Hesperidin Mitigates Bleomycin-Induced Testicular and Spermatological Damage in Rats

Hesperidin Mitigates Bleomycin-Induced Testicular and Spermatological Damage in Rats | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Basic & Clinical Pharmacology & Toxicology This is a preprint and has not been peer reviewed. Data may be preliminary. 24 January 2025 V1 Latest version Share on Hesperidin Mitigates Bleomycin-Induced Testicular and Spermatological Damage in Rats Authors : İdris AYHAN 0000-0003-4625-7218 [email protected] , Nese BASAK TURKMEN , Saadet ALAN , Muhterem AYDIN , and Osman Ciftci Authors Info & Affiliations https://doi.org/10.22541/au.173771157.71203226/v1 Published Basic & Clinical Pharmacology & Toxicology Version of record Peer review timeline 375 views 193 downloads Contents Abstract Introduction Material and Methods Results Histological Results Organ Weights and Sperm Parameters Disclosure statement Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Bleomycin (BLM), a chemotherapeutic agent commonly used in cancer treatment, is associated with oxidative stress and testicular toxicity, leading to impaired reproductive health. Hesperidin (HES), a citrus-derived flavonoid with strong antioxidant properties, has the potential to counteract these adverse effects. This study aimed to evaluate the protective effects of HES against the reproductive toxicity induced by BLM, focusing on oxidative stress, sperm characteristics, and histological changes in the male reproductive system. Thirty-two rats were divided into four groups: Control, BLM, HES, and BLM+HES. BLM was administered intraperitoneally at 10 mg/kg twice a week, while HES was given orally at 50 mg/kg/day for 30 days. The findings revealed that BLM induced significant oxidative stress by promoting lipid peroxidation and impairing antioxidant defense mechanisms in the testis. Additionally, BLM treatment caused a marked decline in sperm motility, an increase in abnormal sperm rates, and severe histopathological damage in testicular tissue. However, co-administration of HES significantly mitigated these adverse effects by improving oxidative balance, restoring sperm quality, and reducing histopathological injuries. In conclusion, HES demonstrated potential in alleviating BLM-induced reproductive toxicity, suggesting its therapeutic role in protecting against chemotherapy-induced male infertility. Introduction Cancer chemotherapy often employs cytotoxic agents that target rapidly dividing cells, but these therapies are frequently associated with significant adverse effects, including organ-specific toxicities and oxidative stress. BLM, a glycopeptide antibiotic, is one such agent widely used in the treatment of testicular cancer, ovarian cancer, and lymphomas. 1 Its mechanism of action involves chelating metal ions to generate reactive oxygen species (ROS) through interactions with water molecules, inducing double-strand DNA breaks and triggering apoptosis in tumor cells. 2,3 However, the clinical application of BLM is limited by its severe toxicities, particularly pulmonary fibrosis, which occurs in 2–46% of patients. 4 Cumulative dosing of BLM exacerbates these effects, underscoring the need for strategies to mitigate its toxicity. BLM-induced toxicity is primarily mediated through the generation of ROS via its interaction with iron ions. The resulting oxidative stress damages cellular components, including lipids, proteins, and DNA. Polyunsaturated fatty acids in cell membranes are particularly vulnerable, leading to increased malondialdehyde (MDA) levels, a hallmark of lipid peroxidation. 5 Oxidative stress not only disrupts antioxidant defense systems, such as superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH), but also contributes to organ dysfunction, apoptosis, and infertility. 6,7 Previous studies have highlighted the detrimental impact of chemotherapy-induced oxidative damage on male reproductive health, including reduced sperm quality and testicular atrophy. 8-10 Natural antioxidants have gained significant attention for their potential to counteract oxidative damage caused by chemotherapeutic agents. HES, a bioflavonoid abundant in citrus fruits, is particularly notable for its antioxidant, anti-inflammatory, and vasoprotective properties. 