In Vitro Toxicity Screening of Different Categories of Two-Dimensional (2D) Nanomaterials for Genotoxicity and Activation of the NLRP3 Inflammasome

Details

• Personal Author:
  Aldinger, Jeffrey A. ; Fraser K ; Kodali VK ; Roach KA ; Roberts, Jennifer R. ; Stefaniak, Aleksandr B. ; Stueckle, Todd A. ; Xin X
• Description:
  Background and Purpose: Two-dimensional (2D) nanomaterials are a large class of engineered nanoparticles with a multitude of applications in electronics, biosensors, and more. Increased demand for these materials, including graphene, montmorillonite nanoclay, transition metal dichalcogenides (TMDs), such as WS2 and MoS2, and hexagonal boron nitride (hBN), has elevated the potential for occupational exposures during manufacturing, notably respiratory exposure. Although graphene has been well investigated, there are relatively few toxicity studies of this class of materials as a whole. Existing studies indicate these materials may have the propensity to induce inflammation and cytotoxicity; however, some results are contradictory and comparison across the entire highly variable class remains difficult. The goal of the current study was to conduct a comparative toxicity study of representative 2D materials for the different categories listed above using high throughput in vitro screening assays. Methods: The five materials were thoroughly characterized including, but not limited to, size, density, surface area, and hydrodynamic diameter. A battery of toxicity assays was performed using human bronchial epithelial cells (BEAS-2B) and human THP-1 macrophages in doses ranging from 1-100 µg/ml for 24 hours. Cytotoxicity and cell viability were assessed using WST-1 and Alamar blue for each cell type. In Beas-2B cells, oxidative stress was assess using CellROX, and genotoxicity was assessed by quantifying changes in cell cycle, H2AX, comet assay, and micronuclei (cytokinesis block assay) in addition to a quantification of cytokine secretion. Using THP-1 cells, activation of the NLRP3 inflammasome was assessed by quantifying the secretion and/or activation of cytokines and caspases. Additionally, changes in phagocytic ability was measured using phrodoGreen uptake. Results: Significant reduction in cell viability was found to occur with graphene at doses ≥ 12.5 µg/ ml. Nanoclay and hBN had significant changes at doses ≥ 25 µg/ml, while little to no changes were seen, even at the highest doses (100 µg/ml) for TMDs. No significant changes in DNA-associated oxidative stress or double stranded DNA damage (H2AX) were observed, though a trend for cell cycle arrest in G0/G1 phase was observed with high dose nanoclay exposure. While most cytokines measured were unchanged, nanoclay did induce a dose-dependent decrease in IL-12p70 in BEAS-2Bs. Furthermore, no significant changes in DNA damage or micronuclei formation was observed, suggesting these materials are unlikely to induce genotoxicity at the doses used in this study. Inflammasome activation was assessed in THP-1 cells. IL-1b was found to be significantly increased at an average of 3.6 (6.25 µg/ml) and 4.8 (25 µg/ml) times the control level following nanoclay exposure, and a significant 1.8-fold change occurred following hBN (6.25 µg/ml) exposure. A 4-fold change in Caspase-1 also resulted from exposure to 25 µg/ ml nanoclay. However, no significant changes were found in cathepsin B or IL-18 release suggesting minimal activation of the NLRP-3 inflammasome for most materials with the exception of nanoclay. Finally, there were minimal difference in phagocytic capacity among treatment groups indicating no particle-specific effect on clearance capacity. Conclusions: These initial findings suggest that the TMD category is relatively less toxic than the other classes of 2D materials and nanoclay may be of greater toxicological concern compared to other materials in this study. However, minimal evidence suggest that these 2D nanomaterials are of overall significant toxicological concern. [Description provided by NIOSH]
• Subjects:
  Cytotoxins Immune System Manufacturing And Industrial Facilities Occupational Health Safety Toxicology
• Keywords:
  Cytokines Cytotoxicity Genotoxicity Immune Reaction In Vitro Study Manufacturing Nanomaterials
• 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 ; RHD - Respiratory Health Division
• Topic:
  Workplace Safety & Health
• Location:
  North America
• Volume:
  198
• NIOSHTIC Number:
  nn:20069335
• Citation:
  Toxicologist 2024 Mar; 198(S1):310
• Federal Fiscal Year:
  2024
• NORA Priority Area:
  Manufacturing
• 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-512:19df3d3c35c872dfb7c3c0ba42649efe9994b712909e7231d32f7edfb9335992e581f85122196f1148f67063abbd6c5f5de9c628549836c16a0755f606dbef24
• Download URL:
  https://stacks.cdc.gov/view/cdc/207835/cdc_207835_DS1.pdf
• File Type:
  [PDF - 540.79 KB ]