A standardized extract of Ginkgo biloba leaves is a well-defined product and contains 24% flavone glycosides and 6% terpene lactones⁶. The studies with the testicular germ cell apoptosis indicate that there is a protective effect of Ginkgo biloba extract 761 (EGb 761) against apoptosis and testicular damage^7,8. It is reported that EGb 761 modulates expression of apoptotic related genes, reduces generation of free radicals and increases activity of antioxidant enzymes different animal tissues^9-11.

In recent years, experimental studies have been conducted showing the protective effect of EGb 761 against toxic agents. However, there was not any study related to the therapeutic effect of the herbal products against DEHP formed testicular toxicity. It was aimed with the present study to investigate the protective effect of EGb 761 as known a powerful antioxidant and the anti-apoptotic properties on DEHP formed sub-acute toxicity in prepubertal rat testis.

DEHP (Acros, New Jersey, USA) was dissolved in corn oil (5 mL/kg) and administered to the animals by gavage at a dose of 2 g/kg/every other day. The dose used in this study were based on previous reports showing the adverse effects of DEHP on male reproductive organ in young rats¹². EGb 761 (Abdi Ibrahim, Istanbul, Turkey) was administered 50 mg/kg¹³ by the same method one hour after DEHP treatment. All treatments were maintained for 28 days. A total of 72 male Wistar albino rats were randomly divided into four experimental groups of 18 animals each. These groups were arranged as follows: Control; treated with 5 mL/kg corn oil during 28 days. DEHP; treated with 5 mL/kg corn oil + DEHP 2 g/kg during the first 14 days; treated with 5 ml/kg corn oil during the last 14 days. DEHP+EGb 761; treated with DEHP 2 g/kg + 50 mg/kg EGb 761 during the first 14 days; treated with 50 mg/kg EGb 761 during the last 14 days. EGb 761; treated with 50 mg/kg EGb 761 during 28 days.

Sample collection and homogenate preparation: The animals were sacrificed under slight ether anaesthesia exactly 24 h after the last administration at the end of 7, 14 and 28th days (into 3 sacrificed groups of six animals each), testes were removed. They were divided into two pieces; the first testis section was used for the histopathological examination, the second testis was stored at -20 °C until biochemical analyses.

Biochemical analysis: Lipid peroxidation level was measured based on the concentration of thiobarbituric acid reactive substances, and the amount of produced malondialdehyde (MDA) was used as an index of LPO¹⁴. Catalase activity was spectrophotometrically determined by measuring the decomposition of hydrogen peroxide (H₂O₂) at 240 nm according to the method of Aebi¹⁵. Reduced glutathione (rGSH) content of the testis homogenate was measured at 412 nm using the method of Sedlak and Lindsay¹⁶. Glutathione peroxidase (GSH-Px) activity was determined according to the method of Lawrence and Burk¹⁷. The protein content in the testis was measured by method of Lowry et al.¹⁸. The serum testosterone level was measured by ELISA method using Elisa testosterone kit (Dia Metra, Italy) according to the manufacturer’s instructions and expressed as ng/mL.

Histological examination: Testis tissues were fixed by Bouin’s solution, embedded in paraffin, and were stained with haematoxylin-eosin and PAS- haematoxylin. A total of 25 seminiferous tubules (ST) were randomly examined per section and, their diameters and germinal cell layer thickness (GCLT; from the basal membrane towards the lumen of the tubule) were measured using an ocular micrometer in a light microscope and, and the mean size of ST and GCLT were calculated. Johnsen’s testicular scoring¹⁹ was used to categorise the spermatogenesis in control and treatment groups. Twenty-five ST from each section were evaluated, and a score between 1 (very poor) and 10 (perfect) was given to each tubule according to Johnsen’s criteria.

Determination of apoptotic cells in testis: The apoptotic germ cells were evaluated by using terminal transferase dUTP nick-end labelling (TUNEL, In Situ Cell Death Detection Kit, POD; Roche, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, tissue sections were deparaffinized and digested with 20 mg/mL proteinase K for 10 min, followed by treatment with 3% H₂O₂ for 5 min to inhibit endogenous peroxidases. After washing with PBS, sections were then incubated with the TUNEL reaction mixture containing TdTenzyme and digoxigenin–11–dUTP in humidified atmosphere at 37 °C for 1 h and then stop/wash buffer was applied for 30 min at 37 °C. Labeling was revealed using 3-amino-9-etilcarbazole substrate. Counterstaining was performed using Mayer’s hematoxylin and sections were dehydrated and mounted with glycerol. For negative controls, sections were incubated with solution without terminal transferase. Apoptotic index was calculated as percentage of TUNEL-positive cells by the following equation: apoptotic cells %= (number of TUNEL-positive cells/total number of cells) × 100²⁰.