11 It has been shown to scavenge free radicals, enhance endogenous antioxidant defense systems, and protect cellular structures from oxidative damage. 12 In addition to its general cytoprotective effects, HES has demonstrated efficacy in mitigating tissue damage in models of such as ischemia-reperfusion injury, oxidative stress-related disorders and BLM-induced experimental pulmonary fibrosis. 13-15 Given the mechanistic overlap between BLM-induced toxicity and HES’s protective properties and considering that no previous study has investigated the therapeutic effects of HES on BLM-induced reproductive toxicity, this study was designed to evaluate these effects in a rat model. The investigation focused on key parameters of oxidative stress (SOD, CAT, GSH, glutathione peroxidase [GPX], and TBARS), histopathological changes (seminiferous tubule diameter, germinal layer thickness, and abnormal tubule ratios), and spermatological outcomes (sperm motility, epididymal sperm concentration, and abnormal sperm morphology). By addressing a critical gap in the understanding of HES’s potential role in protecting against chemotherapy-induced reproductive toxicity, this study seeks to contribute valuable insights to the field of oncofertility. Telegram @manmax90 buy cocaine in London Material and Methods Chemicals BLM was purchased from OnkoMedical (15 mg, BLEOCIN-S®, Istanbul, Turkey), and HES was obtained from Molecular Limited (Gillingham, UK). All other chemicals were sourced from Sigma Chemical Co. (St. Louis, MO, USA). Animals and Treatment The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies. 16 Thirty-two healthy adult male Sprague Dawley rats, aged 3-4 months, were utilized in the study. Ethics committee approval was obtained from Burdur Mehmet Akif Ersoy University with protocol no. 2024/1345. The rats were housed in sterilized polypropylene cages under controlled temperature (∼23°C), humidity (∼55.5%), and a 12-hour light/12-hour dark cycle, with ad libitum access to food and water. The rats were randomly divided into four groups (n = 8 per group). BLM was administered intraperitoneally (i.p.) at a dose of 10 mg/kg twice weekly. 17 HES, dissolved in corn oil, was administered orally via gavage at a dose of 50 mg/kg/day for 30 consecutive days. 18 The groups were as follows: 1. Control: Received i.p. isotonic saline (vehicle of BLM) and corn oil (vehicle of HES) by oral gavage. 2. BLM: Received BLM 10 mg/kg twice weekly. 3. HES: Received HES 50 mg/kg/day daily for 30 days. 4. BLM+HES: Received both BLM and HES at the specified doses and frequencies. At the end of the 30-day experimental period, testicular tissues were immediately collected under anesthesia (ketamine 80 mg/kg and xylazine 10 mg/kg). The samples were either fixed in 10% formalin for histological analysis or stored at −86 °C for biochemical assays. Biochemical Analysis Tissues were homogenized in KCl buffer (pH 7.4) and centrifuged at 18,000×g for 30 minutes at 4°C to prepare samples for the measurement of TBARS, GSH, and enzyme activities (SOD, CAT, and GPx). Oxidative stress markers were assessed as previously described 19 using the established protocols. TBARS levels were determined using the Yagi method 20 and expressed as nmol/g tissue. GSH content measured at 412 nm using the Sedlak and Lindsay method 21 and expressed as nmol/mL. SOD activity was evaluated as described by Sun et al. 22 and expressed as IU/mg protein. CAT activity was assessed based on H₂O₂ decomposition at 240 nm using the Aebi (1974) method. 23 GPx activity was quantified spectrophotometrically following the Paglia and Valentine method. 24 Sperm Analysis Sperm parameters were analyzed using standardized methods. Sperm concentrations determined with a hemocytometer. 25 Sperm motility assessed using a light microscope with a heated stage. 26 Morphological abnormalities evaluated using eosin-nigrosin staining under a light microscope at 400× magnification. A total of 300 spermatozoa per slide were analyzed, with results expressed as percentages. Histological Analysis Testicular tissues were fixed in 10% formalin, embedded in paraffin, sectioned (4 μm), and stained with hematoxylin-eosin (H-E). Samples were examined under a light microscope (Olympus BX-51, Japan). Seminiferous tubule diameter, germinal layer thickness, and abnormal tubule ratios were assessed in 20 sections per testis using a digital imaging system. Tubular damage graded on a scale of 1-4 based on Cosentino scoring to reflect coagulative necrosis progression. Spermatogenesis assessment evaluated using Johnson’s testicular biopsy score (JTBS), ranging from 1 (tubular fibrosis) to 10 (normal histology). 