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Qualitative Assessment of Carbon Nanotube Toxicity to Mouse Liver using Dark‐field Microscopy
Author(s) -
Angtuaco Steven E.,
Savenka Alena,
Fite Todd,
Liu Shijie,
Khodakovskaya Mariya,
Basnakian Alexei
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.04954
Subject(s) - tunel assay , toxicity , colocalization , chemistry , terminal deoxynucleotidyl transferase , biophysics , nanotechnology , materials science , microbiology and biotechnology , medicine , biochemistry , biology , apoptosis
Broad use of nanomaterials may eventually lead to environmental poisoning, including the contamination of food. However, methods for the assessment of nanomaterial toxicity are not well developed. The goal of the current study was to develop a techniques to measure toxicity of multiwall carbon nanotubes (MWCNTs) in vivo . For this, male CD‐1 mice were fed by oral gavage with a water suspension of MWCNTs at varying doses up to 10 mg/kg/day. Twenty‐four hours later, blood chemistry testing showed elevation of aspartate aminotransferase (AST), a liver injury marker, at the highest used dose. The liver damage was confirmed by the terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) assay. Our further analysis included the application of dark‐field microscopy (DFM) for (a) the colocalization between MWCNTs and TUNEL‐positive cells and (b) development of a method to quantify MWCNTs burden in liver tissue. DFM clearly showed some elevation of particulate material in the liver with occasional association with TUNEL‐positive cells. DFM did not seem to allow precise quantification of nanoparticle aggregates due to low sensitivity of DFM at the concentration observed in the liver. However, we discovered that CNT gavage increased the percentage of nuclei colocalized with DF objects by systematically counting the number of liver cell nuclei in contact with dark‐field (DF) objects. TUNEL‐positive nuclei were photographed, analyzed at 100‐fold magnification and sorted by CNT treatment category. The process was repeated for DAPI nuclei to serve as a control. The percentage of nuclei with DF colocalization was recorded per group. Our results indicate that the rate of colocalization between dark‐field objects and nuclei appears to correlate directly with CNT‐gavage treatment. Another important observation, confirmed by an electron microscopy, was that there were many alive TUNEL‐negative cells containing MWCNTs. Overall, our data indicate that the DFM method designed for particle toxicology has only limited potential for measurement of nanoparticle toxicity, and that some mouse liver cells are capable of accumulating MWCNTs without inducing immediate cell death. Support or Funding Information UAMS/ASPET Pharmacology/Toxicology Summer SURF Program USDA subcontract (Basnakian, PI) VA Merit Review grant (Basnakian, PI) COBRE grant (Hauer‐Jensen, PI)To study the effect of carbon nanotube (CNT) ingestion on mouse liver, we systematically counted the number of liver cell nuclei in contact with dark‐field (DF) objects. We found that CNT gavage increased the percentage of nuclei colocalized with DF objects at 100x. Of a total of 38 TUNEL cells recorded in gavage tissue, 22 were touching a DF‐positive object. Livers were extracted from male CD‐1 mice 72 hours after receiving a CNT gavage of 10mg CNT/kg mouse weight.