Genome instability and cell death under the action of the fungicide from the phthalimide class on human lymphocytes in vitro

Introduction. The studies of captan genotoxicity in vitro show inconsistent findings, suggesting both the presence and absence of its capacity to induce DNA damage. The specific types of DNA lesions caused by captan remain unclear.

The aim of this work. To study the genotoxic and cytotoxic effects of captan using the enzyme-modified DNA-comet assay, along with an assessment of different forms of cell death in peripheral blood lymphocytes.

Materials and methods. Genotoxic effects of captan were studied in vitro on human lymphocytes using a modified protocol of the DNA-comet technique that included the treatment of gel slides with DNA repair enzymes (APE1, Fpg, ENDO III (Nth), USER III, OGG1, hAAG1, and T4 PDG). Analysis of lymphocyte death forms in response to captan exposure was carried out using an automated fluorescent cell analyzer.

Results. Study of the trend in DNA lesion accumulation revealed a statistically significant increase in the DNA damage levels compared to the concurrent controls after incubation of lymphocytes for 15–120 minutes with captan at the concentrations ranging from 5 to 25 µg/mL in the absence of metabolic activation. The effect was found to depend on the pesticide concentration and the incubation time. Analysis of the types of DNA alterations using a modified DNA-comet assay revealed that, in addition to DNA strand breaks, oxidized bases, AP-sites, and uracil are also formed after captan exposure in vitro. Treatment of lymphocytes with captan resulted in cytotoxic effects characterized by a statistically significant decrease in viable cell proportions, increase in fractions of early and late apoptotic cells, and necrosis. These effects were dependent on the concentration of captan and exposure duration. After 3 hours of exposure to 25 µg/mL captan, the total proportion of apoptotic cells reached 57%, the proportion of necrotic cells was 6%, and the level of viable lymphocytes was 37%.

Limitations. The study focuses exclusively on assessing the in vitro cytotoxicity and genotoxicity of captan.

Conclusion. In vitro exposure of captan induces to the formation of DNA breaks, oxidized bases, AP-sites, and cytosine deamination. Apoptosis was the predominated type of lymphocyte death due to the effect of captan in vitro.

Compliance with ethical standards. The study was approved by the Ethics Committee of the Federal Scientific Center for Hygiene. F.F. Erisman” of Rospotrebnadzor (Protocol No. 1, September 29, 2020). All participants gave informed voluntary written consent to participate in the study.

Contribution:
Egorova O.V. – concept and design of the study; collecting and processing of material; analysis of the results; writing the text;
Kotnova A.P., Gorenskaya O.V., Averianova N.S. – the collection and processing of material;
Ilyushina N.A. – concept and design of the study; analysis of the results; writing the text.
All authors are responsible for the integrity of all parts of the manuscript and approval of the manuscript final version.

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

Funding. The work was carried out within the framework of state assignment (No. 121090800086-7).

Received: August 7, 2025 / Accepted: October 15, 2025 / Published: November 14, 2025

1. Gordon E.B., Krieger R., ed. Captan and folpet. In: Hayes’ Handbook of Pesticide Toxicology. Elsevier; 2010: 1915–49.

2. Snyder R.D. Effects of Captan on DNA and DNA metabolic processes in human diploid fibroblasts. Environ. Mol. Mutagen. 1992; 20(2): 127–33. https://doi.org/10.1002/em.2850200208

3. Moo-Muñoz A.J., Azorín-Vega E.P., Ramírez-Durán N., Moreno-Pérez P.A. Evaluation of the cytotoxic and genotoxic potential of the captan-based fungicides, chlorothalonil-based fungicides and methyl thiophanate-based fungicides in human fibroblasts BJ. J. Environ. Sci. Health B. 2021; 56(10): 877–83. https://doi.org/10.1080/03601234.2021.1972721

4. Cohen S.M., Gordon E.B., Singh P., Arce G.T., Nyska A. Carcinogenic mode of action of folpet in mice and evaluation of its relevance to humans. Crit. Rev. Toxicol. 2010; 40(6): 531–45. https://doi.org/10.3109/10408441003742903

