Cytotoxic effect of doxorubicin on spermatogenic epithelial cells in vitro

Introduction. It is known that the antitumor drug doxorubicin (Dox) is a reprotoxicant that has a negative effect on reproductive function in mammals. In the male body, doxorubicin leads to significant damage to testicular cells, inducing DNA damage, the formation of double-stranded breaks, crosslinking, and free radicals.

The purpose of this work was to examine the effect of doxorubicin on the development of spermatogenic epithelial cells in vitro.

Material and methods. The study was performed on the primary culture of mouse spermatogenic epithelium. The cytotoxic effect of doxorubicin was studied at concentrations of 0.25, 0.05, and 0.005 µg/ml. The degree of cytotoxicity and viability of cells was assessed by their morphological state, DNA damage, and the dynamics of the number of living cells in culture for 40 days.

Results. 24 hours after exposure to Dox, a dose-dependent statistically significant decrease in cell viability was observed in all the studied samples, with the maximum effect – a decrease in the number of live cells to 50% – observed at a concentration of 0.25 µg/ml, and a decrease to 13% at a concentration of 0.05 µg/ml. The decrease in cell viability persisted in subsequent stages of cultivation, which determined the nature of the monolayer, as well as changes in the morphology of the Sertoli cells.

Limitations. The obtained results refer to a heterogeneous cell culture as the object of research.

Conclusion. During the experimental work, all the studied concentrations of doxorubicin showed a cytotoxic effect on somatic and spermatogenic cells. The most harmful concentrations of doxorubicin for spermatogenic epithelial cells in vitro were 0.25 and 0.05, which led to the cell death within 24 hours and caused DNA damage after four hours of exposure. The study revealed characteristic features of pathomorphological changes in Sertoli cells. Even at low concentrations, doxorubicin can disrupt the formation of the feeder layer.

Compliance with ethical standards. The study was approved by the Bioethics Commission of the Research Institute of General Prophylaxis and Epidemiology of the Federal Medical and Biological Agency of Russia (Minutes No. 2 dated March 28, 2023).The animals were removed from the experiment in accordance with GOST 33215-2014 and in accordance with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (ETS N 123), Directive 2010/63/EC of the European Parliament and of the Council of 22 September 2010 on the Protection of Animals Used for Scientific Purposes.

Authors’ contribution:
Golubentseva Yu.V. – study concept and design, data collection and processing, statistical analysis, text writing;
Popov V.B. – text writing, editing;
Radilov A.S. – writing and editing.
All co-authors are responsible for approving the final version of the article and ensuring the integrity of all parts of the article.

Conflict of interest. The authors declare that they have no apparent or potential conflicts of interest related to the publication of this article.

Funding. The study had no sponsorship.

Received: 28 апреля 2026 /Revised: 12 мая 2026 / Accepted: / Published: 2026

1. Liu S., Wei B., Wei H., Xu A., Sheng L., Sun X., et al. L-cysteine mitigates busulfan-induced testicular injury through modulation of CBS/H2S axis. Front. Cell Dev. Biol. 2025; 13: 1679330. https://doi.org/10.3389/fcell.2025.1679330 https://elibrary.ru/atvfas

2. Hall A., Mattison D., Singh N., Chatzistamou I., Zhang J., Nagarkatti M., et al. Effect of TCDD exposure in adult female and male mice on the expression of miRNA in the ovaries and testes and associated reproductive functions. Front. Toxicol. 2023; 5: 1268293. https://doi.org/10.3389/ftox.2023.1268293 https://elibrary.ru/edpndv

3. Zakhidov S.T., Paranyushkina L.P., Makhran Khoda Kh.A., El’-Saed K.A.-Kh., Golichenkov V.A. Effect of chemical mutagens on spermatogenesis in mammals. A quantitative estimate. Izvestiya Rossiiskoi akademii nauk. Seriya Biologicheskaya. 1994; (6): 870–9. (in Russian)

4. Tahir A., Ijaz M.U., Naz H., Afsar T., Almajwal A., Amor H., et al. Protective effect of didymin against 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin-induced reproductive toxicity in male rats. Naunyn Schmiedebergs Arch. Pharmacol. 2024; 397(4): 2203–14. https://doi.org/10.1007/s00210-023-02763-4 https://elibrary.ru/pubzzr

5. Martini M., Froment P., Franceschini I., Pillon D., Guibert E., Cahier C., et al. Perinatal exposure to methoxychlor affects reproductive function and sexual behavior in mice. Front. Endocrinol. (Lausanne). 2020; 11: 639. https://doi.org/10.3389/fendo.2020.00639 https://elibrary.ru/pfzisk

6. Kulibin A.Yu., Zakhidov S.T., Marshak T.L., Chelombit’ko O.M. Response of the spermatogenic system to chemical mutagen dipin in SAMP1 senescence-accelerated mice. Biology Bulletin. 2008; 35(3): 233–42. https://doi.org/10.1134/S1062359008030023 https://elibrary.ru/lkwtox

