In vivo genotoxicity assessment of a multiwalled carbon nanotube in a mouse ex vivo culture

Our plain-English summary of In vivo genotoxicity assessment of a multiwalled carbon nanotube in a mouse ex vivo culture, by K. Horibata, H. Takasawa, M. Hojo, et al (Genes and Environment, 2022).

The study

• Authors: K. Horibata, H. Takasawa, M. Hojo, Y. Taquahashi, M. Shigano, S. Yokota, N. Kobayashi, K.I. Sugiyama, M. Honma, S. Hamada
• Journal: Genes and Environment
• Published: 2022;44(1):24
• Cited by: 4 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Multiwalled carbon nanotubes (MWCNTs) are suspected lung carcinogens because their shape and size are similar to asbestos. Various MWCNT types are manufactured; however, only MWNT-7 is classified into Group 2B by The International Agency for Research on Cancer. MWNT-7's carcinogenicity is strongly related to inflammatory reactions. On the other hand, inconsistent results on MWNT-7 genotoxicity have been reported. We previously observed no significant differences in both Pig-a (blood) and gpt (lung) mutant frequencies between MWNT-7-intratracheally treated and negative control rats. In this study, to investigate in vivo MWNT-7 genotoxicity on various endpoints, we attempted to develop a lung micronucleus assay through ex vivo culture targeting the cellular fraction of Clara cells and alveolar Type II (AT-II) cells, known as the initiating cells of lung cancer. Using this system, we analyzed the in vivo MWNT-7 genotoxicity induced by both whole-body inhalation exposure and intratracheal instillation. We also conducted an erythrocyte micronucleus assay using the samples obtained from animals under intratracheal instillation to investigate the tissue specificity of MWNT-7 induced genotoxicities.

Results: We detected a significant increase in the incidence of micronucleated cells derived from the cellular fraction of Clara cells and AT-II cells in both MWNT-7-treated and positive control groups compared to the negative control group under both whole-body inhalation exposures and intratracheal instillation. Additionally, the erythrocyte micronucleus assay detected a significant increase in the incidence of micronucleated reticulocytes only in the positive control group.

Conclusions: Our findings indicated that MWNT-7 was genotoxic in the lungs directly exposed by both the body inhalation and intratracheal instillation but not in the hematopoietic tissue.

© 2022. The Author(s).

In plain English

This short report described an animal experiment on MWNT-7, a multiwalled carbon nanotube that the authors noted is suspected as a lung carcinogen because its shape and size are similar to asbestos. Male C57BL/6NcrSlc mice were exposed by whole-body inhalation (2 mg/m3, 2 hours a day for five days) or by a single 0.05 mg dose placed into the windpipe (intratracheal instillation). The authors developed a test that grew lung cells (Clara cells and alveolar Type II cells) outside the body and counted cells with micronuclei, a measure of genetic damage. After inhalation, micronucleated lung cells averaged 0.283% in air-exposed mice versus 1.483% in MWNT-7 mice in a first test, and 0.390% versus 1.330% in a confirmatory test. After instillation they averaged 1.403% with the vehicle (saline containing 0.1% Tween-80) versus 3.517% with MWNT-7. These increases were statistically significant. Blood was tested only after instillation, and there micronucleated reticulocytes rose significantly only in the positive-control group, not with MWNT-7. The authors concluded that MWNT-7 was genotoxic in directly exposed lungs but not in blood-forming tissue. They said they could not yet explain the mechanism.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Animal experiment (short report) using a newly developed lung micronucleus assay with ex vivo cell culture, plus a blood (erythrocyte) micronucleus test done only after intratracheal instillation
• Who was studied: Male C57BL/6NcrSlc mice from Japan SLC, 12 weeks old at the start of dosing. For inhalation, 3 mice per group in the first test and 5 per group in the confirmatory test. For intratracheal instillation, 6 mice per group.
• Exposure: MWNT-7 multiwalled carbon nanotubes, about 100 nm in diameter, with 27.5% of fibers 5 micrometres or longer, dispersed by the Taquann method. Inhalation group: 2 mg/m3 for 2 h/day on 5 consecutive days, with lungs sampled 5 days after the last dose. Instillation group: a single dose of 0.05 mg per animal into the trachea, compared with a vehicle of saline containing 0.1% Tween-80. Ethyl methane sulfonate (EMS), given by injection into the abdomen, was the positive control.
• Main results:
  • Whole-body inhalation, first test: mean percentage of micronucleated lung cells was 0.283 (plus or minus 0.076) in air-exposed mice, 1.483 (plus or minus 0.548) in MWNT-7 mice and 0.567 (plus or minus 0.126) in EMS mice. The MWNT-7 and EMS groups were significantly higher than the control (p less than 0.05, Fisher's exact test).
  • Whole-body inhalation, confirmatory test: mean percentage of micronucleated lung cells was 0.390 (plus or minus 0.119) in air-exposed mice, 1.330 (plus or minus 0.449) in MWNT-7 mice and 0.820 (plus or minus 0.114) in EMS mice. The MWNT-7 and EMS groups were again significantly increased.
  • Intratracheal instillation, lung: mean percentage of micronucleated lung cells was 1.403 (plus or minus 0.457) with vehicle only (saline containing 0.1% Tween-80), 3.517 (plus or minus 0.808) with MWNT-7 and 4.348 (plus or minus 0.936) with EMS. The MWNT-7 and EMS groups showed significant increases.
  • Intratracheal instillation, blood (the only exposure method for which blood was tested): micronucleated reticulocytes increased significantly only in the positive-control (EMS) group. Mean percentages were 0.448 (plus or minus 0.060) with vehicle, 0.418 (plus or minus 0.026) with MWNT-7 and 2.210 (plus or minus 0.319) with EMS.
• The authors' conclusion: The authors concluded that MWNT-7 was genotoxic in the lungs directly exposed by both inhalation and intratracheal instillation, but not in the hematopoietic (blood-forming) tissue. They suspected the mechanism is linked to the local response in directly exposed tissue.
• Limitations the authors state:
  • The authors stated that their results cannot explain the mechanism of MWNT-7-induced genotoxicity in the living animal.
  • The authors noted that the negative control group in the instillation experiment had a slightly higher rate of micronucleated lung cells than the air controls in the inhalation experiment. They suggested the saline and Tween-80 vehicle may have caused a slight inflammatory reaction.

Topics

Medical terms in this study, explained in our Lexicon: Genotoxicity, Lung Cancer.

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