Sage Journals: Discover world-class research

Abstract

Introduction

Doxorubicin (DXR), a chemotherapeutic antibiotic, is widely used as an anticancer drug in clinics. Grape seed extract is known for its potent antioxidant properties. The aim of this study is to investigate the effect of high-antioxidant content Vitis vinifera L. seed extract against DXR-induced testicular and epididymal damage.

Methods

30 male rats were randomly divided into five groups with six animals in each group: Control, Sham, DXR (a single i.p. dose of 15 mg/kg), DXR + VIT (120 mg/kg VIT seed extract via gavage for 14 days and a single i.p. dose of DXR (15 mg/kg) on day 5, VIT (120 mg/kg VIT seed extract via gavage for 14 days). Animals were sacrificed under anesthesia 24 hours after the last drug administration, and blood, testis, and epididymis tissues were collected.

Results

Tissues from the DXR group exhibited atrophic seminiferous tubules, Leydig cell degeneration, tunica albuginea and basal membrane thinning, immature spermatogenic cells, vascular congestion, epididymal atrophy, epithelial cell deletion, decreased sperm count, increased connective tissue, and absence of sperm in the lumen. Serum levels of interleukin 6 (IL-6), interleukin 1β (IL-1β), Tumor Necrosis Factor α (TNF-α), Total Oxidant Status (TOS), Total Antioxidant Status (TAS), and testosterone were increased in the DXR group, while interleukin 10 (IL-10) levels were decreased. The DXR + VIT group showed a near-recovery similar to the control.

Conclusion

DXR increased oxidative stress, apoptosis, and inflammation in the testis and epididymis, whereas VIT exhibited protective effects against these damages.

Keywords

Doxorubicin Vitis vinifera L testes epididymis

Introduction

Cancer is a significant public health issue that causes serious mortality rates worldwide.¹ In 2020, 19.3 million new cancer cases were diagnosed globally, and 10 million cancer-related deaths were recorded.² Various methods are used in cancer treatment, with chemotherapy being the most commonly used. Advances in chemotherapy and chemotherapeutic drugs have increased the life expectancy of cancer patients³; however, the long-term use of anti-tumor drugs has serious toxic effects, which restrict their clinical applications.⁴

Doxorubicin (DOX) is a widely used anticancer drug in clinical practice. However, its dose-dependent toxicity limits its clinical use. Until recently, most studies have focused on DOX-induced cardiotoxicity, hepatotoxicity, and nephrotoxicity. However, there are gaps in our knowledge regarding the adverse effects of DOX on the reproductive system. Experimental studies have shown that DOX leads to infertility by altering sperm production, development, and structural integrity, reducing sperm motility and count.⁵

It has been reported that DOX causes toxicity in various tissues, including the liver and kidneys.⁶ Studies have particularly demonstrated that DOX causes damage to the testes, which are structures of the male reproductive system.⁷ Cancer treatment can lead to decreased testosterone levels and may also result in compensatory damage in the hypothalamic-pituitary-gonadal axis and Sertoli cells.⁸

DOX induces excessive production of free radicals, such as nitric oxide and reactive oxygen species, and increases lipid peroxidation. Consequently, damage occurs in the gonads of both males and females.⁹ Spermatogonia in the seminiferous tubules of the testes are more affected by these drugs, leading to a decrease in sperm count and infertility.¹⁰

Vitis vinifera L. is a perennial plant from the Vitaceae family, commonly used for grape and wine production. Grape seed oil is rich in fatty acids such as linoleic acid (65%–75%), vitamin E (50 mg), and phytosterols, in addition to phenolic compounds like catechins (414 mg), epicatechins (130.4 mg), and gallic acid (77 µg). It is promising as a nutritional compound and is important as a therapeutic agent with active properties for health, as identified primarily through in vitro studies and some in vivo research. The benefits of consuming this oil include regulating the expression of antioxidant enzymes, anti-atherosclerotic and anti-inflammatory effects, and protection against oxidative cell damage and certain types of cancer.¹¹

