Toxicity of antimony, copper, cobalt, manganese, titanium and zinc oxide nanoparticles for the alveolar and intestinal epithelial barrier cells in vitro

Abstract

Heavy metals are found naturally on Earth and exposure to them in the living environment is increasing as a consequence of human activity. The toxicity of six different metal oxide nanoparticles (NP) at different points in time was compared using resazurin assay. After incubating Caco2 and A549 cells with 100 μg/mL of Sb₂O₃, Mn₃O₄ and TiO₂ nanoparticles (NPs) for 24 h no toxic effects were observed while Co₃O₄ and ZnO NPs had moderate effects and CuO NPs were toxic below 100 μg/mL (24 h EC₂₅ = 11 for A549 and 71 μg/mL for Caco2). The long-term monitoring (up to 9 days) of cells to NPs revealed that the toxic effects of Mn₃O₄ and Sb₂O₃ NPs remarkably increased over time. The 9 day EC₅₀ values for Sb₂O₃ NPs were 22 and 48 μg/mL for A549 and Caco2 cells; and for Mn₃O₄ NPs were 47 and 29 μg/mL for A549 and Caco2 cells, respectively. In general, the sensitivity of the cell lines in the resazurin assay was comparable. Trans epithelial electrical resistance (TEER) measurements were performed for both cell types exposed to Co₃O₄, Sb₂O₃ and CuO NPs. In TEER assay, the Caco2 cells were more susceptible to the toxic effects of these NPs than A549 cells, where the most toxic NPs were the Sb₂O₃ NPs: the permeability of the Caco2 cell layer exposed to 10 μg/mL Sb₂O₃ NPs already increased after 24 h of exposure.

Keywords: A549 cells; Caco2 cells; Long term viability; Metal oxide nanoparticles; Non-monotonic dose–response (NMDR); Transepithelial electrical resistance (TEER).

Fig. 1

The 9 days (216 h) of metabolic activity in human lung epithelial cells A549 and human intestinal epithelial cells Caco2 cells after a single exposure to dispersions of MeO NPs at t = 0. Resazurin was used to measure metabolic activity. Concentrations of metal oxides are presented as μg metal/mL. The metabolic activity is presented as a % of the unexposed control cells. See also Table 1 for the respective EC values. The presented data are average values of three independent measurements ±SD

Fig. 2

Time-dependent decrease of the resistance of the epithelial cell layer (% of control) of Caco2 (upper panels) and A549 cells (lower panels) after exposure to Co₃O₄, CuO and Sb₂O₃ NPs over 3 or 12 days (values of TEER measurements are shown in Table S1). The exposure time is indicated on the panels. The data presented are average values of the two independent measurements ±SD

Fig. 3

The 24 h viability of Balb/c 3T3 cells exposed to MeO NP (resazurin assay). The presented values are at concentrations of μg metal/mL

Fig. 4

Balb/c 3T3 cells 7 days after a single exposure to TiO₂ at a concentration of 6 μg metal/mL. The cell layer exposed to TiO₂ is similarly confluent to the normal negative control

Fig. 5

TEM images of cells exposed to MeO NPs for 24 h at concentration EC₂₀. a Balb/c 3T3 cells exposed to Co₃O₄ NP at a concentration of 64.5 μg metal/mL; b A549 cells exposed to Co₃O₄ NP at a concentration of 56.1 μg metal/mL; c Caco2 cell exposed to Sb₂O₃ at a concentration of 44.7 μg metal/mL; d Caco2 cells exposed to CuO at a concentration of 3.1 μg metal/mL (photograph by M. Visnapuu)