5. Final Report on the Safety Assessment of Captan. J. Am. Coll. Toxicol. 1989; 8(4): 643–80. https://doi.org/10.3109/10915818909010526

6. Mouchet F., Gauthier L., Mailhes C., Ferrier V., Devaux A. Comparative evaluation of genotoxicity of captan in amphibian larvae (Xenopus laevis and Pleurodeles waltl) using the comet assay and the micronucleus test. Environ. Toxicol. 2006; 21(3): 264–77. https://doi.org/10.1002/tox.20180

7. Arce G.T., Gordon E.B., Cohen S.M., Singh P. Genetic toxicology of folpet and captan. Crit. Rev. Toxicol. 2010; 40(6): 546–74. https://doi.org/10.3109/10408444.2010.481663

8. Fedorova N.E., Bereznyak I.V., Bondareva L.G., Dobreva N.I., Egorchenkova O.E., Dobrev S.D. Captan: problems associated with its identification in environmental objects and food products. Ways of solution. Khimicheskaya bezopasnost’. 2022: 6(2): 227–42. https://doi.org/10.25514/CHS.2022.2.23015 https://elibrary.ru/aiaqjr (in Russian)

9. Greenburg D.L., Rusiecki J., Koutros S., Dosemeci M., Patel R., Hines C.J., et al. Cancer incidence among pesticide applicators exposed to captan in the Agricultural Health Study. Cancer Causes Control. 2008; 19(10): 1401–7. https://doi.org/10.1007/s10552-008-9187-9

10. Lebailly P., Devaux A., Pottier D., De Meo M., Andre V., Baldi I., et al. Urine mutagenicity and lymphocyte DNA damage in fruit growers occupationally exposed to the fungicide captan. Occup. Environ. Med. 2003; 60(12): 910–7. https://doi.org/10.1136/oem.60.12.910.

11. Presutti R., Harris S.A., Kachuri L., Spinelli J.J., Pahwa M., Blair A., et al. Pesticide exposures and the risk of multiple myeloma in men: An analysis of the North American Pooled Project. Int. J. Cancer. 2016; 139(8): 1703–14. https://doi.org/10.1002/ijc.30218

12. Averianova N.S., Kara L.A., Egorova O.V., Ilyushina N.A. The study of primary DNA damage under the combined action of pesticides. Toksikologicheskii vestnik. 2021; 29(4): 14–21. https://doi.org/10.36946/0869-7922-2021-29-4-14-21 https://elibrary.ru/ozitie (in Russian)

13. Egorova O.V., Ilyushina N.A., Rakitskii V.N. Mutagenicity evaluation of pesticide analogs using standard and 6-well miniaturized bacterial reverse mutation tests. Toxicology in Vitro. 2020; 69: 105006. https://doi.org/10.1016/j.tiv.2020.105006

14. Egorova O.V., Suzina N.E., Ilyushina N.A. Salmonella mutant strains resistant to herbicides – acetohydroxyacid synthase inhibitors and their use at the Ames test. Toxicol. In Vitro. 2023; 93: 105699.

15. Tsareva A.A., Ignatyev S.D., Egorova O.V., Averianova N.S., Kotnova A.P., Ilyushina N.A. Effect of a phthalimide pesticide on human peripheral blood lymphocytes in vitro: induction of DNA damage. Toksikologicheskii vestnik. 2023; 31(6): 376–84. https://doi.org/10.47470/0869-7922-2023-31-6-376-384 https://elibrary.ru/orykxh (in Russian)

16. Gorenskaya O.V., Egorova O.V., Averyanova N.S., Kotnova A.P., Ilyushina N.A. Evaluation of the cytotoxic and genotoxic effects of cyprodinil in vitro. Toksikologicheskii vestnik. 2024: 32(6): 348–56. https://doi.org/10.47470/0869-7922-2024-32-6-348-356 https://elibrary.ru/luveol (in Russian)

