ABSTRACT
Aim:
Carbon tetrachloride (CCI4) is a volatile organic chemical agent that can cause damage to many tissues. Caffeic acid phenethyl ester (CAPE), which is structurally similar to flavonoids, is an active component of honeybee propolis. CAPE is known for its antitoxic, antioxidant, and anti-inflammatory effects. In this study, we aimed to investigate the effects of CAPE against testicular damage caused by CCI4.
Materials and Methods:
Twenty-eight Wistar albino rats were divided into 4 groups (n=7) as, Group 1: control (5% ethanol, 1 mL/day/ip), Group 2: olive oil (0.5 mL/day over/ip), Group 3: CCI4 (0.5 mL/kg over/ip), Group 4: CCI4+CAPE (10 μmol/kg/day/ip). Tissue samples collected at the end of the experiment were detected in 10% formaldehyde and embedded in paraffin. Five-micron-thick sections taken from paraffin blocks were stained with hematoxylin-eosin. To evaluate testicular damage, 100 tubules from each section were randomly examined at 20x magnification under a light microscope and classified as intact, atrophic, and degenerated tubules. Sections were examined by using Leica DFC 280 light microscope and Leica Q Win Image Analysis system (Leica Micros Imaging Solutions Ltd. Cambridge, UK).
Results:
The testicular sections of the control group and the olive oil group had a normal histological appearance. In the CCI4 group, 55.00±4.22% of the seminiferous tubules were intact, 25.00±2.67% were atrophic and 20.00±1.88% were degenerative. In addition, multinucleated giant cells were found in the lumen of some seminiferous tubules. In the CCI4+CAPE group, 72.14±3.91% of the tubules were intact, 16.42±2.10% were atrophic, and 11.42±2.36% were degenerative. While the number of affected tubules significantly increased in the CCI4 group compared to the control group (p<0.05), the number of affected seminiferous tubules decreased significantly in the CCI4+CAPE group compared to the CCI 4 group (p<0.05).
Conclusion:
We think that CAPE may be useful in reducing the damaging effects of CCI4 on the testicle.
INTRODUCTION
Xenobiotic carbon tetrachloride (CCl4) is a colorless liquid with a sweet odor1. CCl4, which is a highly toxic substance with potential carcinogenic effects in humans and animals, is now used extensively in the manufacture of chlorofluorocarbon refrigerants in industry and thus in air conditioning and refrigeration systems. CCl4 has toxic effects on the kidney, heart, lung, brain, and testicular tissues, especially on the liver2,3. Studies have shown that CCl4 causes spermatogenic cell damage, basal membrane separation, seminiferous tubular atrophy, expansion in the interstitial area, and a decrease in sperm count4,5.
Caffeic acid phenethyl ester (CAPE), which is structurally similar to flavonoids, is an active component of honeybee propolis6. CAPE is known for its antitoxic, antioxidant, anti-inflammatory, antiviral, immunomodulatory, neuroprotective, and cytostatic effects7,8. Since it strongly modulates the arachidonic acid cascade compared to other propolis components, its anti-inflammatory effect is more pronounced6. It has been shown to block all reactive oxygen species formed by the xanthine dehydrogenase/xanthine oxidase system at a concentration of 10 μmol/L9.
In this study, we aimed to investigate the histological changes in the testicular tissue of rats with CCl4-induced and the curative effect of CAPE on these changes by histochemical methods.
MATERIALS AND METHODS
Experimental Animals
The 28 3-month-old male Wistar albino rats weighing 200-250 g, which were used in our study, were obtained from İnönü University Experimental Animals Production and Research Center. Approval was obtained from the Experimental Animals Ethics Committee of İnönü University Faculty of Medicine (protocol no: 2012/A-49, date: 21.03.2012). The rats were housed in rooms where the room temperature was between 24 and 27 °C, ventilation conditions were met, and the lighting was 12 hours light and 12 hours dark. The rats were fed standard pellet feed ad libitum throughout the study.
Experimental Groups
Randomly selected subjects were divided into 4 different groups.
