Toxic Effect of Cyclophosphamide on Sperm Morphology, Testicular Histology and Blood Oxidant-Antioxidant Balance, and Protective Roles of Lycopene and Ellagic Acid

Authors


Author for correspondence: Gaffari Türk, Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Fırat University, 23119 Elazığ, Turkey (fax: +90 424 238 81 73, e-mail gturk@firat.edu.tr; gaffariturk@hotmail.com).

Abstract

Abstract:  In this study, the toxic effect of cyclophosphamide (CP) on sperm morphology, testicular histology and blood oxidant–antioxidant balance, and protective roles of lycopene (LC) and ellagic acid (EA) were investigated. For this purpose, 48 healthy, adult, male Sprague-Dawley rats were divided into six groups; eight animals in each group. The control group was treated with placebo. LC, EA and CP groups were given alone LC (10 mg/kg/every other day), EA (2 mg/kg/every other day) and CP (15 mg/kg/week) respectively. One of the last two groups received CP + LC, and the other treated with CP + EA. All treatments were maintained for 8 weeks. At the end of the treatment period, morphological abnormalities of sperm, plasma malondialdehyde (MDA) levels and glutathione (GSH) levels, and GSH-peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) activities in erythrocytes, and testicular histopathological changes were examined. CP administration caused statistically significant increases in tail and total abnormality of sperm, plasma MDA level and erythrocyte SOD activity, and decreases in erythtocyte CAT activity, diameters of seminiferous tubules, germinal cell layer thickness and Johnsen’s Testicular Score along with degeneration, necrosis, immature germ cells, congestion and atrophy in testicular tissue. However, LC or EA treatments to CP-treated rats markedly improved the CP-induced lipid peroxidation, and normalized sperm morphology and testicular histopathology. In conclusion, CP-induced lipid peroxidation leads to the structural damages in spermatozoa and testicular tissue of rats, and also LC or EA have a protective effect on these types of damage.

Most of the chemotherapeutic drugs used in the treatment of neoplastic cells cause various sorts of damage to normal living cells. One of these drugs is cyclophosphamide (CP; C7H17Cl2N2O3P; MW: 279.10 g/mol; N-bis(2-chloroethyl)-1-oxo-6-oxa-2-aza-1λ5-phosphacyclohexan-1-amine hydrate). It has potent anticancer, and as well as immunosuppressive effects for organ transplantation and autoimmune diseases. CP therapy is a common continuing problem in the treatment of a variety of glomerular diseases and leads to gonadal toxicity as a side effect of the drug [1]. Previous studies have shown that CP alters sperm chromatin structure, composition of sperm head basic proteins and increases abnormal sperm rate, and manifest biochemical and histological alterations in testis [2–4]. It has been reported that oxidative stress-induced biochemical and physiological damage is responsible for CP toxicity in testis and spermatozoa [5–7]. The mitochondrial membrane of spermatozoa is more susceptible to lipid peroxidation, as this compartment is rich in polyunsaturated fatty acids and has been shown to contain low amounts of antioxidants [8,9]. Additionally, mitochondria and plasma membranes of morphologically abnormal spermatozoa produce reactive oxygen species (ROS) [10].

Recently, there is growing interest in understanding the roles and mechanisms of the carotenoids and phytochemicals as inhibitors of oxidative stress. Lycopene (LC; C40H56; MW: 536.87; ψ,ψ-Carotene, 2,6,10,14,19,23,27,31-Octamethyl-dotriaconta-2,6,8,10,12,14,16,18,20,22,24,26,30-tridecaene), a carotenoid occurring naturally in tomatoes, has attracted considerable attention as an antioxidant. LC, because of its high number of conjugated double bonds, has been reported to exhibit high singlet oxygen (1O2)-quenching ability and to act as a potent antioxidant, preventing the oxidative damage of critical biomolecules including lipids, proteins and DNA [11]. Ellagic acid (EA; C14H6O8; MW: 302.20; 3,7,8-tetrahydroxy[1]-benzopyrano[5,4,3-cde] [1]benzopyran-5,10-dione) has potent radical scavenging and chemopreventive properties [12,13]. Raspberries, strawberries, walnuts, longan seed, mango kernel [14,15] and pomegranate [16] are rich plants with respect to EA. It contains four hydroxyl groups and two lactone groups in which the hydroxyl group is known to increase antioxidant activity in lipid peroxidation and protect cells from oxidative damage [17]. It has been reported that the therapeutic antioxidant effect of LC [18] and EA [19] on germ cells could serve as promising intervention to oxidative stress-induced infertility problems. In our earlier study [20], we observed that CP led to decreased sperm motility and count, testicular tissue lipid peroxidation and testicular apoptosis, and also LC and EA had protective effects. The present study was also designed to investigate whether LC or EA has possible protective effect against CP-induced alterations in sperm morphology, blood oxidant–antioxidant balance and testicular histology in rats.

