Short-Term Pulmonary Toxicity Assessment of Pre- and Post-incinerated Organomodified Nanoclay in Mice

Abstract

Organomodified nanoclays (ONCs) are increasingly used as filler materials to improve nanocomposite strength, wettability, flammability, and durability. However, pulmonary risks associated with exposure along their chemical lifecycle are unknown. This study's objective was to compare pre- and post-incinerated forms of uncoated and organomodified nanoclays for potential pulmonary inflammation, toxicity, and systemic blood response. Mice were exposed via aspiration to low (30 μg) and high (300 μg) doses of preincinerated uncoated montmorillonite nanoclay (CloisNa), ONC (Clois30B), their respective incinerated forms (I-CloisNa and I-Clois30B), and crystalline silica (CS). Lung and blood tissues were collected at days 1, 7, and 28 to compare toxicity and inflammation indices. Well-dispersed CloisNa caused a robust inflammatory response characterized by neutrophils, macrophages, and particle-laden granulomas. Alternatively, Clois30B, I-Clois30B, and CS high-dose exposures elicited a low grade, persistent inflammatory response. High-dose Clois30B exposure exhibited moderate increases in lung damage markers and a delayed macrophage recruitment cytokine signature peaking at day 7 followed by a fibrotic tissue signature at day 28, similar to CloisNa. I-CloisNa exhibited acute, transient inflammation with quick recovery. Conversely, high-dose I-Clois30B caused a weak initial inflammatory signal but showed comparable pro-inflammatory signaling to CS at day 28. The data demonstrate that ONC pulmonary toxicity and inflammatory potential relies on coating presence and incineration status in that coated and incinerated nanoclay exhibited less inflammation and granuloma formation than pristine montmorillonite. High doses of both pre- and post-incinerated ONC, with different surface morphologies, may harbor potential pulmonary health hazards over long-term occupational exposures.

Keywords: inflammation; life cycle; nanoparticles; pulmonary; toxicity.

Figure 1.

FESEM characterization of dispersed pre- and post-incinerated nanoclays in saline vehicle. (a) Single (white arrow) and stacked platelets of pristine CloisNa and (b) organomodified Clois30B exhibited stacked or exfoliated platelet morphology with occasional detection of colloidal aluminum silicate agglomerates (black arrow). Inset shows exfoliated platelet with 250 nm scale bar. (c) I-CloisNa possessed a large, amorphous particle structure, while (d) I-Clois30B exhibited either a smooth or pocketed structure. (e) Aluminum silicate colloids were observed in all samples. (f) Heat-inactivated crystalline silica.

Figure 2.

Deposition of pre- and post-incinerated organomodified montmorillonite nanoclay (300 μg/mouse) in day 1 postexposure animals. (a) H & E staining of terminal bronchiole (TB) and alveolar duct (AD) of exposed lung. 100× magnification. Green arrows indicate deposited particle associated with alveolar macrophages. (b) Polarized light microscopy of deposited particle in AD and alveoli of exposed lung. Images show particles (white) with polarized filter. 1000× magnification. CloisNa deposited in TB and AD regions as loose, stacked agglomerates or loosely dispersed in alveoli as small clusters and showed low polarization imaging potential (white arrows). FESEM/EDX elemental analysis confirmation of CloisNa within alveolar space (b, upper right). Clois30B was primarily found in TB and AD as large aggregates, with small aggregates in alveoli regions (black arrows). Both ICloisNa and I-Clois30B deposited primarily in TB and AD regions, while crystalline silica was found well-dispersed in alveoli.

Figure 3.

Bronchoalveolar lavage cell differential analysis following pre- and post-incinerated organomodified nanoclay exposure. (a) Total cell counts, (b) macrophages, (c) neutrophils, and (d) eosinophils. Different letters indicate those treatments significantly different from each other (p < 0.05, n = 8).

Figure 4.

Cytotoxicity (LDH activity; a) and airway damage (total protein; b) markers in collected BAL fluid following pre- and post-incinerated organomodified nanoclay pulmonary exposure. Different letters indicate those treatments significantly different from each other (p < 0.05, n = 8).

Figure 5.

Heat map and hierarchical cluster analysis of BAL cytokine expression profiles at day 1 following pre- and post-incinerated organomodified nanoclay pulmonary exposure. Color scale bar represents Log2-fold change values. * indicate those treatments that were significantly different from their time-associated vehicle controls (p < 0.05, n = 8).

Figure 6.

Hierarchical cluster analysis of BAL cytokine expression profiles at day 7 following pre- and post-incinerated organomodified nanoclay pulmonary exposure. (a) Heat map and (b) radar plot depicting high-dose CloisNa, Clois30B, and CS with similar inflammatory and fibrotic tissue signaling profiles. Color scale bar represents Log2-fold change values. * indicate those treatments that were significantly different from their time-associated vehicle controls (p < 0.05, n = 8).

Figure 7.

Hierarchical cluster analysis of BAL cytokine expression profiles at day 28 following pre- and post-incinerated organomodified nanoclay pulmonary exposure. (a) Heat map showing I-Clois30B and CS showing similar inflammatory profiles, while CloisNa and Clois30B showed similar fibrotic tissue signature profiles. Color scale bar represents Log2-fold change values. * indicate those treatments that were significantly different from their time-associated vehicle controls (p < 0.05, n = 8). (b) Radar plots depicting a more inflammatory (left) vs more fibrotic profiles (right).

Figure 8.

Time course of C57BL/6 male lung histopathology response following 300 μg aspiration exposure to pristine (CloisNa) or organomodified (Clois30B) montmorillonite nano-clay. CloisNa exposure (a) caused pulmonary edema on day 1 and robust neutrophil and macrophage inflammatory response that peaked at day 7 characterized by macrophage and type II pneuomocyte hyperplasia and focal lymphocyte infiltrate out to day 28. Conversely, Clois30B exposure resulted in mild neutrophilic inflammation associated with particle-laden macrophages. 400× magnification. Both CloisNa (b; left panel; day 28) and Clois30B (right panel; day 7) exposures caused granulomas comprised of macrophages and nanoclay particulate. 400× magnification. Blue and red arrows indicate free or macrophage engulfed particles, respectively.

Figure 9.

Akt, MAPK, and NFκB signaling at day 1 and day 7 following exposure to pre- and post-incinerated organomodified nanoclay. Western blot and quantitative densitometry analysis of protein bands at (a) and (b) day 1 and (c) and )d) day 7 postexposure. Phosphorylated and basal protein expression was normalized to basal and B-actin protein expression, respectively. * and † indicate significant increase or decrease, respectively, compared to saline-exposed animals (n = 4, p ≤ 0.05).

Figure 10.

Caspase 1, IL-18, and NFκB signaling at day 1 and day 7 following exposure to pre- and post-incinerated organomodified nanoclay. Western blot and quantitative densitometry analysis of protein bands at (a) and (b) day 1 and (c) and (d) day 7 postexposure. Phosphorylated and basal protein expression was normalized to basal and B-actin protein expression, respectively. * and † indicate significant increase or decrease, respectively, compared to saline-exposed animals (n = 4, p ≤ 0.05).