Potential effect of cerium oxide nanoparticles against boldenone-induced oxidative stress, inflammation, and reproductive dysfunction in male albino rats.

Potential effect of cerium oxide nanoparticles against boldenone-induced oxidative stress, inflammation, and reproductive dysfunction in male albino rats - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Search Log in Primary site navigation Search Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Sci Rep . 2026 Jun 3;16:17156. doi: 10.1038/s41598-026-55900-3 Potential effect of cerium oxide nanoparticles against boldenone-induced oxidative stress, inflammation, and reproductive dysfunction in male albino rats Hanaa M Hassan 1 , Honyda S Mohammed 2 , Nuha Anajirih 3 , Safaa S Soliman 2 , Fatma S Mohammed 4 , Ahmed A Aldarmahi 5, 6 , Ahmed Abdeen 7, ✉ , Shrouk Amer 7 , Eman E ELwakeel 8, 9 , Heba M Abdelkareem 10, 11 , Ahmad F Rawan 12, ✉ , Mustafa Shukry 13, ✉ , Samah F Ibrahim 14 , Majed Aldehri 15 , Areej M Alshehri 16 , Mostafa F Abdel-Rahman 2 Author information Article notes Copyright and License information 1 Department of Agricultural Chemistry, Faculty of Agriculture, Minia University, Minia, 61519 Egypt 2 Department of Zoology, Faculty of Science, Minia University, Minia, 61519 Egypt 3 Department of Emergency Medical Services, Faculty of Health Sciences, Umm Al-Qura University, Al-Qunfudhah, Makkah Saudi Arabia 4 Biochemistry Program, Department of Chemistry, Faculty of Science, Minia University, Minia, 61519 Egypt 5 Department of Basic Science, College of Science and Health Professions, King Saud Bin Abdulaziz University for Health Sciences, 21582 Jeddah, Saudi Arabia 6 King Abdullah International Medical Research Centre, National Guard-Health Affairs, 21582 Jeddah, Saudi Arabia 7 Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Benha University, Toukh, 13736 Egypt 8 Department of Anatomy and Embryology, Faculty of Medicine, Benha University, Benha, 13518 Egypt 9 Department of Basic Science, Faculty of Nursing, Riyadh Elm University, Riyadh, Saudi Arabia 10 Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Benha University, Benha, Egypt 11 Department of Medical Biochemistry, Molecular Biology and Physiology, Faculty of Medicine, Mutah University, Mutah, 61710 Jordan 12 Pre-Clinic Department, Faculty of Veterinary Science, Nangarhar University, Jalalabad, 2603 Nangarhar Afghanistan 13 Department of Biomedical Sciences, College of Veterinary Medicine, King Faisal University, P.O. Box 400, 31982 Al-Ahsa, Saudi Arabia 14 Department of Internal Medicine, College of Medicine, Princess Nourah bint Abdulrahman University, P.O. Box 84428, 11671 Riyadh, Saudi Arabia 15 Department of Anatomy, College of Medicine, King Khalid University, 62529 Abha, Saudi Arabia 16 Department of Clinical Anatomy, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia ✉ Corresponding author. Received 2025 Nov 12; Accepted 2026 May 27; Collection date 2026. © The Author(s) 2026 Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ . PMC Copyright notice PMCID: PMC13234341  PMID: 42236878 Abstract Anabolic–androgenic steroids such as boldenone (BOL) are used in veterinary medicine to improve growth, food conversion in food-producing animals, and recently utilized by bodybuilders to increase muscle mass and enhance physical performance. Additionally, they have serious adverse effects on various organs, mostly the testicular tissues. In this context, the purpose of this study was to assess BOL-induced testicular damage and the potential protective benefits of cerium oxide nanoparticles (CeNP). Twenty-four adult male rats were randomly allocated into 4 equal groups: Control, BOL (5 mg/kg), CeNP (100 μg/kg), and a combination of BOL and CeNP-treated groups. All of the treatments were administered intraperitoneally to the rats, twice a week for 8 consecutive weeks. Hematological parameters, oxidative stress markers, pro-inflammatory cytokines, and hormonal profiles were measured using standard biochemical and immunoassay techniques, as well as histopathological changes in the testicular tissue. The results showed that BOL markedly increased testicular oxidative damage as demonstrated by a drastic elevation of lipid peroxidation marker (MDA), NO levels, with a notable decline in GSH concentration, as well as SOD and CAT enzyme activity. Additionally, inflammation has also been elicited, evidenced by elevated levels of testicular proinflammatory cytokines, including TNF-α and IL-6, along with a marked decrease in serum levels of LH, FSH, and testosterone. Interestingly, CeNP diminished the detrimental effects of BOL on testicular tissue by reducing lipid peroxidation, inflammation, and enhancing antioxidant activity. The present findings indicated that CeNP may serve as a promising protective agent against BOL-induced reproductive damage in male rats. Keywords: Boldenone, Cerium oxide nanoparticles, Reproductive toxicity, Oxidative stress, Inflammatory cytokines Subject terms: Biochemistry, Medical research, Physiology, Zoology Introduction Boldenone (BOL; 1,4-androstadiene-17b-ol-3-one) is a well-known anabolic–androgenic steroid (AAS) synthesized through the dehydrogenation of testosterone. It is predominantly produced for veterinary usage, notably for horses, to improve feed conversion efficiency and promote growth in fattening animals 1 . Despite being prohibited for human consumption, BOL remains illegally accessible and is widely abused by bodybuilders, athletes, and non-athletes for fitness 2 – 5 . Its anabolic effects are mediated through stimulation of protein synthesis, decreasing protein breakdown, and increasing water retention, nitrogen, and electrolytes, which consequently improve the positive nitrogen balance 6 , 7 . However, its performance-enhancing properties, accumulating evidence highlights the serious health hazards associated with BOL exposure. The International Agency for Research on Cancer (IARC) has classified BOL as a potential human carcinogen, having a higher carcinogenic potential compared with other anabolic steroids 8 . In addition, BOL misuse has been implicated in a variety of pathological conditions, including liver impairment 9 , cardiovascular abnormalities 1 , renal damage 10 , and testicular disorders 11 . Humans may be negatively impacted by BOL directly through muscle injections or indirectly through the consumption of meat from animals treated with BOL 11 . Experimental investigations have demonstrated that oxidative stress is a key mechanism underlying BOL-induced toxicity. BOL exposure has been shown to increase the generation of reactive oxygen species (ROS) and promote lipid peroxidation, leading to disruption of endogenous antioxidant defense systems in several organs, altering the antioxidant defense systems of various tissue organs in rat models 11 , 12 . In recent years, advances in nanotechnology have introduced innovative therapeutic strategies for the diagnosis and treatment of numerous health issues 13 . Notably, nanoparticles exhibit distinctive physicochemical properties that enable them to mitigate cellular damage induced by free radicals 14 . Among these nanomaterials, cerium oxide nanoparticles (CeNP) have garnered a lot of interest for use in biological applications due to their remarkable redox-based antioxidant activity 13 , 15 , 16 . CeNP exert their protective effects by scavenging ROS, enhancing cellular antioxidant defenses, and mimicking the enzymatic activities of superoxide dismutase (SOD) and catalase (CAT) through reversible switching between reduced (Ce +3 ) and oxidized (Ce +4 ) states, depending on their double redox status 17 . Additionally, CeNP has been reported to improve