Approaches to the in vitro assessment of the genotoxic potential of carbon nanotubes

Introduction. Carbon nanotubes (CNTs) are high-tech materials, but their genotoxic and carcinogenic potential has not been sufficiently studied. To assess the genotoxicity of CNTs, alternative in vitro methods are needed, the development of which is impossible without a preliminary study of DNA damage mechanisms.

Material and methods. The study was conducted on 2D and 3D human respiratory cell cultures. The effects of Russian single-wall (SWCNT TUBALL™, purified and unpurified) and multi-wall (MWCNT Taunit-M) CNTs were investigated. The cytotoxicity of CNTs was assessed using the MTS assay and lactate dehydrogenase (LDH) assay. The genotoxic potential was investigated using the DNA comet method. The localization of CNTs in cells was determined by transmission electron microscopy. To detect oxidative stress, reactive oxygen species (ROS) levels were measured using a DCFDA kit. The pro-fibrogenic and pro-apoptotic activities of CNTs were determined by assessing gene expression (TGF-β1, P53, Bax, Bcl2) using real-time PCR.

Results. Fibroblasts demonstrated the highest sensitivity to CNT cytotoxicity, while A549 cells demonstrated the lowest. CNT genotoxicity was detected in all cells at a concentration of 20 µg/ml. All CNTs penetrated the cells, and MWCNTs were detected in the cell nucleus. Oxidative stress was induced dose-dependently by all types of CNTs, with unpurified SWCNTs producing the most pronounced effect. In BEAS-2B cells, MWCNTs induced an increase in TGF-β1 expression at low concentrations, while SWCNTs induced a similar effect only at high concentrations. In A549 cells, all types of CNTs exhibited a pro-fibrogenic effect at all concentrations. In fibroblasts, no significant increase in TGF-β1 gene expression was observed. In BEAS-2B and A549 cells, CNTs demonstrated the ability to initiate apoptotic signaling pathways, while in MRC5-SV40 cells, no signs of apoptosis in response to CNTs were detected. Studies in 3D cell culture models demonstrated signs of oxidative stress in response to CNTs; no pro-apoptotic or pro-fibrogenic effects of CNTs were detected.

Limitations. The possible role of DNA repair was not assessed in this study. The study covers a limited number of specific types of domestically produced CNTs.

Conclusion. The genotoxicity of CNTs is determined by their type and type of target cells, manifesting itself in a subtoxic concentration range. Key mechanisms include oxidative stress, direct DNA damage, and induction of apoptosis. The results obtained on 3D models demonstrate lower sensitivity, which may indicate a protective role of the tissue organization.

Compliance with ethical standards. The study does not require a biomedical ethics committee opinion or other documents.

Authors’ contribution:
Fatkhutdinova L.M. – study design, analysis of material, editing;
Timerbulatova G.A. – cell culturing, cell testing, summarizing the results, editing;
Gabidinova G.F. – development of approaches for culturing 3D models, cell testing, literature review, statistical data processing, summarizing the results.
All co-authors approved the final version of the article and are responsible for the integrity of all parts of the article.

Conflict of interest. The authors declare no apparent and potential conflicts of interest in relation to the publication of this article.

Funding. The study was supported by the Russian Science Foundation grant No. 22-25-00512. https://rscf.ru/project/22-25-00512/

Received: January 22, 2026 / Accepted: February 2, 2026 / Published: March 18, 2026

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