In one study, the protective effects of grape seed proanthocyanidin extract on cadmium-induced testicular apoptosis, endothelial nitric oxide synthase (eNOS) expression, and toxicity in rats were investigated. The study found that in the testis tissue of the cadmium group treated with grape seed proanthocyanidin extract, there was a reduction in Tunnel staining and eNOS expression. This indicates that grape seed proanthocyanidin extract has a strong protective effect against cadmium-induced testicular toxicity in rats.¹²

In another study examining the toxic effects of formaldehyde on testicular tissue and the protective effects of grape seed extract against these toxic effects, it was found that the SOD and TAS values in formaldehyde-exposed rats did not significantly decrease compared to the control group and the grape seed extract-treated group. However, testosterone levels were statistically significantly higher in the grape seed extract-treated group compared to the control group. Additionally, it was noted that formaldehyde exposure led to a reduction in interstitial edema and expansion of the seminiferous tubule lumen, with the number of germ cells within the seminiferous tubules being close to normal.¹³

The aim of this study is to investigate the effect of grape (Vitis vinifera L.) seed extract against testicular and epididymal damage induced by doxorubicin in rats.

Materials and Methods

Ethical approval

Ethical approval for the study was obtained from the Afyon Kocatepe University Local Ethics Committee for Animal Experiments, with reference number AKUHADYEK-11-23 and decision number 49533702/13, dated 27.02.2023. All experiments were performed in accordance with relevant named guidelines and regulations. All authors complied with the ARRIVE guidelines.

Experiment animals and their features

The rats will be kept at a temperature between 21 and 22°C in a 12-h light and 12-h dark environment. An ad libitum feeding regimen will be applied. The study will use 30 adult male Wistar albino rats. The 30 rats will be randomly divided into five groups, with six rats in each group. The characteristics of the groups are shown in Table 1. Experiments were reported in accordance with the ARRIVE guidelines.

Table 1.

Experimental groups and their characteristics.

Groups	Group Information
Group 1 (control)	Group fed with ad libitum water and food
Group 2 (Sham)	Group receiving distilled water via gavage for 14 days, with intraperitoneal (i.p.) SF (serum physiologic) administration on the 5th day of the experiment
Group 3 (DXR)	Group consisting of rats administered a single dose of 15 mg/kg i.p. Doxorubicin (DXR) on the 5th day of the experiment^a
Group 4 (DXR + VIT)	Group consisting of rats that will receive 120 mg/kg Vitis vinifera L. (VIT)^b seed extract via gavage for 14 days, and a single dose of 15 mg/kg i.p. Doxorubicin (DXR) on the 5th day of the experiment^a
Group 5 (VIT)	Group consisting of rats that will receive 120 mg/kg Vitis vinifera L. (VIT) seed extract via gavage for 14 days^b

^aReference for the DXR dose determination: (M. El-Moselhy & El-Sheikh, 2014).

^bReference for the VIT dose determination: (Evcimen et al., 2020).

^cReferences for determining the number of animals: (Dogan & Dogan, 2020; Festing, 2006).

Experimental procedure

24 hours after the final drug administration, the animals were sacrificed under anesthesia with Ketamine (90 mg/kg) / Xylazine (8-10 mg/kg), and tissue and blood samples were collected. Blood samples for biochemical analysis were centrifuged at 3500 RPM. Serum samples were stored at −80°C for biochemical analyses.

Histochemical analysis

Testis and epididymis tissues were fixed in Bouin’s solution. After 24 hours of fixation, the tissues were washed several times with 70% alcohol. The tissues were dehydrated through a series of increasing alcohol concentrations (70%, 80%, 96%, 100%) and cleared in xylene. The cleared testis and epididymis tissues were embedded in paraffin and sectioned at a thickness of 5 µm using a microtome. The sections obtained were stained with Hematoxylin-Eosin (HE), Periodic Acid Schiff (PAS), and Martius Scarlet Blue (MSB). The stained sections were examined and evaluated using a photomicroscope (Eclipse E−600 Nikon, Japan) and an image analysis system (NIS Elements Nikon, Japan), and photographs were taken. A semi-quantitative evaluation was performed based on the degree and extent of staining as follows: 0 = normal, 1 = mild, 2 = moderate, and 3 = severe.¹⁴