Materials and Methods

Chemicals.  Cyclophosphamide (Endoxan, 500 mg) was purchased from Eczacıbaşı-Baxter (İstanbul, Turkey). LC 10% FS (Redivivo TM, Code 7803) was obtained from DSM Nutritional Products (İstanbul, Turkey). EA was supplied from Fluka (Steinheim, Germany) and the other chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

Animals and experimental design.  Forty-eight healthy adult male Sprague-Dawley rats (8 weeks old) were used in this study. The animals were obtained from Fırat University, Experimental Research Centre (Elazığ, Turkey) and were housed under standard laboratory conditions (temperature 24 ± 3°C, humidity 40–60%, a 12-hr light : dark cycle). A commercial pellet diet (Elazığ Food Company, Elazığ, Turkey) and fresh drinking water were given ad libitum. The protocol for the animal use was approved by the Institutional Review Board of the National Institute of Health and Local Committee on Animal Research.

Cyclophosphamide was administered to the animals at a dose of 15 mg/kg once a week. LC was suspended in corn oil and administered to the animals at a dose of 10 mg/kg/every other day. EA is hardly dissolved under natural condition. Therefore, it was dissolved in alkaline solution (0.01 n NaOH; approximately pH 12). This final solution (pH = 8) after the addition of EA was administered to the animals at a dose of 2 mg/kg/every other day. All treatments were applied by gavage and maintained for 8 weeks. The animals were randomly divided into six experimental groups of eight rats in each. These groups were arranged as follows:

Group 1 – control: treated with placebo – received 0.5 ml/rat slightly alkaline solution + 0.5 ml/rat corn oil every other day.

Group 2 – LC: treated with 0.5 ml/rat slightly alkaline solution + 0.5 ml/rat LC.

Group 3 – EA: received 0.5 ml/rat corn oil + 0.5 ml/rat EA.

Group 4 – CP: received 0.5 ml/rat CP + a mixture of slightly alkaline solution and corn oil (0.5 ml/rat).

Group 5 – CP + LC: treated with 0.5 ml/rat CP + 0.5 ml/rat LC.

Group 6 – CP + EA: treated with 0.5 ml/rat CP + 0.5 ml/rat EA.

Sample collection and preparation of erythrocytes.  The rats were killed under slight ether anaesthesia at the end of 8 weeks. Testes were removed, cleared of adhering connective tissue and fixed in 10% neutral-formalin solution for histopathological examinations. Blood samples were taken into tubes containing anticoagulant (2% sodium oxalate). The samples were centrifuged at 200 × g for 5 min. at 4°C; then the plasma was removed immediately and stored at −20°C until analysis. The buffy coat on top of the erythrocyte layer was carefully removed and 10 ml of isotonic NaCl solution was added. Resuspended erythrocyte was centrifuged at 1000 × g for 10 min. and the upper part removed again. Then, 10 ml of phosphate buffer solution was added and the erythrocytes were centrifuged, and the upper buffer part removed by Pasteur pipette. The erythrocytes were diluted 10 times with ice-cold water, vortexed and stored at −20°C until used.

Assessment of morphologically abnormal sperm.  To determine the per cent of morphologically abnormal spermatozoa in left cauda epididymis, the slides stained with eosin–nigrosin (1.67% eosin, 10% nigrosin and 0.1 m sodium citrate) were prepared. The slides were then viewed under a light microscope at 400× magnification. A total of 300 spermatozoa were examined on each slide (2400 cells in each group), and the head, tail and total abnormality rates of spermatozoa were expressed as per cent [21].