reproductive function by increasing serum luteinizing hormone (LH) and testosterone levels, as well as enhancing sperm quality and quantity, largely attributed to their ROS-scavenging capability 18 . Despite these promising biological properties, the potential chemoprotective effects of CeNP against anabolic–androgenic steroid-induced testicular damage, particularly that caused by BOL, remain largely unexplored. Thus, the current study intends to investigate the protective effect of CeNP against BOL-induced testicular toxicity in a rat model. The study evaluates hematological parameters, oxidative stress biomarkers, hormonal profiles, inflammatory responses, and histopathological alterations in testicular tissue to elucidate the potential role of CeNP in mitigating BOL-induced testicular toxicity. Materials and methods Molecular docking assessment The three-dimensional structures of rats’ superoxide dismutase-1 (SOD1; P07632 ), SOD2 ( P07895 ), SOD3 ( Q08420 ), and catalase (CAT; P04762 ) were sourced from the UniProt ( https://www.uniprot.org/ ) database. The boldenone (13308) three-dimensional structure was retrieved from the PubChem ( https://pubchem.ncbi.nlm.nih.gov/ ) database. Ligand and target proteins were prepared for the docking process using the Chimera 1.18 software, while molecular docking was done by AutoDock Vina software executed in Chimera 1.18. Finally, the molecular interactions of boldenone and target proteins were visualized using the Discovery Studio 2016 Client software. Chemicals The commercial form of BOL, Equigan® vial (Laboratorios Tornel Co., Mexico), was acquired. Each vial contains an oily solution (50 mg BOL/mL vehicle). Ce oxide (CeO 2 ) nanoparticles (CeNP) were acquired from (Sigma-Aldrich in St. Louis, USA). Characterization of Ce oxide nanoparticles The structural properties of the synthesized CeO 2 nanoparticles were investigated using X-ray diffraction (XRD) analysis performed on a Rigaku MiniFlex 600 diffractometer with Cu Kα radiation (λ = 1.5406 Å). Fourier transform infrared spectroscopy (FTIR) measurements were carried out using an Alpha II Bruker FTIR spectrometer to identify the functional groups and Ce–O bonding vibrations. The thermal stability and decomposition behavior were evaluated using a NEXTA thermal analyzer through thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and differential thermal analysis (DTA). The morphology and surface characteristics of the nanoparticles were examined using a JEOL JCM-7000 scanning electron microscope (SEM). Animals and experimental design Twenty-four adult male albino rats (150–165 g, 8-weeks-old) were acquired from the Experimental Animal Care Center, Faculty of Medicine, Minia University, Egypt. Two weeks before the trial’s commencement, the rats were housed in a well-controlled environment with a temperature of 25 °C, a light/dark cycle of 12:12, and a humidity of 60 ± 5%, fed a standard pellet diet, and allowed free access to water. Following acclimation, all rats were sorted into four equal groups (6 rats each); Control group, rats were given saline intraperitoneally; BOL group, animals received BOL intraperitoneally, at a dose of 5 mg/kg b.wt, twice a week 19 , CeNP group, rats were treated with CeNP, given intraperitoneally (100 μg/kg b.wt, twice a week) 20 , and the BOL + CeNP group, rats were treated with the abovementioned dose of BOL and CeNP. All rats received treatment for 8 weeks, and within 24 h of the final treatment, Pentobarbital (100 mg/kg i.p.) was used to anesthetize each group; thereby, two separate blood samples were gathered from the inferior vena cava. The first blood sample was collected in heparinized tubes for hemogram evaluation. The second blood sample was allowed to coagulate at room temperature before being centrifuged at 2000 g for 15 min. Serums were then gathered and stored at − 20 °C for further biochemical examinations. Moreover, each rat’s testicles were taken after scarification and perfused with ice-cold phosphate buffer saline (PBS). One testicle was collected and processed for oxidative stress biomarkers and proinflammatory cytokines evaluation. The other testicle was preserved in a 10% buffered formalin for further histological assessments. All animals were humanely euthanized at the study endpoint by cervical dislocation, in accordance with the approved protocol. Hematological examination The hematological examination, including (RBCs and WBCs count, Hb concentration, PCV %) were estimated using an automatic cell counter (H.A-Vet Clindiage, Belgium) according to Thrall et al. 21 . Tissue oxidative and antioxidant biomarker assays Testicular tissue levels of oxidative stress and antioxidant biomarkers, including malondialdehyde (MDA), nitric oxide (NO), reduced glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT), were determined using commercially available diagnostic kits (Laboratory Bio-diagnostic Co., Giza, Egypt) according to the manufacturer’s instructions. MDA was measured using the thiobarbituric acid reactive substances (TBARS) method (Cat. No. MD2529), based on the reaction between MDA and thiobarbituric acid under acidic conditions to form a pink chromogen, which was quantified spectrophotometrically at 534 nm. Results were expressed as nmol/g tissue. NO levels were determined indirectly by measuring nitrite, a stable end product of nitric oxide metabolism, using Griess reagent (Cat. No. NO2533). The resulting azo dye was measured at 540 nm, and the results were expressed as µmol/g tissue. GSH content was assessed using the DTNB method (Cat. No. GR2511), where GSH reacts with 5,5′-dithiobis-(2-nitrobenzoic acid) to form a yellow-colored chromophore measured at 412 nm. Results were expressed as µmol/g tissue. SOD activity was determined based on its ability to inhibit superoxide-mediated reactions (Cat. No. SD2521), and results were expressed as U/g tissue. CAT activity was measured by monitoring the decomposition rate of hydrogen peroxide (H 2 O 2 ) at 240 nm (Cat. No. CA2517), and results were expressed as U/g tissue. Proinflammatory cytokine and hormonal assays Levels of proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), were measured in testicular tissue homogenates using commercial ELISA kits (Elabscience Biotechnology Co., Wuhan, China; Cat. No. E-EL-R0019 for TNF-α and E-EL-R0015 for IL-6) in accordance with the manufacturer’s instructions. Cytokine levels were expressed as pg/mg protein. In addition, serum levels of reproductive hormones, including luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone, were determined using ELISA kits (Cusabio Biotech Co., Wuhan, China; Cat. No. CSB-E12654r for LH, CSB-E06869r for FSH, and CSB-E05100r for testosterone) following the manufacturer’s protocol. Hormonal levels were expressed as mIU/mL for LH and FSH, and ng/mL for testosterone. All ELISA assays were based on the sandwich ELISA principle, in which the target analyte binds to a specific capture antibody pre-coated onto the microplate wells. A biotinylated detection antibody is subsequently added, followed by enzyme conjugate and chromogenic substrate, producing a colorimetric reaction proportional to the analyte concentration, which was measured at 450 nm. All samples were analyzed in duplicate to ensure accuracy and reproducibility. Histological investigation Testicular tissues from all experimental groups were preserved in 10% phosphate buffered formalin solution for 24 h. Subsequently, tissues were thoroughly washed under running tap water for 10 min, then dehydrated in ascending grades of ethyl alcohol. The specimens were cut into 5-μm paraffin slices using a rotary microtome, after being cleaned in xylene and fixed