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. 2024 Aug 26;28(3):464-470.
doi: 10.5935/1518-0557.20240041.

The protective effect of Ozone on the mice testicular damage induced by methotrexate

Layasadat Khorsandi  1   2 , Negar Varaa  3 , Reza Dadfar  4 , Sadegh Moradi Vastegani  5 , Yousef Asadi-Fard  6 , Akram Ahangarpour  5 , Amirhesam Keshavarz-Zarjani  2   7
Affiliations

The protective effect of Ozone on the mice testicular damage induced by methotrexate

Layasadat Khorsandi et al. JBRA Assist Reprod. .

Abstract

Objective: Methotrexate (MTX) is widely administered for the treatment of various cancers. However, MTX induces male reproductive toxicity. In the current study, the effect of ozone therapy (OT) on reducing the toxic effects of MTX in the mouse testicles has been investigated.

Methods: Twenty-four mice were divided into four groups: control, OT (4 mg/kg ozone), MTX (20 mg/kg), and MTX + OT. Testosterone levels, histological changes, and oxidative stress biomarkers were assessed to evaluate the protective effects of OT.

Results: The results demonstrated that MTX disrupted germinal epithelium, reduced serum testosterone levels, and enhanced oxidative stress in testicular tissue. However, treatment with OT attenuated these adverse effects. OT effectively restored the levels of antioxidant enzymes, such as catalase (CAT), glutathione (GSH), and superoxide dismutase (SOD). OT reduced lipid peroxidation, as indicated by decreased malondialdehyde (MDA) levels. OT preserved normal spermatogenesis, improved morphometric parameters, and reduced histological changes by MTX. Moreover, OT effectively restored testosterone levels.

Conclusions: OT protects against MTX-induced testicular damage by suppressing oxidative stress.

Keywords: chemotherapy; methotrexate; oxidative stress; ozone; testis.

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Figures

Figure 1
Figure 1
Testosterone levels in the different groups (Mean ± SD; n = 7). * & # p<0.05, ** p<0.001. The asterisk and # symbols indicate a comparison to the control and MTX-intoxicated groups, respectively.
Figure 2
Figure 2
Light microscopy of testicular tissue from the control and experimental groups (Arrows show vacuoles in the germinal epithelium); H&E staining; Magnifications: ×250.
Figure 3
Figure 3
Testosterone levels in the different groups (Mean ± SD; n = 7). * & # p<0.05, ** p<0.001. The asterisk and # symbols indicate a comparison to the control and MTX-intoxicated groups, respectively.
Figure 4
Figure 4
Johnsen scored assessments in the different groups. Values expressed as Mean ± SD for six mice. * & # p<0.05, ** p<0.001. The asterisk and # symbols indicate a comparison to the control and MTX-intoxicated groups, respectively.
Figure 5
Figure 5
Seminiferous tubule diameter (STD) and seminiferous epithelium height (SEH) in the different groups. Values expressed as Mean ± SD for six mice. *& # p<0.05, **& ## p<0.01, ***p<0.001. The asterisk and # symbols indicate a comparison to the control and MTX-intoxicated groups, respectively.
Figure 6
Figure 6
Oxidative stress biomarkers in different groups (Mean ± SD; n = 7). *&#p<0.05, **&##p<0.01, ***p<0.001. The asterisk and # symbols indicate a comparison to the control and MTX-intoxicated groups, respectively.
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