Investigation of the Effect of Rosmarinic Acid on Cyclophosphamide-Induced Gonadal Toxicity ()
1. Introduction
Cyclophosphamide (CP) is one of the successful anticancer drugs ever designed. CP is still used as a chemotherapeutic agent for blood and bone marrow transplantation [1]. Although it is used in many clinical studies, it is known that CP has adverse effects such as reproductive toxicity [2]. In case of exposure to CP, significant histological changes such as weight loss in the gonads, degenerated spermatogenesis, oligozoospermia and azoospermia are observed [3]. The cause of gonadal toxicity from CP is unclear. However, studies know that it causes severe oxidative stress, apoptosis and inflammation [4]. The destructive damage that develops after the use of CP is difficult and takes years to recycle [5]. It is thought that the toxic effect of CP decreases after the application of ginseng, which is an antioxidant [6]. In a study, it was seen that ginger and vitamin E prevented gonadal toxicity against cyclophosphamide [7]. Rosmarinic acid (RA), which belongs to the Lamiaceae family, is a natural antioxidant found in most plants [8]. RA has effects such as immunomodulatory, antibacterial and antiviral activities [9]. Due to its antioxidant effect, rosmarinic acid is widely used in cosmetics and foods, as well as medical use. In addition, it has the ability to inhibit RA lipoxygenase and cyclooxygenase activity [10]. In studies, it is known that RA increases the level of serum testosterone [11].
In our study, we aimed to investigate the possible role of RA, which has an important antioxidant and immunomodulatory effect, against the toxic effect of CP on reproductive toxicity.
2. Material and Method
Ethical Approval and Experiment Procedure—Approved by Dicle University Animal Experiments Local Ethics Committee (ethical approval number: 2020/16). The experiment was carried out in Dicle University Experimental Animal Care Unit. In the experimental protocol, 28 animals, 15 - 16 weeks old, 200 - 250 g male Wistar Albino rats were used. Experimental animals were obtained from Dicle University Health Sciences Research and Application Center. This study, which is part of her doctoral study, was supported by Dicle University Scientific Research Projects (BAP). The subjects were housed in a 12-hour dark and 12-hour light cycle at room temperature (22 ± 2˚C), in cages containing 7 animals, with free access to water and food. According to the experimental protocol, daily injections were made administered intraperitoneally (i.p) at the same time each day. Rosmarinic acid (CAS 20283-92-5, Santa Cruz Biotechnology,) was dissolved in 1% alcohol, filtered on filter paper and stored at −20˚C until use.
All experimental groups were subjected to the following procedures for 14 days.
Group 1 (Control) (n = 7): Subjects received only 1 ml of 0.9% saline solution per day intraperitoneally for 14 days.
Group 2 (Rosmarinic Acid) (n = 7): The subjects were given 20 mg/kg Rosmarinic acid intraperitoneally for 14 days.
Group 3 (Cyclophosphamide) (n = 7): Only 1 ml of 0.9% saline solution was administered intraperitoneally to the subjects for the first 7 days. Cyclophosphamide 20 mg/kg per day was administered intraperitoneally for the last 7 days (from the 8th day).
Group 4 (Rosmarinic Acid + Cyclophosphamide) (n = 7): The subjects were given 20 mg/kg Rosmarinic acid daily intraperitoneally for 14 days. From the 8th day of the experiment to the end of the experiment (last 7 days of the experiment), 20 mg/kg cyclophosphamide was given intraperitoneally daily.
Experiment times and model and CP doses were designed based on the studies of Zina et al. [12] and Sabik et al. [13].
Anesthesia and Surgical Procedures
At the end of the experiment, rats were sacrificed under anesthesia of 75 mg/kg intramuscular ketamine hydrochloride and 10 mg/kg xylazine. Testicular tissue was removed by performing lower fixed in abdominal dissection. Subsequently, testicular tissues were %10 formaldehyde.
Histological examination
5-μm thick sections were taken from parafin-embedded testicular tissues. After the deparaffinization process, the sections were passed through alcohol series and taken into distilled water. After the deparaffinized through alcohol series and taken into distilled water Hematoxylin Eosin dye (HE) was applied to sections. The stained sections were washed with distilled water, then passed through alcohol series and dehydrated. They were covered with entellan and examined under a light microscope (Zeiss, Imager A2, Germany).
Statistical analysis
The data obtained in the study was expressed as an arithmetic mean ± standard deviation and the Mean Rank value. Statistical analysis was performed using SPSS 22.0 program. In Multiple comparison, Kruskal-Wallis test was used. p < 0.05 was accepted as significant.
3. Results
Statistical findings
Cyclophosphamide group according to the statistical data obtained body and testicular weights of rats belonging to the group statistically compared to other groups. When the cyclophosphamide group was compared with the other groups, a significant difference was detected (p < 0.05) (Table 1). In addition, when we compared the cyclophosphamide group with the rosmarinic acid and rosmarinic acid + cyclophosphamide group, we found that there was a significant difference. Except for the cyclophosphamide group, it was determined that there was no difference between the other groups (p > 0.05).