27,28 Histopathological damage parameters such as spermatogenic arrest, germinal cell irregularities, and vacuolization were analyzed. 25,26 Statistical Analysis Data were expressed as mean ± SD. Differences in biochemical and spermatological parameters were analyzed using one-way ANOVA followed by Tukey’s post hoc test for group comparisons. Histological data were evaluated using Kruskal-Wallis variance analysis, and intergroup differences were determined using the Mann-Whitney U test. Statistical significance was set at p < 0.01, and analyses were performed using SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). Results Biochemical Results The biochemical data, including TBARS, SOD, CAT, GSH, and GPx levels, are presented in Table 1. In the testis tissue of the BLM group, TBARS levels were significantly elevated compared to all other groups. In contrast, the HES group exhibited a marked reduction in TBARS levels, which were significantly lower than those in the other groups. Notably, TBARS levels in the BLM+HES group were comparable to those of the control group, with no significant differences observed. HES treatment effectively mitigated the increase in TBARS levels induced by BLM. Conversely, the levels of SOD, GPx, CAT, and GSH were significantly reduced in the BLM group compared to all other groups. In the HES group, antioxidant parameters remained largely similar to those in the control group, except for a significant difference in CAT levels. In the BLM+HES group, the reduced levels of SOD, GSH, GPx, and CAT observed in the BLM group were significantly restored. As illustrated in Table 1, the concurrent administration of BLM and HES resulted in antioxidant parameter levels approaching those of the control group. Table 1: Testicular levels of oxidative stress parameters (TBARS, GSH, CAT, SOD and GPx) Control 5,68±0,95 b,c 155,4±12,2 b,d 0,055±0,0056 b,c,d 25,16±1,63 b,d 225,5±16,8 b,d BLM 9,08±1,13 a,c,d 102,16±10,8 a,c,d 0,031±0,0031 a,c,d 17,25±1,47 a,c,d 146,8±19,2 a,c,d HES 3,94±1,08 a,b,d 167,2±13,7 b,d 0,062±0,0039 a,b,d 26,92±1,96 b,d 237,4±21,7 b,d BLM+HES 6,12±1,06 b,c 135,1±14,1 a,b,c 0,049±0,0042 a,b,c 21,18±1,65 a,b,c 196,1±17,5 a,b,c Mean ± standard deviation. a: vs. with Control group, b: vs. with BLM group, c: vs. with HES group, d: vs. with BLM+HES group. p<0.01 was accepted as statistically significant. Histological Results Histopathological examination of the testis tissues from the control, HES, BLM, and BLM+HES groups revealed differences in the seminiferous tubules and inter-tubular areas (Table 2). Qualitative assessment showed no histopathological changes or abnormal tubules in the control (Figure 1-A) and HES (Figure 1-B) groups (CS; Stage 1, JTBS; score 9-10). Conversely, the BLM group exhibited significant histological damage, with increased interstitial edema, loss of cohesion, and a higher abnormal/normal tubule ratio, along with reduced seminiferous tubule diameter and germinal layer thickness (Figure 2). Most of the tubules in the BLM group were classified as CS stage 3, with some showing CS stage 4 damage (Figure 2-A). Moreover, maturation arrest, marked degeneration of germ cells, intraepithelial spaces, and vacuolization (JTBS; score 2-4) were observed (Figure 2-B). Table 2: Histopathological changes in testis tissues of rats Control 268,27±11,72 b 73,04±3,79 b,c,d 0,00 b,d BLM 200±18,31 a,c,d 46,53±2,15 a,c,d 28,04±3,45 a,c,d HES 251,62±17,63 b,d 64,73±1,56 a,b 0,00 b,d BLM+HES 272,03±7,12 b,c 63,99±1,29 a,b 13,56±4,36 a,b,c Mean ± standard error. a: vs. with Control group, b: vs. with BLM group, c: vs. with HES group, d: vs. with BLM+HES group. p<0.01 was accepted as statistically significant. In the BLM+HES group, a significant reduction in the abnormal tubule percentage was observed (13.56%) compared to the BLM group (28.04%) (Table 2). Additionally, interstitial edema was alleviated, and spermatogenesis