in paraffin. For histological examination, serial sections that had been de-waxed were stained with hematoxylin and eosin 22 . The stained sections were examined under a light microscope (Olympus CX 31 microscope, Tokyo, Japan), and photomicrographs were captured using a digital camera system. For the evaluation of histopathological alterations, the severity of lesions was scored as follows: (−) absence of the lesion = 0%, (+) mild > 0–25%, (++) moderate > 25–50%, and (+++) severe > 50–100% of the examined tissue sections 23 . Statistical analyses The variables’ normality was tested whether they were normally distributed at P > 0.05 using Shapiro–Wilk’s test. Data are presented as box-and-whisker plots with individual data points. Homogeneity of variances among groups was evaluated using Levene’s test. One-way ANOVA was used to statistically compare the various groups using GraphPad Prism software, version 10, USA. To compare groups, Duncan’s test was used as a post hoc test. P values below 0.05 were considered to be statistically significant. Multivariate analyses, including principal component analysis (PCA), correlation heatmap, clustering analysis, and variable importance in projection (VIP), were conducted using RStudio software under R version 4.0.2 (specifically version 2025.05.1) on Windows. Results Molecular docking The data in Table 1 and Fig. 1 explored the molecular docking interactions of BOL with rats’ SOD1, SOD2, SOD3, and CAT. BOL interacted with the binding site of SOD1, achieving a binding free energy of − 7.40 kcal/mol through hydrogen bonds (ASN66 and ARG144 residues) and hydrophobic interaction (PRO63 and LYS137 residues) (Fig. 1 A). It is also bound with LEU62, LEU88, LYS89, and PRO169 residues (hydrophobic interaction) in the SOD2 binding site with a binding free energy of − 8.60 kcal/mol (Fig. 1 B). By binding free energy of − 7.30 kcal/mol, BOL bound with PRO137 (hydrophobic interaction) and ASN140 (hydrogen bond) residues in the binding site of SOD3 (Fig. 1 C). Furthermore, it is bound to CAT binding sites with a hydrogen bond (ARG263 residue) and hydrophobic interaction (ALA8, LEU262, ARG263, PHE266, PRO322, and TYR325 residues) by a binding free energy of − 10.00 kcal/mol (Fig. 1 D). Table 1. Molecular docking interactions of BOL with rats’ SOD1, SOD2, SOD3, and CAT. Targets Residues Hydrogen bond Charge Hydrophobic interaction Halogen SOD1 PRO63 0 0 1 0 ASN66 1 0 0 0 LYS137 0 0 1 0 ARG144 1 0 0 0 SOD2 LEU62 0 0 1 0 LEU88 0 0 1 0 LYS89 0 0 1 0 PRO169 0 0 1 0 SOD3 PRO137 0 0 1 0 ASN140 1 0 0 0 CAT ALA8 0 0 1 0 LEU262 0 0 1 0 ARG263 1 0 1 0 PHE266 0 0 1 0 PRO322 0 0 1 0 TYR325 0 0 1 0 Open in a new tab Fig. 1. Open in a new tab Molecular docking interaction of BOL with rats SOD1 ( A ), SOD2 ( B ), SOD3 ( C ), and CAT ( D ). BOL boldenone, CAT catalase, SOD superoxide dismutase. Characterization of CeNP Figure 2 presents the comprehensive physicochemical characterization of bare CeNP using XRD, FTIR, thermal analysis (TGA/DSC/DTA), and SEM techniques. The XRD pattern shown in Fig. 2 A confirms the crystalline nature of CeO 2 with characteristic diffraction peaks centered approximately at 2θ = 28.5°, 33.1°, 47.5°, 56.3°, 59.1°, and 69.4°, corresponding to the (111), (200), (220), (311), (222), and (400) crystal planes of fluorite-type cubic CeO 2 , respectively. The sharp diffraction peaks and the absence of secondary impurity phases indicate the successful formation of highly crystalline CeNP with phase purity. The dominant (111) reflection suggests preferential crystal orientation and agrees well with the standard fluorite cubic structure of ceria (JCPDS No. 34-0394) 24 . The relatively broadened diffraction peaks also imply the nanoscale crystallite dimensions of the prepared particles. Fig. 2. Open in a new tab Physicochemical characterization of bare CeO 2 nanoparticles: ( A ) X-ray diffraction (XRD) pattern confirming the crystalline fluorite cubic structure of CeO 2 , ( B ) Fourier-transform infrared (FTIR) spectrum showing characteristic Ce–O stretching ( C ) thermogravimetric analysis (TGA) profile illustrating thermal stability and weight loss behavior, ( D ) differential scanning calorimetry (DSC) curve, ( e ) differential thermal analysis (DTA) curve, and ( F ) scanning electron microscopy (SEM) micrographs revealing the agglomerated nanoscale morphology of CeO 2 nanoparticles at × 4000, and × 7000. The FTIR spectrum in Fig. 2 B further verifies the chemical structure of CeNP. A broad absorption band observed around 3400 cm −1 is attributed to the stretching vibration of surface hydroxyl groups and adsorbed water molecules 25 . The weak band near 1630 cm −1 corresponds to the bending vibration of molecular water 26 . More importantly, the strong absorption bands located below 700 cm −1 , particularly around 500–600 cm −1 27 , are assigned to the Ce–O stretching vibration 28 , confirming the formation of cerium oxide lattice bonds. The presence of surface hydroxyl groups may enhance the redox activity and surface reactivity of CeNP through the Ce 3+ /Ce 4+ transition mechanism. Moreover, the thermal behavior of CeNP was evaluated by TGA, DSC, and DTA analyses, as displayed in Fig. 2 C–E. The TGA profile reveals a very small overall weight loss (~ 6–7%) up to 1000 °C, indicating the excellent thermal stability of the synthesized CeNP. The initial weight loss below approximately 200 °C can be attributed to the evaporation of physically adsorbed moisture and residual volatile species. The gradual weight reduction at higher temperatures may arise from the removal of surface hydroxyl groups and decomposition of trace residual organic species. The DSC and DTA curves exhibit broad thermal events without sharp decomposition peaks, further confirming the structural stability and absence of significant phase transformation within the investigated temperature range. The broad endothermic behavior observed at lower temperatures is associated with dehydration and desorption processes, whereas the relatively stable thermal response at elevated temperatures reflects the high thermal resistance of CeNP, which is an important feature for biomedical and antioxidant applications under physiological and oxidative environments. Furthermore, the SEM micrographs shown in Fig. 2 F demonstrate that the CeNP possess an agglomerated nanostructured morphology composed of irregularly shaped fine particles with porous-like clustered architecture. The observed aggregation behavior is commonly associated with the high surface energy and nanoscale dimensions of CeNP. Despite partial agglomeration, the particles remain within the nanoscale range (~ 28 nm) and provide a relatively high surface area, which can facilitate enhanced surface redox reactions and reactive oxygen species scavenging activity. Such morphological characteristics are advantageous for biological and antioxidant applications because they increase the density of active surface oxygen vacancies and improve the reversible Ce 3+ /Ce 4+ redox cycling capability. Hematological parameters The effects of BOL and/or CeNP treatment on hematological parameters of rats are displayed in Fig. 3 . BOL intoxication significantly increased ( P < 0.05) RBCs count, Hb concentration, and PCV % besides an increase in WBCs count relative to the control group. However, CeNP supplementation notably showed an improvement in all blood parameters. Fig. 3. Open in a new tab Dot-box plot presents the effect of BOL and/or CeNP on hematological profile, including RBC ( A ), Hb ( B ), PCV% ( C ), and WBCs ( D ). Data are presented as box plots, where the box represents the interquartile range (IQR, 25th to 75th percentile), the horizontal line