Principles for assessing the genotoxicity of carbon nanomaterials in vitro (on the example of carbon nanotubes) (literature review)

Introduction. Genotoxicity of nanomaterials (NM) is becoming a major concern when investigating new NM for their safety. Each mutagen is considered to be potentially carcinogenic, therefore a genotoxicity assessment is necessary. However, a clear strategy for assessing the genotoxic effect of NM has not yet been developed.

Material and methods. The material for the analysis have included literature sources from the bibliographic databases PubMed, Scopus, RSCI.

Results. Physicochemical characterization of NM is carried out using high-resolution microscopic and light scattering methods. Before testing for genotoxicity, it is necessary to know the cytotoxicity of the tested NM in order to select the appropriate concentration range. The most important and significant tests are based on the cell viability. MTT assay is a colorimetric test that evaluates the metabolic activity of cells. In addition, viability can be determined using microscopy, flow cytometry, determination of lactate dehydrogenase. Genotoxicity evaluation can be carried out only after the preliminary steps. The strategy should include genotoxicity endpoints: DNA damage, gene mutations, chromosomal damage. The in vitro mammalian gene mutation test, usually performed using mouse lymphoma cells, detects a wide range of genetic damage, including gene deletions. The most common test for detecting chromosomal damage is an in vitro micronucleus assay. DNA strand breaks are most often assessed using the comet DNA assay.

Conclusion. Compulsory stages in the study of the genotoxicity of nanomaterials should be preliminary studies, including physicochemical characterization and assessment of cytotoxicity, as well as the study of the endpoints of genotoxicity and potential mechanisms.

1. https://www.transparencymarketresearch.com/pressrelease/carbon-nano-tubes-market.htm (Дата обращения: 24.10.2021 г.)

2. Liu Z., Tabakman S., Welsher K., Dai H. Carbon nanotubes in biology and medicine: In vitro and in vivo detection, imaging and drug delivery. Nano Res. 2009; 2: 85. https://doi.org/10.1007/s12274-009-9009-8

3. Chen R.J., Bangsaruntip S., Drouvalakis K.A. et al. Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors. Proc. Nat. Acad. Sci. USA. 2003; 100: 4984-9. https://doi.org/10.1073/pnas.0837064100

4. Masato Naya, Norihiro Kobayashi, Kohei Mizuno, Kyomu Matsumoto, Makoto Ema, Junko Nakanishi. Evaluation of the genotoxic potential of single-wall carbon nanotubes by using a battery of in vitro and in vivo genotoxicity assays. Regulatory Toxicology and Pharmacology. 2011; 61(2): 192-8. https://doi.org/10.1016/j.yrtph.2011.07.008

5. DeMarini D.M. The role of genotoxicity in carcinogenesis. In: Baan RA, Stewart BW, Straif K, editors. Tumour Site Concordance and Mechanisms of Carcinogenesis. Lyon (FR): International Agency for Research on Cancer; 2019. (IARC Scientific Publications, No. 165.) Chapter 12.

6. Grosse Y., Loomis D.,kio Guyton K.Z., Lauby-Secretan B., El Ghissassi F., Bouvard V., Benbrahim-Tallaa L., Guha N., Scoccianti C., Mattock H., Straif K. International Agency for Research on Cancer Monograph Working Group. Carcinogenicity of fluoro-edenite, silicon carbide fibres and whiskers, and carbon nanotubes. Lancet Oncol. 2014; 15(13): 1427-8. https://doi.org/10.1016/S1470-2045(14)71109-X

7. Toyokuni S. Genotoxicity and carcinogenicity risk of carbon nanotubes. Adv Drug Deliv Rev. 2013; 65(15): 2098-110. https://doi.org/10.1016/j.addr.2013.05.011

8. Li Z., Hulderman T., Salmen R., Chapman R., Leonard S.S., Young S.H., Shvedova A., Luster M.I., Simeonova P.P. Cardiovascular effects of pulmonary exposure to single-wall carbon nanotubes. Environ Health Perspect. 2007; 115(3): 377-82. https://doi.org/10.1289/ehp.9688

9. Shvedova A.A., Kisin E., Murray A.R., Johnson V.J., Gorelik O., Arepalli S., Hubbs A.F., Mercer R.R., Keohavong P., Sussman N., Jin J., Yin J., Stone S., Chen B.T., Deye G., Maynard A., Castranova V., Baron P.A., Kagan V.E. Inhalation vs. aspiration of single-walled carbon nanotubes in C57BL/6 mice: inflammation, fibrosis, oxidative stress, and mutagenesis. Am J Physiol Lung Cell Mol Physiol. 2008; 295(4): L552-65. https://doi.org/10.1152/ajplung.90287.2008