Statistical analyses: Data are presented as mean ± SEM. The degree of significance was set at P<0.05. Nonparametric Kruskal Wallis Varyans Analyses were used to determine the differences between the groups with respect to all parameters. Also, Mann Whitney-U test was used to determine the groups that causing this differences. In addition, the differences between the groups in terms of ST diameter, GCLT parameters, Johnsen testicular score, and TUNEL positive apoptotic cell index were determined with ANOVA (One Way of Analysis Variance) using Duncan test. The SPSS for Windows version 15.0 program (SPSS, Chicago, IL, USA) was used for statistical analyses.

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Table 1: Mean±SEM values of body (BW) and testis weights (TW) in different treatment groups. (DEHP= Di-(2-ethylhexyl) phthalate, EGb 761= Ginkgo biloba extract 761).

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Table 2: Mean±SEM values of diameters ST, GCLT, Johnsen testicular score, and TUNEL positive apoptotic cell indices in different treatment groups. (DEHP= Di-(2-ethylhexyl) phthalate, EGb 761= Ginkgo biloba extract 761).

The histopathological changes in the testes of all experimental groups were given in Table 3. It was observed that histological appearences of testes of control and EGb 761 groups were normal in the 7th day (Figure 1A and D). The histopathological changes such as necrosis, degeneration, desquamation, vacuolization, disorganization and reduction in germinal cells, atrophy in tubules were observed in DEHP group (Figure 1G). These types of histopathological damage were more milder in DEHP+EGb 761 group (Figure 1J).

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Figure 1: (A-F) Normal histological appearance of seminiferous tubules, Sertoli cell (arrow), and Leydig cells (arrowheads) in control and EGb 761 groups of 7th, 14th and 28th days. (G) Vacuolization in germinal cells (arrowheads) in DEHP group of 7th day. (H) Severe atrophy, degeneration and necrosis (stars) in seminiferous tubules in DEHP group of 14th day. (I) Severe Leydig cell hyperplasia (arrowheads) in DEHP group of 28th day. (J-L) Gradually regeneration in seminiferous tubules in DEHP + EGb 761 groups during the experiment periods (H&E).

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Table 3: The existence of some pathological lesions in testes of different treatment groups. (DEHP= Di-(2-ethylhexyl) phthalate, EGb 761= Ginkgo biloba extract 761).

It was observed that ST was smooth of morphology and started of spermatogenesis in control and EGb 761 groups in the 14^th day. (Figure 1B and E). In DEHP group pointed out which deepened the tubular atrophy due to necrosis in ST and continued the degeneration (Figure 1H). In addition to multinucleated giant cell formations in some tubules, Leydig cell hyperplasia, interstitial edema and capillary congestion was observed significantly. In DEHP + EGb 761 group it was seen that the intensive atrophic and necrotic changes replaced the mild degenerative changes. Furthermore, the presence of regeneration and spermatogenesis in many of the ST was detected in this group. And compared with DEHP treated group, interstitial regions showed a near normal appearence, but mild Leydig cell hyperplasia was found. (Figure 1K).

In the 28th day, the histological structures of control and EGb 761 groups were normal and were formed a complete spermatogenesis in ST (Figure 1C and F, 3A and B). In DEHP group continued the necrotic and degenerative changes together with more intensive Leydig cell hyperplasia (Figure 1I), and thickening of ST basal membrane were seen as the most evidently in this period (Figure 3C). In DEHP+EGb 761 group, there were only mild degenerative changes in ST (Figure 1L). It was determined that the basal membrane and Leydig cells were normal in appearence compared with those in DEHP group (Figure 3D).

Figure 2 illustrates apoptosis, demonstrated by TUNEL-staining in the testes of control and treated groups (Figure 2). The apoptotic cell index of DEHP group was significantly (P<0.05) higher than that of the control and other groups. However, a significant (P<0.05) decrease was observed in apoptotic cell indices of DEHP+EGb 761 group compared with that of the DEHP group only (Table 2).

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Figure 2: (A-F) TUNEL-staining in control and EGb 761 groups of 7th, 14th and 28th days. TUNEL-staining (G-I) in DEHP groups and (J-L) in DEHP + EGb 761 groups of experiment periods. Apoptotic spermatocytes (arrowheads) and Sertoli cell (arrow) indicate (TUNEL).

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Figure 3: (A-B) Normal histological appearance in basal membrane of seminiferous tubules and elongated spermatids (arrowheads) in control and EGb 761 groups. (C) Prominent thickening in basal membrane of seminiferous tubules (arrowheads) and severe Leydig cell hyperplasia (star) in DEHP group. (D) Normal histological appearance in basal membrane of seminiferous tubules in DEHP + EGb 761 group (PAS & H).

Biochemical findings and testosterone levels of all groups were given in Table 4. It was determined that MDA, GSH-Px and CAT levels significantly increased in DEHP and DEHP+EGb 761 groups than control and EGb 761 groups. It was determined that rGSH and serum testosterone levels decreased in DEHP group during experimental period (P<0.05). Additionally, It was seen marked increase in the serum testosterone levels in DEHP+EGb 761 group compared to DEHP group in the 28th days (P<0.05).

