Cytotoxic chemotherapy has increased the survival rates of a number of diseases, most notably, cancers of the testicles. However, there is considerable morbidity associated with treatment, and one of the most frequent long-term side effects of this medication is testicular dysfunction.1 The highly effective platinum-based antineoplastic agent cisplatin (CIS; also known as cisplatinum) is used to treat a variety of tumors, including those of the testis, bladder, stomach, lung, endometrium, cervix, ovary, neck, and head2–6 as well as prostate cancer, nasopharyngeal carcinoma, gynecological cancers, and breast cancer.7
Cisplatin binds to DNA strands, blocking their replication and thereby preventing the production of new proteins. Additionally, some studies4,8,9 have revealed that CIS is a DNA-damaging agent that can cross-link with purine bases on DNA, interfering with the repair mechanisms of the DNA, damaging DNA, and forming CIS-DNA adducts that cause cell death through a variety of mechanisms, ultimately causing cancer cells to undergo apoptosis.
Furthermore, it has been widely known that the biochemical underpinnings of CIS toxicity are associated with oxidative stress via the production of reactive oxygen species (ROS). These oxidants are thought to exert their effects by directly harming target cells.10–13
Despite being a very effective chemotherapeutic agent, the use of CIS can result in toxicity (particularly testicular damage) and nephrotoxicity, as well as a host of unfavorable side effects, including allergic reactions, weakened immunity to infections, bleeding, and gastrointestinal issues.4 The mechanism behind CIS-induced testicular damage is brought on by oxidative stress and ROS production.14 In addition, CIS promotes lipid peroxidation and lowers protective enzymes against oxidative damage in testes.15 This drug specifically targets spermatogenic cells due to their strong mitotic activity.16
Therefore, it stands to reason that increasing antioxidant capacity may be a useful strategy for reducing testicular damage following CIS. Herbs are sources of various natural antioxidants and have free radical–scavenging capacity.17 Licorice is a medicinal ornamental herb that can be utilized for therapy of different diseases,18 spanning from colds to hepatic disturbances and even cancer.19
Licorice possesses numerous significant pharmacological characteristics and has anti-inflammatory, anticancer, antioxidant, antimicrobial, antiviral, antiatherosclerotic, antihepatitis, antinephritic, cardioprotective, hepatoprotective, and immunomodulatory effects.20,21 Moreover, licorice does not have any major adverse effects when used regularly.22 Many potent phytochemicals with antioxidant and anti-inflammatory qualities can be found in licorice. The primary components of licorice are saponins, which include 1 molecule of glycyrrhetinic acid and 2 molecules each of glucuronic acid, polysaccharides, polyphenols, and glycyrrhetinic acid. The active parts of licorice are abundant in bioflavonoids and phenolic compounds, which are the active forms of antioxidants.23
Glycyrrhizin (GLZ), which is extracted from the root of licorice (Glycyrrhiza glabra), is thought to be the primary active ingredient in this herb. The food and cosmetics sectors currently use it. Furthermore, GLZ has demonstrated a range of health-promoting effects, including antitumor, antioxidant, and antiulcerative benefits, even though the mechanism of action is still largely unknown.24
Herbal remedies could potentially reduce the cytotoxic effects of manufactured pharmaceuticals while also offering wide safety margins. The regenerative potential of damaged cells and tissues caused by toxic chemicals might be enhanced by the therapeutic component of herbal medicine25; thus, to lessen the adverse effects on various organs, the use of herbal remedies has equaled that of chemical drugs.26
The current study set out to evaluate the potential for oxidative damage, apoptosis, and testicular toxicity linked to CIS intoxication, as well as the potential contribution of GLZ to the reduction of the toxicological consequences of CIS.
Methods
Chemicals and kits
Cisplatin was supplied by EIMC United Pharmaceutical as a 50-mg/50-mL vial of Unistin. Before use, a powder form of GLZ (Sigma-Aldrich) was dissolved in distilled water and stored at 2 to 4 °C.
Catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) kits were purchased from Bio Diagnostic Co. Furthermore, kits for luteinizing hormone (LH), testosterone, and follicle-stimulating hormone (FSH) were purchased from MyBioSource.
Animals and housing
In the present study, 40 apparently healthy mature male Wistar albino rats with an average age of 3 months and weight of 200 ± 20 g were obtained from the animal facility at the Faculty of Veterinary Medicine, Zagazig University. The animals were reared under standard conditions (12-hour light/12-hour dark cycle, constant temperature of 20 to 23 °C, and humidity at 50 ± 5%) for at least 1 week before the experiment for accommodation and to avoid transportation stress; those conditions were also preserved to the end of the experiment. The rats were housed in clear polypropylene cages with 10 rats/cage, providing them with free access to water and dry rat pellets for food under sanitary conditions.
Ethical approval
The research protocol was reviewed and approved by the Zagazig University IACUC (approval No. ZU-IACUC/2/F/206/2023).
Study design and treatments
A total of 40 rats were randomly divided into the following 4 equal groups (n = 10 each): the control group (G1), CIS-treated group (G2), GLZ-treated group (G3), and GLZ plus CIS group (G4). Glycyrrhizin in G3 and G4 was given once daily by oral gavage for 60 days at the dose of 25 mg/kg body weight. This dose was selected based on a previous study27 revealing both antioxidant and hepatorenal protective potentials. The G2 rats received a single IP injection of CIS at the dose of 7 mg/kg body weight. On the 11th day of GLZ treatment, G4 received a single IP injection of CIS (7 mg/kg). The dose of CIS to induce testicular toxicity was chosen based on a prior study.28 The rats in G1 and G3 were injected IP with normal saline once on the 11th day from the beginning of treatments.
