Evaluation of protective effects of gallic acid on cisplatin‐induced testicular and epididymal damage

Cisplatin is an effective chemotherapeutic drug used to treat many types of tumours. However, it may cause male reproductive toxicity. Gallic acid exhibits beneficial effects such as antioxidant, anti‐inflammatory and antitumor. The current study investigated the beneficial effects of gallic acid against testis and epididymis toxicity induced by cisplatin. Male rats were divided into 4 groups as follows (n = 7): Control, cisplatin (a single dose of 8 mg/kg), Gallic acid (50 mg/kg) and cisplatin +Gallic acid groups. Testis was examined morphometrically by stereological methods. In addition, apoptosis, DNA damage, oxidative stress parameters in testis and testosterone in serum were measured. Epididymis was histopathologically evaluated. As a result, a significant decrease was observed in the number of spermatogonia, Leydig and Sertoli cells, testicular volume, height of germinal epithelial, Bcl‐2 immunopositive cell number, activity of CAT, GSH and SOD enzymes and serum testosterone levels compared with the cisplatin group control group, while a significant increase was observed in the number of Caspase‐3, Bax and 8‐OHdG immunopositive cells and the MDA levels. However, Gallic acid significantly restored these parameters. Our study reveals that Gallic acid may improve Cisplatin‐induced male reproductive toxicity by reducing oxidative stress, suppressing apoptosis and DNA damage and restoring structural and functional deterioration.


| INTRODUC TI ON
Cisplatin (CP) is a powerful antineoplastic drug widely used to treat various cancers such as bladder, lung, and testis cancer (Azarbarz et al., 2020). The anti-tumour effect of CP, a DNA-alkylating molecule, prevents the replication and transcription of DNA by breaking the DNA double strands (Ahmed et al., 2011). However, CP has side effects such as ototoxicity, nephrotoxicity, neurotoxicity and testicular toxicity, as well as significant anti-tumour effects (Elrashidy & Hasan, 2020). It has been determined that oxidative stress, inflammation and ischaemic injury are pathological conditions caused by CP toxicity in tissues (Eren et al., 2020). The testis has become the target of chemotherapeutic agents due to the spermatogenesis and spermiogenesis in which testicular cell division and morphological changes occur, and these agents can easily damage the testis (Ceylan et al., 2020). The permanent side effects of CP cause disruption of the Sertoli cell and seminiferous epithelium that results in the impairment of spermatogenesis (Whirledge et al., 2015). CP causes Leydig cell dysfunction leading to impaired testosterone secretion (El-shafaei et al., 2018). In addition, CP causes epididymal toxicity by resulting in decreased epididymal sperm count (Kohsaka et al., 2020). Thus, infertility occurs in parallel with CP application (Mercantepe et al., 2018). Although the underlying mechanism of CP toxicity on the testis and epididymis is not fully understood, one of its basic mechanisms is estimated to be related to its induction of reactive oxygen species (ROS) production and the decrease in the level of testicular antioxidants (Eren et al., 2020). Thus, the use of natural products that can scavenge ROS with anti-oxidative capacity is gradually increasing in the treatment of diseases and protection of tissues against intoxication of toxic substances. Various cytoprotective, antioxidant and functional foodstuffs have been applied to reduce or prevent CP toxicity (Kohsaka et al., 2020).
Gallic acid (GA), a polyphenol compound found in many vegetables and fruits such as bananas, strawberries, sumac, green tea and oak bark, exhibits antioxidant, anti-inflammatory, anticancer and antiviral effects (Zhu et al., 2019). GA that is widely used in medicine, cosmetics and foods attracts the attention of researchers because of its useful effects (Dehghani et al., 2020). The antioxidant effect of GA ensures that the functions of biological systems are carried out properly, and the balance between the oxidant and antioxidant mechanism is maintained (Shruthi et al., 2018). Also, its anti-inflammatory effect prevents or reduces the expression of inflammatory molecules and, thus, can be used in the treatment of inflammatory diseases (Kim et al., 2006). It has been reported that GA exhibits protective effects against cyclophosphamide-induced toxicity in testis and epididymis . Previous studies have shown that GA can protect the reproductive system of rats incumbent on manganese and AFB1-induced oxidative and inflammatory damage (Owumi et al., 2020). It has been also reported that GA exhibits curative effects on CP-induced nephrotoxicity and bone marrow genotoxicity (Akomolafe et al., 2014;Dehghani et al., 2020;Shruthi et al., 2018).
It is very important to protect male reproductive health against CP toxicity in patients undergoing chemotherapy. However, no adequate study has been found on the effects of GA on CP-induced testis and epididymis toxicity. Therefore, in our study, we investigated the mechanism underlying the effects of GA in CP-induced male reproductive toxicity in detail by stereological immunohistochemical and biochemical evaluation.

