Analysis of cyto- and genotoxicity of aqueous extracts of peat smoke in vitro

Introduction. Wildfire smoke (WFS) is a complex mixture of particulate matter (PM) and toxic gases. Nevertheless, the contribution of each fraction to the overall toxicity remains poorly understood. Peat smoke, characterized by prolonged smoldering and a specific chemical composition, is of particular interest.

Materials and methods. Peat smoke extracts (PSE) were obtained by bubbling smoke through a DMEM culture medium, followed by separation into unfiltered (UF-PSE), containing PM and soluble gas components, and filtered (F-PSE), containing only water-soluble gases. The obtained extracts in dilutions of 1:3, 1:1, 3:1 and without dilution were used for exposure of 3T3-SV40 mouse fibroblast culture. Cytotoxicity was assessed using the dye exclusion method, cellular metabolic activity was assessed using the MTT assay, and fragmentation and whole-genome DNA methylation were assessed using the comet assay. The qualitative composition of volatile compounds in UF-PSE was determined using gas chromatography-mass spectrometry.

Results. No statistically significant differences in cell viability, fragmentation, or whole-genome DNA methylation were detected between the experimental and control groups. However, a trend toward a dose-dependent decrease in viability and metabolic activity was noted under UF-PSE, which was absent for F-PSE. The level of whole-genome DNA methylation under UF-PSE exposure was 1.6 times higher than under F-PSE. Volatile aldehydes C1–C4, primarily acetaldehyde and formaldehyde, were identified in the chemical composition of UF-PSE. No other volatile organic compounds were detected.

Limitations. The study is limited to the use of a single 24-hour exposure to F-PSE and UF-PSE on 3T3-SV40 with a qualitative chemical analysis of smoke components, a study of its cytotoxicity, genotoxicity, ability to introduce epigenetic changes and influence the metabolic activity of cells.

Conclusion. Although there were no statistically significant differences, the identified trends indicate a possible contribution of PM to the cytotoxicity and epigenetic effects of peat smoke, which requires further study, including chronic exposure and its quantitative chemical analysis.

Compliance with ethical standards. The study was approved by the local ethics committee of the East-Siberian Institute of Medical and Ecological Research (Protocol No.7 of 12/15/2023), conducted in accordance with the European Convention for the Protection of Vertebrate Animals Used for Experiments or for Other Scientific Purposes (ETS N 123), Directive 2010/63/EC of the European Parliament and the Council of 22.09.2010 on the protection of animals used for scientific purposes.

Contribution:
Abramova V.A. – concept and design of research, data collection and processing, statistical data processing, text writing, editing;
Vasyuhina P.O. – data collection and processing;
Alekseenko A.N. – concept and design of research, data collection and processing; Vokina V.A. – concept and design of research, data collection and processing;
Sosedova L.M. – concept and design of research, editing.
All authors are responsible for the integrity of all parts of the manuscript and approval of the manuscript final version.

Conflict of interest. The authors declare no conflict of interest.

Funding. The study had no sponsorship.

Received: April 8, 2026 / Accepted: June 18, 2026 / Published: September 28, 2026

1. Chen L.W., Moosmüller H., Arnott W.P., Chow J.C., Watson J.G., Susott R.A., et al. Emissions from laboratory combustion of wildland fuels: emission factors and source profiles. Environ. Sci. Technol. 2007; 41(12): 4317–25. https://doi.org/10.1021/es062364i

2. McMeeking G.R., Kreidenweis S.M., Baker S., Carrico C.M., Chow J.C., Collett Jr. J.L., et al. Emissions of trace gases and aerosols during the open combustion of biomass in the laboratory. J. Geophys. Res. 2009; 114(D19): D19210. https://doi.org/10.1029/2009JD011836 https://elibrary.ru/naaslh

3. Reid C.E., Brauer M., Johnston F.H., Jerrett M., Balmes J.R., Elliott C.T. Critical review of health impacts of wildfire smoke exposure. Environ. Health Perspect. 2016; 124(9): 1334–43. https://doi.org/10.1289/ehp.1409277 https://elibrary.ru/xtgufv

4. Black C., Tesfaigzi Y., Bassein J.A., Miller L.A. Wildfire smoke exposure and human health: Significant gaps in research for a growing public health issue. Environ. Toxicol. Pharmacol. 2017; 55: 186–95. https://doi.org/10.1016/j.etap.2017.08.022 https://elibrary.ru/yjckqe