Sample collection
Rats were weighed after 60 days and subsequently euthanized under anesthesia (ketamine, 200 mg/kg body weight, IP). A midline scrotal incision was performed and the testes were collected and weighed on a digital scale. For the purpose of histological analysis, the testes were fixed with Bouin solution. Furthermore, blood samples were taken via heart puncture into sterile tubes without anticoagulant to separate serum, followed by centrifugation at 3,000 X g 10 minutes. The serum was kept for various biochemical and hormonal investigations at –80 °C. Data for the biochemical and hormonal investigations performed are outside of the scope of this study and are not reported at the present time.
Antioxidant enzyme assays
The antioxidant enzymes GPx, SOD, and CAT were assessed in serum to detect the characteristics of oxidative stress.
Serum hormonal assay
As directed by the manufacturer, ELISA kits (MyBioSource) were used to measure the activities of testosterone, LH, and FSH.
Histological and histochemical processing
After being fixed in Bouin solution for 48 hours, small fragments from the fixed testes were moved and postfixed in neutral-buffered 10% formalin. The samples were then dehydrated in increasing ethanol grades, cleaned in xylene, and embedded in paraffin wax to create paraffin blocks. Sections measuring 5 µm in thickness were prepared and stained with Harris H&E stain for standard histological investigations; blue Masson trichrome stain was used to illustrate collagen fibers, Mercuric bromophenol blue was used to assess proteins, and periodic acid–Schiff was used to identify glycogen and neutral mucopolysaccharides. All of these histological and histochemical stains were carried out in accordance with Suvarna et al.29
Anti-caspase 3 immunohistochemical reactivity
For immunohistochemical labeling, 5-μm-thick, formalin-fixed, paraffin-embedded sections from testes were mounted on glass slides. Deparaffinized sections were stained by an indirect immunoperoxidase technique30 in which, after deparaffinization and rehydration, heat-induced antigen retrieval was applied using a microwave (600 W) after immersion of the testicular sections for 10 minutes in 0.1 M citrate buffer (pH 6). The sections were incubated in absolute methanol (containing 3% hydrogen peroxide) for 30 minutes at room temperature to eliminate the endogenous peroxidase activity. Then, the sections were incubated in blocking solution (PBS containing 10% normal goat serum and 0.1% Triton-X-100) for 1 hour at room temperature to block the nonspecific labeling. After blocking, the sections were incubated overnight at 4 °C with specific primary antibodies diluted in the blocking solution using anti-caspase 3 (ab184787) and rabbit monoclonal antibodies as a marker of apoptosis at 1:1,000 dilution (abcam). The sections were washed with PBS (3 times for 5 minutes each). Next, the sections were incubated with specific biotinylated secondary antibodies at room temperature for 1 hour. Then, sections were washed with PBS (3 times/5 min each) and subsequently incubated with streptavidin-peroxidase at room temperature for 30 minutes. The positive reactions were visualized by adding 3,3'-diaminobenzidine tetrahydrochloride–hydrogen peroxide solution for 3 minutes. Next, the nuclei were counterstained with Harris H&E. Finally, the sections were dehydrated by ethanol, cleared in xylene, coverslipped, and examined under a microscope with a magnification of 40X, 100X, 400X, 1,000X (ocular lens = 10 X objective lens = 4, 10, 40, 100).30
Sperm morphology and abnormalities (methyl violet)
The semen samples were obtained from the caudal region of the rats’ epididymis (cauda epididymis) and then diluted immediately in 2.9% sodium citrate buffer to reach an appropriate quantity of sperm cells in each microscopic field. On a glass slide, a drop of diluted semen was placed, and then the drop was distributed using the tip of another slide to create a film. The slide was heat fixed and then submerged for 4 to 5 minutes in a freshly made mixture consisting of 9 parts 1% aqueous methyl violet and 1 part 1% aqueous sodium carbonate solution. Next, the slides were cleaned with distilled water and the stain was poured out. The slides were then dried by running them over a flame 2 or 3 times after being wiped between filter sheets without cleaning the films. To facilitate the distinction, neutral Canada balsam mounting medium was used to mount the slides. Using 40X and 100X objective lenses, the films were inspected, and the sperm morphology and anomalies were evaluated under a light microscope. On methyl violet–stained slides, the proportion of aberrant sperm was found and quantified per 100 sperms.31,32 Finally, the sperm cells took on a violet hue against the background that was not dyed.
Statistical analysis
The SEM was used to present the findings. The effects of the 4 treatment groups on the various biochemical markers were assessed with a 1-way ANOVA with the Duncan multiple test as a post hoc test. Statistical significance was determined with a P value less than .05. All analyses and charts were created with SPSS Statistics (version 24.0; IBM Corp) and Prism (version 8.0.2; GraphPad Software Inc).
Results
Body weight and testis weight
The body weight of the treated groups did not differ statistically significantly from that of the controls (212.8 ± 2.71a). However, in contrast to G1 (2.91 ± 0.027a), a statistically significant decrease in testicular weight (net weight, both right and left) was noted in G2 (2.31 ± 0.0182b; P < .05; Figure 1; Supplementary Table S1).
