SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Abstract:  Cisplatin and other platinum complexes are important chemotherapeutic agents and useful in the treatment for several cancers such as prostate, ovarian and testis. However, severe side effects including reproductive toxicity of cisplatin and other platinum complex cause limitations in their clinical usage. In this context, we aimed to compare the damage in testis caused by cisplatin and a novel platinum-N-heterocyclic carbene complex (Pt–NHC). To this end, 35 Sprague–Dawley rats were divided randomly into five equal groups (n = 7 in each group). Cisplatin and Pt–NHC were intraperitoneally administered as a single dose of 5 mg/kg or 10 mg/kg, and the rats were then killed 10 days after this treatment. The testicular tissues and serum samples were taken from all rats for the determination of reproductive toxicity. The results showed that cisplatin and Pt–NHC caused toxicity on the reproductive system via increased oxidative and histological damage, decreased serum testosterone levels and negatively altered sperm characteristics in a dose-dependent manner (p < 0.05). At the same dose levels, cisplatin generally caused lower toxicity on the reproductive system compared with Pt–NHC. In conclusion, these results suggest that Pt–NHC has more toxic effects on the male reproductive system than cisplatin, and in terms of clinical usage, Pt–NHC may be unsafe compared with cisplatin.

Cisplatin is the most widely used antitumour drug, especially for the treatment for testicular tumours. Also, it is effective against several other diseases such as ovarian, cervical and bladder cancer [1–3]. However, cisplatin has deleterious side effects including nephrotoxicity, ototoxicity, cardiotoxicity, hepatotoxicity, gastrointestinal and reproductive dysfunction [4,5]. Previous experimental studies [6,7] have shown that testicular toxicity related to cisplatin administration is an important problem in males owing to high mitotic activity of spermatogenic cells. It was determined that cisplatin exposure may lead to azoospermia, abnormalities in sperm, impaired spermatogenesis and decrease in testosterone levels in rats [8,9]. Besides, it caused oxidative stress characterized by an increase in lipid peroxidation and decrease in the antioxidant system as well as histological changes in testis tissue [10]. The cellular mechanism of reproductive toxicity induced by cisplatin is poorly understood. Traditionally, it was suggested that cisplatin exposure disrupted the redox balance of tissues including testis, and this resulted from oxidative stress. It was clearly shown that oxidative stress and reactive oxygen species play an important role in the pathogenesis of cisplatin toxicity [11,12].

The clinical success of cisplatin is limited by severe side effects and resistance. Therefore, much attention has focused on designing new platinum compounds with improved pharmacological and toxicological properties. Many studies have shown that N-heterocyclic carbenes (NHC), as a carrier for metal complexes including platinum II, exert antitumour and antimicrobial activity. Although the anticancer potential of novel platinum-N-heterocyclic carbene complexes (Pt–NHC) was determined by Skander et al. [13], their toxicity in the reproductive system has not been examined.

In this context, we aimed to evaluate the reproductive toxicity of Pt–NHC and to compare these toxic effects with those of cisplatin. For this purpose, we determined the levels of oxidative, histological, hormonal and spermatological damage in testis tissues of rats.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Chemicals  Cisplatin (10 mg/10 mL, Code 1876A) was obtained from Faulding Pharmaceuticals Plc (Warwickshire, UK). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The novel Pt–NHC complex was synthesized at Inonu University, Organometallic Research Laboratory, according to Ciftci et al. [5] (Scheme 1).

image

Figure Scheme 1..  Chemical structure of Pt–NHC complex.

Download figure to PowerPoint

Animals and treatment  A total of 35 healthy, adult male Sprague–Dawley rats (2–3 months old; 200–250 g) were obtained from the Experimental Animal Institute, Malatya, Turkey, for this experiment. The animals were housed in sterilized polypropylene rat cages and kept in a 12-hr light/dark cycle at an ambient temperature of 21°C. Food and water were provided ad libitum. The experiments performed were based on the animal ethics guidelines of the Institutional Animals Ethics Committee.