Immunohistochemical analysis

After paraffin blocks were sectioned to a thickness of 5 µm, the sections were deparaffinized and boiled in citrate buffer solution for 15 minutes. Following treatment with H2O2 (TA-060-HP, Lab. Vision Corporation, USA) and Ultra V Block (TA-125-UB, Thermo Scientific, UK) solutions for 5 minutes, the sections were incubated with antibodies against HSP 70 (sc-32,239, Santa Cruz, USA), NF-κB-p65 (E-AB-32,233, Elabscience, USA), TNF-α (sc-52,746, Santa Cruz, USA), and Caspase-3 (E-AB-66,940, Elabscience, USA). Subsequently, the sections were incubated with a secondary antibody (Biotinylated Goat Anti-Polyvalent TP-125-BN, Thermo Scientific, UK) and Streptavidin HRP (Horse Radish Peroxidase) (TS-125-HR, Thermo Scientific, UK) at room temperature for 30 minutes. After incubation with DAB (3,3′-diaminobenzidine) (TA-125 HD, Thermo Scientific, UK), the tissues were counterstained with Mayer’s hematoxylin and mounted with Entellan (HX85172161, Merck, Germany). All preparations were examined and evaluated using a photomicroscope (Eclipse E−600 Nikon, Japan) and an image analysis system (NIS Elements Nikon, Japan), and photographs were taken. Based on the degree and extent of immunostaining, a semi-quantitative evaluation was performed as follows: 0 = normal, 1 = mild, 2 = moderate, and 3 = severe.¹⁴

Biochemical analysis

Rat blood samples were placed in additive-free chemistry tubes and centrifuged at 3500 rpm for 10 minutes. After centrifugation, blood serum samples were taken. Serum levels of IL-6, IL-10, IL-1β, and TNF-α were measured using ELISA with BT-LAB (Bioassay Technology Laboratory, Zhejiang, China) kits. Serum Total Oxidant Status (TOS) and Total Antioxidant Status (TAS) measurements were performed using Rel Assay Diagnostics TOS and TAS measurement kits (Mega Tıp San ve Tic Ltd Şti, Şahinbey/Gaziantep/Turkey). Absorbance readings were taken using a Thermo Scientific Multiskan FC ELISA reader (Thermo Fisher Scientific (Shanghai) Instruments Co. Ltd, Qin Qiao Road, PuDong, Shanghai, China). Results were reported as ng/L for IL-6 and TNF-α, pg/ml for IL-10, and ng/ml for IL-1β; and as µmol/L for TOS and mmol/L for TAS.¹⁵

Statistical analysis

Biochemical, histochemical, and immunohistochemical results were analyzed using GraphPad Prism (v.6.0) and one-way ANOVA with the SPSS v. 24 (SPSS Inc., Chicago, IL, USA) statistical software. Tukey’s multiple comparison test was used to analyze differences between group means. Values are presented as mean ± SD, and p < 0.05 was considered statistically significant.

Results

Histopathological Examination Results of testis tissue

Normal testis histology is observed in the Control, Sham, and VIT groups (a1, a2, a3, b1, b2, b3, e1, e2, e3). In the DXR group, normal-appearing seminiferous tubules (red star) are present alongside atrophic seminiferous tubules (black star). There is also separation and disruption in the interstitial area and Leydig cells (green arrowhead) (c1). Notable features include thinning and separation of the tunica albuginea (thick green arrow), immature Sertoli cells (blue star), and vascular congestion (yellow star) (c2). Thinning and invagination of the basal membrane (thin red arrow) are significant findings in the DXR group (c3). In the treatment group, DXR + VIT, significant improvement is observed (d1, d2, d3) (Figure 1).

Figure 1.