Effect of cisplatin (CIS) and glycyrrhizin (GLZ) on the body weight; testicular weight; levels of serum testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and some antioxidant enzymes (catalase [CAT], superoxide dismutase [SOD], and glutathione peroxidase [GPx]); seminiferous tubular diameters; and sperm abnormality percentages for all experimental groups. According to the Duncan multiple test, the different letters (a–d) are significantly different between treatments at the P < .05 level. G1 = Control group (G1). G2 = CIS-treated group. G3 = GLZ-treated group. G4 = GLZ plus CIS.
Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.10.0288
Serum hormonal levels
A comparison of G2 blood testosterone (2.4 ± 0.0413d), LH (31.241 ± 0.534d), and FSH levels (5.82 ± 0.0631d) to G1 values (3.5412 ± 0.0621a, 41.271 ± 0.314a, 7.41 ± 0.0241a, respectively) revealed a substantial drop. Conversely, in contrast to G2, G4 exhibited a statistically significant increase in serum levels of testosterone. (3.0031 ± 0.0534c), LH (37.313 ± 0.45392c), and FSH (6.425 ± 0.0583c; P < .05; Figure 1; Supplementary Table S1).
Antioxidant enzymes
Glutathione peroxidase (230.41 ± 2.08151c), SOD (190.34 ± 3.1349c), and CAT (15.154 ± 0.46102c) levels were significantly lower in G2 than in G1 (258.55 ± 2.451b, 214.67 ± 4.213ab, and 18.293 ± 0.46592b, respectively). In contrast, the GPx, SOD, and CAT levels in G4 were significantly higher than those in G2 (251.77 ± 2.82251b, 201.31 ± 5.34169bc, and 17.41 ± 0.41673b, respectively; P < .05). These abnormalities were greatly ameliorated by GLZ and CIS coadministration in G4 (Figure 1; Supplementary Table S1).
Seminiferous tubular diameters and percentage of sperm abnormalities
The mean seminiferous tubular diameters in G2 (197.44 ± 0.661d) were considerably smaller than the control (249.64 ± 0.346a). Additionally, the sperm abnormality percentage was considerably higher in the animals in G2 (16.76 ± 0.843a) than in G1 (12.34 ± 0.311bc; P < .05). Meanwhile, GLZ and CIS coadministration in G4 ameliorated all these abnormalities (Figure 1; Supplementary Table S1).
Testicular histopathology
The mature male rats in G1 had testes that were histologically examined. The results showed that the testicular parenchyma was normal and intact, primarily consisting of 2 parts: the intertubular part (a significant amount of highly vascularized interstitial connective tissue) and the tubular part (many rounded or oval seminiferous tubules; Figure 2). Higher magnification revealed that the seminiferous tubules were lined with intact stratified seminiferous epithelium, which consisted of fewer, nondivided pyramidal Sertoli cells encircled by several rows of proliferating, normally organized, highly divided spermatogenic cells, which were represented by spermatogonia, spermatocytes I and II, spermatids, and sperms, and they rested on a thin basal lamina. The highly vascularized intertubular connective tissue appeared containing 2 types of cells: polygonal Leydig cells with spherical nuclei and flat myoid cells with flat nuclei (Figure 2).
A photomicrographs of mature male rats testes of G1 (A and B). A—Normal, intact testicular parenchyma of tubular part (oval or rounded seminiferous tubules [S]) and intertubular part (a considerable amount of highly vascularized intertubular connective tissue housing Leydig and myoid cells, arrowhead). B—Higher magnification of A, showing normal, intact seminiferous tubules lining stratified seminiferous epithelium; pyramidal Sertoli cells (dashed arrow) surrounded with several rows of normal, organized, proliferated, and highly divided spermatogenic cells (inside square) rested on a thin basal lamina; and normal polygonal Leydig cells (arrow) and flat myoid cells (arrowhead) in the intertubular part. C through K—Photomicrographs of mature male rat's testes of G2. C—Severe thickening of the testicular capsule (double arrowheads). D—Severe fibrosis of the testicular capsule (arrow). E—Subcapsular blood vessel dilatation with congestion (arrow). F—Severe degenerative changes of the seminiferous tubules lining epithelium with loss of their normal organization and distribution of numerous spermatogenic cells with a pyknotic nucleus, with severe aggregation of round spermatids at the lumen of seminiferous tubules with complete loss of any sperms (arrow). G—Germ cell depletions (arrowheads). H and I—Severe coagulative necrosis of spermatogenic cells, especially spermatocytes and spermatids (arrow). J and K—Severe hydropic degeneration and vacuolizations of spermatogenic cells, especially spermatogonia and primary spermatocytes (arrowhead). H&E (A through K) and blue Masson trichrome (D) stains are shown. Bar = 300 µm (A) and 50 µm (B through K).
Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.10.0288
In the meantime, the testes of G2 showed severe thickening and fibrosis of the testicular capsule and tunica albuginea together with significant subcapsular blood vessel dilatation and congestion (Figure 2). At the level of the tubular part, the seminiferous tubules lining the epithelium showed severe degenerative changes with loss of normal organization. Additionally, a large number of spermatogenic cells with pyknotic nuclei were distributed, and aggregations of round spermatids that filled the lumen of seminiferous tubules were observed, completely devoid of sperm. Moreover, the depletion of germ cells was observed (Figure 2), along with severe coagulative necrosis of spermatogenic cells, particularly spermatocytes and spermatids, which were evident by the tubular architecture, loss of cellular features, and total loss of sperm. A few sections under examination revealed the loss of multiple spermatogenic cell types, including secondary spermatocytes, spermatids, and sperms. Only 2 cell types were found: primary spermatocytes with a pyknotic nucleus and spermatogonia resting on a thick basal lamina. Additionally, the lumen of the seminiferous tubules widened and became empty of sperm. Severe hydropic degeneration (vacuolizations) manifested in the form of vacuoles with varying shapes and sizes within the cytoplasm of spermatogenic cells, notably spermatogonia and primary spermatocytes (Figure 2).