17. Collins A.R., Duthie S.J., Dobson V.L. Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis. 1993; 14(9): 1733–5. https://doi.org/10.1093/carcin/14.9.1733

18. Muruzabal D., Collins A., Azqueta A. The enzyme-modified comet assay: Past, present and future. Food Chem. Toxicol. 2021; 147: 111865. https://doi.org/10.1016/j.fct.2020.111865

19. FAO/WHO. Pesticide residues in food. Captan; 1982. Available at: https://inchem.org/documents/jmpr/jmpmono/v82pr07.htm

20. Rocchi P., Perocco P., Alberghini W., Fini A., Prodi G. Effect of pesticides on scheduled and unscheduled DNA synthesis of rat thymocytes and human lymphocytes. Arch. Toxicol. 1980; 45(2): 101–8. https://doi.org/10.1007/BF01270907

21. Eastmond D.A. Factors influencing mutagenic mode of action determinations of regulatory and advisory agencies. Mutat. Res. Rev. Mutat. Res. 2012; 751(1): 46–63. https://doi.org/10.1016/j.mrrev.2012.04.001

22. Couch R.C., Siegel M.R. Interaction of captan and folpet with mammalian DNA and histones. Pestic. Biochem. Physiol. 19997; 7: 531–46.

23. Dillwith J.W., Lewis R.A. Inhibition of DNA polymerase by captan. Biochim. Biophys. Acta. 1982; 696(3): 245–52. https://doi.org/10.1016/0167-4781(82)90054-9

24. Rahden-Staron I. The inhibitory effect of the fungicides captan and captafol on eukaryotic topoisomerases in vitro and lack of recombinagenic activity in the wing spot test of Drosophila melanogaster. Mutat. Res. 2002; 518(2): 205–13. https://doi.org/10.1016/s1383-5718(02)00107-9

25. Fernandez-Vidal A., Arnaud L.C., Maumus M., Chevalier M., Mirey G., Salles B., et al. Exposure to the fungicide captan induces DNA base alterations and replicative stress in mammalian cells. Environ. Mol. Mutagen. 2019; 60: 286–97. https://doi.org/10.1002/em.22268

26. Scariot F.J., Jahn L., Delamare A.P.L., Echeverrigaray S. Necrotic and apoptotic cell death induced by Captan on Saccharomyces cerevisiae. World J. Microbiol. Biotechnol. 2017; 33(8): 159. https://doi.org/10.1007/s11274-017-2325-3

27. Inoue T., Kinoshita M., Oyama K., Kamemura N., Oyama Y. Captan-induced increase in the concentrations of intracellular Ca2+ and Zn2+ and its correlation with oxidative stress in rat thymic lymphocytes. Environ. Toxicol. Pharmacol. 2018; 63: 78–83. https://doi.org/10.1016/j.etap.2018.08.017

28. Suzuki T., Nojiri H., Isono H., Ochi T. Oxidative damages in isolated rat hepatocytes treated with the organochlorine fungicides captan, dichlofluanid and chlorothalonil. Toxicology. 2004; 204(2–3): 97–107. https://doi.org/10.1016/j.tox.2004.06.025

29. Reuter S., Gupta S.C., Chaturvedi M.M., Aggarwal B.B. Oxidative stress, inflammation, and cancer: how are they linked? Free Radic. Biol. Med. 2010; 49(11): 1603–16. https://doi.org/10.1016/j.freeradbiomed.2010.09.006

30. Bartsch H., Nair J. Chronic inflammation and oxidative stress in the genesis and perpetuation of cancer: role of lipid peroxidation, DNA damage, and repair. Langenbecks Arch. Surg. 2006; 391(5): 499–510. https://doi.org/10.1007/s00423-006-0073-1

31. Klaunig J.E., Wang Z., Pu X., Zhou S. Oxidative stress and oxidative damage in chemical carcinogenesis. Toxicol. Appl. Pharmacol. 2011; 254(2): 86–99. https://doi.org/10.1016/j.taap.2009.11.028