| Experimental animals and drug treatment
The protocol of our study was approved by Van Yuzuncu Yil University Animal Experiments Local Ethics Committee (decision number: 2021/02-25), and all methods were performed to the guidelines of the committee. In this study, a total of 28 adult male Wistar rats weighing 200 ± 20 g were used, and the animals were obtained from the Van Yüzüncü Yıl University Experimental Animals Research and Application Center. Rats were randomly divided into four groups (n = 7). The control (C) group was received 0.5 ml saline by intraperitoneal (IP). CP group was applied a single dose of 8 mg kg -1 day -1 CP (IP) on the 5th day of study. GA group was received 50 mg/kg GA by gastric gavage for 10 days. CP +GA group was received 50 mg/kg GA by gavage for 10 days and a single dose of 8 mg/kg CP (IP) on the 5th day of the study. The doses of CP and GA were designed according to a previous study (Abarikwu et al., 2020;Tomar et al., 2017). Rats were housed under standard conditions of temperature (25 ± 2ºC), relative humidity (50 ± 10%) and 12 hr light/12 hr dark cycle. The animals were fed with a standard pellet diet (ad libitum).

| Stereological analysis
To evaluate stereological parameters, testis was fractionated into fractions (f) by the systematic random sampling rules (Cahill et al., 1996). For this, the testis was divided into four equal parts (f1 = 1/4). One of the four pieces of the testis was randomly selected and then embedded in paraffin. 4 µm thickness sections were cut for stereological examination. The first section was taken randomly, and the next sections were taken every 50 steps (f2 = 1/50). An average of 10-13 sections was taken. After the sections taken were stained with haematoxylin-eosin (H-E), examined by light microscopy (Olympus BX53) using a microscopy camera (Olympus DP27) and microscope imaging systems (Olympus CellSens Entry).

| Calculation of the total number of cells
The physical dissector counting method was used to calculate the total number of spermatogonia, Sertoli and Leydig cells according to previous studies (Altindağ & Özdek, 2021). The following formula was used to calculate the total cell number: is the numerical density of the cell of interest (cells/unit volume) and V final is the total volume of the testis (Gundersen, 1986;Mayhew & Gundersen, 1996).

| Estimation of the total volume
The point-counting method of the Cavalieri principle was used to calculate the total volume of the testis, germinal epithelial volume, and interstitial volume. For this, the total numbers of points hitting all testis, germinal epithelial and interstitial area were counted using the point-counting probe. The following formula was used to calculate the volume: where 'V' is the volume of the structure, '∑P' is the total number of points hitting the structure, 'a/p' is the area covered by one point and 'h' is the height of dissector. To calculate the total volume of the tissue, the reference volume is multiplied by the fraction ratios (f1 and f2) and the shrinkage rate. After the histological tissue processing stages, the volume of tissue is usually shrunk. The testis shrinkage rate was calculated according to previous studies (Altindağ & Özdek, 2021).