1. Control group: Rats in this group were administered 5% ethanol intraperitoneally (i.p) 1 mL/day for 10 days.
2. Olive oil group: 0.5 mL/day extra olive oil was administered i.p to the rats in this group for 10 days.
3. CCI4 group: 0.5 mL/kg CCI4 was administered i.p to the rats in this group for 10 days.
4. CCI 4+CAPE group: 0.5 mL/kg prepared by dissolving in olive oil was administered i.p to rats in this group for 10 days by dissolving in CCI4 followed by dissolving in 10 µmol/kg CAPE (Sigma, St. Louis, MO).
The study’s 11th 5 mg/kg xylazine and 50 mg/kg ketamine i.p. were administered to the rats, and their abdomens were opened under general anesthesia with a midline incision. The testicular tissue samples were collected for histological examination.
Histological Analyses
Detection and follow-up of tissues were started for histopathological evaluation. Then, 10% formaldehyde was added to ensure good fixation of the tissues. The specimens were then divided into smaller pieces of 3-4 mm, placed in plastic tissue follow-up cassettes, and fixed in formaldehyde for 24 hours. After the fixation process was completed, the parts were rinsed in running tap water for 24 hours. They were then dehydrated in graduated alcohols, made transparent in, and embedded in paraffin. Five-micron sections were taken from paraffin blocks using a Leica RM2145 microtome. The sections were stained with hematoxylin and eosin to observe the general histological structure. To evaluate testicular damage, 100 tubules from each section were randomly examined under a light microscope at 20x magnification and classified as intact, atrophic, and degenerated tubules. Sections were examined by using Leica DFC 280 light microscope and Leica Q Win Image Analysis system (Leica Micros Imaging Solutions Ltd. Cambridge, UK).
Statistical Analysis
Statistical analyses were conducted via the Statistical Package for the Social Sciences (SPSS) program (SPSS for Windows version 13) and MedCalc (2007, Belgium) statistical software. All results are expressed as arithmetic mean ± standard error. The measurable variables in all groups did not have a normal distribution according to the Shapiro-Wilk normality test (p>0.05). For this reason, the Kruskal-Wallis analysis of variance, one of the non-parametric tests, was used for the general comparison of the groups in terms of all variables, whereas the Connover test was used for the pairwise comparison of the groups. The results were considered significant at p<0.05.
RESULTS
Histopathological Evaluation
When the testicular sections of the control and olive oil groups were examined, the seminiferous tubules had a normal histological appearance. The tubules consisted of a seminiferous epithelium sitting on a distinct basal lamina. Sertoli cells and spermatogenic serial cells in the seminiferous epithelium were clearly distinguishable. While spermatogonia were located just above the basal membrane and had a round or oval shape, spermatids were observed in the lumen (Figure 1A, 1B).
CCI4: Carbon tetrachloride, CAPE: Caffeic acid phenethyl ester
In the CCI4 group, degenerative cells with eosinophilic cytoplasm were found in some tubules, which arrested spermatocytes in different stage of division (Figure 1C). In addition, multinucleated giant cells were observed in the lumen of some seminiferous tubules (Figure 1D). In this group, 55.00±4.22% of the seminiferous tubules were intact, 25.00±2.67% were atrophic and 20.00±1.88% were degenerative. In the CCI4+CAPE group, 72.14±3.91% of the tubules were intact, 16.42±2.10% were atrophic, and 11.42±2.36% were degenerative (Figure 1E, 1F). While the number of affected tubules significantly increased in the CCI4 group compared to the control group (p<0.05), the number of affected seminiferous tubules significantly decreased in the CCI4+CAPE group compared to the CCI4 group (p<0.05) (Table 1).
CCI4: Carbon tetrachloride, CAPE: Caffeic acid phenethyl ester
DISCUSSION
Exposure to toxic substances in the environment and/or workplace is considered to be one of the main factors responsible for sperm quality decline10. The persistence of toxic substances in our environment and all their effects on reproductive health, especially general health, make them a public health concern.