Microscopic Images of Testis Tissue. Thick Red Arrow: Spermatogonium, Thick Black Arrow: Primary Spermatocyte, Thick Blue Arrow: Secondary Spermatocyte, Red Arrowhead: Sertoli Cell, Blue Arrowhead: Early Stage Spermatid, Black Arrowhead: Late Stage Spermatid, Green Arrowhead: Leydig Cell Cluster, Black Star: Sperm Cluster in Lumen, Thick Green Arrow: Tunica Albuginea, Thin Red Arrow: Basal Membrane. a1, b1, e1 Scale bar = 50 µm, x 400; c1, d1 Scale bar = 100 µm, x 200; Hematoxylin-Eosin (HE). a2, b2, c2, d2, e2 Scale bar = 100 µm, x 200; Martius Scarlet Blue (MSB). a3, b3, d3, e3 Scale bar = 100 µm, x 200; e3 Scale bar = 50 µm, x 400; PAS (Periodic Acid–Schiff).

Histopathological examination results of Epididymis Tissue

Normal epididymis histology is observed in the Control, Sham, and VIT groups (A1, B1, E1). In the DXR group, atrophic epididymis (red star), deletion in epithelial cells (thick red arrow), decreased and separated stereocilia (thick black arrow) were observed (C1). Additionally, increased connective tissue (red arrowhead) and absence of sperm in the lumen (green star) were noted (C2). Separation and disruption of the basal membrane were significant findings (C3). In the DXR + VIT group, a nearly normal appearance was observed, indicating that the treatment had a positive effect (D1, D2, D3) (Figure 2).

Figure 2.

Microscopic Images of Epididymis Tissue. Thick Black Arrow: Stereocilia, Black Arrowhead: Basal Cell, Green Arrowhead: Columnar Cell, Black Star: Sperm Cluster in Lumen, Green Star: Lumen Without Sperm, Red Star: Immature Epididymis, Thin Red Arrow: Dense Connective Tissue. A1, B1, C1, D1, E1 Scale bar = 100 µm, x 200; Hematoxylin-Eosin (HE). A2, B2, C2, D2, E2 Scale bar = 100 µm, x 200; Martius Scarlet Blue (MSB). A3, C3 Scale bar = 100 µm, x 200; B3, D3, E3 Scale bar = 50 µm, x 400; PAS (Periodic Acid–Schiff).

Immunohistochemical results

Immunohistochemical examination results of HSP 70, NF-κB-p65, TNF-α, and Caspase-3 in Testis Tissue

In the testis tissue of the Control, Sham, and VIT groups, no immunoreactivity for HSP 70, NF-κB-p65, TNF-α, and Caspase-3 was observed. In the DXR group, there was intense (+++) immunoreactivity for HSP 70, NF-κB-p65, TNF-α, and Caspase-3 in spermatogenic series cells (red star). Notably, strong staining was observed particularly in late-stage spermatids and spermatozoa (red arrow). Additionally, no immunoreactivity for HSP 70, NF-κB-p65, TNF-α, and Caspase-3 was seen in the interstitial area and Leydig cells (green arrowhead). In the treatment group, DXR + VIT, mild (+) immunoreactivity for HSP 70, NF-κB-p65, TNF-α, and Caspase-3 in spermatids and spermatozoa (red arrow) was a notable finding (Figures 3 and 5).

Figure 3.

Microscopic Images of Testis Tissue Showing Immunoreactivity for HSP 70, NF-κB-p65, TNF-α, and Caspase-3 in Control, Sham, DXR, DXR + VIT, and VIT Groups. Red Star: Spermatogenic series cells, Red Arrow: Spermatids and spermatozoa, Green Arrowhead: Leydig cell cluster, Negative Control (b1, b2, b3, and b4), HSP 70 (a1, c1, d1, e1, f1), NF-κB-p65 (a2, c2, d2, e2, f2), TNF-α (a3, c3, d3, e3, f3), Caspase-3 (a4, c4, d4, e4, f4), DAB x 40, Scale bar = 50 µm.