| Estimation of the height of the germinal epithelium
The height of the germinal epithelium (HGE) was estimated according to previous studies (Altindağ & Özdek, 2021). For this, the following formulas were used: where '∑I' is the total number of intersection points of the luminal surface of the epithelium and the test lines, '∑P' is the total number of points hitting testis and 'I/p' is the length of a test line. The total surface area is calculated by multiplying the surface density by the total volume (Altindağ & Özdek, 2021;Noorafshan, 2014).

| Histological preparation
At the end of the experiment, rats were anaesthetised by xylazine (10 mg/kg) and ketamine (50 mg/kg) IP. For stereological, immunohistochemical and histological examination, the testis and epididymis tissues were removed by an incision made from the scrotal region and fixed in 10% buffered formalin. Testis and epididymis were embedded in paraffin after the tissue processing stages. Sections of 4 µm thickness taken from paraffin blocks were stained with haematoxylin-eosin (H&E).
The histological examination was made according to the Johnsen score (Johnsen, 1970) For this, 15-20 areas were evaluated by random sampling in 8-10 sections taken from each animal in the groups.

| Immunohistochemical preparation
The sections of 4 µm thickness taken from paraffin blocks were deparaffinised and dehydrated. After sections were incubated in 3% Hydrogen peroxide (H 2 O 2 ), in citrate buffer (ph 6.1), in Ultra V Block, Cell numerical density was estimated by counting Caspase-3, Bax, Bcl-2 and 8-OHdG positive cells using the physical dissector counting method, which is a stereological method by a light microscope (Olympus BX53) and cellSens Software imaging systems (Olympus).

TA B L E 1 Effect of GA on stereological parameters in CP-induced rats
The formula below was used for this:Nv = the total number of immunopositive cells counted in all the dissector, ∑P is the total number of counting frames, a/frame is the area of counting frame and h is the height of dissector.

| Measurements of serum testosterone levels
At the end of the study, blood samples taken from the heart with an injector were centrifuged at +4ºC at 3,000 rpm for 10 min. Serum testosterone was evaluated in an autoanalyzer (Abbott Architect ci16200, ) using commercial kits (Abbott Architect 2nd Generation Testosterone Reagent Kit).  (Dubovskiy et al., 2008;Lartillot et al., 1988).

| Measurement of parameters related to oxidative stress in testis
GSH (Beutler et al., 1963) and SOD (Sun et al., 1988) were similarly performed according to previous studies.

F I G U R E 1
Light microscope images of testis stained by haematoxylin and eosin (H-E) staining. x200. Testis has normal histological architecture in control (a) and GA (d) groups. In the CP group (b), atrophy (arrow) in the seminiferous tubules, loss of germ cells and a decrease in sperm count in the lumen of the testis was observed. GA improved testicular structural deterioration caused by CP (c)

| Statistical analysis
Statistical analyses were accomplished by using SPSS 21.0 software. Differences between groups were evaluated by the one-way analysis of variance (ANOVA) followed by the Tukey post hoc test.
p ≤ 0.05 was accepted as statistically significant. All data were expressed as mean ± standard deviations.

| Total number of testicular cells
As in Table 1, the total number of spermatogonia, Sertoli and Leydig cells was significantly decreased in rats of the CP group compared with the control group, but these parameters were significantly increased in the CP + GA group compared with the CP group (p < 0.05). GA group was observed to be similar to the control group.

| The total volume of testicular parameters
Compared with the control group, total testicular volume and total seminiferous tubule volume were significantly decreased in the CP group (p < 0.05), while no significant difference was observed in the total interstitial area volume. Compared with the CP group, a significant increase was observed in the total testicular volume and total seminiferous tubule volume in the CP + GA group (p < 0.05) However, no significant change was seen in the GA group compared with the control group. (Table 1).

| Height of germinal epithelium
The height of germinal epithelium significantly decreased in the CP group compared with the control group. Nevertheless, a significant increase was observed in the CP + GA group compared with the CP group (p < 0.05) But, the GA group was similar to the control group (Table 1).