CCl4 is a colorless, volatile, and toxic industrial substance that is rapidly absorbed by humans and animals after being released into the air, water, and soil through toxic emissions. Studies have shown that CCI4 causes liver3, lung11, kidney12-14 and testicular damage15 in experimental animals. Studies have reported that CCl4 administration damages the reproductive system by causing oxidative toxicity in male rats4,14,16.
Given the changes caused by the adverse effects of many drugs and chemical agents, the possible effects of healing agents on different tissues of the body need to be investigated. Propolis is a sticky substance with very strong antiviral, antibacterial, and antifungal effects consisting of a mixture of various oils, pollens, special resins, and waxy substances collected by honeybees from the cones and barks of trees and from buds and sprouts of plants17. CAPE is an active component of propolis and has been shown to be a pharmacologically safe compound with anti-inflammatory, antimitogenic, anticarcinogenic, antioxidant, and immunomodulatory effects18-22. This study was designed to histologically evaluate the therapeutic effects of CAPE on testicular injury induced by CCI4.
Previous experimental studies have reported that CCl4 exposure causes structural and functional damage in the male reproductive system23. Moreover, similar previous studies have shown that histopathological changes occur in testicular tissues due to CCl4 toxicity24. In the CCI4 study conducted by Türk et al.5 with rats, it was reported that they observed degeneration in germ cells, edema, and congestion in the interstitial area. Similarly, in the study conducted by Khan and Ahmed4 with rats, degeneration, basal membrane separation, seminiferous tubule atrophy, and expansion in the interstitial area were reported in the spermatogenic serial cells of CCl4. In their study with rats, Horn et al.16 reported that they observed only Sertoli cells in the seminiferous tubules as a result of loss of germ cells, vacuolization in the germinal epithelium, and interruption of meiosis with the administration of CCI4. In our study, we classified the tubules according to the presence of degenerative cells and their atrophy and histopathologically showed the damage caused by CCl4 to the testis. In our study, we found atrophic tubules in the testicular tissue, after CCI4 administration, and degenerated cells with eosinophilic cytoplasm in some tubules, which paused at certain stages of meiosis and were observed in different ways. We also detected the presence of multinucleated giant cells in the lumen of some seminiferous tubules.
In our study, it was obeserved that the histological damage to the testis with the administration of CCI4 decreased with CAPE treatment. CAPE has these protective effects on the basis of antioxidant actions, but the exact mechanisms of anti-oxidant properties of CAPE are not known yet. However, it has been speculated that CAPE may affect transcription and/or translation of genes and gene products of anti-oxidant enzymes25. In addition, the protective effects of CAPE may be caused by its ability to block the bioactivation of CCl4 by inhibiting CYP2E1 activity, in combination with its ability to scavenge free radicals26. In a study by Atik et al.27, investigating the effects of CAPE against testicular damage caused by ischemia/reperfusion in rats, it was reported that the effect of histopathological damage decreased with CAPE application. Abdallah and El-Refaei28 reported that the widespread inflammation, necrosis, and hemorrhage they observed in the testis when cadmium was administered to rats was alleviated by the administration of CAPE. In our study, we found that the number of affected seminiferous tubules decreased significantly in the CCI4+CAPE group compared to the CCI4 group.
Study Limitations
The most important limitation of our study is that it is not supported by functional recovery and biochemical parameters.
CONCLUSION
In conclusion, our study investigated the possible effect of CAPE on testicular damage by CCl4. The results of our study show that CCl4 causes testicular damage and that this testicular damage improves to some extent when CCl4 and CAPE are given together. In view of these results, we think that the use of antioxidant and anti-inflammatory agents such as CAPE may have beneficial effects in the treatment of testicular damage.
Ethics
Ethics Committee Approval: Approval was obtained from the Experimental Animals Ethics Committee of İnönü University Faculty of Medicine (protocol no: 2012/A-49, date: 21.03.2012).
Informed Consent: Animal experiment.
Peer-review: Internally peer-reviewed.
Authorship Contributions
Concept: E.T., Design: E.T., Data Collection or Processing: B.G., N.V., A.T., H.E., Analysis or Interpretation: E.T., Literature Search: B.G., Writing: B.G.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: The authors declared that this study received no financial support.