inside the box denotes the median, whiskers extend to the minimum and maximum values within 1.5 × IQR, and individual data points are overlaid as circles to display the full distribution (n = 6; * P < 0.05, ** P < 0.01, *** P < 0.001). BOL boldenone, CeNP cerium oxide nanoparticles, Hb hemoglobin, PCV packed cell volume, RBC red blood cells, WBCs white blood cells. Effect of BOL and/or CeNP on oxidant/antioxidant status The testicular oxidant/antioxidant state was significantly disrupted by BOL insults, as displayed in Fig. 4 . There was a drastic elevation ( P < 0.05) in the levels of MDA and NO, with a notable decline of GSH concentration, as well as enzyme activities of SOD and CAT in the testicular tissues with respect to the controls. Remarkably, when CeNP and BOL were administered together, the antioxidant levels were significantly modulated and kept within the physiological zone ( P < 0.05). Fig. 4. Open in a new tab Dot-box plot presents the effect of BOL and/or CeNP on the oxidative stress biomarkers in testicular tissue, including GSH ( A ), MDA ( B ), NO ( C ), SOD ( D ), and CAT ( E ). Data are presented as box plots, where the box represents the interquartile range (IQR, 25th to 75th percentile), the horizontal line inside the box denotes the median, whiskers extend to the minimum and maximum values within 1.5 × IQR, and individual data points are overlaid as circles to display the full distribution (n = 6; *** P < 0.001). BOL boldenone, CAT catalase, CeNP cerium oxide nanoparticles, GSH reduced-glutathione, MDA malondialdehyde, NO nitric oxide, SOD superoxide dismutase. Effect of BOL and/or CeNP on pro-inflammatory cytokine and hormonal levels As illustrated in Fig. 5 , compared to the control group, BOL triggered an inflammatory reaction evidenced by a significant ( P < 0.05) rise in TNF-α and IL-6 levels in the testicular tissue homogenate. Furthermore, serum levels of LH, FSH, and testosterone were considerably ( P < 0.05) reduced by BOL exposure. Interestingly, the BOL-exposed rats treated with CeNP showed a substantial decrease in the harmful effect of BOL, expounded by significant ( P < 0.05) modulation of all pro-inflammatory cytokines and serum levels of reproductive hormones. Fig. 5. Open in a new tab Dot-box plot presents the effect of BOL and/or CeNP on inflammatory cytokines and hormonal assay, including TNF-α ( A ), IL-6 ( B ), LH ( C ), FSH ( D ), and testosterone ( E ). Data are presented as box plots, where the box represents the interquartile range (IQR, 25th to 75th percentile), the horizontal line inside the box denotes the median, whiskers extend to the minimum and maximum values within 1.5 × IQR, and individual data points are overlaid as circles to display the full distribution (n = 6; * P < 0.05, ** P < 0.01, *** P < 0.001). BOL boldenone, CeNP cerium oxide nanoparticles, FSH follicle stimulating hormone, LH luteinizing hormone, TNF-α tumor necrosis factor-alpha. Histopathological alteration of the testicular tissue after treatment with BOL and/or CeNP The histological changes in testicular tissues after BOL and/or CeNP treatment were evaluated to validate the previously acquired results. The control and CeNP-treated rats displayed a normal structure of the seminiferous tubules (normal arrangement of germ cells and Sertoli cells, the spermatogonia line the periphery of the seminiferous tubules, the primary spermatocytes are situated above the spermatogonia, and the spermatids are situated higher in the epithelium. Additionally, some Sertoli cells were observed near a normal basement membrane in seminiferous tubules. Sperms were plentiful in the lumen of the seminiferous tubules. There were several Leydig cells in the interstitial tissue, as exhibited in Fig. 6 . Contrariwise, BOL-intoxicated rats exhibited abnormalities in the organization of the germ cells and a reduction in the quantity of germ cells with black nuclei and vacuolated cytoplasm. These alterations also caused the tubules to lack sperm in their lumen. There were a few Leydig cells and dilated interstitial tissue. Additionally, there were dilated, congested blood vessels and inflammatory cell infiltration. Alternatively, the concurrent exposure to BOL + CeNP showed an improvement in their epithelium. Furthermore, the interstitial tissue between the tubules was enhanced, and sperms were detected in the lumen, with the exception of a few vacuolation in the cytoplasm of some germ cells. Interestingly, the semiquantitative lesion scoring shown in Table 2 affirming that the BOL-intoxicated group exhibited the highest injury severity along all detected lesions in comparison to other treated groups. While, the CeNP cotreated BOL-injured rats indicated lesser damage than those treated with BOL alone. Fig. 6. Open in a new tab Histopathological analysis of testicular tissues in different treatment groups. Control and CeNP-treated groups show normal testicular architecture. BOL-intoxicated group exhibits severe damage, including germ cell depletion, cytoplasmic vacuolation (yellow arrows), pyknotic nuclei (green arrows), widened interstitial spaces (IS) with reduced Leydig cells (L), a dilated congested blood vessel (red star) with inflammatory cells (blue arrow), and a lack of sperm (blue star). BOL + CeNP co-treated group shows significant histological recovery, with restored seminiferous tubules (ST), reduced vacuolation, visible Sertoli cells (SE) and basement membrane (BM), reappearance of sperm (S) in the lumen, and normal interstitial spaces (IS) containing Leydig cells (L). H&E-stained sections; scale bars = 20 µm (400× magnification). Table 2. Comparative scoring of the testicular histopathological changes in each treatment group. Lesions Experimental groups Control CeNP BOL BOL + CeNP Tubular lesions Lack of sperms in lumen − − +++ − Tubular vacuolation − − ++ + Tubular necrosis − − +++ + Germinal cells desquamation − − +++ + Atrophied tubules − − ++ + Interstitial lesions Interstitial edema − − ++ + Diffuse interstitial proliferation − − ++ + Inflammatory cell infiltration − − ++ + Vascular lesions Congestion − − +++ + Thrombus formation − − + + Endothelial proliferation − − ++ + Hypertrophy of tunica muscularies − − ++ + Open in a new tab (−): Normal histology, (+): Mild, (++): Moderate, (+++): Severe. Multivariate analyses As illustrated in Fig. 7 A, the correlation heatmap between all of the variables, indicating whether the measured values were strongly or negatively associated. Additionally, the clustering heatmap in Fig. 7 B provided a clear visual representation of all the data. It emphasized the main variations in the concentration levels of each variable among the different treated groups. In comparison to the other groups, BOL-induced testicular injury was more likely to exhibit damage based on the given parameters. All the assessed parameters showed intermediate color intensities in the groups that received both BOL and CeNP, suggesting that animals receiving BOL responded favorably to the protective effect of the CeNP treatment. A volcano plot is used to effectively illustrate the substantial difference between the groups, as seen in Fig. 7 C and D. This shows the negative log[10]-transformed P values displayed against the log2-fold change in expression in a scatter plot. Fig. 7. Open in a new tab Multivariate analyses of all data sets after BOL and/or CeNP intervention. ( A ) The correlation heatmap illustrates the inter-relationship among the studied variables. ( B ) The clustering heatmap indicates the concentration values of the tested parameters among different groups. ( C , D ) Volcano plots of BOL vs the control group and BOL versus the BOL + CeNP group, respectively. The