10. Elespuru R., Pfuhler S., Aardema M.J., Chen T., Doak S.H., Doherty A., Farabaugh C.S., Kenny J., Manjanatha M., Mahadevan B., Moore M.M., Ouédraogo G., Stankowski L.F. Jr., Tanir J.Y. Genotoxicity Assessment of Nanomaterials: Recommendations on Best Practices, Assays, and Methods. Toxicol Sci. 2018; 164(2): 391-416. https://doi.org/10.1093/toxsci/kfy100

11. Kohl Y., Rundén-Pran E., Mariussen E., Hesler M., El Yamani N., Longhin E.M., Dusinska M. Genotoxicity of Nanomaterials: Advanced In Vitro Models and High Throughput Methods for Human Hazard Assessment-A Review. Nanomaterials (Basel). 2020; 10(10): 1911. https://doi.org/10.3390/nano10101911

12. Clift M.J.D., Raemy D.O., Endes C., Ali Z. Lehmann A.D., Brandenberger C., Petri-Fink A., Wick P., Parak W.J., Gehr P. et al. Can the Ames test provide an insight into nano-object mutagenicity? Investigating the interaction between nano-objects and bacteria. Nanotoxicology. 2013; 7: 1373-85. https://doi.org/10.3109/17435390.2012.741725

13. Zhisong Lu, Yan Qiao, Xin Ting Zheng, Chan-Park M.B., Chang Ming Li. Effect of particle shape on phagocytosis of CdTe quantum dot-cystine composites. Med. Chem. Commun. 2010; 1:84-6. https://doi.org/10.1039/c0md00008f

14. Park Ki Ho, Chhowalla M., Iqbal Z., Sesti F. Single-walled Carbon Nanotubes Are a New Class of Ion Channel Blockers. Journal of Biological Chemistry. 2003; 278(50): 50212-6. https://doi.org/10.1074/jbc.M310216200

15. Peter P. Fu, Qingsu Xia, Huey-Min Hwang, Paresh C. Ray, Hongtao Yu. Mechanisms of nanotoxicity: Generation of reactive oxygen species. Journal of Food and Drug Analysis. 2014; 22(1): 64-75. https://doi.org/10.1016/j.jfda.2014.01.005

16. Birch M.E., Ruda-Eberenz T.A., Chai M., Andrews R., Hatfield R.L. Properties that influence the specific surface areas of carbon nanotubes and nanofibers. Ann Occup Hyg. 2013; 57(9): 1148-66. https://doi.org/10.1093/annhyg/met042

17. Yuan X., Zhang X., Sun L., Wei Y., Wei X. Cellular Toxicity and Immunological Effects of Carbon-based Nanomaterials. Part Fibre Toxicol. 2019; 16(1): 18. https://doi.org/10.1186/s12989-019-0299-z

18. Colvin V. The potential environmental impact of engineered nanomaterials. Nat Biotechnol. 2003; 21: 1166-1170. https://doi.org/10.1038/nbt875

19. Wick P. et al. The degree and kind of agglomeration affect carbon nanotube cytotoxicity. Toxicol. Lett. 2007; 168(2): 121-31. https://doi.org/10.1016/j.toxlet.2006.08.019

20. Partikel K., Korte R., Mulac D., Humpf H.U., Langer K. Serum type and concentration both affect the protein-corona composition of PLGA nanoparticles. Beilstein J Nanotechnol. 2019; 10: 1002-15. https://doi.org/10.3762/bjnano.10.101

21. Goodman C.M. et al. Toxicity of gold nanoparticles functionalized with cationic and anionic side chains. Bioconjug. Chem. 2004. 15(4): 897-900. https://doi.org/10.1021/bc049951i

22. Hoshino A. et al. Physicochemical properties and cellular toxicity of nanocrystal quantum dots depend on their surface modification. Nano Lett. 2004; 4(11): 2163-9. https://doi.org/10.1021/nl048715d.s001

23. Lacerda L. et al. Dynamic imaging of functionalized multi-walled carbon nanotube systemic circulation and urinary excretion. Adv. Mater. 2008; 20(2): 225-30. https://doi.org/10.1002/adma.200702334

24. Moore T.L., Rodriguez-Lorenzo L., Hirsch V., Balog S., Urban D., Jud C., Rothenutishauser B., Lattuada M., Petri-Fink A. Nanoparticle colloidal stability in cell culture media and impact on cellular interactions. Chem. Soc. Rev. 2015; 44, 6287-305.