A few of the sections under examination showed significant damage to the basal lamina of the seminiferous tubules and sloughing of its lining epithelium into the tubular lumen and numerous spermatid giant cells forming in the tubular lumen with necrotic Sertoli cells. Furthermore, significant atrophy of the seminiferous tubules with increasing intertubular spaces was shown, and spherical spermatids with localized coagulative necrosis were seen (Figure 3).
Photomicrographs of mature male rat testes of G2. A—Severe damage of the seminiferous tubules basal lamina with sloughing of its lining epithelium into the tubular lumen (arrowhead). B through D—Formation of multiple spermatids giant cells in the tubular lumen (arrow) and necrotic Sertoli cells (arrowhead). E and F—Atrophy of the seminiferous tubules (arrow) with increasing of intertubular spaces. H&E stain (A through F) is shown. Bar = 50 µm (A, B, D, E, and F) and 10 µm (C).
Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.10.0288
At the level of the intertubular component, multiple pathological changes were presumed, mimicking severe intertubular edema: rise of the intertubular fluid, which reacted positively with periodic acid–Schiff stain, combined with severe Leydig cell hyperplasia. Moreover, severe dilatation of the intertubular blood vessels with severe congestion and engorgement with blood was observed in conjunction with severe thickening and fibrosis of the blood vessel wall (Figure 4).
Photomicrographs of mature male rats testes of G2 (A through F). A and B—Severe intertubular edema (arrow). C—Strongly periodic acid–Schiff positive reactivity of the intertubular fluid (arrow). D and E—Severe Leydig cells hyperplasia (arrowhead). F through H—Severe intertubular blood vessel dilatation with severe congestion, engorged with blood (arrow). I—Severe thickening and fibrosis of the blood vessel wall (arrow). H&E (A, D, F, G, and H), Mercuric bromophenol blue (B), periodic acid–Schiff (C and E), and blue Masson trichrome (I) stains are shown. Bar = 50 µm (A through E and H) and 300 µm (F and G).
Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.10.0288
The testes of G3 showed intact testicular parenchyma of the intact tubular part with normal, organized lining epithelium and normal intertubular part that resembled the testes of G1 without any pathological changes (Figure 5).
A and B—Photomicrographs of mature male rats testes of G3. A—Intact testicular parenchyma of normal tubular part (S) and intertubular part (arrowhead) that appeared normal resembling the normal control group without any pathological changes. B—Higher magnification of A, showing intact seminiferous tubules lining stratified seminiferous epithelium with normal, organized, spermatogenic cells (arrowhead), with normal intertubular part (arrow). C through H—Photomicrographs of mature male rats testes of G4. C—Intact tubular part (S) and intertubular part (arrowhead). D—Higher magnification of C, showing intact seminiferous tubules lining epithelium (arrowhead), with normal intertubular part (arrow) in most of the examined sections. E—Mild intertubular edema (arrow), with mild degenerative changes and disorganization of the seminiferous tubules lining epithelium (arrowhead) in some examined sections. F—Mild to moderate Leydig cell hyperplasia (arrowhead). G—Moderate intertubular blood vessel dilatation with congestion (arrow). H—Moderate fibrosis of the intertubular blood vessel wall (arrow). H&E stain (A through E and G), periodic acid–Schiff (F), and blue Masson trichrome (H) stains are shown. Bar = 300 µm (A and C) and 50 µm (B and D through H).
Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.10.0288
In the meantime, the histological analysis of the testes of G4 revealed the most desirable and successful therapeutic interference. The majority of the examined sections of this group displayed normal, intact tubular parts with normal lining epithelium and intertubular parts that appeared to be normal; however, very few sections showed mild to moderate pathological changes, resembling mild degenerative changes of the seminiferous tubules lining epithelium, mild intertubular edema, mild Leydig cell hyperplasia, and intertubular blood vessel congestion, along with mild fibrosis of the intertubular blood vessel wall (Figure 5). The testicular histopathology lesion scores were semiquantitative and documented for each experimental group and are provided (Supplementary Table S2).
With regard to the immunohistochemical reactivity of the testes against the anti-caspase 3 antibody in each experimental group, the majority of positive signals were seen in the nuclei of morphologically discernible apoptotic cells. In the testicular parenchyma, the lining epithelium of the seminiferous tubules and Leydig cells of G1 showed total negative expression against anti-caspase 3 antibody. Meanwhile, G2 exhibited diffuse strongly positive immunolocalizations against anti-caspase 3 antibodies that were widely expressed in most of the testicular parenchyma, confirming widespread apoptosis, especially in secondary spermatocytes and spermatids, sperms, and Leydig cells. Additionally, in the testicular parenchyma, G3 exhibited total negative expression against the anti-caspase 3 antibody; seminiferous tubules lining the epithelium and Leydig cells resembled those of G1. Furthermore, nearly all of the seminiferous tubules lining epithelium and Leydig cells that were examined had a negative expression of G4. However, only specific tubules demonstrated mild to moderately positive immunolocalization against anti-caspase 3 antibodies, particularly at the level of secondary spermatocytes and spermatids (Figure 6).