y-axis shows whether that change is significant or not, while the x-axis represents the fold change in expression. Variables with statistically significant differential expression lie above a horizontal threshold (the dotted line). Closer to zero indicates less significance, while moving away from zero in either direction indicates more significance. BOL boldenone, CeNP cerium oxide nanoparticles. Principal component analysis (PCA) was employed to ascertain the association between the various interventions, as shown in Fig. 8 A. In all measurements, 75.7% of the variance was explained by two main dimensional components (Component 1 and Component 2). Component 1 explained 70.4% of the variation, Component 2 explained 5.3%, and Component 1 explained the majority of the characteristics. According to the PCA biplot, BOL-intoxicated rats were clearly different from the BOL + CeNP and control groups and clustered on the left side. Furthermore, according to the variable importance in projection (VIP), the most important variables in differentiating BOL-treated animals from the others were NO, MDA, WBCs, PCV, Hb, TNF-α, IL-6, and RBCs (Fig. 8 B). Fig. 8. Open in a new tab Biplot of principal component analysis ( A ) and variable importance project (VIP) scores ( B ) after BOL and/or CeNP treatments. The contribution strength is indicated by a colored scale with the highest (red color) and lowest (blue color) values. At the top of the gradient scale is dark red (the highest value), and at the bottom is blue (the lowest value). BOL boldenone, CeNP cerium oxide nanoparticles. Discussion Anabolic androgenic steroids (AAS) represent a broad class of synthetic compounds that mimic the actions of male sex hormones, like testosterone and dihydrotestosterone. Despite a well-documented adverse health effect, particularly those of BOL, the abuse of AAS by athletes to improve physical performance has significantly expanded globally in recent years. In parallel, nanotechnology has become a viable method for disease investigation and treatment. In this context, the present study provides comprehensive evidence that BOL-induced testicular damage is triggered by a complicated pathogenic cascade involving oxidative stress, inflammatory reactions, and disruption of the hypothalamic–pituitary–gonadal (HPG) axis. Crucially, the current findings demonstrate that CeNP exerts a marked protective effect against these deleterious alterations, emphasizing its potential as a promising nanotherapeutic approach for reducing the reproductive harm caused by anabolic steroids. The hematological disturbances associated with BOL exposure can be attributed to the erythropoietic effects of BOL. Previous studies have demonstrated that BOL enhances erythropoietin production and activates bone marrow function, resulting in increased erythrocyte mass, hemoglobin concentration, and packed cell volume 29 – 31 . Such hematological alterations may increase blood viscosity and alter tissue oxygenation dynamics, potentially contributing to systemic complications associated with prolonged AAS misuse 32 . In parallel, leukocytosis observed after BOL administration may represent an inflammatory or immune-mediated response to steroid-induced tissue injury, as reported previously by Urhausen et al. 33 and Neamat-Allah 30 , who revealed a sharp rise in the total leucocytic count in response to BOL. Accumulating evidence indicates that oxidative stress represents a major toxic mechanism underlying BOL-induced tissue damage 6 , 34 – 39 . While the primary cause of male infertility is still unknown, between 30 and 80% of infertility cases were shown to be caused by excessive levels of ROS 40 . The testis’s high lipid content and rapid O 2 consumption make it a vulnerable organ to lipid peroxidation 41 . Overproduction of ROS and nitrogen species disrupts redox homeostasis, resulting in lipid peroxidation, protein oxidation, and DNA damage, which collectively compromise cellular integrity and function 42 – 44 . In line with this concept, the present study demonstrated marked oxidative damage in the testicular tissue following BOL exposure, characterized by enhanced lipid peroxidation and NO, along with substantial reductions in GSH concentration, as well as enzymatic activities of SOD and CAT in the testicular tissues. These results are consistent with previously published studies 6 , 8 , 11 . GSH is the main endogenous antioxidant that is widely present in all biological systems because it scavenges ROS directly, acting as a nonenzymatic antioxidant 45 , 46 . The first line of defense against free radicals is provided by endogenous enzymes SOD and CAT. The superoxide radical anion is dismutated to H 2 O 2 and O 2 by SOD, whereas H 2 O 2 is broken down to O 2 and H 2 O by CAT 47 . In addition to the harmful effects of ROS, MDA itself can cause substantial cellular damage by drastically changing the DNA integrity, cellular proteins, and mitochondrial membrane potential 46 . Moreover, free radicals like nitric oxide can combine with superoxide to generate peroxynitrite (ONOO − ), which can seriously harm the tissues 6 . Surprisingly, iNOS increases the production of NO and toxic peroxy-nitrite species, which intensifies necroptosis and inflammation 46 , 48 . Collectively, these oxidative mechanisms may contribute to impaired testicular function and infertility 49 . Oxidative stress is also closely linked to inflammatory activation. Increased intracellular signaling cascades that boost proinflammatory gene expression and cytokine release can be triggered by elevated ROS levels, resulting in long-lasting inflammatory reactions 50 . In the current investigation, elevated levels of TNF-α and IL-6 suggest activation of inflammatory pathways following BOL exposure. In the pathophysiology of inflammation, TNF-α is the most significant and early inflammatory mediator 51 . TNF-α and NF-κB/p65 pathways activate proinflammatory cytokines, such as IL-6, and adhesion molecules, which promote leukocyte recruitment 52 . Histopathological evidence of inflammatory cell infiltration further supports the involvement of inflammation in BOL-induced testicular injury. Consistent with these findings, several studies have previously reported an increase in the expression of several inflammation-related genes following BOL exposure 35 , 53 , 54 . Disruption of the HPG axis represents another critical consequence of BOL exposure. Remarkably, it is well known that any disruption in the HPG axis, which is primarily associated with male and female reproductive efficiency, may result in decreased or total infertility 34 , 55 . The observed suppression of LH, FSH, and testosterone reflects impaired endocrine regulation of testicular function. Testosterone is essential for the ultimate maturation of sperm and is also controlled by LH, which is secreted from the anterior pituitary gland. Meanwhile, the preservation of testicular gametogenic activity depends on FSH. The reduction of reproductive hormones in our study may be explained by the severe damage caused to Leydig and Sertoli cells by higher production of free radicals and oxidative stress caused by BOL, as one of the potential pathways for the damage of Leydig cells triggered by anabolic–androgenic steroids. Lipid peroxidation of Leydig cell mitochondrial membranes, which are the main location for testosterone biosynthesis, prevents cholesterol from moving to the inner mitochondrial membrane, hence suppressing the steroidogenesis process 56 , 57 . Additionally, excessive ROS may disrupt the hypothalamic release of gonadotrophic hormones, such as LH and FSH, by crossing the blood–brain barrier 58 . These mechanisms are