Biochemical analyses.  Plasma malondialdehyde (MDA) concentration, the end-product of lipid peroxidation, was measured according to the method described by Satoh [22] and expressed as nmol/ml. The packed cells were used for the analysis of glutathione (GSH), GSH-peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD). Erythrocyte GSH was estimated by the method described by Beutler [23], using dithio-bis-nitrobenzoic acid and expressed as nmol/gHb. Erythrocyte GSH-Px (EC 1.11.1.9) activity was measured by the method described by Beutler [23] in which cumene hydroperoxide was used as substrate. Oxide GSH produced by the action of erythrocyte GSH-Px and cumene hydroperoxide, was reduced by GSH reductase (GSH-az) and nicotinamide adenine dinucleotide phosphate reduced form (NADPH). The decrease in the concentration of NADPH was measured at 340 nm. The enzyme activity was expressed as U/gHb. CAT (EC 1.11.1.6) enzyme converts hydrogen peroxide (H2O2) into H2O and 1/2 O2. The CAT activity was measured by the method described by Aebi [24]. The principle of this method was based on the hydrolyzation of H2O2 and decreasing absorbance at 240 nm. The conversion of H2O2 into H2O and 1/2 O2 in 1 min. under standard condition was considered to be the enzyme reaction velocity. The enzyme activity was expressed as k/gHb. The SOD (EC 1.15.1.1) enzyme, which catalyses the dismutation of the superoxide anion (O2−˙) into H2O2 and molecular oxygen, is one of the most important antioxidative enzymes. SOD activity determination was based on SOD’s inhibition of the reaction of O2−˙, from xanthine by xanthine oxidase and the reduction of nitroblue tetrazolium [25]. The enzyme activity was expressed as U/gHb.

Testicular histopathology.  Ten sections were taken from each testis. Fixed testicular tissues in 10% neutral-formalin were then embedded in paraffin, washed in graded alcohol series, sectioned at 5 μm and were stained with haematoxylin and eosin [26]. Light microscopy with ocular micrometer was used to measure the diameters of seminiferous tubules and germinal cell layer thicknesses (GCLT) and to evaluate the damages in testicular tissue [21]. Johnsen’s Testicular Score [27] was performed for control and treatment groups. All cross-sectioned tubules were evaluated systematically, and a score between 1 (very poor) and 10 (excellent) was given to each tubule according to Johnsen’s criteria. Twenty-five tubules were evaluated for each animal.

Statistical analysis.  All values are presented as mean ± S.E.M. Differences were considered to be significant at p < 0.05. One-way anova and post hoc Tukey-HSD test were used to determine differences between the groups. The spss/pc program (Version 10.0; SPSS, Chicago, IL, USA) was used for the statistical analysis.

Results

Sperm abnormality rates in response to various treatments for 8 weeks of treatment are presented in table 1. While alone LC and EA treatments did not affect the whole portion of the spermatozoa, only CP administration caused statistically significant (p < 0.01) increases in tail and total abnormality of sperm in comparison with the control group. A significant (p < 0.01) decrease in tail abnormality was observed in CP + LC and CP + EA groups as compared with alone CP group. Although the values of total abnormality were brought near values to control by LC or EA administrations to CP-treated rats, these administrations could not improve significantly this parameter when compared with the alone CP group.

Table 1. 
Mean ± S.E.M. values of abnormal sperm rate, DST, GCLT and Johnsen’s Testicular Score.
Parameters
Abnormal sperm rate (%)
GroupsHeadTailTotalDST (μm)GCLT (μm)Johnsen’s Testicular Score (0–10)
  1. The mean differences between the values bearing different superscript letters within the same column are statistically significant (A and B: p < 0.01; a, b, c and d: p < 0.05).

  2. DST, diameter of seminiferous tubules; GCLT, germinal cell layer thickness; LC, lycopene; EA, ellagic acid; CP, cyclophosphamide.

Control2.28 ± 0.313.78 ± 0.78A6.06 ± 2.01A223.6 ± 2.20a76.40 ± 0.98a9.67 ± 0.21ac
LC2.16 ± 0.391.83 ± 0.37A3.99 ± 1.67A225.1 ± 2.00a75.67 ± 1.19a10.00 ± 0.00a
EA1.89 ± 0.412.55 ± 0.31A4.44 ± 0.72A224.5 ± 1.90a74.73 ± 0.99a10.00 ± 0.00a
CP3.39 ± 0.626.50 ± 1.03B9.89 ± 3.28B185.8 ± 2.93b55.20 ± 0.72b7.83 ± 0.40b
CP + LC3.33 ± 0.293.39 ± 0.35A6.72 ± 0.72AB216.0 ± 2.14a63.60 ± 0.60d9.17 ± 0.31ac
CP + EA3.11 ± 0.673.75 ± 0.62A6.86 ± 1.90AB217.6 ± 11.28a68.20 ± 0.83c9.00 ± 0.26c