Photomicrographs of the immunohistochemical staining of the testis against anti-caspase 3 antibody in all experimental groups where the positive signal is mostly expressed in the nuclei of morphologically identifiable apoptotic cells. A and B—G1 showed completely negative expression against anti-caspase 3 antibody within the testicular parenchyma; seminiferous tubules lining epithelium (arrowhead) and Leydig cells (arrow). C through G—G2 showed diffuse strongly positive immunolocalizations against anti-caspase 3 antibodies that were widely expressed in almost all of the testicular parenchyma confirming widespread apoptosis especially secondary spermatocytes and spermatids (arrowhead) in C and D, sperms (dashed arrow) in E, and Leydig cells (arrows) in F and G. H—G3 showed completely negative expression against anti-caspase 3 antibody within the testicular parenchyma; seminiferous tubules lining epithelium (arrowhead) and Leydig cells (arrow) resembling the control group. I through K—G4 showed negative expression in almost all of the examined seminiferous tubules lining epithelium (arrowheads) and Leydig cells (dashed arrows), but only individual tubules showed mild to moderate positive immunolocalization against anti-caspase 3 antibody, especially at the level of secondary spermatocytes and spermatids (arrow). Immunohistochemical stain against anti-caspase 3 antibody (A through K) is shown. Bar = 70 µm (A, H, I, and K), 50 µm (B through D, F, and J), and 10 µm (E and G).
Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.10.0288
Sperm morphology and abnormalities
Sperms with normal morphology were seen in the middle, principal, and end sections of G1 methyl violet–stained semen smears, which showed no abnormalities. The sperm also had a normal hooked head. When compared with G1, G2 revealed a notable increase in sperm abnormalities in both the head and tail. It showed different forms and shapes of sperm abnormalities, such as a flattened hookless head, banana head, detached head, abnormal angulation of head with neck with severe coiled principle piece of tail, S-shaped bent middle piece with bent principle piece, sharply bent or broken tail, principle piece with a distal protoplasmic droplet, detached tail, single tight tail coiling in the middle piece with S-shaped bent principle piece, sharp bent tail in both principle and end pieces, short tail, sharp curved (zigzag-shaped) principle piece with no clear end piece, looped principle piece, and coiled principle piece. Nevertheless, sperm with normal morphology and no anomalies were seen in G3. Furthermore, most of the smears evaluated by G4 revealed sperms with normal morphology; however, a few smears displayed mild abnormalities, particularly in the tail, such as bent middle pieces (Supplementary Figure S1).
Discussion
A potent and effective anticancer drug, CIS is used to treat a variety of malignancies of the testicles, ovaries, uterus, lung, head, and neck. However, due to its numerous adverse effects including testicular toxicity that may have lasting effects on spermatogenesis and fertility, the therapeutic uses of CIS are limited.33 According to Cherry et al,34 CIS can also result in sperm chromosomal abnormalities, decreased spermiogenesis, and temporary or permanent azoospermia. Because GLZ lessens the toxicity to the testicles following CIS, it has recently garnered increased attention.
The current study demonstrated that the testicular weight of the CIS-treated group was significantly lower than that of the control and other treated groups but that there were no appreciable differences in body weight between the control and any of the treated groups. The findings of Türk et al,12 Ilbey et al,16 and Sherif et al,35 who saw a significant decrease in testicular weight in G2 compared with the G1 but no discernible decrease in body weight between these groups, support these results. In addition, the reductions in the testicular weight were related to severe parenchymal atrophy that was reported in our histological findings. Furthermore, testis weight (left and right) as well as the length and width of both testes was statistically significantly reduced in the male rats receiving the CIS treatment.36 Moreover, rather than being the consequence of general toxicity, the effects of CIS on the testis may be attributed to its particularly harmful effects on the target organ.37 When CIS is given to male rats, the decrease in testicular weight serves as a highly reliable predictor of gonadal toxicity. This is most likely the result of aberrant ROS generation38 and high cytotoxicity-induced tubular atrophy.14
According to reports, the testosterone levels of the CIS-treated rats were significantly lower than those of the control group. Impaired Leydig cells may be the cause of the reduced testosterone levels in CIS-treated groups.16,35,37 Furthermore, a publication39 has confirmed our findings by linking the alterations in testosterone caused by CIS to a decrease in the number of LH receptors on Leydig cells. In contrast, the G4 had a statistically significant increase in serum levels of FSH, LH, and testosterone in comparison to the G2. The results of Aldhahrani et al26 supported these conclusions by elucidating how G glabra extract (licorice extract) prevented a drop in serum testosterone as well as a rise in IL-1β and IL-6 levels.