supported by previous reports demonstrating significant reductions in LH, FSH, and testosterone following BOL exposure 6 , 11 , 59 . In the same data frame, these results are consistent with those of Mohammed et al. 60 and Oda and El‐Ashmawy 61 , who revealed that BOL injections significantly decreased the level of testosterone hormone. Histological alterations observed in the present study further corroborate the association between endocrine disruption and testicular structural damage. Notably, CeNP treatment markedly attenuated BOL-induced testicular injury by modulating hematological parameters, restoring redox balance, suppressing inflammatory responses, and improving reproductive hormone levels. The antioxidant activity of CeNP is mainly attributed to its unique ability to undergo reversible redox cycling between Ce 3+ and Ce 4 ⁺ oxidation states 15 . This dynamic surface chemistry allows CeNP to function as a regenerative antioxidant 62 . Specifically, Ce 3+ sites can donate electrons to neutralize superoxide radicals (O ·− ), mimicking superoxide dismutase activity, whereas Ce 4+ facilitates the decomposition of hydrogen peroxide (H 2 O 2 ), resembling catalase-like activity 62 . Importantly, this redox cycling is self-renewing, enabling sustained antioxidant activity without rapid depletion, unlike conventional antioxidants 15 . These findings are consistent with previous reports suggesting improved antioxidant status and reproductive hormone levels following CeNP supplementation 18 , 63 . Restoration of redox balance by antioxidative agents, such as CeNP, may therefore indirectly preserve HPG axis integrity by protecting hypothalamic and pituitary cells from ROS-mediated dysfunction. In addition to their antioxidant effects, CeNP exhibited pronounced anti-inflammatory properties, as evidenced by reduced levels of TNF-α and IL-6 and diminished inflammatory cell infiltration in testicular tissue. These observations align with previous reports showing that CeNP may reduce apoptotic cell death, monocyte/macrophage infiltration, and proinflammatory cytokine production, including TNF-α and IL-6 64 . Therefore, collectively, the protective effects of CeNP on testicular tissue may be attributed to their dual role in restoring redox balance and suppressing inflammation, which in turn preserves the integrity of the hypothalamic–pituitary–gonadal axis and improves hormonal regulation. Interestingly, considering all of the previously mentioned findings, multivariate statistical analysis was conducted using PCA, correlation and clustering heatmaps, volcano plots, and VIP scores to evaluate the degree of testicular damage brought on by BOL exposure. All of the previously obtained data were supported by the correlation heatmap, which verified a significant appropriate correlation between all parameters under study. The clustering heatmap of the same data demonstrated the differences in each variable’s concentration levels across the various treatment groups. Rats exposed to BOL had a higher probability of exhibiting more testicular damage, according to the heatmap. Furthermore, the VIP scores demonstrated that the main modulators of BOL-induced testicular damage were oxidative stress and inflammation. According to the VIP scores, the most important variables in response to BOL and/or CeNP treatments were NO, MDA, WBCs, PCV, Hb, TNF-α, and IL-6. Thus, the CeNP could mitigate these harmful effects of BOL. Figure 9 illustrates the underlying molecular pathway of the protective effect of CeNP upon BOL-induced testicular injury. Fig. 9. Open in a new tab Schematic diagram summarizes the underpinning molecular pathway of the protective effect of CeNP upon BOL-induced testicular damage. Conclusions Based on the current findings, BOL-induced testicular injury is driven by interconnected oxidative, inflammatory, and endocrine disruption mechanisms. Pretreatment with CeNP appears to attenuate these pathological mechanisms, highlighting their potential protective role against anabolic steroid-induced reproductive toxicity. Despite these promising findings, the current study is limited by the use of a single BOL dose and treatment duration in a preclinical animal model. In addition, direct fertility-related endpoints, such as sperm parameters and reproductive performance, were not evaluated. Future research is advised to study the dose–response relationships, long-term fertility outcomes to further elucidate CeNP-mediated protection and its potential translational relevance. Acknowledgements The authors extend their appreciation to the Deanship of Scientific Research and the Postgraduate at King Khalid University for funding this work through a Large Group Research Project under grant number (RGP2/529/45). Author contributions Conceptualization, investigation, and methodology; H.M.H, H.S.M., S.S.S., F.S.M., A.A., S.A., E.E.E., and M.F.A. Formal analysis, data curation, validation, and visualization; A.A.A., A.A., N.A., H.M.A., A.F.R., M.S., S.F.I., M.A., and A.M.A. Supervision; A.A. Writing original draft, review, and editing; all authors. All authors have approved the published version of this manuscript. Funding This research was funded by the Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2026R127), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. This work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Research, King Faisal University, Saudi Arabia (Grant No. KFU260379). Data availability Upon request, the corresponding authors can supply the data that was utilized to confirm the study’s findings. Declarations Competing interests The authors declare no competing interests. Institutional review board statement All animal procedures strictly adhered to the UK Animals Act 1986 (ASPA) and the 3Rs principles (replacement, reduction, refinement). Ethical approval was granted by the Minia University Animal Ethical Committee, Egypt (Approval No. MU/FA002/01/25). ARRIVE guidelines statement This study aligns with the ARRIVE guidelines to ensure rigorous, transparent, and detailed reporting of experimental design, methodologies, and results in animal research. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Contributor Information Ahmed Abdeen, Email: ahmed.abdeen@fvtm.bu.edu.eg. Ahmad F. Rawan, Email: afaridrawan@gmail.com Mustafa Shukry, Email: matta@kfu.edu.sa. References 1. Tousson, E. et al. Physiological and biochemical changes after boldenone injection in adult rabbits. Toxicol. Ind. Health 32 , 177–182 (2016). [ DOI ] [ PubMed ] [ Google Scholar ] 2. Sabra, H. M., M, E. S. R., Abosalem, M., Nabila, M. A. & Ibrahim, S. S. Ameliorative effects of lycopene against boldenone undecylenate toxicity in albino rats. Benha Vet. Med. J. 732 , 237–249 (2018). [ Google Scholar ] 3. Park, M. et al. Determination of boldenone in postmortem specimens including blood and urine samples using LC–MS/MS. J. Pharm. Biomed. Anal. 169 , 111–115 (2019). [ DOI ] [ PubMed ] [ Google Scholar ] 4. Jamal, M. et al. Anabolic–androgenic steroid use in a young body-builder: A case report and review of the literature. Ann. Med. Surg. 83 , 104567 (2022). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 5. Saber, T. M. et al. Early postmortem biochemical, histological, and immunohistochemical alterations in skeletal muscles of rats exposed to boldenone undecylenate: Forensic implication. J. Forensic Leg. Med. 83 , 102248 (2021). [ DOI ] [ PubMed ] [ Google Scholar ] 6. Mowaad, N. A., El, M. E. A. & Yasser, S. The behavioral and neurochemical changes induced by boldenone and/or tramadol in adult male rats. Neurochem. Res. 48 , 1320–1333 (2023). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 7. Hassan, H. et al. Mitigating effects of ginseng bulk on hepatic and immunological toxicity induced by boldenone undecylenate in rats. J. Adv. Vet. Res. 15 , 233–237 (2025). [ Google Scholar ] 8. Aly, M. A. S., El-Sayed El-Shamarka, M., Soliman, T. N. & Elgabry, M. A. E. Protective effect of nanoencapsulated curcumin against boldenone-induced testicular toxicity and oxidative stress in male albino rats. Egypt. Pharm. J. 20 , 72–81 (2021). [ Google Scholar ] 9. Ziaolhagh, S. J., Khojasteh, L., Ziaolhagh, S. S. & Yahyaei, B. The effect of Boldenone anabolic steroid, and endurance and resistance training on liver damage markers in rats. Feyz Med. Sci. J. 22 , 143–152 (2018). [ Google Scholar ] 10. Barakat, L. A., Tousson, E., Ibrahim, W. & El-Hakeem, A. Role of propolis in improving hepatic and renal damage in boldenone undecylenate in male rats. Am. J. Biol. Chem. 3 , 8–15 (2015). [ Google Scholar ] 11. Behairy, A. et al. The modulatory role of vitamin C in boldenone undecylenate induced testicular oxidative damage and androgen receptor dysregulation in adult male rats. Antioxidants 9 , 1053 (2020). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 12. Behairy, A. et al. Boldenone undecylenate-mediated hepatorenal impairment by oxidative damage and dysregulation of heat shock protein 90 and androgen receptors expressions: Vitamin C preventive role. Front. Pharmacol. 12 , 651497 (2021). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 13. Elshony, N., Nassar, A. M. K., El-sayed, Y. S. & Samak, D. Ameliorative role of cerium oxide nanoparticles against Fipronil impact on brain function, oxidative stress, and apoptotic cascades in albino rats. Front. Neurosci. 15 , 1–18 (2021). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 14. Beshbishy, A. M., Batiha, G.E.-S., Yokoyama, N. & Igarashi, I. Ellagic acid microspheres restrict the growth of Babesia and Theileria in vitro and Babesia microti in vivo. Parasit. Vectors 12 , 1–13 (2019). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 15. Das, S. et al. Cerium oxide nanoparticles: Applications and prospects in nanomedicine. Nanomedicine 8 , 1483–1508 (2013). [ DOI ] [ PubMed ] [ Google Scholar ] 16. Kyosseva, S. V. & McGinnis, J. F. Cerium oxide nanoparticles as promising ophthalmic therapeutics for the treatment of retinal diseases. World J. Ophthalmol. 5 , 23–30 (2015). [ Google Scholar ] 17. Brandão Da Silva Assis, M., Nestal De Moraes, G. & De Souza, K. R. Cerium oxide nanoparticles: Chemical properties, biological effects and potential therapeutic opportunities (review). Biomed. Rep. 20 , 48 (2024). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 18. Kobyliak, N. M. et al. Antioxidative effects of cerium dioxide nanoparticles ameliorate age-related male infertility: optimistic results in rats and the review of clinical clues for integrative concept of men health and fertility. EPMA J. 6 , 1–22 (2015). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 19. Tousson, E., Hafez, E., Massoud, A. & Elfeky, A. A. Ameliorating effect of propolis and moringa extract against equigan induced neurotoxicity and oxidative stress on rat hippocampus. J. Biosci. Appl. Res. 2 , 30–37 (2016). [ Google Scholar ] 20. Adebayo, O. A., Akinloye, O. & Adaramoye, O. A. Cerium oxide nanoparticles attenuate oxidative stress and inflammation in the liver of diethylnitrosamine-treated mice. Biol. Trace Elem. Res. 193 , 214–225 (2020). [ DOI ] [ PubMed ] [ Google Scholar ] 21. Thrall, M. A., Weiser, G., Allison, R. W. & Campbell, T. W. Veterinary Hematology and Clinical Chemistry (Wiley, 2012). [ Google Scholar ] 22. Bancroft, J. D. & Gamble, M. Fixatives. In Theory and Practice of Histological Techniques 6th edn 72–75 (Churchill Livingstone, Elsevier, 2008). [ Google Scholar ] 23. Tohamy, H. G. et al. Ameliorative effects of boswellic acid on fipronil-induced toxicity: Antioxidant state, apoptotic markers, and testicular steroidogenic expression in male rats. Animals 11 , 1302 (2021). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 24. Ramanjaneyulu, V. & Narsaiah, T. B. Hydrothermal synthesis of CeO 2 /rGO nano composite and its characterization. REEN Process Des. Sustain. Chem. Manuf. 6 , 76–78 (2025). [ Google Scholar ] 25. Reports, S. Unveiling the atomic-scale mechanism of water-induced wettability transition in ZnO nanopods: A combined experimental and molecular dynamics study IN. Sci. Rep. 10.1038/s41598-026-48983-5 (2026). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 26. Chen, X., Schweitzer-Stenner, R., Krimm, S., Mirkin, N. G. & Asher, S. A. N-methylacetamide and its hydrogen-bonded water molecules are vibrationally coupled. J. Am. Chem. Soc. 10.1021/ja00103a033 (1994). [ Google Scholar ] 27. Ta, K. M. et al. Infrared and Raman diagnostic modeling of phosphate adsorption on ceria nanoparticles. J. Phys. Chem. C 127 , 20183–20193 (2023). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 28. Valsaraj, P. V. & Divyarthana,. Structural, optical and antimicrobial properties of green synthesized cerium oxide nanoparticles. AIP Conf. Proc. 2162 , 20022 (2019). [ Google Scholar ] 29. Nafeaa, A. A., Abdel-Magid, A. D. & Aita, S. A. Biochemical and physiological influences of boldenoe undecylate as a growth promotor on different ages of male white New Zeland rabbits. Glob. J. Adv. Res. 3 , 441–451 (2016). [ Google Scholar ] 30. Neamat-Allah, A. N. Effect of boldenone undecylenate on haematological and biochemical parameters in veal calves. Glob. Vet. 13 , 1092–1096 (2014). [ Google Scholar ] 31. Heiland, C. E., Schickel, Y., Lehtihet, M., Börjesson, A. & Ekström, L. Supra-physiological doses of anabolic androgenic steroids impact erythropoietin and blood parameters. Drug Test. Anal. 15 , 599–604 (2023). [ DOI ] [ PubMed ] [ Google Scholar ] 32. Borowiec, A., Waluszewska, I., Jurkiewicz, M. & Szczurek-Wasilewicz, W. Impact of anabolic–androgenic steroid abuse on the cardiovascular system: Molecular mechanisms and clinical implications. Int. J. Mol. Sci. 26 , 11037 (2025). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 33. Urhausen, A., Torsten, A. & Wilfried, K. Reversibility of the effects on blood cells, lipids, liver function and hormones in former anabolic-androgenic steroid abusers. J. Steroid Biochem. Mol. Biol. 84 , 369–375 (2003). [ DOI ] [ PubMed ] [ Google Scholar ] 34. Mowaad, N. A., Asaad, G. F., El-shamarka, M. E. A. & Khalil, S. Cross-talk between down-regulation of steroidogenic genes expression and oxidative and apoptotic biomarkers in testes induced by administration of tramadol and boldenone and their combination in male albino rats. Iran. J. Basic Med. Sci. 25 , 808 (2022). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 35. El‐Shamarka, M. E., Eliwa, H. A. & Ahmed, M. A. E. Inhibition of boldenone‐induced aggression in rats by curcumin: Targeting TLR4/MyD88/TRAF‐6/NF‐κB pathway. J. Biochem. Mol. Toxicol. 36 , e22936 (2022). [ DOI ] [ PubMed ] [ Google Scholar ] 36. Tousson, E., Elgharabawy, R. M. & Elmasry, T. A. Grape seed proanthocyanidin ameliorates cardiac toxicity induced by boldenone undecylenate through inhibition of NADPH oxidase and reduction in the expression of NOX2 and NOX4. Oxid. Med. Cell. Longev. 2018 , 9434385 (2018). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 37. Tousson, E., Zahran, F. & Shalapy, M. A. Oral supplementation of aqueous Moringa and Ginkgo leaf extracts abates oxidative stress and testicular injury associated with boldenone injection in rats. Pharmacol 7 , 381–389 (2016). [ Google Scholar ] 38. Dornelles, G. L. et al. Biochemical and oxidative stress markers in the liver and kidneys of rats submitted to different protocols of anabolic steroids. Mol. Cell. Biochem. 