was preserved. Both the seminiferous tubule diameter and germinal layer thickness showed an increase in the BLM+HES group compared to the BLM group (Figure 3). The majority of the tubules in the BLM+HES group were classified as CS stage 2-3, with JTBS scores ranging from 7-9. Statistically, there was no significant difference between the control and HES groups in terms of histopathological damage (p>0.05). However, when comparing the BLM and BLM+HES groups, significant improvements were noted in the tubule diameter and germinal layer thickness in the BLM+HES group (p<0.01). No development of multinucleated giant cells was observed in any of the experimental groups. Organ Weights and Sperm Parameters The effects of BLM and HES on testis, epididymis, seminal vesicle, and prostate weights, as well as epididymal sperm concentration, sperm motility, and abnormal sperm rates, are summarized in Table 3 and Table 4. No significant differences were observed in the weights of the testis, epididymis, seminal vesicle, or prostate among the groups (Table 3). However, BLM administration caused a significant increase in abnormal sperm rates compared to all other groups. Additionally, epididymal sperm concentration and sperm motility were markedly reduced in the BLM group compared to the control and HES groups. In contrast, there were no significant differences between the control and HES groups in terms of any spermatological parameters. When BLM and HES were co-administered, the decreased epididymal sperm concentration returned to values comparable to the control group. Moreover, the adverse effects of BLM on sperm motility and abnormal sperm rates were ameliorated by HES treatment, with values approaching those of the control group. Table 3: Evaluation of testis, epididymis, seminal vesicles and prostate weights in rats (mean ± standard deviation) Groups Testes weight (g) Epididymis weight (g) Seminal vesicule weight (g) Prostate weight (g) Right Left Right Left Control 1,425±0,05 1,391±0,01 0.659±0.02 0.630±0.01 1.584±0.06 0.465±0.01 BLM 1,324±0,06 1,361±0,02 0.665±0.03 0.609±0.02 1.019±0.05 0.493±0.03 HES 1,366±0,03 1,366±0,02 0.608±0.03 0.628±0.02 1,240±0.09 0.529±0.02 BLM+HES 1,326±0,05 1,363±0,03 0.625±0.06 0.614±0.01 1,250±0.12 0.461±0.02 Table 4: Assessment of sperm motility, epididymal sperm concentration, and abnormal sperm rates Groups Parameters Sperm motility (%) Epididymal sperm concentration (million/g tissue) Abnormal sperm rate (%) Head Tail Total Control 92.15±2.06 b 334.28±6.34 b 3.85±0.26 b 3.00±0.21 b 6.85±0.34 BLM 66.69±3.54 a,c 234.85.±15,82 a,c,d 8.57±0.29 a,d 8.28±0.35 a,d 16.85±0.55 HES 93.09±2.09 c,d 342.85±8.08 b,d 3.00±0.43 d 3.00±0.37 d 6.00±0.72 BLM+HES 78,34±2.88 c 305.42±11.60 b,c 5.42±0.36 b,c 5.28±0.48 b,c 10,71±0.62 Mean ± standard deviation. a: vs. with Control group, b: vs. with BLM group, c: vs. with HES group, d: vs. with BLM+HES group. p<0.01 was accepted as statistically significant. Discussion BLM is an effective chemotherapeutic agent used in treating various cancers, including testicular cancer. However, its use is limited due to significant side effects, particularly on the male reproductive system. These side effects, including oxidative stress, histological damage, and sperm abnormalities, can impair fertility and overall reproductive health. Protecting testicular tissue and maintaining sperm quality are critical goals in mitigating BLM toxicity. The current study demonstrated that HES treatment significantly alleviated the toxic effects of BLM on the testicular tissue and sperm parameters. HES was shown to reduce oxidative stress, improve histological integrity, and enhance sperm quality when administered alongside BLM. Antineoplastic drugs such as BLM, disrupt the oxidant-antioxidant balance in testicular tissue, leading to oxidative damage. This imbalance manifests as lipid peroxidation, a key marker of cellular damage, and reductions in antioxidant defense mechanisms. 29,30 In the present study, BLM treatment significantly increased TBARS levels, a reliable indicator of lipid peroxidation, while markedly reducing antioxidant enzyme activities (SOD, CAT, GPx) and GSH levels. These findings are consistent with previous studies demonstrating the role of oxidative stress in BLM-induced toxicity in both lung and testicular tissues. 