Here, a substantial drop in the levels of GPx, SOD, and CAT in G2 relative to the control values demonstrated testicular oxidative stress. These findings were consistent with earlier research conducted following the statements made by Salem et al36 and Amin et al40 that treatment of male rats with CIS led to a significant increase in testicular tissue levels of malondialdehyde, a byproduct of lipid peroxidation, while there was a significant decrease in glutathione levels; SOD, GPx, and CAT activities and plasma testosterone. In contrast, the GPx, SOD, and CAT levels in G4 were significantly higher than those in G2. When GLZ and CIS were administered before coadministration in G4, these deficits were dramatically reduced. These results were validated by the findings of Aldhahrani et al,26 who emphasized that the prior administration of G glabra extract (licorice extract) raised GPx, glutathione, and catalase levels and their activities in testis and blood. In addition, a significant rise was observed in SOD and CAT activities together with a drop in malondialdehyde levels following treatment with licorice extract.41
The testes of G2 showed severe hydropic degeneration, vacuolization within the cytoplasm of spermatogenic cells (specifically spermatogonia and primary spermatocytes), atrophy of the seminiferous tubules with increasing intertubular spaces, severe intertubular edema accompanied by severe Leydig cell hyperplasia, and severe coagulative necrosis of spermatogenic cells, especially spermatocytes and spermatids. In addition, severe dilatation of the intertubular blood vessels with severe congestion was described, accompanied by severe thickening and fibrosis of its wall. These results were validated by the findings of Ilbey et al,16 who emphasized that rats given CIS alone experienced significant degeneration, necrosis, and reduction in seminiferous tubules combined with a decrease in germinal cell thickness. Furthermore, compared to controls, all CIS-treated rats exhibited considerable maturation arrest, irregular seminiferous tubules with a few spermatogonia, and a depletion of germ cells. These findings were reported by Sherif et al35 and Ilbey et al.37 Intertubular tissue hyalinization and perivascular fibrosis were also identified. Additionally, compared to G1, the mean seminiferous tubular diameters in G2 were significantly smaller.10,29 According to Cherry et al,34 testicular histology analyses further show that CIS significantly damages the populations of germ cells, Leydig, and Sertoli. In addition, the results of Yucel et al42 provided further clarification that the group that received CIS treatment exhibited a maturational loss in germinal cells, cessation of spermatogenesis at the primary spermatocyte stage, mild perivascular fibrosis, intertubular connective tissue disorganization, and hyalinization. However, in a small number of sections, mild to moderate pathological changes were observed that resembled mild degenerative changes of the seminiferous tubules lining epithelium, mild intertubular edema, and mild Leydig cell hyperplasia. These findings illuminated the preferable and effective therapeutic interference. The histological examination of the testes of G4 revealed this particular combination of normal, intact tubular parts with normal lining epithelium and intertubular parts that appeared normal. According to Sakr et al,41 rats with testicular injury that received licorice extract (G glabra extract) showed a small number of damaged cells, but the majority of the tubules appeared intact and showed a normal spermatogenic development. Additionally, typical spermatozoa were seen to be filling the lumen. Additionally, the licorice extract reduced the hazardous group's percentage of seriously injured tubules from 51% to 18%. Furthermore, Sakr et al41 found that by scavenging free radicals and promoting the activities of antioxidant enzymes, the licorice aqueous extract reduced testicular damage.
The present work discussed that the protective action of GLZ may be explained by the fact that it can prevent the cellular damage generated as a result of oxidative stress in spermatogenic cells of seminiferous tubules and Leydig cells of the intertubular connective tissue. Furthermore, there is a notable rise in testosterone levels in rats treated with CIS after receiving GLZ. The spermatogenic suppression clarified in CIS-treated rats in this investigation may be caused by free radical products that are formed in the testicular tissue and have a deleterious effect on spermatogenesis, in addition to decreased testosterone levels. As a result, the GLZ's antioxidant and free radical–scavenger qualities may be linked to the rats’ improved spermatogenesis and seminiferous tubules lining epithelium.
The testicular parenchyma expressed anti-caspase 3 antibody widely, and G2 showed diffuse strongly positive immunolocalizations against it. This confirmed the widespread apoptosis, particularly in secondary spermatocytes and spermatids, sperms, and Leydig cells. According to reports,34 germ cell apoptosis is significant in the G2 induction of testicular damage, which is consistent with our findings.
The CIS-treated group demonstrated a notable increase in the proportion of sperm abnormalities of the head and tail, including a flattened hookless head, banana head, S-shaped bent middle piece, principal piece with distal protoplasmic droplet, and sharply curved (zigzag-shaped) principle piece, using methyl violet–stained semen smears. The findings of Ateşşahin et al,10 Salem et al,36 and Yucel et al42 corroborated our findings that when compared to the control rat, the male rats treated with CIS exhibited a significantly higher percentage of head, tail, and total sperm abnormalities, along with a significantly lower percentage of sperm concentration and sperm motility. In the meantime, the majority of the smears evaluated in G4 revealed sperms with normal morphology; however, some minor abnormalities, particularly in the tail, were noted. Our findings were corroborated by the findings of Aldhahrani et al,26 who showed that preadministration of licorice extract considerably reduced the changes in the spermogram and improved and lowered sperm defects and abnormalities that were reported in the toxic group.
We can draw the conclusion that testicular tissues are subjected to severe histopathological damage, apoptosis, cytotoxic impact, and strong oxidative stress due to CIS. Additionally, due to its strong cytoprotective, antiapoptotic, antioxidant, and hormone-regulating potentials, GLZ pre-coadministration and posttreatment have been shown to be useful for prophylaxis and significantly mitigating the testicular toxicity caused by CIS.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
The authors extend their appreciation to the Deanship of Scientific Research and Graduate Studies at King Khalid University for funding this work through the Large Research Project under grant number RGP2/61/45.
We would like to thank the Deanship of Scientific Research, Qassim University, Saudi Arabia for funding this research.
We would like to thank AlMaarefa University, Riyadh, Saudi Arabia for supporting this research.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.
Funding
The authors extend their appreciation to the Deanship of Scientific Research and Graduate Studies at King Khalid University for funding this work through the Large Research Project under grant number RGP2/61/45.