5. Hargrove M.M., Kim Y.H., King C., Wood C.E., Gilmour M.I., Dye J.A., et al. Smoldering and flaming biomass wood smoke inhibit respiratory responses in mice. Inhal. Toxicol. 2019; 31(6): 236–47. https://doi.org/10.1080/08958378.2019.1654046

6. Kim Y.H., Warren S.H., Krantz Q.T., King C., Jaskot R., Preston W.T., et al. Mutagenicity and lung toxicity of smoldering vs. flaming emissions from various biomass fuels: implications for health effects from wildland fires. Environ. Health Perspect. 2018; 126(1): 017011. https://doi.org/10.1289/ehp2200

7. Young T.M., Black G.P., Wong L., Bloszies C.S., Fiehn O., He G., et al. Identifying toxicologically significant compounds in urban wildfire ash using in vitro bioassays and high-resolution mass spectrometry. Environ. Sci. Technol. 2021; 55(6): 3657–67. https://doi.org/10.1021/acs.est.0c06712 https://elibrary.ru/spdtbg

8. Deering-Rice C.E., Nguyen N., Lu Z., Cox J.E., Shapiro D., Romero E.G., et al. Activation of TRPV3 by wood smoke particles and roles in pneumotoxicity. Chem. Res. Toxicol. 2018; 31(5): 291–301. https://doi.org/10.1021/acs.chemrestox.7b00336 https://elibrary.ru/ygwfcp

9. Garcia-Hurtado E., Pey J., Borrás E., Sánchez P., Vera T., Carratalá A., et al. Atmospheric PM and volatile organic compounds released from Mediterranean shrubland wildfires. Atmos. Environ. 2014; 89: 85–92. https://doi.org/10.1016/j.atmosenv.2014.02.016

10. Junquera V., Russell M.M., Vizuete W., Kimura Y., Allen D. Wildfires in eastern Texas in August and September 2000: emissions, aircraft measurements, and impact on photochemistry. Atmos. Environ. 2005; 39(27): 4983–96. https://doi.org/10.1016/j.atmosenv.2005.05.004

11. Reisen F., Brown S., Cheng M. Air toxics in bushfire smoke: firefighter’s exposure during prescribed burns. For. Ecol. Manag. 2006; 234(Suppl.): S144. https://doi.org/10.1016/j.foreco.2006.08.191

12. Migliaccio C.T., Kobos E., King Q.O., Porter V., Jessop F., Ward T. Adverse effects of wood smoke PM(2.5) exposure on macrophage functions. Inhal. Toxicol. 2013; 25(2): 67–76. https://doi.org/10.3109/08958378.2012.756086

13. Roscioli E., Hamon R., Lester S.E., Jersmann H.P.A., Reynolds P.N., Hodge S. Airway epithelial cells exposed to wildfire smoke extract exhibit dysregulated autophagy and barrier dysfunction consistent with COPD. Respir. Res. 2018; 19(1): 234. https://doi.org/10.1186/s12931-018-0945-2 https://elibrary.ru/gggcwn

14. Danielsen P.H., Møller P., Jensen K.A., Sharma A.K., Wallin H., Bossi R., et al. Oxidative stress, DNA damage, and inflammation induced by ambient air and wood smoke particulate matter in human A549 and THP-1 cell lines. Chem. Res. Toxicol. 2011; 24(2): 168–84. https://doi.org/10.1021/tx100407m

15. Vokina V.A, Abramova V.A., Sosedova L.M. Method for assessing the toxicity of biomass combustion products. Patent RF №2855752 C1; 2025. (in Russian)

16. Abramova V.A., Vokina V.A., Sosedova L.M. Evaluation of cytotoxicity of peat smoke extract on 3T3-SV40 cell culture. Gigiena i Sanitaria (Hygiene and Sanitation, Russian journal). 2025; 104(11): 1549–53. https://doi.org/10.47470/0016-9900-2025-104-11-1549-1553 https://elibrary.ru/hskznr (in Russian)

17. Durnev A.D., Zhanataev A.K., Anisina E.A., Sidneva E.S., Nikitina V.A., Oganesyants L.A., et al. Application of the alkaline gel electrophoresis method of isolated cells for assessing the genotoxic properties of natural and synthetic compounds: Methodological recommendations. Moscow; 2006. https://elibrary.ru/sevvqt (in Russian)

18. Durnev A.D., Merkulov V.A., Zhanataev A.K., Nikitina V.A., Voronina E.S., Seredenin S.B. Methodological recommendations for the assessment of DNA damage by alkaline gel electrophoresis of individual cells in pharmacological studies. In: Guidelines for Conducting Preclinical Studies of Medicinal Products. Part 1 [Rukovodstvo po provedeniyu doklinicheskikh issledovanii lekarstvennykh sredstv. Chast’ 1]. Moscow: Grif i K; 2012: 115–28. https://elibrary.ru/wxwgpv (in Russian)