17,31,32 The testicular tissue is particularly vulnerable to oxidative damage due to its high polyunsaturated fatty acid content and reliance on antioxidant mechanisms for protection. 33 Our results confirm that BLM-induced oxidative imbalance contributes to structural and functional damage in the testes, including alterations in seminiferous tubules and germ cell degeneration. HES treatment effectively counteracted these effects by reducing TBARS levels and restoring antioxidant defenses. HES, a potent flavonoid with strong antioxidative properties, likely protects cell membranes by stabilizing fatty acid composition and reducing free radical production. These findings align with prior studies that reported the antioxidative effects of HES in preventing cisplatin- and ischemia-reperfusion-induced testicular damage. 34,35 BLM is widely recognized for its detrimental effects on testicular function and its ability to cause significant histological disruptions. 32,36 Although the histotoxicity of BLM alone on testicular tissue has not been extensively studied, its combination with other agents such as etoposide and cisplatin (commonly used in BEP therapy for testicular cancer) is known to cause significant structural damage. 37,38 Consistent with these findings, our study demonstrated that BLM treatment alone leads to notable histological alterations in the testis, including an increased percentage of abnormal tubules, reduced seminiferous tubular diameter, and decreased germinal layer thickness. Additionally, BLM induced maturation arrest, pronounced germ cell degeneration, spermatocytic arrest, intraepithelial spaces, and vacuolization. In contrast, co-administration of HES with BLM significantly mitigated these histological damages, restoring testicular structure closer to normal. Our findings also revealed that HES treatment alone did not result in histopathological changes in the testis, highlighting its safety profile. The histopathological damage observed in BLM-treated groups is likely attributable to the oxidative stress-induced imbalance between oxidant and antioxidant systems within testicular tissue. This imbalance not only compromises testicular integrity but also reduces sperm function, which can lead to male infertility. Therefore, the ability of HES to alleviate oxidative stress in the testis is particularly significant, as it not only protects testicular structure but also potentially improves fertility outcomes. Sperm abnormalities and impaired motility are well-documented side effects of chemotherapeutic agents, including BLM. 17,36 In this study, BLM administration significantly reduced sperm motility and concentration while increasing the percentage of abnormal sperm. However, no significant changes were observed in the weights of reproductive organs (testes, vesicular seminalis, and prostate), consistent with limited literature on this topic. HES co-administration reversed BLM-induced sperm abnormalities, improving motility and concentration while reducing abnormal sperm percentages. To the best of our knowledge, this is the first study to evaluate the protective effects of HES on BLM-induced sperm toxicity. These findings are supported by previous research demonstrating the protective role of HES in preserving male reproductive health against various toxicants. 34,35 Our results indicate that HES’s ameliorative effects on sperm parameters may be attributed to its antioxidative properties, which mitigate oxidative damage and restore cellular integrity in testicular tissue. By reducing free radical production and stabilizing antioxidant defenses, HES appears to play a crucial role in preserving sperm function and male fertility in the context of BLM toxicity. The findings of this study highlight the detrimental impact of BLM on the male reproductive system, emphasizing the importance of developing protective strategies to mitigate these effects. HES demonstrated significant potential as a protective agent, reducing oxidative stress, preserving testicular histology, and improving sperm quality. The novelty of this study lies in its demonstration of HES’s protective effects against BLM-induced testicular and spermatological damage. While previous studies have explored the