7. Mark M., Jacobs H., Oulad-Abdelghani M., Dennefeld C., Féret B., Vernet N., et al. STRA8-deficient spermatocytes initiate, but fail to complete, meiosis and undergo premature chromosome condensation. J. Cell Sci. 2008; 121(Pt. 19): 3233–42. https://doi.org/10.1242/jcs.035071

8. Ma H.T., Niu C.M., Xia J., Shen X.Y., Xia M.M., Hu Y.Q., et al. Stimulated by retinoic acid gene 8 (Stra8) plays important roles in many stages of spermatogenesis. Asian J. Androl. 2018; 20(5): 479–87. https://doi.org/10.4103/aja.aja_26_18 https://elibrary.ru/ippbyx

9. Skinner R., Mulder R.L., Kremer L.C., Hudson M.M., Constine L.S., Bardi E., et al. Recommendations for gonadotoxicity surveillance in male childhood, adolescent, and young adult cancer survivors: a report from the international late effects of childhood cancer guideline harmonization group in collaboration with the PanCareSurFup consortium. Lancet Oncol. 2017; 18(2): e75–90. https://doi.org/10.1016/s1470-2045(17)30026-8

10. Yang F., Teves S.S., Kemp C.J., Henikoff S. Doxorubicin, DNA torsion, and chromatin dynamics. Biochim. Biophys. Acta. 2014; 1845(1): 84–9. https://doi.org/10.1016/j.bbcan.2013.12.002 https://elibrary.ru/yeahxb

11. Sah S.K, Khatiwada S., Chaudhary D., Jha Ch.B., Bhattacharya S. Doxorubicin induced histomorphometric changes in testes of albino rats. Nepal J. Biotechnol. 2015; 3(1): 10–4. https://doi.org/10.3126/njb.v3i1.14223

12. Savchenkova I.P., Vasileva S.A. Cultivation of boar spermatogonia on Sertoli cells. Cell and Tissue Biology. 2016; 10(3): 242–9. https://doi.org/10.1134/S1990519X16030123 https://elibrary.ru/vkauvl

13. Volkova N.A., Korzhikova S.V., Kotova T.O., Vetokh A.N., Volkova L.A., Zinovieva N.A. Isolation and characterization of rooster (Gallus Gallus) spermatogonia. Sel’skokhozyaistvennaya biologiya. 2016; 51(4): 450–8. https://doi.org/10.15389/agrobiology.2016.4.450rus https://elibrary.ru/wkcfqt (in Russian)

14. Drugbank. Doxorubicin. Available at: https://go.drugbank.com/drugs/DB00997

15. Rahman A., Ganjei A., Neffe J.R. Comparative immunotoxicity of free doxorubicin and doxorubicin encapsulated in cardiolipin liposomes. Cancer Chemother. Pharmacol. 1986; 16(1): 28–34. https://doi.org/10.1007/bf00255282

16. Silva R.C., Britto D.M.C., de Fátima Pereira W., Brito-Melo G.E.A., Machado C.T., Pedreira M.M. Effect of short- and medium-term toxicity of doxorubicin on spermatogenesis in adult Wistar rats. Reprod. Biol. 2018; 18(2): 169–76. https://doi.org/10.1016/j.repbio.2018.03.002

17. Habas K., Anderson D., Brinkworth M.H. Germ cell responses to doxorubicin exposure in vitro. Toxicol. Lett. 2017; 265: 70–6. https://doi.org/10.1016/j.toxlet.2016.11.016

18. Brilhante O., Okada F.K., Sasso-Cerri E., Stumpp T., Miraglia S.M. Late morfofunctional alterations of the Sertoli cell caused by doxorubicin administered to prepubertal rats. Reprod. Biol. Endocrinol. 2012; 10: 79. https://elibrary.ru/mragtd

19. Hoeijmakers J.H.J. DNA repair mechanisms. Maturitas. 2001; 38(1): 17–22. https://doi.org/10.1016/S0378-5122(00)00188-2

20. Litvinov S.V. Main repair pathways of double-strand breaks in the genomic DNA and interactions between them. Cytol. Genet. 2014; 48(3): 189–202. https://doi.org/10.3103/S0095452714030062

21. Bächler S. Detection of γH2AX foci in animal tissue. Report; 2012. Available at: https://static1.squarespace.com/static/559921a3e4b02c1d7480f8f4/t/598062f09f745638bf643db5/1501586162696/Baechler.pdf

22. Hamer G., Roepers-Gajadien H.L., van Duyn-Goedhart A., Gademan I.S., Kal H.B., van Buul P.P., et al. DNA double-strand breaks and ɤH2AX signaling in the testis. Biol. Reprod. 2003; 68(2): 628–34. https://doi.org/10.1095/biolreprod.102.008672

23. Iksanova A.G., Bondar’ O.V., Balakin K.V. Methods of Cytotoxicity Research in Drug Screening [Metody issledovaniya tsitotoksichnosti pri skrininge lekarstvennykh preparatov]. Kazan’; 2016 (in Russian)