The rats were randomly divided into five equal groups (n = 7 in each group). We selected cisplatin dose according to upper (10 mg/kg) and lower (5 mg/kg) ranges of a popular side effect dose (7 mg/kg) for this experiment [5,6]. Similarly, because the therapeutic dosage of Pt–NHC was not reported, we used the same dose for Pt–NHC. All drugs were intraperitoneally (i.p.) given via only one administration after all chemicals had been dissolved in dimethylsulfoxide. In the first (control) group, dimethylsulfoxide was administered to the rats by a single injection. In the second and third (low dose) groups, cisplatin and Pt–NHC were administered to the rats at a dose of 5 mg/kg. In the fourth and fifth (high dose) groups, cisplatin and Pt–NHC were administered to the rats at a dose of 10 mg/kg. The animals were killed under ether anaesthesia after 10 days of drug administration. For biochemical analysis, the right testis was immediately dissected and weighed. Under anaesthesia, blood samples were collected from the left ventricle with an injector. Serum was obtained after whole-blood centrifugation (3000 × g, 20 min., 4°C). Tissue and serum samples were frozen at −45°C until analysis.

Biochemical assay  The homogenization of tissue has been described in our previous study [14]. The levels of thiobarbituric acid-reactive substances (TBARS), total glutathione levels and catalase, superoxide dismutase and glutathione peroxidase activities were determined by spectrophotometric methods, and these methods have been outlined in our previous studies [14–20].

Histological examination  For the histological study, the left testis was weighed and fixed in 10% formalin, dehydrated in ethyl alcohol, cleared in xylol and embedded in paraffin wax. Sections of 5 μm thickness were cut and stained with haematoxylin and eosin to determine histopathological changes. The diameter of the seminiferous tubule and germinative cell layer thickness from twenty different areas of each testis were measured using a Leica Q Win Plus Image Analysis System (Leica Micros Imaging Solutions Ltd, Cambridge, UK) at 10×. The testicular damage severity was semiquantitatively assessed for each of the parameters as follows: spermatogenic arrest (ceased the spermatogenesis), disintegration in the spermatogenic layer, disorganization in germinal cells, multi-nucleated giant cell formation, degeneration, desquamation and vacuolization of spermatogenic cells. The degree of damage was identified as absent (0), slight (1), moderate (2) and severe (3). Maximum score was noted as 21, and the microscopic score of each section was calculated as the sum of the scores. Sections were examined by a Leica DFC 280 light microscope at 40× and 100× magnification for overall histological examination.

Evaluation of sperm parameters  The epididymal sperm concentration was determined with a haemocytometer using a modified method briefly described by Ciftci et al. [14]. The percentage of forward progressive sperm motility was evaluated using a light microscope with heated (37°C) stage as described by Ciftci et al. [21]. To determine the percentage of morphologically abnormal spermatozoa, the slides stained with eosin–nigrosin were prepared. A total of 300 sperm cells was examined on each slide (2100 cells in each group), and the head, tail and total abnormality rates of spermatozoa were expressed as percentages [13,21].

Hormonal analysis  Serum testosterone level was determined by enzyme-linked immunosorbent assay (ELISA) using anti-rat ELISA commercial kits from Cayman Chemical Company (Ann Arbor, MI, USA) using the CA-2000 ELISA microplate reader (CIOM Medical Co., Ltd., Changchun, China). Testosterone quantities in the samples were calculated from standard curves of testosterone using a linear regression method.