ORCID
W. A. M. Ghonimi https://orcid.org/0000-0003-1324-0655
References
- 1.↑
Howell SJ, Shalet SM. Testicular function following chemotherapy. Hum Reprod Update. 2001;7(4):363–369. doi:10.1093/humupd/7.4.363
- 2.↑
Colpi GM, Contalbi GF, Nerva F, Sagone P, Piediferro G. Testicular function following chemo-radiotherapy. Eur J Obstet Gynecol Reprod Biol. 2004;113(suppl 1):S2–S6. doi:10.1016/j.ejogrb.2003.11.002
- 3.
Howell SJ, Shalet SM. Spermatogenesis after cancer treatment: damage and recovery. J Natl Cancer Inst Monogr. 2005;34(34):12–17. doi:10.1093/jncimonographs/lgi003
- 4.↑
Dasari S, Tchounwou PB. Cisplatin in cancer therapy: molecular mechanisms of action. Eur J Pharmacol. 2014;740:364–378. doi:10.1016/j.ejphar.2014.07.025
- 5.
Adejuwon SA, Femi-Akinlosotu OM, Omirinde JO. Cisplatin-induced testicular dysfunction and its amelioration by Launaea taraxacifolia leaf extract. Andrologia. 2015;47(5):553–559. doi:10.1111/and.12302
- 6.↑
Zhang C, Xu C, Gao X, Yao Q. Platinum-based drugs for cancer therapy and anti-tumor strategies. Theranostics. 2022;12(5):2115–2132. doi:10.7150/thno.69424
- 7.↑
Kiss RC, Xia F, Acklin S. Targeting DNA damage response and repair to enhance therapeutic index in cisplatin-based cancer treatment. Int J Mol Sci. 2021;22(15):8199. doi:10.3390/ijms22158199
- 8.↑
Wang D, Lippard SJ. Cellular processing of platinum anticancer drugs. Nat Rev Drug Discov. 2005;4(4):307–320. doi:10.1038/nrd1691
- 9.↑
Romani AMP. Cisplatin in cancer treatment. Biochem Pharmacol. 2022;206:115323. doi:10.1016/j.bcp.2022.115323
- 10.↑
Ateşşahin A, Karahan I, Türk G, Gür S, Yilmaz S, Ceribaşi AO. Protective role of lycopene on cisplatin-induced changes in sperm characteristics, testicular damage and oxidative stress in rats. Reprod Toxicol. 2006;21(1):42–47. doi:10.1016/j.reprotox.2005.05.003
- 11.
Ateşşahin A, Sahna E, Türk G, et al. Chemoprotective effect of melatonin against cisplatin-induced testicular toxicity in rats. J Pineal Res. 2006;41(1):21–27. doi:10.1111/j.1600-079X.2006.00327.x
- 12.↑
Türk G, Ateşşahin A, Sönmez M, Ceribaşi AO, Yüce A. Improvement of cisplatin-induced injuries to sperm quality, the oxidant-antioxidant system, and the histologic structure of the rat testis by ellagic acid. Fertil Steril. 2008;89(5)(suppl):1474–1481. doi:10.1016/j.fertnstert.2007.04.059
- 13.↑
Awadalla E. Ameliorative effect of the crude oil of the Nigella sativa on oxidative stress induced in rat testes by cisplatin treatment. Biomed Prev Nutr. 2012; 2(4):265–268. doi:https://doi.org/10.1016/j.bionut.2012.08.005
- 14.↑
Azu O, Duru FIO, Osinubi AA, et al. Histomorphometric effects of Kigelia africana (Bignoniaceae) fruit extract on the testis following short-term treatment with cisplatin in male Sprague-Dawley rats. Middle East Fertil Soc J. 2010;15(3):200–208. doi:10.1016/j.mefs.2010.07.001
- 15.↑
Antunes LM, Darin JD, Bianchi NL. Effects of the antioxidants curcumin or selenium on cisplatin-induced nephrotoxicity and lipid peroxidation in rats. Pharmacol Res. 2001;43(2):145–150. doi:10.1006/phrs.2000.0724
- 16.↑
Ilbey YO, Ozbek E, Cekmen M, Simsek A, Otunctemur A, Somay A. Protective effect of curcumin in cisplatin-induced oxidative injury in rat testis: mitogen-activated protein kinase and nuclear factor-kappa B signaling pathways. Hum Reprod. 2009;24(7):1717–1725. doi:10.1093/humrep/dep058
- 17.↑
Al-Attar AM, Elnaggar MHR, Almalki EA. Protective effect of some plant oils on diazinon induced hepatorenal toxicity in male rats. Saudi J Biol Sci. 2017;24(6):1162–1171. doi:10.1016/j.sjbs.2016.10.013
- 18.↑
Wang ZY, Nixon DW. Licorice and cancer. Nutr Cancer. 2001;39(1):1–11. doi:10.1207/S15327914nc391_110.1207/S15327914nc391_1
- 19.↑
Parlar A, Arslan SO, Çam SA. Glabridin alleviates inflammation and nociception in rodents by activating BKC a channels and reducing NO levels. Biol Pharm Bull. 2020;43(5):884–897. doi:10.1248/bpb.b20-00038
- 20.↑
Kim SC, Byun SH, Yang CH, Kim CY, Kim JW, Kim SG. Cytoprotective effects of Glycyrrhizae radix extract and its active component liquiritigenin against cadmium-induced toxicity (effects on bad translocation and cytochrome c-mediated PARP cleavage). Toxicology. 2004;197(3):239–251. doi:10.1016/j.tox.2004.01.010
- 21.↑
Wang X, Zhang H, Chen L, Shan L, Fan G, Gao X. Liquorice, a unique “guide drug” of traditional Chinese medicine: a review of its role in drug interactions. J Ethnopharmacol. 2013;150(3):781–790. doi:10.1016/j.jep.2013.09.055
- 22.↑
Shin S, Jang JY, Choi BI, et al. Licorice extract does not impair the male reproductive function of rats. Exp Anim. 2008;57(1):11–17. doi:10.1538/expanim.57.11
- 23.↑
Shirwaikar A, Malini S, Kumari SC. Protective effect of Pongamia pinnata flowers against cisplatin and gentamicin induced nephrotoxicity in rats. Indian J Exp Biol. 2003;41(1):58–62.