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Table 4: Mean±SEM values of malondialdehyde (MDA), reduced glutathione (rGSH), glutathione-peroxidase (GSH-Px) and catalase (CAT) activities and serum testosterone levels in different treatment groups. (DEHP= Di-(2-ethylhexyl) phthalate, EGb 761= Ginkgo biloba extract 761).

The lipid peroxidation (LPO) induced with toxic substances indicates the presence of oxidative stress in tissue with high concentrations of MDA, low concentrations of GSH^9,26. The previous studies related to DEHP toxicity indicated that it was free radical trigger which caused increase in GSH-Px and CAT enzymes together with decrease in SOD and GSH levels^2,27. In this study, biochemically the determination of increase in the concentration of MDA, decrease in GSH level, increase in the enzymes levels such as GSH-Px and CAT indicate that our findings were similar to the findings in the literature.

Many studies stated that DEHP toxication negatively affected testosterone production of Leydig cells^22,28,29. Miura et al.³⁰ indicated that the concentration of testosterone significantly decreased in association with Leydig cell dysfunction in DEHP treated animals. In addition, it was reported that DEHP toxicity caused to a decrease in the antioxidant enzyme activities, and an increase in the ROS and lipid peroxides²². Leydig cells are sensitive to the extracellular sources of ROS leading to ultrastructural changes in their structures³¹. In accordance with literature data given above, it is thought that the increase in the lipid peroxide levels and the inhibition of the testicular steroidogenesis caused Leydig cell dysfunction. Akingbemi et al.²¹ reported that Leydig cell hyperplasia induced as a result of DEHP exposure and this was associated with serum testosteron and LH levels. During experiment of our study, it was observed that there was a significant decrease in testosterone levels of DEHP treated group compared to the others. But, it was seen an increase in 28th day compared to previous experiment periods. It has been thought that these changes in testosterone levels which may be depend on Leydig cells dysfunction and hyperplasia.

A repeated administration of DEHP was reported to cause testicular DNA damage^5,12,23. The apoptotic cell indices of DEHP groups were found to be significantly higher than those of the control groups in our study.

Ablake et al.³² reported that DEHP firstly caused the spermatogenic deterioration and focal degeneration. Then, It was determined spermatogenic arrest found in Sertoli cells together with just a few spermatogoniums, but there was regeneration in the some tubules at the end of the experimental process. Additionally, while Park et al.¹² suggested that DEHP toxicity caused edema in interstitial region and giant cells with the multinucleate with germ cell origin, the other researchers argued that DEHP induced hyperplasia in Leydig cells^21,33,34. Similar to the literature findings in our experimental study; it was observed that the testicular damage and interstitial edema began in 7th day of the treated, this damage was still more deepened and multinucleate giant cell formation seen on 14th day. It was also observed that degenerative effects and Leydig cell hyperplasia to be more intense on 28th day. But there were a few tubule regeneration. Testicular damage observed in this work may be elucidated with the direct or indirect effect of DEHP, which later induces lipid peroxidation that is a chemical mechanism capable of corrupting the structure and function of testis.

EGb 761 has eliminated the testicular damage with variety mechanisms such as stabilized the membranes and prevention LPO³⁵. Toxicity studies showed that EGb 761 protected testis by suppressing ROS production and combating antioxidant enzyme deactivation against oxidative stress. In other words, it was determined that EGb 761 treatment neutralized the abnormalities in both MDA and the GSH levels, also transformed to normal the antioxidant enzyme activities such as SOD, GSH-Px and CAT^8,36. In this study significant decreases in MDA level, GSH-Px and CAT activities and marked increase in rGSH level of EGb 761 treated rats was observed. These findings demonstrate that EGb 761 has a potent antioxidative effect.

It was reported that EGb 761 was a herbal agent which has a suppressive role against the sexual function dysfunction³⁷ and increased the sexual performance by increasing their testosterone levels in the young rats³⁸. In addition, it was stated that the testosterone level decreasing the result of Leydig cell damage in diabetic rats gradually increased with EGb 761³⁹. This study showed that DEHP induced the decreased testosterone levels increased in EGb 761 treatment.

Kanter⁴⁰ demonstrated that the decrease in ST diameter and germinal cell degeneration composed with ischemia / reperfusion injury approached to the control group with EGb 761. EGb 761 administration to DEHP treated rats significantly increased in ST diameter and in GCLT in parallel with the literatures in our study. This result may be associated with beneficial effects of EGb 761 being the spermatic cell proliferation in tubules.

The previous studies indicated that EGb 761 has antiapoptic properties^13,40. Yeh et al.⁸ stated that EGb 761 pressured doxorubicin induced germ cell apoptosis and had antiapoptotic effect on testis. Also, it was determined that the ischemia / reperfusion and the testicular torsion increased the germ cell apoptosis and decreased these negative effects with EGb 761^7,13,40. In this study, it was determined that DEHP induced testicular apoptosis gradually improved depending on antiapoptotic properties of EGb 761.

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