Statistical analysis  All values are presented as mean ± S.E.M. Differences were considered to be significant at p < 0.05 for biochemical, hormonal and spermatological changes. The computer program SPSS 11.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. For biochemical values, statistical analyses were performed using one-way anova and post hoc Tukey’s honestly significant difference test. Histological results were compared with Kruskal–Wallis variance analysis. Where differences between the groups were detected, group means were compared using the Mann–Whitney U test.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Biochemical evaluation

Catalase, superoxide dismutase, glutathione peroxidase activities, TBARS and glutathione levels in testis tissue of rats exposed to 5 mg/kg (low dose) or 10 mg/kg (high dose) doses of cisplatin and Pt–NHC are given in table 1. In all groups, a significant decrease in superoxide dismutase, catalase, glutathione peroxidase activities and glutathione levels was observed in the testicular tissues of rats compared with those of the control group. However, the TBARS levels in testis tissue of rats were significantly increased in the experimental groups compared with those of the control groups. The present study revealed that the administration of cisplatin and Pt–NHC changed superoxide dismutase, catalase, glutathione peroxidase activities, TBARS as well as glutathione levels in a dose-related manner. Generally, the high dose of Pt–NHC and cisplatin caused a decrease in antioxidant enzymes and an increase in TBARS levels in testicular tissue compared with low-dose application.

Table 1.    Changes in superoxide dismutase, catalase, glutathione peroxidase activities and glutathione and TBARS levels in testis tissue of rats administered cisplatin and Pt–NHC complex (value ± SEM; n = 7).
GroupsControlCisplatinPt–NHC
Low dose (5 mg/kg)High dose (10 mg/kg)Low dose (5 mg/kg)High dose (10 mg/kg)
  1. *Means significantly different from control group (< 0.05).

  2. aSignificant difference between low- and high-dose treatment in the same drug (< 0.05).

  3. bSignificant difference between same dose treatments of cisplatin and Pt–NHC (< 0.05).

TBARS (nmol/g tissue)12.3 ± 0.3316.3 ± 0.66*a22.9 ± 0.51*b17.0 ± 0.60*a26.7 ± 0.38*
Reduced glutathione (nmol/ml)24.1 ± 1.1118.2 ± 0.84*a12.2 ± 0.62*16.0 ± 1.42*a11.7 ± 0.68*
Catalase (k/mg protein)0.78 ± 0.180.54 ± 0.13*ab0.36 ± 0.10*0.45 ± 0.21*a0.31 ± 0.17*
Superoxide dismutase (U/mg protein)39.8 ± 1.4635.8 ± 1.41*ab26.1 ± 0.92*29.4 ± 1.15*a23.8 ± 1.98*
Glutathione peroxidase (U/mg protein)687.6 ± 15.0539.3 ± 16.7*ab433.0 ± 7.10*b495.1 ± 9.7*a349.8 ± 7.5*

On the other hand, low-dose Pt–NHC group’s superoxide dismutase, catalase and glutathione peroxidase activities were significantly decreased compared with those of the low-dose cisplatin group. However, there were no significant changes in TBARS and glutathione levels between Pt–NHC and cisplatin at low-dose application. In the high-dose administration groups, superoxide dismutase and glutathione peroxidase activities significantly changed but catalase activities did not change at the same dose application. Besides, TBARS levels of the high-dose Pt–NHC treatment group were significantly increased compared with those of the cisplatin treatment with the same dose.

Hormonal results

Serum testosterone levels are shown in fig. 1. We determined that serum testosterone levels in both doses of cisplatin and Pt–NHC were decreased significantly compared with the control groups. Besides, the testosterone levels were significantly changed in a dose-dependent manner in all groups. Also, there were significant changes between the same doses of cisplatin and Pt–NHC.

image

Figure 1.  Serum testosterone level in rats treated with cisplatin and Pt–NHC. *Means significantly different from control group (< 0.05); a, significant difference between low- and high-dose treatment in the same drug (< 0.05); b, significant difference between same dose treatment of cisplatin and Pt–NHC (< 0.05).