- 24.↑
Damle M. Glycyrrhiza glabra (liquorice) - a potent medicinal herb. Int J Herb Med. 2014;2:132–136.
- 25.↑
Bedi O, Bijjem KRV, Kumar P, Gauttam V. Herbal induced hepatoprotection and hepatotoxicity: a critical review. Indian J Physiol Pharmacol. 2016;60(1):6–21.
- 26.↑
Aldhahrani A, Soliman MM, Althobaiti F, et al. The modulatory impacts of Glycyrrhiza glabra extract against methotrexate-induced testicular dysfunction and oxidative stress. Toxicol Res (Camb). 2021;10(4):677–686. doi:10.1093/toxres/tfab049
- 27.↑
Karimani A, Heidarpour M, Moghaddam Jafari A. Protective effects of glycyrrhizin on sub-chronic diazinon-induced biochemical, hematological alterations and oxidative stress indices in male Wistar rats. Drug Chem Toxicol. 2019;42(3):300–308. doi:10.1080/01480545.2018.1497053
- 28.↑
Mesbahzadeh B, Hassanzadeh-Taheri M, Aliparast MS, Baniasadi P, Hosseini M. The protective effect of crocin on cisplatin-induced testicular impairment in rats. BMC Urol. 2021;21(1):117. doi:10.1186/s12894-021-00889-2
- 29.↑
Suvarna SK, Layton C, Bancroft JD. Bancroft's Theory and Practice of Histological Techniques. 8th ed. Churchill Livingstone; 2018:83–92.
- 30.↑
Polak JM, Van Noorden S. Introduction to Immunocytochemistry. 3rd ed. BIOS Scientific Publishers; 2003:13–81.
- 31.↑
Filler R. Methods for evaluation of rat epididymal sperm morphology. Methods Toxicol. 1993;3:334–343. doi:10.1016/B978-0-12-461207-5.50025-0
- 32.↑
Seed J, Chapin RE, Clegg ED, et al. Methods for assessing sperm motility, morphology, and counts in the rat, rabbit, and dog: a consensus report. Reprod Toxicol. 1996;10(3):237–244. doi:10.1016/0890-6238(96)00028-7
- 33.↑
Silici S, Ekmekcioglu O, Eraslan G, Demirtas A. Antioxidative effect of royal jelly in cisplatin-induced testes damage. Urology. 2009;74(3):545–551. doi:10.1016/j.urology.2009.05.024
- 34.↑
Cherry SM, Hunt PA, Hassold TJ. Cisplatin disrupts mammalian spermatogenesis, but does not affect recombination or chromosome segregation. Mutat Res. 2004;564(2):115–128. doi:10.1016/j.mrgentox.2004.08.010
- 35.↑
Sherif IO, Abdel-Aziz A, Sarhan OM. Cisplatin-induced testicular toxicity in rats: the protective effect of arjunolic acid. J Biochem Mol Toxicol. 2014;28(11):515–521. doi:10.1002/jbt.21593
- 36.↑
Salem EA, Salem NA, Maarouf AM, Serefoglu EC, Hellstrom WJG. Selenium and lycopene attenuate cisplatin-induced testicular toxicity associated with oxidative stress in Wistar rats. Urology. 2012;79(5):1184.e1–1184.e6. doi:10.1016/j.urology.2011.12.006
- 37.↑
Ilbey YO, Ozbek E, Simsek A, Cekmen M, Otunctemur A, Somay A. Chemoprotective effect of a nuclear factor-kappaB inhibitor, pyrrolidine dithiocarbamate, against cisplatin-induced testicular damage in rats. J Androl. 2009;30(5):505–514. doi:10.2164/jandrol.108.006270
- 38.↑
Boekelheide K. Mechanisms of toxic damage to spermatogenesis. J Natl Cancer Inst Monogr. 2005;34(34):6–8. doi:10.1093/jncimonographs/lgi006
- 39.↑
Maines MD, Sluss PM, Iscan M. cis-platinum-mediated decrease in serum testosterone is associated with depression of luteinizing hormone receptors and cytochrome P-450scc in rat testis. Endocrinology. 1990;126(5):2398–2406. doi:10.1210/endo-126-5-2398
- 40.↑
Amin A, Hamza AA, Kambal A, Daoud S. Herbal extracts counteract cisplatin-mediated cell death in rat testis. Asian J Androl. 2008;10(2):291–297. doi:10.1111/j.1745-7262.2008.00379.x
- 41.↑
Sakr SA, Shalaby SY. Carbendazim-induced testicular damage and oxidative stress in albino rats: ameliorative effect of licorice aqueous extract. Toxicol Ind Health. 2014;30(3):259–267. doi:10.1177/0748233712456059
- 42.↑
Yucel C, Arslan FD, Ekmekci S, et al. Protective effect of all-trans retinoic acid in cisplatin-induced testicular damage in rats. World J Mens Health. 2019;37(2):249–256. doi:10.5534/wjmh.180105