antioxidative properties of HES in other contexts, this is the first to investigate its role in mitigating BLM-induced reproductive toxicity. These results provide a strong foundation for future research exploring the clinical applicability of HES as an adjunctive therapy in cancer treatment regimens involving BLM. Further research is needed to explore the molecular mechanisms behind HES’s protective effects and to evaluate its clinical efficacy. Studies focusing on fertility outcomes and potential interactions with other chemotherapeutic agents would enhance the translational potential of HES. While our thirty-day duration study effectively assessed acute responses, extending the duration could provide deeper insights into the sustained impacts of BLM and HES on reproductive health and sperm quality. Telegram @manmax90 buy cocaine in London In conclusion, this study demonstrated the toxic effects of BLM on the male reproductive system, characterized by increased oxidative stress, histological alterations, and impaired sperm parameters. HES treatment effectively alleviated these effects, likely through its antioxidative properties. These findings suggest that HES may serve as a promising therapeutic agent to protect male reproductive health during BLM therapy. Telegram @manmax90 buy cocaine in London Disclosure statement The authors do not declare any conflicts of interest/competing interests. Table List Table 1: Testicular levels of oxidative stress parameters (TBARS, GSH, CAT, SOD and GPx) 6 Table 2: Histopathological changes in testis tissues of rats 7 Table 3: Evaluation of testis, epididymis, seminal vesicles and prostate weights in rats 8 Table 4: Assessment of sperm motility, epididymal sperm concentration, and abnormal sperm rates 8 Figure List Figure 1: Normal testicular seminiferous tubules in the control and hesperidin groups 12 Figure 2: Histopathological alterations in the bleomycin group. 13 Figure 3: Bleomycin+hesperidin group, showing spermatogenesis. 13 Figure 1: Normal testicular seminiferous tubules in the control (A) and hesperidin (B) groups, with spermatogenesis and germinal cells (thin arrows). H-E; x20, scale bars: 100 µm (A and B). Figure 2: Histopathological alterations in the bleomycin group, including total aplasia and basal membrane thickening in the testicular seminiferous tubules (A), and maturation arrest, severe epithelial vacuolization, shrinkage, and tubular degeneration in Sertoli cells (B, thin arrow). A: H-E; x10, B: H-E; x20. Scale bars: 200 µm (A) and 100 µm (B). Figure 3: Bleomycin+hesperidin group, showing spermatogenesis (thin arrow), intraepithelial vacuolization, and degeneration (thick arrow) in seminiferous tubules (A and B). Presence of spermatogenesis (thin arrow) and intraepithelial vacuolization (thick arrow) in seminiferous tubules (C). Evidence of spermatogenesis and increased mitotic activity (thin arrow) (D). A, B and C: H-E; x40, D: H-E; x100. Scale bars: 50 µm (A, B and C) and 25 µm (D). Telegram @manmax90 buy cocaine in London REFERENCES 1. Mohammed SM, Al-Saedi HFS, Mohammed AQ, et al. Mechanisms of bleomycin-induced lung fibrosis: A review of therapeutic targets and approaches. Cell Biochemistry and Biophysics . 2024: 1-26. 2. Heydari-Bafrooei E, Amini M, Saeednia S. Electrochemical detection of DNA damage induced by Bleomycin in the presence of metal ions. Journal of Electroanalytical Chemistry . 2017; 803: 104-110. 3. Della Latta V, Cecchettini A, Del Ry S, Morales MA. Bleomycin in the setting of lung fibrosis induction: from biological mechanisms to counteractions. Pharmacological research . 2015; 97: 122-130. 4. Hay J, Shahzeidi S, Laurent G. Mechanisms of bleomycin-induced lung damage. Archives of toxicology . 1991; 65: 81-94. 5. Abidi A, Kourda N, Feki M, Ben Khamsa S. Protective effect of Tunisian flaxseed oil against bleomycin-induced pulmonary fibrosis in rats. Nutrition and cancer . 2020; 72(2): 226-238. 6. Kinnula VL, Fattman CL, Tan RJ, Oury TD. Oxidative stress in pulmonary fibrosis: a possible role for redox modulatory therapy. American journal of respiratory and critical care medicine . 2005; 172(4): 417-422. 7. Zini A, De Lamirande E, Gagnon C. Reactive oxygen species in semen of infertile patients: levels of superoxide dismutase‐and catalase‐like activities in seminal plasma and spermatozoa. International journal of andrology . 