Effects of Methandienone on the ovaries of rabbits in relation to the aromatase gene (CYP19A1) and oxidative markers and the possible protective role of selenium

Introduction

Anabolic-androgenic steroids (AAS), such as methandienone, are synthetic derivatives of testosterone which are widely used to enhance physical performance and muscle growth (Vazhat et al., 2024). AAS have potent anabolic properties and utilized to treat senile osteoporosis and various kinds of anemia, as well as their masculinizing effects (Barbonetti et al., 2020), prolonged or excessive use is associated with a number of adverse psychological and biochemical reactions, including cardiovascular dysfunction, hepatotoxicity, endocrine imbalances and reproductive disorders (Albano et al., 2021). The female reproductive system, especially the ovaries, is particularly sensitive to hormonal disturbances and oxidative damage as a result of exposure to exogenous steroids (Matyja et al., 2023).

Within the ovary, aromatase, which is encoded by CYP19A1, is the key enzyme in steroidogenesis, which catalyzes the conversion of androgens to oestrogens by removing Carbon-19 atom from testosterone. This aromatic process is essential for follicular development, ovulation and maintenance of normal ovarian circulation (Magnani et al., 2017). Alterations in the expression of CYP19A1 gene or in its enzyme activity may alter the physiological balance of accumulation of androgens and reduce oestrogens synthesis, potentially leading to affects follicular development and oocyte maturation and infertility (El Fouikar et al., 2024). Elevated androgen levels are also strongly associated with follicular cysts in animals (Kalhori et al., 2018) and polycystic ovarian syndrome (PCOS), a hormonal disorder characterized by masculinity, anovulation, menstrual irregularities and multiple ovarian cysts, which often leads to infertility and widespread metabolic disorders (Morales-Ledesma et al., 2017).

The AAS methandienone has been shown to induce oxidative stress by inducing over production of reactive oxygen species (ROS) and reducing the activity of antioxidant enzymes, mitochondrial dysfunctions (Abed, 2020) and stimulating pro-inflammatory cytokines (TNF-α, IL-6) (Petrovic et al., 2022). This oxidative imbalance is a key pathogenic pathway mediating cytotoxicity and multiple organ apoptotic effects of anabolic-androgenic steroids (Abed, 2020). In ovarian tissue, increased levels of ROS impair mitochondrial integrity, increase lipid peroxidation and induce apoptosis of granulocytes, thereby reducing both follicular development and steroidogenic synthesis (Dutta et al., 2024).

A compound which plays a central role in maintaining the balance of redox and preserving mitochondrial integrity in the body is selenium (Se), a basic micronutrient a basic micronutrient and a critical component of several selenoproteins such as glutathione peroxidase (GPx) and thioredoxin reductase (ThiR) and Selenoprotein P (SelP). Experimental data suggest that selenium supplementation reduces oxidative damage by activating the Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, regulates steroidogenic enzyme expression and improves mammalian reproduction (Rabah et al., 2023). Selenium is also essential in a number of biological processes, including electron transport, regulates immune cell proliferation, hydroxylation and oxidative metabolism of aromatic compounds in the metabolism of animals (Stolwijk et al., 2020).

Previous studies have shown that methandienone may modify gene expression and oxidative status in brain tissues (Abed and Al-Azawi, 2020), with a focus on potential systemic effects. However, the effect on ovarian function and on reproductive tissues is to date largely unknown. Understanding these effects is essential, given the role of CYP19A1 in steroidogenesis and the potential for ovarian dysfunction due to oxidative stress so that the aim of this study was to identify these effects and to try to reduce them by supplementing selenium, which compensates for these disturbances by strengthening antioxidant protection and promoting normal steroidogenic function. Exploring these interactions in the rabbit model may provide valuable insights into the contribution of anabolic and androgenic steroids to ovarian dysfunction and may inform strategies for reducing their reproductive adverse effect.

Materials and methods

In this study, we used twenty young adult female rabbits, aged between 8 and 14 weeks, weighing roughly 850 to 1,200 grams. All the rabbits had the same breed and genetic background and had been taken from Baghdad Cancer Research Center. The animals were matched in age and weight and randomly assigned to the experimental groups. The study was conducted by randomised, controlled, post-test design, and a control group was included for comparison. These rabbits were kept in the Physiology Laboratory at the Veterinary Medicine College in Fallujah University, randomly divided them into four groups, with five rabbits in each. for the T1 group gave a dose of methandienone (Black dragon pharma, Thailand) orally, which was 0.35 mg/kg body weight Selenium yeast supplied by (21st Century^® (USA)) and administered to T2 group at a dose of 3 μg/kg body weight (Abed and Al-Azawi, 2020). Methandienone and selenium were administered orally (T3) to a combined group of rabbits (0.35 mg per kg bodyweight, 3 μg per kg bodyweight). All animals received oral administration every day for 30 days. After 30 days of the experiment, blood samples were taken from each rabbit by cardiac puncture for estrogen evaluation, and at the end of the study, Animal sacrifice was carried out in accordance with approved ethical standards. and ovary were isolated for gene expression evaluation. All animal experimental procedures, including anesthesia administration and blood sampling, were performed in accordance with American Veterinary Medical Association (AVMA) guidelines (Underwood and Raymond, 2013). The estradiol test is a one-step immunoassay used to evaluate the value estradiol in serum of mammals using a universal assay protocol called chemiflex (Fu et al., 2020).

