Assessment of DNA damage according to the data of γН2АХ immunofluorescence analysis in the spermatogenic epithelium cells of DBA/2 and C57Bl/6 mice under conditions of isolated exposure to doxorubicin in vivo and after preconditioning with β-naphthoflavone

Introduction. Doxorubicin (Dox) is an anthracycline antibiotic whose cytotoxic action arises from its intercalating and alkylating effects upon interaction with DNA, leading to double-strand breaks. β-naphthoflavone (βNF) is a synthetic flavonoid, an agonist of the aryl hydrocarbon receptor (AhR) that promotes the transactivation of cytochromes CYP1A, including CYP1A1 – cytochrome, involved in the metabolism of polyaromatic hydrocarbons (PAHs). The activity of CYP1A1 determines the rate of formation of reactive PAH metabolites and the degree of DNA damage in cells. Dox metabolism is believed to involve multiple enzymes, including CYP3A4 and CYP2D6. Inbred C57Bl/6 and DBA/2 mice are characterized by AhR features that determine the differences in the response of animals of different lines to the toxic effects of PAHs.

The aim of the study was to assess the degree of DNA damage in spermatogenic epithelial cells according to immunofluorescence analysis of phosphorylated histone γН2АХ in male mice of two inbred strains differing in AhR inducibility when exposed to an AhR agonist (βNF) and Dox.

Material and methods. The toxic effects of Dox on the spermatogenic epithelium after a single intraperitoneal injection (i. p.) at a dose of 10 mg/kg were evaluated in male mice of the DBA/2 and C57Bl/6 strains. Half of the experimental animals of each strain received βNF (50 mg/kg, i. p.) for 4 consecutive days before treatment with Dox. Assessment of DNA damage in testicular cells was performed based on the results of immunofluorescence analysis for the γН2АХ biomarker.

Results. According to the results of qualitative and quantitative assessment of DNA damage in the cells of the spermatogenic epithelium, based on the immunofluorescence analysis of γН2АХ biomarker, differences in the response of the two murine strains used to the effects of Dox were shown. Preconditioning of animals using βNF contributes to a statistically significant increase in the proportion of cells with double DNA breaks in the testes of males of the C57Bl/6 strain, and does not affect the increase in the proportion of such cells in males of DBA/2.

Limitations. The study was performed using immunofluorescence analysis for the purpose of qualitative and quantitative assessment of DNA damage in the cells of the spermatogenic epithelium after exposure to toxic agents in experimental mice in vivo and does not allow taking into account all possible effects of the substances studied on the processes occurring in the mouse testes.

Conclusion. The results of the study suggest that there is dependence between preconditioning of animals with βNF and an increase in the ratio of cells with DNA damage in the testes of C57Bl/6 mice. No such relationship was found for the DBA/2 mice. Due to the multiplicity of βNF influence on the activity of the xenobiotic metabolism enzymes, the revealed differences in the response of two different strains to Dox exposure after pre-treatment with βNF can be explained not only by the reduced inducibility of AhR in DBA/2 mice, but also by non-obvious effects associated with the effects of βNF on other protein targets in genetically different animals.

Compliance with ethical standards. A positive conclusion was received from the Commission on Biomedical Ethics of the Scientific Research Institute of Hygiene, Occupational Pathology and Human Ecology of the FMBA of Russia. (Protocol No. 2 dated December 16, 2022).

Authors’ contribution:
Golubentseva Yu.V. – collecting material, statistical analysis, text writing;
Popov V.B. – research concept and design, text writing, editing;
Beltyukov P.P. – data analysis and interpretation, text 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 interests. The authors declare that there is no conflict of interests.

Funding. The study had no sponsorship.

Received: August 21, 2025 / Revised: September 16, 2025 / Accepted: October 2, 2025 / Published: November 19, 2025

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