Download figure to PowerPoint

Organ weight and sperm parameters

The effects of cisplatin and Pt–NHC on testis, epididymis, seminal vesicles, prostate weight, epididymal sperm concentration, sperm motility and abnormal sperm rate are presented in table 2. The results show that treatment with cisplatin and Pt–NHC at low and high doses did not change the weight of epididymis, seminal vesicles and prostate compared with the control group. However, the low and high doses of cisplatin and Pt–NHC caused a significant decrease in testis weight compared with the control group. Additionally, low and high doses of cisplatin and Pt–NHC caused a significant decrease in epididymal sperm concentration and sperm motility and an increase in abnormal sperm rate compared with the control group. Also, it was determined that these changes in epididymal sperm concentration, sperm motility and abnormal sperm rate were generally dose-dependent, and these effects were induced with increasing dose. Besides, in the high-dose Pt–NHC group, sperm motility was significantly decreased compared with the high-dose cisplatin group. Similarly, the high-dose Pt–NHC group’s abnormal sperm rate was significantly increased compared with that of the high-dose cisplatin group.

Table 2.    Reproductive organ weights, epididymal sperm concentration, sperm motility and abnormal sperm rate in rats after treatment with cisplatin and Pt–NHC (n = 7).
Examined organsControlCisplatinPt–NHC
Low dose (5 mg/kg)High dose (10 mg/kg)Low dose (5 mg/kg)High dose (10 mg/kg)
  1. *Means significantly different from control group (< 0.05).

  2. aSignificant difference between low- and high-dose treatment of the same drug (< 0.05).

  3. bSignificant difference between same dose treatment of cisplatin and Pt–NHC (< 0.05).

Testes Weight (g)
 Right1.589 ± 0.0371.425 ± 0.065*1.371 ± 0.053*1.437 ± 0.039*1.425 ± 0.030*
 Left1.578 ± 0.0351.381 ± 0.129*1.398 ± 0.040*1.420 ± 0.044*1.385 ± 0.011*
Epididymis Weight (g)
 Right0.502 ± 0.0450.537 ± 0.0070.495 ± 0.0190.525 ± 0.0090.520 ± 0.021
 Left0.552 ± 0.0210.592 ± 0.0170.473 ± 0.0130.546 ± 0.0140.523 ± 0.023
Seminal Vesicles (g)1.185 ± 0.2161.233 ± 0.0211.103 ± 0.4121.081 ± 0.1481.093 ± 0.104
Prostate (g)0.215 ± 0.0300.250 ± 0.0520.198 ± 0.0220.238 ± 0.0440.216 ± 0.011
Sperm Conc. (mil./cauda)76.10 ± 2.8958.10 ± 5.20*a49.60 ± 9.94*61.80 ± 1.09*a50.60 ± 3.55*
Sperm Motility (%)79.33 ± 1.2458.66 ± 0.91*a45.99 ± 2.33*b61.66 ± 0.66*a41.46 ± 2.85*
Abnormal Sperm Rate (%)
 Head5.40 ± 0.247.30 ± 0.208.40 ± 0.247.00 ± 0.449.40 ± 0.24
 Tail6.40 ± 0.247.10 ± 0.379.40 ± 0.248.20 ± 0.378.80 ± 0.20
 Total11.80 ± 0.4814.40 ± 0.40*a17.80 ± 0.20*b15.20 ± 0.37*a18.20 ± 0.37*

Histological changes

In the sections of control rats, germinal cells were organized in concentric layers and seminiferous tubules containing all stages of spermatogenesis (fig. 2C). However, in the cisplatin and Pt–NHC groups of 5 mg/kg, more obvious damage was present such as spermatogenic arrest and disorganization of germinal cells (fig. 2A,B). On the other hand, histopathological evidence was more clearly observed in the high-dose (10 mg/kg) groups rather than in the low-dose groups (5 mg/kg). Seminiferous tubules containing arrested spermatogenic cells at various stages of division and degenerative changes in germinal cells were observed in these groups (fig. 2D). Tubules with round spermatid exhibiting vacuolated cytoplasm were more conspicuous in the high-dose Pt–NHC group (fig. 2E). Multi-nucleated giant cell formation was not observed in any of the groups. Spermatogonia, the stem cells of the germinal epithelium, are located in the basal compartment, whereas primary and secondary spermatocytes and spermatids occupy the adluminal compartment. Spermatogonia undergo mitotic division to form more spermatogonia as well as primary and secondary spermatocytes, which migrate from basal to adluminal compartment. We have shown that spermatogonia were less affected than spermatocytes and spermatids. Moreover, decreased mean seminiferous tubule diameter and decreased germinal cell layer thickness were prominent in the groups given high doses. Diameters of seminiferous tubules, germinal cell layer thickness and histological score are given in table 3.