19. Wentzel J.F., Gouws C., Huysamen C., van Dyk E, Koekemoer G., Pretorius P.J. Assessing the DNA methylation status of single cells with the comet assay. Anal. Biochem. 2010; 400(2): 190–4. https://doi.org/10.1016/j.ab.2010.02.008

20. Alekseenko A.N., Zhurba O.M., Vokina V.A., Merinov A.V., Shayakhmetov S.F. Methodological features of chromato-mass spectrometric identification of organic compounds in the air during landscape fires. Gigiena i Sanitaria (Hygiene and Sanitation, Russian journal). 2022; 101(9): 1103–10. https://doi.org/10.47470/0016-9900-2022-101-9-1103-1110 https://elibrary.ru/txfhki (in Russian)

21. Dong T.T.T., Hinwood A.L., Callan A.C., Zosky G., Stock W.D. In vitro assessment of the toxicity of bushfire emissions: A review. Sci. Total Environ. 2017; 603-604: 268–78. https://doi.org/10.1016/j.scitotenv.2017.06.062

22. Wegesser T.C., Franzi L.M., Mitloehner F.M., Eiguren-Fernandez A., Last J.A. Lung antioxidant and cytokine responses to coarse and fine particulate matter from the great California wildfires of 2008. Inhal. Toxicol. 2010; 22(7): 561–70. https://doi.org/10.3109/08958370903571849

23. Sada-Ovalle I., Chávez-Galán L., Vasquez L., Aldriguetti S., Rosas-Perez I., Ramiréz-Venegas A., et al. Macrophage exposure to polycyclic aromatic hydrocarbons from wood smoke reduces the ability to control growth of Mycobacterium tuberculosis. Front. Med. (Lausanne). 2018; 5: 309. https://doi.org/10.3389/fmed.2018.00309

24. Corsini E., Budello S., Marabini L., Galbiati V., Piazzalunga A., Barbieri P., et al. Comparison of wood smoke PM2.5 obtained from the combustion of FIR and beech pellets on inflammation and DNA damage in A549 and THP-1 human cell lines. Arch. Toxicol. 2013; 87(12): 2187–99. https://doi.org/10.1007/s00204-013-1071-z https://elibrary.ru/hvwesb

25. Leonard S.S., Wang S., Shi X., Jordan B.S., Castranova V., Dubick M. A. Wood smoke particles generate free radicals and cause lipid peroxidation, DNA damage, NFkappaB activation and TNF-alpha release in macrophages. Toxicology. 2000; 150(1–3): 147–57. https://doi.org/10.1016/s0300-483x(00)00256-0

26. Heßelbach K., Kim G.J., Flemming S., Häupl T., Bonin M., Dornhof R., et al. Disease relevant modifications of the methylome and transcriptome by particulate matter (PM2.5) from biomass combustion. Epigenetics. 2017; 12(9): 779–92. https://doi.org/10.1080/15592294.2017.1356555

27. Jalava P.I., Salonen R.O., Hälinen A.I., Penttinen P., Pennanen A.S., Sillanpää M., et al. In vitro inflammatory and cytotoxic effects of size-segregated particulate samples collected during long-range transport of wildfire smoke to Helsinki. Toxicol. Appl. Pharmacol. 2006; 215(3): 341–53. https://doi.org/10.1016/j.taap.2006.03.007

28. Ihantola T., Di Bucchianico S., Happo M., Ihalainen M., Uski O., Bauer S., et al. Influence of wood species on toxicity of log-wood stove combustion aerosols: a parallel animal and air-liquid interface cell exposure study on spruce and pine smoke. Part. Fibre Toxicol. 2020; 17(1): 27. https://doi.org/10.1186/s12989-020-00355-1 https://elibrary.ru/btjlvs

29. Brooks P.J., Zakhari S. Acetaldehyde and the genome: beyond nuclear DNA adducts and carcinogenesis. Environ. Mol. Mutagen. 2014; 55(2): 77–91. https://doi.org/10.1002/em.21824

30. O’Brien P.J., Siraki A.G., Shangari N. Aldehyde sources, metabolism, molecular toxicity mechanisms, and possible effects on human health. Crit. Rev. Toxicol. 2005; 35(7): 609–62. https://doi.org/10.1080/10408440591002183