| Histopathological evaluations
Testis and ductus epididymis were observed to have a normal histological structure in the control group (Figure 1a and Figure 2a). GA application improved these changes in both testis and epididymis ( Figure 1c and Figure 2c). In addition, according to the Johnsen testicular biopsy score results, a lower testicular score was observed in the CP group compared with the control group. On the other hand, the testicular biopsy score of the CP + GA group was higher than the CP group (Figure 7b).

| Immunohistochemical evaluations of Caspase-3, Bax, Bcl-2 and 8-OHdG
As given in Table 2, just a few Caspase-3, Bax and 8-OHdG immunopositive cells were observed in the control and GA groups.

| Serum testosterone levels
Compared with the control group, testosterone level significantly decreased in animals of the CP group (p < 0.05). However, the testosterone level significantly increased in the CP + GA group compared with the CP group (p < 0.05). The testosterone level of animals in the GA group was similar to that of the control group ( Figure 7a).

| Effects of GA on measurement of parameters related to oxidative stress in testis
When compared with the control group, it was observed that the

F I G U R E 3 Light microscopic images of Caspase-3 expressions in the testis. x200. Arrows indicate immunopositive cells in groups.
Control (a) and GA (d) groups are in normal histological architecture. CP triggered upregulation of Caspase-3 (b). GA caused downregulation of Caspase-3 (c)