Immunohistochemical Examination Results of HSP 70, NF-κB-p65, TNF-α, and Caspase-3 in epididymis tissue

In the epididymis tissue of the Control, Sham, and VIT groups, no immunoreactivity for HSP 70, NF-κB-p65, TNF-α, and Caspase-3 was observed. In the DXR group, intense (+++) immunoreactivity for HSP 70, NF-κB-p65, TNF-α, and Caspase-3 was seen in the epididymal epithelium consisting of basal and columnar cells (red arrow). Additionally, strong (+++) immunoreactivity for these markers was noted in the interstitial area and where dense connective tissue is present (red star). In the treatment group, DXR + VIT, mild (+) immunoreactivity for HSP 70, NF-κB-p65, TNF-α, and Caspase-3 in both the epithelium and connective tissue areas was observed (Figures 4 and 5).

Figure 4.

Microscopic Images of Epididymis Tissue Showing Immunoreactivity for HSP 70, NF-κB-p65, TNF-α, and Caspase-3 in Control, Sham, DXR, DXR + VIT, and VIT Groups. Red Arrow: Epididymal epithelium consisting of columnar and basal cells, Red Star: Interstitial area and dense connective tissue, Negative Control (B1, B2, B3, and B4), HSP 70 (A1, C1, D1, E1, F1), NF-κB-p65 (A2, C2, D2, E2, F2), TNF-α (A3, C3, D3, E3, F3), Caspase-3 (A4, C4, D4, E4, F4), DAB x 40, Scale bar = 50 µm.

Figure 5.

Immunohistochemical findings scores for HSP 70, NF-κB-p65, TNF-α, Caspase-3, on testis and epididymis tissues. DXR: Doxorubicin, VIT: Vitis vinifera L. “*” indicates p < 0.05, “**” indicates p < 0.01, “***” indicates p < 0.001.

Biochemical results

Serum levels of Interleukin 6 (IL-6), Interleukin 10 (IL-10), Interleukin 1β (IL-1β), Tumor Necrosis Factor α (TNF-α), Total Oxidant Status (TOS), and Total Antioxidant Status (TAS) were examined and evaluated. When comparing IL-1β, IL-6, and TNF-α levels between the Control group and the DXR group, a significant difference was observed (p < 0.05). Additionally, a significant difference was found between the DXR and the treatment group, DXR + VIT (p < 0.001). IL-10 levels were significantly reduced in the DXR group compared to the Control group (p < 0.05), whereas they were increased in the DXR + VIT group (p < 0.001). This result suggests that VIT exhibits anti-inflammatory effects in response to DXR-induced inflammation. TOS levels were higher in the DXR group, but significantly decreased in the DXR + VIT group (p < 0.001). TAS levels were highest in the VIT group (p < 0.001). Furthermore, the DXR + VIT group showed a significant increase in TAS levels compared to the DXR group (p < 0.001). This indicates that VIT has antioxidant properties. Serum testosterone levels were significantly lower in the DXR group compared to the Control group (p < 0.05). Conversely, they were significantly higher in the DXR + VIT group (p < 0.05). This result demonstrates that VIT helps to re-regulate and increase serum testosterone levels (Figure 6).

Figure 6.

The biochemical results of interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), total oxidant status (TOS), and total antioxidant status (TAS) in serum. DXR: Doxorubicin, VIT: Vitis vinifera L. “*” indicates s p < 0.05, “**” indicates p < 0.01, “***” indicates p < 0.001.

Discussion

Cancer is known to be the second leading cause of death worldwide, following heart diseases.¹⁶ Cytotoxic drugs are used in cancer treatment, but their mechanisms of action are not fully understood. Studies have shown that these drugs, in particular, cause toxicity to the reproductive system.^17,18

Doxorubicin (DXR) is a potent and effective anti-neoplastic drug that causes damage to the male reproductive system, particularly testicular tissue, and leads to infertility.¹⁹ DXR has been shown to significantly increase levels of inflammatory markers such as TNF-α, IL-1β, NF-κB, and IL-6. Inflammatory markers in the testicular tissue of DXR-treated rats were elevated. Inflammation is one of the primary causes of male infertility.²⁰ NF-κB activation is crucial for the expression of pro-inflammatory cytokines (IL-6, TNF-α, COX-2, and IL-1β) associated with severe inflammation and other ROS-related disorders . In our study, serum parameters of rats treated with DXR showed significant increases in TNF-α, IL-1β, and NF-κB levels. Additionally, when DXR and VIT were administered together, VIT’s anti-inflammatory effects led to a significant reduction in inflammatory indices.

