL-8) and ROS generation were higher when ZnO NPs were combined with CeO₂ NPs, compared to cells that were treated with ZnO alone. The release of monocyte chemoattractant protein-1 (MCP-1) was reduced in the cells that were treated with higher doses of ZnO and CeO₂. Thus, the presence of CeO₂ enhanced the toxicity of ZnO in A549 cells at non-toxic levels of CeO₂. This suggests an additive toxicity of these two nanomaterials.

Keywords: cerium oxide; lung epithelial cells; nanoparticles; nanotoxicology; tissue culture; zinc oxide.

Figure 1

CeO₂ XPS survey. (A) Full spectrum of CeO₂ containing O s1 and Ce 3d; (B) detailed survey of Ce 3 d/2 region. CPS = counts per second.

Figure 2

Dose-response curve of ZnO NPs after treating A549 cells for 24 h. ZnO showed a loss in viability of the cells in a dose-dependent manner. N = 8 for each treatment.

Figure 3

Viability of A549 cells after treatment with either individual NPs (two groups) or combined NPs (four groups). The plot shows the mean ± SEM of the percent AlamarBlue reduction in A549 cells (n = 5). For individual NPs, CeO₂ alone showed no loss in viability, whereas ZnO showed a dose-dependent loss in viability. For the combined groups, combining each of the six doses of CeO₂ with 7.8 μg/mL of ZnO did not affect the viability (orange). When each of the six doses of CeO₂ was combined with 15.6 μg/mL of ZnO, the loss in viability at the highest concentration of CeO₂ was more than that when 15.6 μg/mL of ZnO alone was used (pink). When 31.3 μg/mL of ZnO was added to the increasing doses of CeO₂, 62.5 μg/mL of CeO₂ caused the viability to be significantly lower than when 31.3 μg/mL of ZnO alone was used (purple). When 62.5 μg/mL of ZnO alone was compared with 62.5 μg/mL of ZnO plus 31.3 μg/mL or 62.5 μg/mL of CeO₂, no significant difference was observed, but a loss in viability was notable. (**) Significantly different (p < 0.01) from the control. ($) Significantly different (p < 0.05) from 15.6 μg/mL of ZnO alone. (#) Significantly different (p < 0.05) from 31.3 μg/mL of ZnO alone.

Figure 4

Release of lactate dehydrogenase from cells after treatment with either individual NPs (two groups) or combined NPs (for groups) for a 24-hour treatment. The graph represents the mean ± SEM of at least 10 treatments. The LDH was normalized for cell viability, LDH (% of control)/viability (% of control). The CeO₂ group (gray) had no effect on cell viability. Increasing the doses of ZnO only caused an increase in LDH release in a dose-dependent manner. The first two groups of the combined NPs (orange and pink) did not show any increases in LDH leakage. When 31.3 μg/mL of ZnO was combined with CeO₂, a significant elevation in LDH release was observed (purple). When 62.5 μg/mL of ZnO was combined with increasing doses of CeO₂, the LDH release was significantly higher than when 62.5 μg/mL of ZnO alone was used (teal). (*) Significantly different (p < 0.05) from the control. (**) Significantly different (p < 0.01) from the control. (#) Significantly different (p < 0.05) from 31.3 μg/mL of ZnO alone. (##) Significantly different (p < 0.01) from 31.3 μg/mL of ZnO alone. (+) Significantly different (p < 0.05) from 62.5 μg/mL of ZnO alone. (++) Significantly different (p < 0.01) from 62.5 μg/mL of ZnO alone.

Figure 5

Generation of ROS after treating A549 cells with NPs for 24 h. The plot shows the mean ± SEM of ROS generation (n = 7) in A549 cells treated with ZnO, CeO₂, or a mixture of both. ROS was normalized for cell viability, ROS (% of control)/viability (% of control). The CeO₂ group (gray) had no effect on ROS. Increasing doses of ZnO only caused an increase in ROS generation. The mixture of both NPs caused more toxicity than ZnO alone. The presence of CeO₂ augmented ZnO toxicity. (&) Significantly different (p < 0.05) from 7.8 μg/mL of ZnO alone. (++) Significantly different (p < 0.01) from 62.5 μg/mL of ZnO alone.

Figure 6

The effects of ZnO and CeO₂ NPs in individual and mixture forms on the release of IL-8 from A549 cells. The plot shows the mean ± SEM of IL-8 release of six treatments of A549 with ZnO, CeO₂, or mixtures of both. Levels of IL-8 were normalized for cell viability, IL-8 (% of control)/viability (% of control). The CeO₂ group (gray) had no effect on IL-8 release. Increasing doses of ZnO only caused an increase in IL-8 release. Mixtures of both NPs caused more toxicity than ZnO alone. The presence of CeO₂ augmented ZnO toxicity. (*) Significantly different (p < 0.05) from the control. (**) Significantly different (p < 0.01) from the control. (#) Significantly different (p < 0.05) from 31.3 μg/mL of ZnO alone. (++) Significantly different (p < 0.01) from 62.5 μg/mL of ZnO alone.

Figure 7

The effects of ZnO and CeO₂ NPs in individual and mixture forms on the release of MCP-1 from A549 cells. The graph presents the mean ± SEM of MCP-1 release from six treatments of A549 cells with ZnO, CeO₂, or mixtures of both. Levels of MCP-1 were normalized for cell viability, MCP-1 (% of control)/viability (% of control). CeO₂ did not cause an elevation in MCP-1 release (gray). Increasing the doses of ZnO affected MCP-1 release only at 62.5 μg/mL. The mixture of 62.5 μg/mL of ZnO and 62.5 μg/mL of CeO₂ caused a significant reduction in MCP-1 (teal). (**) Significantly different (p < 0.01) from the control. (+) Significantly different (p < 0.05) from 62.5 μg/mL of ZnO alone.