25. Wang, L., Castranova, V., Mishra, A. et al. Dispersion of single-walled carbon nanotubes by a natural lung surfactant for pulmonary in vitro and in vivo toxicity studies. Part Fibre Toxicol. 2010; 7: 31. https://doi.org/10.1186/1743-8977-7-31

26. Youssry M., Al-Ruwaidhi M., Zakeri M. et al. Physical functionalization of multi-walled carbon nanotubes for enhanced dispersibility in aqueous medium. Emergent mater. 2020; 3: 25-32. https://doi.org/10.1007/s42247-020-00076-3

27. Jos A., Pichardo S., Puerto M., Sánchez E., Grilo A., Cameán A.M. Cytotoxicity of carboxylic acid functionalized single wall carbon nanotubes on the human intestinal cell line Caco-2. Toxicology in Vitro. 2009; 23(8): 1491-6. https://doi.org/10.1016/j.tiv.2009.07.001

28. Al-Jamal K.T., Kostarelos K. Assessment of cellular uptake and cytotoxicity of carbon nanotubes using flow cytometry. Methods Mol Biol. 2010; 625: 123-34. https://doi.org/10.1007/978-1-60761-579-8_11 PMID: 20422386.

29. Dalibor Breznan, Dharani Das, Christine MacKinnon-Roy, Benoit Simard, Premkumari Kumarathasan, Renaud Vincent. Non-specific interaction of carbon nanotubes with the resazurin assay reagent: Impact on in vitro assessment of nanoparticle cytotoxicity. Toxicology in Vitro. 2015; 29(1): 142-7. https://doi.org/10.1016/j.tiv.2014.09.009

30. Timerbulatova G.A., Dunaev P.D., Dimiev A.M., et al. Comparative characteristics of various fibrous materials in in vitro experiments. Kazan medical journal. 2021; 102(4): 501-9. https://doi.org/10.17816/KMJ2021-501

31. Siegrist K.J., Reynolds S.H., Kashon M.L. et al. Genotoxicity of multi-walled carbon nanotubes at occupationally relevant doses. Part Fibre Toxicol. 2014; 11:6. https://doi.org/10.1186/1743-8977-11-6

32. Fakhrullin R., Nigamatzyanova L., Fakhrullina G. Dark-field/hyperspectral microscopy for detecting nanoscale particles in environmental nanotoxicology research. Sci. Total Environment. 2021; 772: 145478. https://doi.org/10.1016/j.scitotenv.2021.145478

33. Rubio L., El Yamani N., Kazimirova A., Dusinska M., Marcos R. Multi-walled carbon nanotubes (NM401) induce ROS-mediated HPRT mutations in Chinese hamster lung fibroblasts. Environ. Res. 2016; 146: 185-90. https://doi.org/10.1016/j.envres.2016.01.004

34. Cheng T.F., Patton G.W., Muldoon-Jacobs K. Can the L5178Y Tk+/- mouse lymphoma assay detect epigenetic silencing? Food Chem. Toxicol. 2013; 59: 187-90. https://doi.org/10.1016/j.fct.2013.06.007

35. Demir E, Marcos R. Toxic and genotoxic effects of graphene and multi-walled carbon nanotubes. J Toxicol Environ Health A. 2018; 81(14): 645-60. https://doi.org/10.1080/15287394.2018.1477314 Epub 2018 Jun 6. PMID: 29873610.

36. Shibai-Ogata A., Kakinuma C., Hioki T., Kasahara T. Evaluation of high-throughput screening for in vitro micronucleus test using fluorescence-based cell imaging. Mutagenesis. 2011; 26: 709-19. https://doi.org/10.1093/mutage/ger037

37. Migliore L., Saracino D., Bonelli A., Colognato R., D’Errico M.R., Magrini A., Bergamaschi A., Bergamaschi E. Carbon nanotubes induce oxidative DNA damage in RAW 264.7 cells, Environ. Mol. Mutagen. 2010; 51: 294-303.

38. Kisin E.R., Murray A.R., Keane M.J., Shi X.C., Schwegler-Berry D., Gorelik O., Arepalli S., Castranova V., Wallace W.E., Kagan V.E., Shvedova A.A. Single-walled carbon nanotubes: geno- and cytotoxic effects in lung fibroblast V79 cells. J. Toxicol. Environ. Health A. 2007; 70: 2071-9.