425 , 181–189 (2017). [ DOI ] [ PubMed ] [ Google Scholar ] 39. Bueno, A. et al. Impacts of dose and time of boldenone and stanazolol exposure in inflammatory markers, oxidative and nitrosative stress and histopathological changes in the rat testes. Theriogenology 90 , 101–108 (2017). [ DOI ] [ PubMed ] [ Google Scholar ] 40. Tremellen, K. Oxidative stress and male infertility—A clinical perspective. Hum. Reprod. Update 14 , 243–258 (2008). [ DOI ] [ PubMed ] [ Google Scholar ] 41. Hassan, E., Kahilo, K., Kamal, T., El-Neweshy, M. & Hassan, M. Protective effect of diallyl sulfide against lead-mediated oxidative damage, apoptosis and down-regulation of CYP19 gene expression in rat testes. Life Sci. 226 , 193–201 (2019). [ DOI ] [ PubMed ] [ Google Scholar ] 42. Elgazzar, D., Abdeen, A. & Aboubakr, M. Gentamicin potentiate tigecycline induced cardiotoxicity in rats: The cardiac susceptibility to oxidative stress. Benha Vet. Med. J. 43 , 36–40 (2022). [ Google Scholar ] 43. Antar, S. A. et al. Modulatory role of autophagy in metformin therapeutic activity toward doxorubicin-induced nephrotoxicity. Toxics 11 , 273 (2023). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 44. Marques Neto, S. R., Silva, A. H., Santos, M. C. P., Ferraz, E. F. & Nascimento, J. H. M. The blockade of angiotensin AT 1 and aldosterone receptors protects rats from synthetic androgen‐induced cardiac autonomic dysfunction. Acta Physiol. 208 , 166–171 (2013). [ DOI ] [ PubMed ] [ Google Scholar ] 45. Allam, A. et al. N-acetylcysteine alleviated the deltamethrin-induced oxidative cascade and apoptosis in liver and kidney tissues. Int. J. Environ. Res. Public Health 19 , 1–13 (2022). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 46. Abdel-Daim, M. M. et al. Fucoidan supplementation modulates hepato-renal oxidative stress and DNA damage induced by aflatoxin B1 intoxication in rats. Sci. Total Environ. 768 , 144781. 10.1016/j.scitotenv.2020.144781 (2021). [ DOI ] [ PubMed ] [ Google Scholar ] 47. Althobaiti, F. et al. Exploring the NRF2/HO-1 and NF-κB pathways: Spirulina nanoparticles as a novel approach to combat diabetic nephropathy. ACS Omega 9 , 23949–23962 (2024). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 48. Owumi, S. E. et al. Apigeninidin-rich Sorghum bicolor (L. Moench) extracts suppress A549 cells proliferation and ameliorate toxicity of aflatoxin B1-mediated liver and kidney derangement in rats. Sci. Rep. 12 , 7438 (2022). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 49. Ebokaiwe, A. P. et al. Nigerian bonny-light crude oil induces alteration in testicular stress response proteins and caspase-3 dependent apoptosis in albino wistar rats. Environ. Toxicol. 30 , 242–252 (2015). [ DOI ] [ PubMed ] [ Google Scholar ] 50. Owumi, S., Najophe, E. S., Farombi, E. O. & Oyelere, A. K. Gallic acid protects against Aflatoxin B 1 -induced oxidative and inflammatory stress damage in rats kidneys and liver. J. Food Biochem. 44 , e13316 (2020). [ DOI ] [ PubMed ] [ Google Scholar ] 51. Jang, D. I. et al. The role of tumor necrosis factor alpha (Tnf-α) in autoimmune disease and current Tnf-α inhibitors in therapeutics. Int. J. Mol. Sci. 22 , 1–16 (2021). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 52. Giridharan, S. & Srinivasan, M. Mechanisms of NF-κB p65 and strategies for therapeutic manipulation. J. Inflamm. Res. 11 , 407–419. 10.2147/JIR.S140188 (2018). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 53. Mayada, R. F., Taghred, M. S. & Haytham, A. A. Boldenone-induced apoptotic, structural, and functional alterations in the liver of rabbits. World Rabbit Sci. 23 , 39–46 (2015). [ Google Scholar ] 54. Ibrahim, S. S. & Said, A. M. Effect of lycopene in amelioration of testicular and renal toxicity induced by boldenone undecylenate in male albino rats. Int. J. Med. Biomed. Stud. 3 , 1–9 (2019). [ Google Scholar ] 55. Faccio, L. et al. Serum levels of LH, FSH, estradiol and progesterone in female rats experimentally infected by Trypanosoma evansi . Exp. Parasitol. 135 , 110–115 (2013). [ DOI ] [ PubMed ] [ Google Scholar ] 56. Deng, B., Shen, W.-J., Dong, D., Azhar, S. & Kraemer, F. B. Plasma membrane cholesterol trafficking in steroidogenesis. FASEB J. 33 , 1389 (2018). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 57. Huang, X., Liang, Y., Qing, Y., Chen, D. & Shi, N. Proteasome inhibition by MG-132 protects against deltamethrin-induced apoptosis in rat hippocampus. Life Sci. 220 , 76–83 (2019). [ DOI ] [ PubMed ] [ Google Scholar ] 58. Adeyemi, W. J. et al. Elevated reproductive toxicity effects of diclofenac after withdrawal: Investigation of the therapeutic role of melatonin. Toxicol. Rep. 6 , 571–577 (2019). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 59. Elmasry, T. A., Al-Shaalan, N. H., Tousson, E., El-Morshedy, K. & Al-Ghadeer, A. Star anise extracts modulation of reproductive parameters, fertility potential and DNA fragmentation induced by growth promoter Equigan in rat testes. Braz. J. Pharm. Sci. 54 , e17261 (2018). [ Google Scholar ] 60. Mohammed, H. H., Badawi, M. E., El-Tarabany, M. S. & Rania, M. Effects of boldenone undecylenate on growth performance, maintenance behaviour, reproductive hormones and carcass traits of growing rabbits. Pol. J. Vet. Sci. 10.1515/pjvs-2016-0031 (2016). [ DOI ] [ PubMed ] [ Google Scholar ] 61. Oda, S. S. & El-Ashmawy, I. M. Adverse effects of the anabolic steroid, boldenone undecylenate, on reproductive functions of male rabbits. Int. J. Exp. Pathol. 93 , 172–178 (2012). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 62. Pirmohamed, T. et al. Nanoceria exhibit redox state-dependent catalase mimetic activity. Chem. Commun. 46 , 2736–2738 (2010). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 63. Saleh, H. et al. Chemo-protective potential of cerium oxide nanoparticles against fipronil-induced oxidative stress, apoptosis, inflammation and reproductive dysfunction in male white albino rats. Molecules 10.3390/molecules25153479 (2020). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 64. Davoudi-Kiakalayeh, A., Mohammadi, R., Pourfathollah, A. A., Siery, Z. & Davoudi-Kiakalayeh, S. Alloimmunization in thalassemia patients: New insight for healthcare. Int. J. Prev. Med. 8 , 1–9 (2017). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Associated Data This section collects any data citations, data availability statements, or supplementary materials included in this article. Data Availability Statement Upon request, the corresponding authors can supply the data that was utilized to confirm the study’s findings. Articles from Scientific Reports are provided here courtesy of Nature Publishing Group ACTIONS View on publisher site PDF (4.1 MB) Cite Collections Permalink RESOURCES Similar articles Cited by other articles Links to NCBI Databases Follow NCBI NCBI on X (formerly known as Twitter) NCBI on Facebook NCBI on LinkedIn NCBI on GitHub NCBI RSS feed Connect with NLM NLM on X (formerly known as Twitter) NLM on Facebook NLM on YouTube National Library of Medicine 8600 Rockville Pike Bethesda, MD 20894 Web Policies FOIA HHS Vulnerability Disclosure Help Accessibility Careers NLM NIH HHS USA.gov Back to Top

Related documents

• Estradiol mitigates lipid accumulation, oxidative stress, apoptosis, and MAPKs activation in NASH rat model with bilateral orchidectomy.#636421
• Oxidative Stress and Male Reproductive Health—First Edition#314785
• Protective Effects of Cocoa Ingestion Over Healthy Males Plasma Lipids Subjected to Peroxidative Conditions#1171220
• Oxidative Stress and Male Reproductive Health-First Edition#478651
• Macrobiotic Diet and Flax Seed: Effects on Estrogens, Phytoestrogens, & Fibrinolytic Factors#914001