Malondialdehyde (MDA) assay was determined by colorimetric method (Yalçin et al., 2010), using malondialdehyde colorimetric assay kit (Elabscience china) and assay, protein carbonyl content was determined by colorimetric method (Reznick and Packer, 1994) using protein carbonyl colorimetric assay kit (Elabscience, china).

The total RNA was extracted with a magnatronic nanosorbent kit as specified by the manufacturer (RealBest Extraction 100, Russia). The RNA integrity and purity were verified by nanodrop spectrophotometry. We used a reverse transcriptase (M-MLV) template from the Synthol (Russia) and appropriate primers to reverse transcript the RNA to cDNA for a real-time PCR (RT). Table 1 shows the primer sequences purchased all from Bioneer (Korea). We examined the expression of these genes (the genes of interest) using GAPDH as a control gene. Start ExicyclerTM 96 real-time PCR-based thermal block device and load as follows: The PCR method was used for one hour of reverse transcriptase at 50°C, five minutes at 95°C, and 47 cycles at 96°C, 46 seconds at 62°C, all repeated 45 times. Delta-delta CT has previously been reported to be used to assess the relative amount of expression of a specific gene (Livak and Schmittgen, 2001). Statistical analysis was performed with version 21.0 of SPSS software. Data were expressed as mean ± standard error of the mean (SE) and compared to groups using (One-way) analysis of variance (ANOVA) with a P ≤ 0.05 (Larsen et al., 1973) and then tested using by Tukey's HDS test. All experimental procedures have been carried out in accordance with institutional guidelines on the care and use of laboratory animals.

Results

Discussion

This study showed that administration of methandienone resulted in a marked decrease in mRNA expression of CYP19A1 in ovarian tissues (Table 2) and a concurrent decrease in serum oestrogen concentrations in treated rabbits (T1 group) compared to control groups. These findings may indicate that methandienone has a suppression effect on aromatase activity, resulting in a decrease in the conversion of androgens to oestrogens. The inhibition of aromatase expression observed in this study is consistent with previous studies suggesting that AAS exposure influences the hypothalamic-pituitary-gonadal (HPG) axis and ovarian steroidogenesis through the suppression of CYP19A1 transcription (Barros-Oliveira et al., 2021; El Fouikar et al., 2024).

Notably, supplementation with selenium (T2 group) significantly restored CYP19A1 expression and serum oestrogen concentrations compared with the methandienone-treated group. This improvement highlights the modulatory role of selenium in maintaining steroidogenic enzyme function and its influence on Nrf2, a key regulator of cellular antioxidant protection and a key driver of oxidative stress reduction. Its activation helps to maintain the redox balance and mitochondrial integrity, which is of particular importance to the understanding of the potential effects of selenium in this study (Abed, 2020) and its ability to counter the oxidative stress induced by AAS. Selenium is known to increase the activity of selenoproteins, including glutathione peroxidase and thioredoxin reductase, thereby reducing the accumulation of reactive oxygen species (ROS) and protecting the cellular macromolecules from oxidative damage (Zhang et al., 2023). Thus, the observed upregulation of CYP19A1 following selenium supplementation may reflect the preservation of mitochondrial, endoplasmic reticulum function and transcription of antioxidant enzymes, both of which are essential for steroid hormone synthesis. These findings are consistent with Rabah et al., (2023), who demonstrated that selenium supplementation enhances ovarian function and steroidogenesis under conditions of oxidative stress.

The significant increase in oxidative markers, including malondialdehyde (MDA) and protein carbonyl (PC) levels in methandienone (T1) (Table 3) further highlights the role of oxidative stress in ovarian dysfunction. Increased lipid peroxidation and protein oxidation, associated with decreased antioxidant protection, and when oestrogen levels fall in this group, loss of oestrogenic antioxidant protection (Ruiz-Romero et al., 2025), reflect a disruption in the balance between reactive species generation and the body’s scavenging capacity. These oxidative insults may result in apoptosis of granulosa cells, mitochondrial dysfunction and reduced follicular viability (Daraghmeh et al., 2025). Furthermore, testosterone and its derivatives are considered to be pro-oxidants which may have adverse effects on primary biomolecules (Andrés Juan et al., 2021). Since testosterone increases metabolic activity to promote protein synthesis, it may increase mitochondrial respiration and ATP production, thereby increasing the production of reactive oxygen species (ROS) and contributing to oxidative stress (Stallone and Oloyo, 2023).

Partial normalization of these oxidative markers in the group of selenium-protected (T3) supports the hypothesis that selenium provides cytoprotection by stabilizing the redox homeostasis and increasing the activity of antioxidant enzymes. Although the oxidative indices in the protection group were still higher than in the controls, their decrease compared to T1 indicates that oxidative damage caused by AAS is attenuated rather than reversed.

Conclussion

These findings together highlight a dual pathogenic mechanism of methandienone toxicity: (1) direct endocrine disruption through aromatase inhibition and CYP19A1 downregulation; and (2) indirect oxidative damage by oxidative over production and oxidative depletion of antioxidant defences. The ability of selenium to inhibit both pathways reflects its integral role in maintaining cellular homeostasis, modulating redox signaling and stabilizing steroidogenic gene transcription.

Ethical considerations

This study was conducted in full compliance with the ethical guidelines approved by the Scientific Committee on Research Ethics of the University of Fallujah Faculty of Veterinary Medicine (Approval No: 7; 13/10 2025) (Shahooth et al., 2025).