Plasma MDA levels and antioxidant enzyme activities in erythrocytes are presented in table 2. Alone LC, but not EA significantly (p < 0.05) reduced the GSH levels, CAT and SOD activities in comparison with the control group. While alone CP administration significantly (p < 0.05) increased the MDA levels and SOD activities, it significantly decreased the CAT activities when compared with the control group. CP treatment did not significantly alter the GSH levels and GSH-Px activities. LC and EA administration to CP-treated rats significantly (p < 0.05) reduced the increased MDA levels in comparison with the only CP group. A trend towards to the normalization in CAT and SOD activities was observed in both CP + LC and CP + EA groups.

Table 2. 
Mean ± S.E.M. values of plasma malondialdehyde (MDA) levels, and erythrocyte glutathione (GSH) levels and GSH-peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) activities (LC, lycopene; EA, ellagic acid; CP, cyclophosphamide).
Biochemical parameters
GroupsMDA (nmol/ml)GSH (nmol/gHb)GSH-Px (U/gHb)CAT (k/gHb)SOD (U/gHb)
  1. The mean differences between the values bearing different superscript letters within the same column are statistically significant (a, b, c, d and e: p < 0.05).

Control5.80 ± 0.31a0.56 ± 0.07a44.57 ± 10.5441.4 ± 4.8a1.45 ± 0.08b
LC5.53 ± 0.63a0.19 ± 0.01b42.35 ± 3.5824.7 ± 2.7b0.73 ± 0.05a
EA4.62 ± 0.50a0.64 ± 0.11a25.08 ± 5.0242.6 ± 3.9a1.98 ± 0.25bc
CP7.19 ± 0.39b0.59 ± 0.14a41.82 ± 8.3627.4 ± 3.63b3.00 ± 0.32e
CP + LC5.74 ± 0.56a0.6 ± 0.15a31.28 ± 6.5034.3 ± 3.59ab2.75 ± 0.15de
CP + EA4.96 ± 0.06a0.79 ± 0.12a33.70 ± 2.7133.8 ± 2.19ab2.25 ± 0.15cd

When the structure of testes was histopathologically examined; it was observed that histological appearances of testicular tissues of control (fig. 1F), LC (fig. 1G) and EA (fig. 1H) groups were normal. The histopathological changes such as necrosis, degeneration, desquamation, disorganization and reduction in germinal cells, atrophy in tubules, vacuolization in Sertoli cells, multi-nucleated giant cell formation, interstitial oedema and congestion were observed in alone CP and CP + EA groups (table 3). These types of histopathological damage were more severely in alone CP (figs. 1A–C) group than CP + EA (fig. 1E) group. In other words, EA administration to CP-treated rats provided a moderate improvement. However, LC administration to CP-treated rats caused a pivotal amelioration in testicular histological view compared with the alone CP group (fig. 1D). Significant (p < 0.05) decreases in diameters of seminiferous tubules, GCLT and Johnsen’s Testicular Score were observed in the alone CP group compared with the control group. However, both LC and EA administrations to CP-treated animals significantly (p < 0.05) prevented the CP-induced decreases in these parameters (table 1).

Figure 1.

 (A) Multi-nucleated giant cell formation in alone cyclophosphamide (CP) group [haematoxylin and eosin, 100×]. (B) Severe necrosis, degeneration, disorganization in germinal cells and interstitial oedema in alone CP group (haematoxylin and eosin, 100×). (C) Arrows show Sertoli cell vacuolization in alone CP group (haematoxylin and eosin, 40×). (D) Pivotal amelioration in testicular view in CP + lycopene (LC) group (haematoxylin and eosin, 100×). (E) Moderate amelioration in testicular view along with some spilled germinal cells and interstitial oedema in CP + ellagic acid (EA) group (haematoxylin and eosin, 40×). (F) Normal histological appearance of seminiferous tubules in control group (haematoxylin and eosin, 40×). (G) Normal histological appearance of seminiferous tubules in alone LC group (haematoxylin and eosin, 40×). (H) Normal histological appearance of seminiferous tubules in alone EA group (haematoxylin and eosin, 40×).