1993; 16(3): 183-188. 8. de Almeida AL, Fortuna A, Sousa M, Sá R. A systematic review of bleomycin-induced gonadotoxicity: mechanistic implications for male reproductive health and fertility. Reproductive Toxicology . 2024: 108721. 9. Kopp H-G, Kuczyk M, Classen J, et al. Advances in the treatment of testicular cancer. Drugs . 2006; 66: 641-659. 10. Ayhan İ, Turkmen NB, Taslidere A, Aydin M, Ciftci O. Protective Effect of Nerolidol on Paclitaxel-Induced Reproductive Toxicity in Rats: Oxidative Stress and Inflammation. Basic & clinical pharmacology & toxicology . 2025; 136(2): e14126. 11. Garg A, Garg S, Zaneveld L, Singla A. Chemistry and pharmacology of the citrus bioflavonoid hesperidin. Phytotherapy research . 2001; 15(8): 655-669. 12. Kim JY, Jung KJ, Choi JS, Chung HY. Hesperetin: a potent antioxidant against peroxynitrite. Free radical research . 2004; 38(7): 761-769. 13. Zhou Z, Kandhare AD, Kandhare AA, Bodhankar SL. Hesperidin ameliorates bleomycin-induced experimental pulmonary fibrosis via inhibition of TGF-beta1/Smad3/AMPK and IkappaBalpha/NF-kappaB pathways. EXCLI journal . 2019; 18: 723. 14. Park WS, Park MS, Kang SW, et al. Hesperidin shows protective effects on renal function in ischemia-induced acute kidney injury (Sprague-Dawley rats). Elsevier; 2019:2838-2841.15. Pari L, Shagirtha K. Hesperetin protects against oxidative stress related hepatic dysfunction by cadmium in rats. Experimental and toxicologic pathology . 2012; 64(5): 513-520. 16. Tveden-Nyborg P, Bergmann TK, Jessen N, Simonsen U, Lykkesfeldt J. BCPT 2023 policy for experimental and clinical studies. Basic & clinical pharmacology & toxicology . 2023; 133(4): 391-396. 17. Amirshahi T, Najafi G, Nejati V. Protective effect of royal jelly on fertility and biochemical parameters in bleomycin-‎ induced male rats. Iranian journal of reproductive medicine . 2014; 12(3): 209. 18. Çetin A, Çiftçi O, Otlu A. Protective effect of hesperidin on oxidative and histological liver damage following carbon tetrachloride administration in Wistar rats. Archives of medical science . 2016; 12(3): 486-493. 19. Türkmen NB, Yüce H, Taşlıdere A, et al. 18β-glycyrrhetinic acid attenuates global cerebral ischemia/reperfusion-induced cardiac damage in C57BL/J6 mice. Brazilian Journal of Pharmaceutical Sciences . 2023; 58: e21219. 20. Yagi K. Simple assay for the level of total lipid peroxides in serum or plasma. Free radical and antioxidant protocols . 1998: 101-106. 21. Sedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Analytical biochemistry . 1968; 25: 192-205. 22. Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clinical chemistry . 1988; 34(3): 497-500. 23. Aebi H. Catalase. Methods of enzymatic analysis . Elsevier; 1974:673-684.24. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. The Journal of laboratory and clinical medicine . 1967; 70(1): 158-169. 25. Başak Türkmen N, Ayhan I, Taşlıdere A, Aydın M, Ciftci O. Investigation of protective effect of ellagic acid in phthalates-induced reproductive damage. Drug and Chemical Toxicology . 2022; 45(4): 1652-1659. 26. Ciftci O, Beytur A, Cakir O, Gurbuz N, Vardi N. Comparison of reproductive toxicity caused by cisplatin and novel platinum‐N‐heterocyclic carbene complex in male rats. Basic & clinical pharmacology & toxicology . 2011; 109(5): 328-333. 27. Karagoz MA, Doluoglu OG, Ünverdi H, et al. The protective effect of Papaverine and Alprostadil in rat testes after ischemia and reperfusion injury. International braz j urol . 2018; 44(3): 617-622. 28. Mirhoseini M, Amiri FT, Malekshah AAK, Gatabi ZR, Ghaffari E. Protective effects of melatonin on testis histology following acute torsion-detorsion in rats. International Journal of Reproductive Biomedicine . 2017; 15(3): 141. 29. Nagai K, Fukuno S, Oda A, Konishi H. Protective effects of taurine on doxorubicin-induced acute hepatotoxicity through suppression of oxidative stress and apoptotic responses. Anti-cancer drugs . 2016; 27(1): 17-23. 30. Peng X, Gandhi V. ROS-activated anticancer prodrugs: a new strategy for tumor-specific damage. Therapeutic delivery . 2012; 3(7): 823-833. 31. Beigh S, Rashid H, Sharma S, Parvez S, Raisuddin S. Bleomycin-induced pulmonary toxicopathological changes in rats and its prevention by walnut extract. Biomedicine & Pharmacotherapy . 