Histopathological findings
Hematoxylin-eosin impressions of the groups are shown in Figure 1. Seminiferous tubule structure in the testes of the control group is normal in hematoxylin-eosin stained sections. Spermatogonia appear normal towards the lumen. Sertoli cells were observed to have broad bases and an upward triangular structure (Figure 1(a)). When we examined the rosmarinic acid group (Figure 1(b)), in the transversal section of the seminiferous tubules, spermatogonia close to the basal lamina were found to be chromatin-rich and oval in shape. It was determined that Sertoli cells had a triangular structure and that the maturing spermatogonium
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Table 1. Shows experimental groups with their body weight ± standard deviation (SD) and testicular weight ± standard deviation SD.
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Figure 1. (a) Control group: Seminiferous tubule (s), basal membrane (red arrow) (b) Rosmarinic acid group: Normal appearance spermatogonia (black arrow). (c) Cyclophosphamide group: Disruption of basement membrane integrity (blue arrow), degenerated spermatogonia (green arrow) (d) Cyclophosphamide and rosmarinic acid group: Basement membrane (orange arrow), regularly arranged spermatogonia (s) (H & E, 50 µm).
was normal in development. When the hematoxylin-eosin stained sections belonging to the cyclophosphamide group are examined (Figure 1(c)). It is observed that the structural integrity of the basement membrane of the seminiferous tubules has been disrupted and the basement membrane has completely disappeared in some areas. The spermatogonia in the seminiferous tubule are degenerated and pyknosis is seen in their nuclei. A marked cell inflammation and edema are observed within the intertubular spaces. When the sections belonging to the cyclophosphamide and rosmarinic acid groups are examined (Figure 1(d)); despite the toxic effect of cyclophosphamide, the integrity of the basement membrane appears to be preserved. It is seen that the spermatogonia in the seminiferous tubule are partially degenerated, but overall integrity is provided. Sertoli and Leydig cells were found to have a normal appearance. It is seen that inflammation decreased in the intertubular area. It was determined that rosmarinic acid was protective on testes against tissue damage of cyclophosphamide.
4. Discussion
Cyclophosphamide (CP), an effective anticancer drug, was approved by the FDA in 1959 [14]. However, as a result of studies, it has been determined that CP has toxic effects on cardiac, testicular, kidney and liver [15]. It has been determined that there are deteriorations in the sperm structure in patients who use CP for more than 4 months [16]. There are data that cyclophosphamide also causes apoptosis [17]. In a study conducted on male rats in 2002, it was found that CP had an effect on decreasing testicular weight [18]. In our study, it was determined that cyclophosphamide was effective on testicular weight and there was a statistically significant difference when compared with other groups (Table 1). When Sung et al. compared testes and body weight in the CP group with the control group in their study in 2016, they found that there was a decrease in both testes and body weight in the cyclophosphamide group compared to the control group [19]. Our study shows parallelism with this study. In our study, we found that Rosmarinic acid has a protective effect on testicles and body weight, despite the toxic effect of CP (Figure 1). In a study conducted in 2016, it was determined that tissue integrity was impaired and germ cells were damaged in the testes of rats administered cyclophosphamide [20]. When the hematoxylin-eosin stained sections of the experimental study we conducted were examined, it was determined that the tissue integrity was impaired, and Sertoli cells and germ cells were damaged in the testes of rats administered cyclophosphamide (Figure 1(c)). As a result of many experimental studies, it is known that CP has a toxic effect on the reproductive organs [21]. The findings we obtained with the toxic damage caused by CP in the testicles are similar to the findings obtained in other similar studies [22]. In experimental studies induced by cyclophosphamide, tissue integrity damage and apoptosis are observed [23]. There are also many reports that RA has an effect on inflammation. Studies have shown that RA inhibits complement activation both in vivo and in vitro [24]. However, there are no studies in the literature on whether rosmarinic acid is protective against the efficacy of CP. In this respect, it is important to investigate whether RA has prophylactic and anti-inflammatory effects in our study. In the study of Khaki et al. in 2012, it was observed that RA protects testicular tissue integrity and prevents apoptosis in rats exposed to electromagnetic field [11]. In our study, we obtained results showing that RA protects tissue integrity against the toxic effect of cyclophosphamide (Figure 1(d)). When we look at the findings of the cyclophosphamide group, disruptions in the basement membrane structure, pyknosis in Sertoli and germ cells were observed. These findings are similar to the work of Ghosh et al. [25]. As a result of our study, we observed that RA has a protective effect against toxic damage.
5. Conclusion
It was observed that cyclophosphamide inhibited spermiogenesis in gonads and Sertoli cell adhesion was affected. We think that RA has an antioxidant effect against CP toxicity. Despite the toxic effect of CP, rosmarinic acid is thought to have a significant curative effect on the spermatogenetic process and seminiferous tubule structure in the gonads.
Acknowledgements
This study is a part of Fırat ŞAHİN’s doctoral thesis and was supported Research Projects (DUBAP) with the by Dicle University Scientific project number TIP.21.003.