| D ISCUSS I ON
The current study revealed that CP treatment caused a decrease in the total number of Sertoli, Leydig and spermatogonia cells and total testicular volume, as well as structural deterioration in the testis and epididymis. It also caused a significant increase in apoptosis, oxidative stress and DNA damage indexes and a decrease in testosterone secretion. However, GA significantly improved the side effects of CP by reducing testicular structural deformities, apoptosis, DNA damage, oxidative stress and improving testosterone secretion.
Chemotherapeutic agents are considered to be among the highrisk factors of infertility (Nna et al., 2020). Typically, infertility occurs after chemotherapy and radiotherapy. CP, an antineoplastic agent, is widely used to treat tumours. But, it causes cytotoxic effects in proliferating cells by damaging the binding of DNA base pairs. Excessive cell proliferation in spermatogenesis makes the testis more sensitive to these agents (Mercantepe et al., 2018). Therefore, protecting the testis from CP-induced toxicity has become an important issue in chemotherapy with CP (Afsar et al., 2017). Previous studies have reported that CP-induced testicular toxicity can be prohibited with the treatment of antioxidants such as melatonin, vitamin C and resveratrol (Almeer & Abdel Moneim, 2018;El-shafaei et al., 2018;Reddy et al., 2016). Additionally, it has been published that GA should protective effects against CP-induced nephrotoxicity, ototoxicity and genotoxicity of the bone marrow (Akomolafe et al. 2014;Shruthi et al., 2018;Kilic et al., 2019). However, in our literature review, no study was found on the effects of GA on testicular and epididymal damage induced by CP.
It has been reported that CP increases ROS production, lipid peroxidation and denaturation of structural proteins additionally decreases the activity of the antioxidant system and promotes cell apoptosis by activating p53 (Dasari & Tchounwou, 2014;Kohsaka et al., 2020). Oxidative stress is accepted as the result of the dis-  (Olusoji et al., 2016). In addition, Oyagbemi et al., (2016) reported that GA significantly improved MDA levels, nitrite and H 2 O 2 levels and decreased SOD, GSH in cyclophosphamide-induced testicular and epididymal damage.
According to their role in apoptosis, apoptotic proteins are classified as pro-apoptotic and anti-apoptotic proteins (Abotaleb et al., 2019). Bax protein, a pro-apoptotic protein, increases under oxidative stress conditions and subsequently releases the cytochrome C that impairs the mitochondrial membrane permeability (Fouad et al., 2017). Cytochrome C binds to apoptotic proteaseactivating factor 1 (Apaf-1) and activates it. Activated Apaf-1 forms the apoptosome. Apoptosis is maintained by caspases formed in response to cell death stimuli. The apoptotic protease-activating factor 1 (Apaf-1) controls caspase activation. As a result, activation of Caspase-3 that plays a key role in the execution of apoptosis is stimulated (Riedl et al., 2005). Caspase-3 is frequently used in studies to evaluate apoptosis, because the activation of Caspase-3 is an irreversible stage that induces apoptosis (Mercantepe et al., 2018;Nna et al., 2020). Proteins in the Bcl-2 families control cell death and survival by regulating the mitochondrial apoptotic pathway. Bcl-2, an anti-apoptotic protein, maintains mitochondrial membrane integrity (Fouad et al., 2017). Previous studies have reported that CP (a single dose of 6 mg/kg and 7 mg/kg) causes upregulation of Caspase-3 and downregulation of Bcl-2 (Kohsaka et al., 2020;Nna et al., 2020).
Similarly, Aly and Eid (2020) have reported that CP treatment (a single dose of 7.5 mg/kg) increased the activity of Caspase-3 and Bax, while decreased the activity of Bcl-2 in testis. They also showed that resveratrol decreased the activity of Caspase-3 and Bax, but increased the activity of Bcl-2. In another study, Eren et al., (2020) reported that CP increased the caspase-3 immunoreactivity in the germinal epithelium and Leydig cells. However, they also revealed that amifostine and melatonin decreased the Caspase-3 immunoreactivity in the germinal epithelium and Leydig cells. The present study revealed that CP treatment significantly increased the expres- increased ROS production caused by CP (Köroğlu et al., 2019). Also, quantitative data of our study obtained using stereological methods demonstrated that total volume of the testis, the total volume of seminiferous tubule and germinal epithelial height significantly decreased in animals that received CP compared with control groups.
We estimate that the decrease in these parameters of our study may have been due to the decrease in the total number of testicular cells caused by CP. These changes in testicular morphometric parameters confirmed that CP treatment causes testicular toxicity. However, GA treatment inhibited the total volume of the testis, the total volume of seminiferous tubule and germinal epithelial height.   found that CP treatment significantly decreased the testosterone levels but taurine significantly increased the testosterone levels in CP plus taurine group compared with the CP group. In our study, serum testosterone levels significantly decreased in rats which are applied CP. According to the findings of our study, it is estimated that the decrease in serum testosterone level caused by CP application may be related to the decrease in Leydig cell number as a result of increased apoptosis in Leydig cells. Because testosterone secretion is directly related to Leydig cell function and number (Gholami F I G U R E 7 Effects of GA on testosterone (a), Johnsen score (b), MDA (c), CAT (d), GSH (e) and SOD (f) in CPinduced rats. Values are expressed as means ± SD. Significant differences as compared with the Control group at a P < 0.05, Significant differences as compared with the CP group at b P < 0.05 Jourabi et al., 2021). Decreased testosterone levels caused by CP may disrupt the spermatogenesis process and thus may cause male infertility.

| CON CLUS ION
Our findings demonstrated that CP may cause male reproductive damage through various mechanisms, such as oxidative stress, DNA damage, apoptosis, structural and functional defects. Furthermore, our findings revealed that GA may prevent CP-induced male reproductive toxicity by inhibiting oxidative stress, DNA damage, apoptosis and restoring the structural and functional defects in the testis and epididymis. According to the findings of this study, it is suggested that GA can be used as a protective agent against the side effects of CP treatment in testis and epididymis.

ACK N OWLED G EM ENTS
This study was carried out with the resources provided by the

Department of Histology and Embryology, Faculty of Medicine and
Vocational Higher School of Healthcare Studies, Van Yüzüncü Yıl University.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest.

D at a Av a i l a b i l it y St ate m e nt
The data of this study are available from the author upon reasonable request.