image

Figure 2.  (A) Moderate damage of seminiferous epithelium in the cisplatin 5 mg/kg group. Degeneration of germinal cells and spermatogenic arrest (arrows) are evident, H-E ×132. (B) Moderate damage of seminiferous epithelium in the Pt–NHC 5 mg/kg group. Degeneration of germinal cells and spermatogenic arrest (arrows) are evident, H-E ×132. (C) Testis showing normal testicular morphology with regular arrangement of germinal cells in the control group H-E ×330. (D) Arrested spermatocytes in different stage of division (arrows), and intraepithelial spaces (*) owing to lack of spermatocytes and spermatids in the cisplatin 10 mg/kg group, H-E ×330. (E) Degenerating germinal cells with vacuolated cytoplasm are seen in Pt–NHC 10 mg/kg group. Note the sloughing of germinal cells into the tubular lumen (arrows), H-E ×330.

Download figure to PowerPoint

Table 3.    Levels of diameters of seminiferous tubules (DST), germinal cell layer thickness (GCLT) and histological score (HIS).
 ControlCisplatinPt–NHCp values
Low dose (5 mg/kg)High dose (10 mg/kg)Low dose (5 mg/kg)High dose (10 mg/kg)
  1. *Means significantly different from control group (< 0.05).

  2. aSignificant difference between low- and high-dose treatment of the same drug (< 0.05).

  3. bSignificant difference between same dose treatment of cisplatin and Pt–NHC (< 0.05).

DST207.3 ± 5.8204.3 ± 7.2193.3 ± 5.0210.2 ± 8.0a187.5 ± 3.20.02
GCLT44.8 ± 1.240.7 ± 1.7a30.9 ± 1.4*b37.5 ± 1.2*37.9 ± 1.0*≤0.006
HIS0.6 ± 0.25.6 ± 1.2*a12.2 ± 0.9*7.4 ± 1.4*10.8 ± 0.9*≤0.008

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Cisplatin is the most widely used antitumour drug, especially in the treatment for testicular cancers, but its usage is limited by its toxic effects on the reproductive system. For this reason, the present study aimed to determine whether Pt–NHC is less toxic on the reproductive system or not. The results show that Pt–NHC is a more toxic compound than cisplatin in terms of testicular damage in rats. This may be due to its superior anticancer properties as revealed by cell culture studies [13], and this finding might also suggest that a less therapeutic dose of novel platinum complex is needed when used in chemotherapy.

TBARS generated with peroxidation by reactive oxygen species of fatty acids is regarded as an indicator of lipid peroxidation and leads to irreversible cell damage [22]. However, superoxide dismutase, catalase, glutathione peroxidase (enzymatic) and glutathione (non-enzymatic) members of antioxidant defence systems, which protect against oxidative damage, may cause free radical products in normal physiological conditions and molecules such as TBARS [23]. Oxidative stress is a condition of an imbalance between the TBARS and the antioxidant defence system. In this study, it was determined that cisplatin and Pt–NHC induced lipid peroxidation via an increase in TBARS levels and reduced the antioxidant defence system via a decrease in superoxide dismutase, catalase, glutathione peroxidase activity and glutathione levels in a dose-dependent manner. Our findings are in agreement with and confirmed by many previous studies [9,10]. Owing to the fact that Pt–NHC is a novel compound, there are no studies available about its toxic effects on the testis. On the other hand, it is known that cisplatin treatment significantly causes an increase in TBARS levels and a decrease in superoxide dismutase, catalase, glutathione peroxidase and glutathione levels in testis tissue of rats [6,7]. Additionally, it is determined that Pt–NHC causes more testicular damage than cisplatin when used at the same doses. According to our results, these two compounds, at both high and low doses, may cause a significant oxidative damage owing to imbalance between lipid peroxidation and the antioxidant defence system. We believe that the oxidant/antioxidant status in testis tissue plays a key role in testicular toxicity caused by cisplatin and Pt–NHC in rats.

