Analysis of Serum Metabolome of Rats Following Intratracheal Instillation of Multi-Walled Carbon Nanotubes

Details

• Personal Author:
  Antonini JM ; Boyce G ; Fraser K ; Hettick JM ; Powell MJ ; Roach KA ; Roberts, Jennifer R. ; Shrivastava I
• Description:
  Background and Purpose: While there has been increasing application of nanotechnology in variety of industrial applications, there have been relatively few health effects studies in workers. One of the primary routes of worker exposure to nanomaterials is inhalation. The aim of this study is to identify metabolic phenotypes which can be used to establish biomarkers for adverse outcomes and better understand adverse outcome pathways related to disease following exposure to multi-walled carbon nanotubes (MWCNT). Methods: Male Sprague Dawley rats (approx. 10 weeks old, approx. 300 g) were administered a single intratracheal (IT)-instillation to a low effect (10 µg) and high effect (500 µg) dose of a well-characterized MWCNT, Mitsui-7, or dispersion media as vehicle control (DM). Following exposure, rats were fasted overnight and humanely euthanized at 7 d, 1 m and 3 m. Serum was collected, and bronchoalveolar lavage was performed on the right lung and histopathology was performed on the left lung of rats. A battery of cytokines and proteins were evaluated in lavage fluid. Serum samples were prepared for high-performance liquid chromatography with tandem mass spectrometry (LC-MS)for metabolome analyses. Hydrophilic interaction liquid chromatography (HILIC) was performed. The mass spectrometer was operated in negative electrospray ionization mode. Following spectra acquisition, the raw data was analyzed using Compound Discover (CD) version 2.3 software (ThermoFisher Scientific) for small molecule identification. Metabolites were matched to metabolic pathways in the Metabolika database within CD. The small molecules with associated names and chemical formula were also mapped to the Kyoto encyclopedia of genes and genomes (KEGG) database. The positively identified metabolites were then subject to pathway analysis using Ingenuity Pathway Analysis (IPA) to connect metabolic changes and disease-relevant pathways, and to generate a list of potential metabolomic biomarkers for further studies. Four comparisons were performed in IPA as follows: (i) vehicle control (DM), 7 d low-dose, 7 d high dose, (ii) DM, 1 m low-dose, 1 m high dose, (iii) DM, 3 m low-dose, 3 m high-dose and (iv) low and high doses for all 3 time-points. Results: Pathology analysis showed early onset granulomatous fibrosis in the high dose only scored as moderate on d 7 and 1 m and mild at 3 m. Cytokine evaluation in the lung showed persistent increases in proteins in the high dose group that have been positively associated with inflammatory disease, including IL-1β, IL-18, IP-10, TNF-a, MIP-2, and RANTES. Only MIP-2 and TNF-a were elevated at multiple time points in the low dose. Approximately 3000-3500 small molecules were identified for each of the three time-points. Metabolika analysis mapped approximately half of the small molecules to 230-270 metabolic pathways depending on time and treatment groups. In this analysis, the top mapped pathway for the high dose at 7 d differed from that of DM and the low dose. This was the superpathway of lipoxygenase which is associated with inflammatory disease. For analysis in IPA, of the small molecules with associated names and chemical formula, 178 (7 d), 173 (1 m) and 161 (3 m) were positively identified in the KEGG database. The greatest difference in the activation/inhibition of upstream regulators in the disease and function categories in IPA analysis occurred when comparing the high and low doses to control at 7d, with the high and low dose following a similar pattern to each other for many of the pathways. At 1 m, pathways related to disease and function categories of cell viability and uptake of 2-deoxyglucose were activated at both doses compared to control and lipid peroxidation pathway was activated at high dose when compared to control. At month 3, pathways related to cellular infiltration by macrophages and cell movement of antigen presenting cells were activated at high dose in comparison to control. When comparing high and low dose to each other at 7 d, there were also a greater number of differences than that observed for high versus low dose comparisons at 1 or 3 m. In this comparison, the greatest differences were in activated pathways related to categories of apoptosis, necrosis, immune cell activation, lipid metabolism, production of reactive oxygen species, and inflammatory cell activation among others. Conclusions: The metabolomics analysis showed a consistent pattern with activation/inhibition trends for majority of IPA pathways following MWCNT being opposite to control. Metabolites common to both doses over time may serve as candidates for biomarkers of exposure. The greatest differences in upstream regulators in serum and cytokines in lavage between the low and high dose occurred at 7 d, the time point where inflammation begins to resolve for the low dose but progresses toward disease in the high dose, suggesting this time point may be best suited for development of biomarkers of disease. It is important to note that many statistically significant metabolites identified by compound discoverer could not be incorporated into pathway analyses databases resulting significant data gaps. Further analyses of the metabolome are needed to better delineate the metabolic pathways involved in adverse outcome pathways. [Description provided by NIOSH]
• Subjects:
  Biomarkers Energy Metabolism Laboratories Occupational Health Respiratory System Safety Toxicology
• Keywords:
  Cytokines Inhalation Laboratory Animals Metabolites Multi-walled Carbon Nanotubes MWCNT
• 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
• Pages in Document:
  313-314
• Volume:
  198
• NIOSHTIC Number:
  nn:20069336
• Citation:
  Toxicologist 2024 Mar; 198(S1):313-314
• CAS Registry Number:
  Carbon nanotubes (CAS RN 308068-56-6)
• 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:fdca2bd125a76da5116789a2b1eeba6475ce5a9d8b0aef24b45fe01f1f0d9adbbfd5fe358cbdb184a002b0045971d9fc30fd641c1982541fa7c4883993b50f1a
• Download URL:
  https://stacks.cdc.gov/view/cdc/207836/cdc_207836_DS1.pdf
• File Type:
  [PDF - 557.94 KB ]