Aerosolized Nano-Zinc Oxide (nZnO) Exposure Alters the Cellular Composition of the Fully Differentiated Human Airway Epithelium

Details

• Personal Author:
  Bodas M ; Derk R ; Rojanasakul L ; Stueckle, Todd A.
• Description:
  Background and Purpose: Engineered nanomaterials such as metal-oxide nanoparticles (NP) are widely used in numerous industrial and daily use applications, which makes their exposure a common occupational hazard. Nano-zinc oxide particles (nZnO) are used in many industries such as food packaging, rubber, textiles, and leather. Exposure leads to pulmonary inflammation and oxidative stress, which may result in development of lung disease or worsening of pre-existing lung disease. The adult human airway epithelium is comprised of four major cell types, basal, club, goblet, and ciliated cells. Any change in the numbers of these cell types is termed as 'airway remodeling', which is a disease-related phenotype in several lung diseases. Thus, the aim of this study was to investigate if aerosolized nZnO exposure leads to changes in the cellular composition of the airway epithelium, using air-liquid interface (ALI)-differentiated primary human bronchial epithelial cells. Methods: Normal human bronchial epithelial cells (NHBEpC) (at ≥ 90% viability) were seeded on 12-well porous 0.4 µm- cell culture inserts in basal cell culture medium. On the next day, the media from the apical and basal chambers was removed, and 1 mL complete ALI Medium containing necessary supplements was added to the basal chamber. On the same day, the cells in the apical chamber were exposed to air (airlift) to create the ALI and allowed to differentiate for 28 days. Optimal differentiation was verified by qPCR and immunofluorescent (IF) staining of cell-type specific markers. Stock suspensions of nZnO were sonicated to produce a stable suspension and characterized for hydrodynamic diameter using dynamic light scattering (DLS) analysis. Fully differentiated cells were exposed to aerosolized nZnO for 6 minutes, at the indicated doses, using the VitroCell® Cloud Alpha 12 system. Deposited dose was measured using a quartz crystal microbalance (QCM) integrated in the VitroCell® system. Transmission electron microscopy (TEM) was used to demonstrate dispersion of aerosolized NP on the inserts using TEM grids. Cells were harvested at 24 hours or 7 days post exposure, to investigate changes in (a) inflammation, (b) oxidative stress-related, and (c) cell-type specific markers using qPCR and/or IF staining. Cellular toxicity was quantified by measuring lactate dehydrogenase (LDH) activity in the 'apical wash' and basolateral chamber media. Results: qPCR analysis of the differentiation time-course shows a steady increase in markers of terminally differentiated cells, such as club (SCGB1A1), goblet (MUC5AC) and ciliated cells (FoxJ1). The basal cell (stem/progenitor cell) marker (KRT5) shows an initial increase in expression, but did not vary much during differentiation. The presence of all four major cell types at protein level was confirmed by IF staining of the fully differentiated airway epithelium. DLS analysis showed that nZnO particles suspended in water (vehicle), had a mean hydrodynamic size (Z avg) of 236.6 +/- 18.8 (nm+/-SD), and a Polydispersity index of 0.27 +/- 0.03 (PdI+/-SD), suggesting a constant uniform dispersion. Cellular toxicity analysis showed that the higher dose of nZnO (Mean +/- SD) 4.25 +/- 0.41 µg/cm2 induced a significant increase in LDH activity (in apical wash and basolateral chamber media) at both 24 hours and 7 days' time points, as compared to the low dose nZnO (0.514 +/- 0.09 µg/cm2), and vehicle control. Exposure to high dose of nZnO induced a significant decrease in expression of club (SCGB1A1), goblet (MUC5AC) and ciliated cells (FoxJ1) markers at mRNA levels. This effect was observed at both 24 hours and 7 days' time points, suggesting an early blockage in the cellular differentiation process, which requires further evaluation. In parallel, the expression of basal cell marker(KRT5) was partially modulated by nZnO exposure. Since these cells reside at the bottom of the pseudostratified airway epithelium, it's possible that they were not directly impacted by nZnO exposure, at the doses/time points used. Mechanistically, we found that expression of oxidative stress related gene, heme-oxygenase-1 (HMOX1), was significantly elevated in nZnO-exposed cells at both doses and time points. Additionally, nZnO exposure induced a significant increase in expression of interleukin-1β, a pro-inflammatory cytokine. Conclusions: These results suggest that acute exposure to aerosolized nZnO could alter the cellular composition of the adult airway epithelium, possibly via oxidative stress and inflammation-related mechanisms. Further studies are underway to decipher the specific mechanisms which might regulate nZnO-mediated changes in the numbers of different cell types of the adult airway epithelium, airway remodeling, and the potential for lung disease development upon chronic exposure. [Description provided by NIOSH]
• Subjects:
  Aerosols Lung Diseases Occupational Health Respiratory Tract Diseases Safety
• Keywords:
  Cell Differentiation Gene Expression Lung Disease Nanoparticles Oxidative Stress Zinc Oxide
• ISSN:
  1096-6080
• Document Type:
  Text
• Genre:
  Abstract or Summary
• Place as Subject:
  OSHA Region 3 ; West Virginia
• CIO:
  National Institute for Occupational Safety and Health (NIOSH)
• Division:
  HELD - Health Effects Laboratory Division
• Topic:
  Workplace Safety & Health
• Location:
  North America
• Volume:
  198
• NIOSHTIC Number:
  nn:20069334
• Citation:
  Toxicologist 2024 Mar; 198(S1):309
• CAS Registry Number:
  Zinc oxide (ZnO) (CAS RN 1314-13-2)
• Federal Fiscal Year:
  2024
• Peer Reviewed:
  False
• Source Full Name:
  The Toxicologist. Society of Toxicology 63rd Annual Meeting & ToxExpo, March 10-14, 2024, Salt Lake City, Utah
• Collection(s):
  National Institute for Occupational Safety and Health
• Main Document Checksum:
  urn:sha-512:58e20854cbab8d8a134e1f934fbb65c03bebacaee63062dae3c33e12c869caa0dd2d9cb95a723995d08d85396f82d9b915cb9ab06eabef5f38653d5a48dd45ca
• Download URL:
  https://stacks.cdc.gov/view/cdc/207834/cdc_207834_DS1.pdf
• File Type:
  [PDF - 558.91 KB ]