39. Kato T., Totsuka Y., Ishino K., Matsumoto Y., Tada Y., Nakae D., Goto S., Masuda S., Ogo S., Kawanishi M., Yagi T., Matsuda T., Watanabe M., Wakabayashi K. Genotoxicity of multi-walled carbon nanotubes in both invitro and in vivo assay systems. Nanotoxicology. 2013; 7(4): 452-61. https://doi.org/10.3109/17435390.2012.674571

40. AsakuraM., Sasaki T., Sugiyama T., Takaya M., Koda S., Nagano K., Arito H., Fukushima S. Genotoxicity and cytotoxicity of multi-wall carbon nanotubes in cultured Chinese hamster lung cells in comparison with chrysotile A fibers, J. Occup. Health. 2010; 52: 155-66.

41. García-Rodríguez A., Rubio L., Vila L., Xamena N., Velázquez A., Marcos R., Hernández A. The Comet Assay as a Tool to Detect the Genotoxic Potential of Nanomaterials. Nanomaterials. 2019; 9(10): 1385. https://doi.org/10.3390/nano9101385

42. Collins A., El Yamani N., Dusinska M. Sensitive detection of DNA oxidation damage induced by nanomaterials. Free Radic. Biol. Med. 2017; 107: 69-76. https://doi.org/10.1016/j.freeradbiomed.2017.02.001

43. Samadian H., Salami M.S., Jaymand M., Azarnezhad A., Najafi M., Barabadi H., Ahmadi A. Genotoxicity assessment of carbon-based nanomaterials; Have their unique physicochemical properties made them double-edged swords? Mutation Research/Reviews in Mutation Research. 2020; 783: 108296. https://doi.org/10.1016/j.mrrev.2020.108296

44. Jacobsen N.R., Pojana G., White P., Møller P., Cohn C.A., Smith Korsholm K., Wallin, H. Genotoxicity, cytotoxicity, and reactive oxygen species induced by single-walled carbon nanotubes and C60fullerenes in the FE1-Muta™Mouse lung epithelial cells. Environmental and Molecular Mutagenesis. 2008; 49(6): 476-87. https://doi.org/10.1002/em.20406

45. Hevia L.G., Fanarraga M.L. Microtubule cytoskeleton-disrupting activity of MWCNTs: applications in cancer treatment. J Nanobiotechnol. 2020; 18: 181. https://doi.org/10.1186/s12951-020-00742-y

46. Sargent L.M. et al. Single-walled carbon nanotube-induced mitotic disruption. Mutation research vol. 2012; 745(1-2): 28-37. https://doi.org/10.1016/j.mrgentox.2011.11.017

47. Evans S.J., Clift M.J.D., Singh N., De Oliveira Mallia J., Burgum M., Wills J.W., Wilkinson T.S., Jenkins G.J.S., Doak S.H. Critical review of the current and future challenges associated with advanced in vitro systems towards the study of nanoparticle (secondary) genotoxicity. Mutagenesis. 2017; 32: 233-41. https://doi.org/10.1093/mutage/gew054

48. Pfuhler S., van Benthem J., Curren R., Doak S.H., Dusinska M., Hayashi M., Heflich R.H., Kidd D., Kirkland D., Luan Y. et al. Use of in vitro 3D tissue models in genotoxicity testing: Strategic fit, validation status and way forward. Report of the working group from the 7th International Workshop on Genotoxicity Testing (IWGT). Mutat. Res. Toxicol. Environ. Mutagen. 2020; 850-1.

49. Nelson B.C., Wright C.W., Ibuki Y., Moreno-Villanueva M., Karlsson H.L., Hendriks G., Sims C.M., Singh N., Doak S.H. Emerging metrology for high-throughput nanomaterial genotoxicology. Mutagenesis. 2017; 32: 215-32. https://doi.org/10.1002/adma.200702334

50. Elespuru R., Pfuhler S., Aardema M.J., Chen T., Doak S.H., Doherty A., Farabaugh C.S., Kenny J., Manjanatha M., Mahadevan B. et al. Genotoxicity assessment of nanomaterials: Recommendations on best practices, assays, and methods. Toxicol. Sci. 2018; 164: 391-416. https://doi.org/10.1093/toxsci/kfy100

51. Barosova H., Karakocak B.B., Septiadi D., Petri-Fink A., Stone V., & Rothen-Rutishauser B. An In Vitro Lung System to Assess the Proinflammatory Hazard of Carbon Nanotube Aerosols. International Journal of Molecular Sciences. 2020: 21(15): 5335. https://doi.org/10.3390/ijms21155335

52.