A study has shown that DXR significantly increases the tubular lumen diameter and interstitial spaces; and decreases the seminiferous tubular diameter, epithelial height, tunica propria thickness, and germ cell count.^20,21 Our histopathological findings also revealed atrophic structures in the seminiferous tubules and degeneration in spermatogenic series cells, consistent with this study. It has been reported that DXR reduces testosterone levels, which in turn decreases the germ cell count and causes histopathological damage.²¹ In our study, serum testosterone levels were significantly reduced in the DXR group but increased in the DXR + VIT group, confirming that VIT regulates DXR-induced damage. An imbalance between pro-oxidants and antioxidants results in excessive production of ROS, leading to histopathological damage. Reduction in testosterone levels is one of the main causes of failed spermatogenesis.²² Our study indicates that the DXR + VIT group exhibited regulation of testicular and epididymal damage due to the anti-apoptotic, antioxidant, anti-inflammatory, and androgenic properties of VIT.

Excessive ROS production disrupts sperm plasma membrane fluidity, leading to the loss of fatty acids. This damages the structural integrity of the sperm plasma membrane and thus impairs sperm-egg fusion.²³ Excessive ROS can lead to infertility by reducing sperm motility and increasing sperm apoptosis.²⁴

Although the exact mechanism by which DXR induces testicular toxicity is not fully understood,²⁵ studies have indicated that DXR leads to increased oxidative stress, resulting in lipid peroxidation and cellular apoptosis.²⁶ Caspases, a group of cysteine-aspartic protease enzymes, are normally inactive in the cytoplasm and organelles. Caspase-3 plays a crucial role in initiating and executing cellular apoptosis.²⁷ The increased level of cytochrome C, which leads to cell death or apoptosis, is activated by Caspase-3.²⁸ Our study observed that DXR increased apoptosis, but VIT administration reduced Caspase-3 expression due to its anti-apoptotic properties.

In this study, the DOX group exhibited atrophy in some seminiferous tubules, degeneration of spermatogenic cells, clotting in the luminal content of seminiferous tubules, and abnormal spermatids in the lumen of seminiferous tubules.²⁹ Our immunohistochemical results indicated a significant increase in HSP 70, NF-κB-p65, and TNF-α immunoreactivities in the DXR group. This suggests that DXR increases oxidative stress and inflammation. Our results are consistent with previous studies.

Another study demonstrated that DXR increases inflammatory cytokines, particularly interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the testis.³⁰ Additionally, it has been reported that DXR disrupts the antioxidant/pro-oxidant balance by affecting catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GPx).³¹ The decrease in serum testosterone levels, along with biochemical parameters, disrupts the hypothalamic-pituitary-gonadal axis and affects multiple organ systems.³² Our study has also revealed findings similar to those of other studies. The decrease in serum testosterone levels in the DXR group, particularly through the impact on the hypothalamus-pituitary-gonadal axis, suggests that it may lead to serious long-term damage to the reproductive system. Additionally, the study demonstrated that Vitis vinifera L. has protective effects against oxidative stress, inflammation, and apoptosis, and that it regulates the damage induced by DXR, protecting both the testis and epididymis.

Conclusions

Our study has demonstrated that grape (Vitis vinifera L.) seed extract has a therapeutic effect against DXR-induced testicular and epididymal damage, as shown through histochemical, immunohistochemical, and biochemical assessments. We anticipate that while DXR increases oxidative stress, apoptosis, and inflammation, VIT could be used as a protective agent against these damages. Although our research findings are significant, further studies are needed to explore the effects on the testis and epididymis in more detail.