Histopathological results have shown that cisplatin and the novel Pt–NHC complex have caused testicular damage such as spermatogenic arrest and disorganization of germinal cells in a dose-dependent manner. Besides, it was shown that novel Pt–NHC caused more histological damage in testis tissue than cisplatin at the same dose. As in our study, previous studies have determined that testicular damage was established with cisplatin therapy in rats [9,24]. It is thought that testicular toxic effects of Pt–NHC and cisplatin may be due to oxidative damage caused by these agents and may lead to infertility in rats.

It has previously been shown that treatment with cisplatin can lead to a decrease in reproductive organ weight such as the testis and accessory organs [6,7]. Ilbey et al. [7] indicated that the administration of cisplatin in rats reduced testis weight at a dose of 7 mg/kg. Similarly, Atessahin et al. [6] claimed that cisplatin treatment caused a decrease in both testis and accessory organ weight in rats. In the present study, it was determined that cisplatin and pt–NHC treatment significantly reduced testis weight but did not affect the weight of epididymis, seminal vesicles and prostate compared with the control group. Besides, it was shown that there was no significant change between cisplatin and Pt–NHC in terms of organ weight. Our results are paralleled by previous studies in terms of testis weight. On the other hand, there was a difference between our study and the study by Atessahin et al. [6] regarding accessory organ weight, and this may be due to exposition time and dose of cisplatin and Pt–NHC. Also, in the current study, it was determined that the administration of cisplatin and Pt–NHC significantly decreased epididymal sperm concentration, sperm motility and testosterone level and increased abnormal sperm rate in rats in a dose-dependent manner. However, the toxicity of cisplatin and Pt–NHC on sperm characteristics did not change the difference between experimental groups at the same dose. On the other hand, Pt–NHC caused a larger decrease in the testosterone levels than cisplatin at the same dose. The adverse effects of cisplatin on sperm characteristics and serum testosterone level have previously been documented, but there are no studies examining how Pt–NHC affects these parameters. Previous studies [9,25] have indicated that treatment with cisplatin at 7 mg/kg caused a decrease in sperm count and motility and an increase in abnormal sperm rate in rats. Additionally, Ilbey et al. [7] showed that testosterone levels were significantly lowered with cisplatin treatment. Previous studies [7,9,25] paralleled and confirmed our findings. It is known that oxidative and histological damage in testis tissue leads to toxicity in sperm characteristics and testosterone levels. In this context, it was thought that a decrease in sperm count, motility and testosterone level and an increase in abnormal sperm rate are attributable to oxidative effects (increase in TBARS and decrease in antioxidant status) of antitumour agents. These results indicate that cisplatin and Pt–NHC may lead to infertility in rats.

Conclusion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

In conclusion, it has been clearly determined that both cisplatin and novel Pt–NHC complex induce oxidative and histological damage in rat testis tissue. Additionally, they may cause infertility via an increase in abnormal sperm rate and a decrease in epididymal sperm concentration, sperm motility and testosterone level. However, Pt–NHC caused more oxidative stress, testicular damage and reproductive toxicity in testis tissue than cisplatin. This may also suggest that therapeutic effects of Pt–NHC, as an anticancer drug, may be superior to cisplatin. Therefore, further experimentation is warranted to elucidate the potential efficiency of Pt–NHC in cancer treatment.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

We acknowledge the support of TUBITAK (Scientific and Technical Research Council of the Turkish Republic) under Grant 109T540.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References