Citrullination as early-stage indicator of cell response to single-walled carbon nanotubes

Abstract

Single-walled carbon nanotubes (SWCNTs) have been widely explored as potential technologies for information systems and medical applications. The impact of SWCNTs on human health is of prime concern, if SWCNTs have a future in the manufacturing industry. This study proposes a novel, inflammation-independent paradigm of toxicity for SWCNTs, identifying the protein citrullination process as early-stage indicator of inflammatory responses of macrophages (THP-1) and of subtle phenotypic damages of lung epithelial (A549) cells following exposure to chemically-treated SWCNTs. Our results showed that, while most of the cellular responses of A549 cells exposed to SWCNTs are different to those of similarly treated THP-1 cells, the protein citrullination process is triggered in a dose- and time-dependent manner in both cell lines, with thresholds comparable between inflammatory (THP-1) and non-inflammatory (A549) cell types. The cellular mechanism proposed herein could have a high impact in predicting the current risk associated with environmental exposure to SWCNTs.

Figure 1. Citrullination levels in THP-1 and A549 cells exposed to chemically-treated SWCNTs (p-SWCNTs, p-SWCNTs/BSA, f-SWCNTs, f-SWCNTs/BSA and Mal-SWCNTs/BSA) for 6 and 24 h.

(A) Graphical tables (heatmaps) reflect the citrullination levels ranging from dark green (lower than 15% change from the maximum value measured) to bright green (30%), yellow (50%), bright orange (60%), dark orange (75%) and finally red (higher than 75% change from the maximum value). (B) Representative fluorescent images of untreated (N/T), SWCNTs-treated and PAD-treated (P/T) THP-1 and A549 cells after 24 h exposure. Cells were immunostained for citrulline expression (in green) and nuclei (in blue). SWCNTs sample: p-SWCNTs; scale bar: 40 μm (10× magnification).

Figure 2. Graphical tables (heatmaps) of the cytotoxicity data of THP-1 and A549 cells exposed to p-SWCNTs, p-SWCNTs/BSA, f-SWCNTs, f-SWCNTs/BSA and Mal-SWCNTs/BSA for 3, 6 and 24 h.

Colorimetric gradient tables reflect the changes in cell count reduction, cell membrane permeability, lysosomal mass/pH, nuclear area and nuclear intensity. Colours range from dark green (values lower than 15% change from the maximum value measured) to bright green (30%), yellow (50%), bright orange (60%), dark orange (75%) and finally to red (values higher than 75% change from maximum value). Heatmap values are normalised to the percentages of the positive control (P/C) and Z-score is calculated as described in the statistical analysis section. Data represent two independent experiments performed in triplicate samples.

Figure 3. Release of (A) TNF-α and (B) IL-6 from THP-1 cells after 6 h (light grey) and 24 h (dark grey) exposure to p-SWCNTs, p-SWCNTs/BSA, f-SWCNTs, f-SWCNTs/BSA and Mal-SWCNTs/BSA at various concentrations (1, 5 and 10 μg/mL).

The symbols (*), (**) and (***) indicate significant time-dependent changes (p < 0.05, p < 0.01 and p < 0.001, respectively).

Figure 4. Release of (A) TNF-α and (B) IL-6 from A549 cells after 6 h (light grey) and 24 h (dark grey) exposure to p-SWCNTs, p-SWCNTs/BSA, f-SWCNTs, f-SWCNTs/BSA and Mal-SWCNTs/BSA at various concentrations (1, 5 and 10 μg/mL).

The symbols (*) and (***) indicate significant time-dependent changes (p < 0.05 and p < 0.001, respectively).

Figure 5. Schematic of the purification and covalent functionalization of pristine SWCNTs.

Boxes highlight the SWCNTs samples tested in this study. Figure adapted from Knyazev et al..