A Comparative Analysis of In Vitro Toxicity of Synthetic Zeolites on IMR-90 Human Lung Fibroblast Cells

Abstract

Broad industrial application of zeolites increases the opportunity of inhalation. However, the potential impact of different types and compositions of zeolite on cytotoxicity is still unknown. Four types of synthetic zeolites have been prepared for assessing the effect on lung fibroblast: two zeolite L (LTL-R and LTL-D), ZSM-5 (MFI-S), and faujasite (FAU-S). The cytotoxicity of zeolites on human lung fibroblast (IMR-90) was assessed using WST1 cell proliferation assay, mitochondrial function, membrane leakage of lactate dehydrogenase, reduced glutathione levels, and mitochondrial membrane potential were assessed under control. Intracellular changes were examined using transmission electron microscopy (TEM). Toxicity-related gene expressions were evaluated by PCR array. The result showed significantly higher toxicity in IMR-90 cells with FAU-S than LTL-R, LTL-D and MFI-S exposure. TEM showed FAU-S, spheroidal zeolite with a low Si/Al ratio, was readily internalized forming numerous phagosomes in IMR-90 cells, while the largest and disc-shaped zeolites showed the lowest toxicity and were located in submembranous phagosomes in IMR-90 cells. Differential expression of TNF related genes was detected using PCR arrays and confirmed using qRT-PCR analysis of selected genes. Collectively, the exposure of different zeolites shows different toxicity on IMR-90 cells.

Keywords: IMR-90; cytotoxicity; fibroblast; glutathione; lung; zeolite.

Figure 1

Scanning electron micrographs of the following as synthesized zeolite particles: (A) LTL-R, (B) LTL-D, (C) MFI-S, and (D) FAU-S.

Figure 2

Powder X-ray diffraction patterns of the following as synthesized zeolite samples: FAU-S (purple), MFI-S (green), LTL-D (red), and LTL-R (blue). The patterns are arbitrarily offset along the y-axis for visual clarity.

Figure 3

Dynamic assessment of the viability of IMR-90 cells exposed to zeolite particles. Exposing IMR-90 cells to different concentrations of zeolite particles (10, 50, and 100 μg/mL). The CI values were normalized at the time of the zeolite treatment for the following crystal structures: (A) LTL-R, (B) LTL-D, (C) MFI-S, and (D) FAU-S. Control cells cultured in zeolite-free media were run parallel to treatment group. Experiments were performed in quadruplet and presented as mean ± SD (n = 3). CI, cell index.

Figure 4

Cell viability of IMR-90 cells in the culture treated with 10, 50, and 100 μg/mL of (A) LTL-R, (B) LTL-D, (C) MFI-S, and (D) FAU-S for 72 h. All data are represented as the mean ± SD compared to the control. Symbols *, **, and *** indicate the statistically significant difference with p values <0.05, <0.01, and <0.001, respectively.

Figure 5

Cytotoxic effect of zeolites on lung fibroblast IMR-90 cells. Cells were treated with different concentration of zeolites for 72 h. (A) LDH leakage in medium. The percent of LDH activity was calculated by dividing the amount of acivity in the medium by the total activity (medium and cell lysate). Controls were cultured in zeolite free media were run in parallel to the treatment group. (B) Effect of zeolite on mitochondrial function determined by MTT reduction in IMR-90 human lung fibroblasts. The cells were incubated for 72 h with various concentrations (0, 10, 50, 100, 250 μg/mL) of zeolite LTL-R, LTL-D, MFI-S and FAU-S. Ferrous sulfate (FeSO${}_4$) was used as positive control. The results are presented as mean values ± SD versus unexposed cells to zeolites. The data are expressed as mean ± SD of three independent experiments. * indicates a statistically significant differences compared to control.

Figure 6

(A) Effect of zeolites on mitochondrial membrane potential in lung fibroblast IMR-90 cells. Cells were exposed to different concentration of zeolites for 72 h. The data are expressed as mean ± SD of three independent experiments. * indicate statistically significant differences compared to control. Morphologic chracterization of mitochondrial disruption by MitoTracker Red staining: (B) control, (C) cells with 50 μg/mL FAU-S, and (D) cells with 100 μg/mL FAU-S were visualized using cofocal laser microscopy. Scale bar: 50 μm.

Figure 7

Effect of zeolites on GSH levels in lung fibroblast (IMR-90 cells). Cells were treated with zeolite LTL-R, LTL-D, MFI-S, FAU-S for 72 h. At the end of the exposure, cells were washed with PBS, and GSH (control: 61 ± 5 nmol/mg protein). Control cells cultured in zeolite-free media were run in parallel to treatment groups. The data are expressed as mean ± SD of three independent experiments. * indicates a statistically significant difference compared to controls (p < 0.05).

Figure 8

Ultrastructural features of zeolite particle uptake by IMR-90 cells. The transmission electron micrographs show thin sections with zeolite particle-exposed IMR-90 cells. The cells were cultured in the presence of (A) 50 μg/mL LTL-R, (B) 50 μg/mL MFI-S, (C) 50 μg/mL LTL-D, and (D) 50 μg/mL FAU-S for 72 h. The zeolite particles (arrow) were located as (A,B) aggregates in intracytoplasmic phagosome, (C) aggregates in submembranous phagosome, and (D) small aggregates in numerous cytoplasmic phagosomes.

Figure 9

Gene expression profiles in IMR-90 cells after exposure to 50 μg/mL of zeolites (A) Clustergram of gene expression exposed to 50 μg/mL of LTL-R, LTL-D, MFI-S and FAU-S using PCR arrays. (B) Significantly altered gene expression with more than 2-fold change is shown in Venn diagrams organized by zeolite type. (C) Gene list of PCR array panel.