Footnotes

Author contributions

ES: Design and execution of experiments, including the writing of protocols, conducting histochemical and immunohistochemical analyses, interpretation of results, and documentation of findings. HBK: Execution of biochemical analyses, including interpretation of data and documentation of results.All Authors read and approved the final version of the manuscript. The authors declare that all data were generated in-house and that no paper mill was used.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Scientific Research Projects Coordination Unit of Afyonkarahisar Health Sciences University 23.KARIYER.007.

ORCID iD

Emine Sarman

References

Mirzaei

Gholami

Zabolian

, et al. Resveratrol augments doxorubicin and cisplatin chemotherapy: a Novel therapeutic strategy. Curr Mol Pharmacol 2023; 16(3): 280–306.

Sung

Ferlay

Siegel

, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA A Cancer J Clin 2021; 71(3): 209–249.

Gatta

Botta

Rossi

, et al. Childhood cancer survival in Europe 1999-2007: results of EUROCARE-5--a population-based study. Lancet Oncol 2014; 15(1): 35–47.

Cao

Liu

Jia

, et al. Rare earth fluorescent nanoprobes with minimal side effects enable tumor microenvironment activation for chemotherapy. J Rare Earths 2022; 42(2): 256–262.

Afsar

Razak

Almajwal

, et al. Doxorubicin-induced alterations in kidney functioning, oxidative stress, DNA damage, and renal tissue morphology; Improvement by Acacia hydaspica tannin-rich ethyl acetate fraction. Saudi J Biol Sci 2020; 27(9): 2251–2260.

Song

Chu

Liang

, et al. Protective effects of dioscin against doxorubicin-induced hepatotoxicity via regulation of Sirt1/FOXO1/NF-κb signal. Front Pharmacol 2019; 1: 2.

Shati

Khalil

. Acylated ghrelin suppresses doxorubicin-induced testicular damage and improves sperm parameters in rats via activation of Nrf2 and mammalian target of rapamycin. J Cancer Res Therapeut 2023; 19(5): 1194–1205.

Ghafouri-Fard

Shoorei

Abak

, et al. Effects of chemotherapeutic agents on male germ cells and possible ameliorating impact of antioxidants. Biomed Pharmacother 2021; 142: 112040.

Çeribaşı

Sakin

Türk

, et al. Impact of ellagic acid on adriamycin-induced testicular histopathological lesions, apoptosis, lipid peroxidation and sperm damages. Exp Toxicol Pathol 2012; 64(7-8): 717–724.

10.

Meistrich

. Relationship between spermatogonial stem cell survival and testis function after cytotoxic therapy. Br J Cancer Suppl 1986; 7: 89–101.

11.

Di Pietro Fernandes

CSL

Dos Santos

Fernandes

DSHP

, et al. Nutraceutical potential of grape (Vitis vinifera L.) seed oil in oxidative stress, inflammation, obesity and metabolic alterations. Molecules 2023; 28(23): 7811.

12.

Sönmez

Tascioglu

. Protective effects of grape seed extract on cadmium-induced testicular damage, apoptosis, and endothelial nitric oxide synthases expression in rats. Toxicol Ind Health 2016; 32(8): 1486–1494.

13.

Uysal

Can

Özcan

. Protective effect of grape seed extract (Vitis vinifera) on the damage of formaldehyde to rat testes. Balıkesir Sağlık Bilim Derg 2022; 11(3): 487–494.

14.

Sarman

Gulle

Ilhan

. Histochemical, immunohistochemical, and biochemical investigation of the effect of resveratrol on testicular damage caused by methotrexate (MTX). Reprod Sci 2023; 30(11): 3315–3324.

15.

Savas

Gultekin

. Positive effects of meal frequency and calorie restriction on antioxidant systems in rats. North Clin Istanb 2017; 26(4): 109–116.

16.

Rhee

Joseph

Pandit

, et al. Increasing trauma deaths in the United States. Ann Surg 2014; 260(1): 13–21.

17.