2017; 94: 418-429. 32. Kilic T, Ciftci O, Cetin A, Kahraman H. Preventive effect of chrysin on bleomycin-induced lung fibrosis in rats. Inflammation . 2014; 37: 2116-2124. 33. Chainy G, Samantaray S, Samanta L. Testosterone‐induced changes in testicular antioxidant system. Andrologia . 1997; 29(6): 343-349. 34. Celik E, Oguzturk H, Sahin N, Turtay MG, Oguz F, Ciftci O. Protective effects of hesperidin in experimental testicular ischemia/reperfusion injury in rats. Archives of Medical Science . 2016; 12(5): 928-934. 35. Kaya K, Ciftci O, Cetin A, Doğan H, Başak N. Hesperidin protects testicular and spermatological damages induced by cisplatin in rats. Andrologia . 2015; 47(7): 793-800. 36. Kilarkaje N, Al-Bader M. Effects of antioxidants on drugs used against testicular cancer-induced alterations in metastasis-associated protein 1 signaling in the rat testis. Toxicology and Industrial Health . 2016; 32(1): 89-97. 37. Bagheri-Sereshki N, Hales BF, Robaire B. The effects of chemotherapeutic agents, bleomycin, etoposide, and cisplatin, on chromatin remodeling in male rat germ cells. Biology of reproduction . 2016; 94(4): 81, 1-9. 38. Ishihara M, Satoh K, Hanaoka M, et al. Cerebral venous sinus thrombosis following cisplatin-based chemotherapy for testicular tumor. No Shinkei geka Neurological Surgery . 2017; 45(5): 417-422. Information & Authors Information Version history V1 Version 1 24 January 2025 Peer review timeline Published Basic & Clinical Pharmacology & Toxicology Version of Record 23 Sep 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Basic & Clinical Pharmacology & Toxicology Authors Affiliations İdris AYHAN 0000-0003-4625-7218 [email protected] Pamukkale Universitesi View all articles by this author Nese BASAK TURKMEN Inonu Universitesi View all articles by this author Saadet ALAN Ankara Dr Abdurrahman Yurtaslan Onkoloji Egitim ve Arastirma Hastanesi View all articles by this author Muhterem AYDIN Firat Universitesi View all articles by this author Osman Ciftci Pamukkale Universitesi View all articles by this author Metrics & Citations Metrics Article Usage 375 views 193 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation İdris AYHAN, Nese BASAK TURKMEN, Saadet ALAN, et al. Hesperidin Mitigates Bleomycin-Induced Testicular and Spermatological Damage in Rats. Authorea . 24 January 2025. DOI: https://doi.org/10.22541/au.173771157.71203226/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . Format Please select one from the list RIS (ProCite, Reference Manager) EndNote BibTex Medlars RefWorks Direct import Tips for downloading citations document.getElementById('citMgrHelpLink').addEventListener('click', function() { popupHelp(this.href); return false; }); $(".js__slcInclude").on("change", function(e){ if ($(this).val() == 'refworks') $('#direct').prop("checked", false); $('#direct').prop("disabled", ($(this).val() == 'refworks')); }); View Options View options PDF View PDF Figures Tables Media Share Share Share article link Copy Link Copied! Copying failed. Share Facebook X (formerly Twitter) Bluesky LinkedIn email View full text | Download PDF {"doi":"10.22541/au.173771157.71203226/v1","type":"Article"} Now Reading: Share Figures Tables Close figure viewer Back to article Figure title goes here Change zoom level Go to figure location within the article Download figure Toggle share panel Toggle share panel Share Toggle information panel Toggle information panel Go to previous graphic Go to next graphic Go to previous table Go to next table All figures All tables View all material View all material xrefBack.goTo xrefBack.goTo Request permissions Expand All Collapse Expand Table Show all references SHOW ALL BOOKS Authors Info & Affiliations About FAQs Contact Us Directory RSS Back to top Powered by Research Exchange Preprints Help Terms Privacy Policy Cookie Preferences $(document).ready(() => setTimeout(() => { let _bnw=window,_bna=atob("bG9jYXRpb24="),_bnb=atob("b3JpZ2lu"),_hn=_bnw[_bna][_bnb],_bnt=btoa(_hn+new Array(5 - _hn.length % 4).join(" ")); $.get("/resource/lodash?t="+_bnt); },4000)); (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'a00faa224f6806fb',t:'MTc3OTY2MDk4NA=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();