Patel

Kaufmann

. How does doxorubicin work? Elife 2012; 18(1): e00387.

18.

Pont

Albrecht

. Fertility after chemotherapy for testicular germ cell cancer. Fertil Steril 1997; 68(1): 1–5.

19.

Ijaz

Yaqoob

Hamza

, et al. Apigetrin ameliorates doxorubicin prompted testicular damage: biochemical, spermatological and histological based study. Sci Rep 2024; 14(1): 9049.

20.

Dutta

Sengupta

Slama

, et al. Oxidative stress, testicular inflammatory pathways, and male reproduction. Int J Mol Sci 2021; 22(18): 10043.

21.

Chouhan

Yadav

Prakash

, et al. Increase in the expression of inducible nitric oxide synthase on exposure to bisphenol A: a possible cause for decline in steroidogenesis in male mice. Environ Toxicol Pharmacol 2015; 39(1): 405–416.

22.

Jin

Sha

, et al. Polystyrene microplastics induced male reproductive toxicity in mice. J Hazard Mater 2021; 5(401): 123430.

23.

Dutta

Majzoub

Agarwal

. Oxidative stress and sperm function: a systematic review on evaluation and management. Arab J Urol 2019; 17(2): 87–97. DOI: 10.1080/2090598X.2019.1599624.

24.

Bisht

Faiq

Tolahunase

, et al. Oxidative stress and male infertility. Nat Rev Urol 2017; 14(8): 470–485.

25.

Rizk

Zaki

Mina

. Propolis attenuates doxorubicin-induced testicular toxicity in rats. Food Chem Toxicol 2014; 67: 176–186.

26.

Trivedi

Tripathi

Jena

. Hesperetin protects testicular toxicity of doxorubicin in rat: role of NFκB, p38 and caspase-3. Food Chem Toxicol 2011; 49: 838–847.

27.

Jin

Zhu

, et al. TBK1 suppresses RIPK1-driven apoptosis and inflammation during development and in aging. Cell 2018; 174(6): 1477–1491.

28.

Lindqvist

Heinlein

Huang

, et al. Prosurvival Bcl-2 family members affect autophagy only indirectly, by inhibiting Bax and Bak. Proc Natl Acad Sci USA 2014; 111(23): 8512–8517.

29.

Alafifi

Wahdan

Elhemiely

, et al. Modulatory effect of liraglutide on doxorubicin-induced testicular toxicity and behavioral abnormalities in rats: role of testicular-brain axis. Arch Pharmacol. 2023;396(11):2987–3005.

30.

Kabel

Mahmoud

Kholy

. Ameliorative potential of gemfibrozil and silymarin onexperimentally induced nephrotoxicity in rats. 19. Afr J Urol 2013; 19(4): 171–178.

31.

Fouad

Refaie

Abdelghany

. Naringenin palliates cisplatin and doxorubicin gonadal toxicity in male rats. Toxicol Mech Methods 2019; 29(1): 67–73.

32.

Ahles

Saykin

. Candidate mechanisms for chemotherapy-induced cognitive changes. Nat Rev Cancer. 2007; 7(3): 192–201. https://doi.org/10.1038/nrc2073

Effect of grape seed extract on doxorubicin-induced testicular and epididymal damage in rats

Abstract

Introduction

Methods

Results

Conclusion

Keywords

Introduction

Materials and Methods

Ethical approval

Experiment animals and their features

Experimental procedure

Histochemical analysis

Immunohistochemical analysis

Biochemical analysis

Statistical analysis

Results

Histopathological Examination Results of testis tissue

Histopathological examination results of Epididymis Tissue

Immunohistochemical results

Immunohistochemical examination results of HSP 70, NF-κB-p65, TNF-α, and Caspase-3 in Testis Tissue

Immunohistochemical Examination Results of HSP 70, NF-κB-p65, TNF-α, and Caspase-3 in epididymis tissue

Biochemical results

Discussion

Conclusions

Footnotes

Author contributions

Declaration of conflicting interests

Funding

ORCID iD

References