Table 3. 
The existence of some pathological lesions in testicular tissues of different treatment groups (LC, lycopene; EA, ellagic acid; CP, cyclophosphamide).
Groups
ParametersControlLCEACPCP + LCCP + EA
Necrosis in germinal cells++
Atrophy in seminiferous tubules++
Degeneration in germinal cells++
Desquamation in germinal cells++
Vacuolization in Sertoli cells++
Reduction in germinal cell counts++
Disorganization in germinal cells++
Interstitial oedema and capillary congestion++
Multi-nucleated giant cell formation++

Discussion

In this study, to determine the toxic effect of CP and possible protective roles of LC and EA on reproductive functions of healthy male rats, we examined the changes in sperm morphology, histopathological status of testis, plasma lipid peroxidation level and erythrocyte antioxidant enzyme activities.

It has been shown that spermatocytes and spermatids (pachytene spermatocytes, round and elongated spermatids) are able to generate low levels of ROS (in particular, O2˙) that are essential to many of the physiological processes such as capacitation, hyperactivation and sperm–oocyte fusion. Because plasma membranes of spermatozoa contain large quantities of polyunsaturated fatty acids and their cytoplasm contains low concentrations of scavenging enzymes, they are particularly susceptible to the damage induced by excessive ROS [8,9]. ROS can attack the unsaturated bonds of the membrane lipids in an autocatalytic process, with the genesis of peroxides, alcohol and lipidic aldehydes as by-product of the reaction. Thus, the increase of free radicals in cells can induce the lipid peroxidation by oxidative breakdown of polyunsaturated fatty acids in membranes of cells. Obviously, peroxidation of sperm lipids destroys the structure of lipid matrix in the membranes of spermatozoa, and it is associated with rapid loss of intracellular ATP leading to axonemal damage, decreased sperm viability and increased mid-piece morphological defects, and even it completely inhibits spermatogenesis in extreme cases [21]. Tripathi and Jena [28] have reported that CP treatment at the doses of 100 and 200 mg/kg significantly enhances the abnormality in sperm head morphology in a dose-dependent manner. In studies by Selvakumar et al. [2] and İlbey et al. [4], it was shown that treatment of male rats with CP causes a significant increase in dead and abnormal spermatozoa. In this study, CP administration caused statistically significant increases in tail and total abnormality of sperm in comparison with the control group. Our findings are in agreement with the above reports. Lifestyle, medical conditions/treatments and environmental factors increase ROS production. Mitochondria and plasma membranes of morphologically abnormal spermatozoa produce ROS through the NADP-dependent and NAD-dependent oxido-reductase systems respectively [10]. Increased morphological defects and production of abnormal sperms also may be as a result of the direct toxicity of CP, because cellular DNA is a primary target of CP in its anti-neoplastic and toxic activity [4]. An other mechanism for CP toxicity on sperm morphology may also be peroxidation of polyunsaturated fatty acids in plasma membranes of spermatozoa by free radicals.

Cyclophosphamide causes histopathological reduction in size and number of the seminiferous tubules, degeneration and vacuolation in spermatogonia, spermatocytes and less number of germ cells, irregular seminiferous tubules, reduced seminiferous epithelial layers, significant maturation arrest, perivascular fibrosis and hyalinization of intertubular tissue [4,28–31]. Necrosis, degeneration, desquamation, disorganization and reduction in germinal cells, atrophy in tubules, vacuolization in Sertoli cells, multi-nucleated giant cell formation, interstitial oedema and congestion, reduced diameters of seminiferous tubules, GCLT and Johnsen’s Testicular Score were observed in histological structure of CP-treated rats in the present study. The damage observed in the histological structure of testis in this work may be elucidated with the direct or indirect effect of CP, which later induces lipid peroxidation that is a chemical mechanism capable of disrupting the structure and function of testis.

The degree of excessive ROS production-induced oxidative stress in the organism can be determined with direct or indirect measurement of free radicals and enzymatic/non-enzymatic antioxidants. The determination MDA and/or thiobarbituric acid reactive substance, which are by-products of lipid peroxidation, is one of the indirect measurement methods for oxidative stress [32]. Cells have mechanisms to combat ROS production partially or totally through antioxidant mechanisms, enzymatic or vitamin complexes to prevent excessive peroxidation of substrates. The endogenous antioxidant enzymes such as SOD, GSH-Px and CAT are mainly responsible for the elimination of excessive ROS. GSH-Px uses GSH as a substrate [33]. Generally, it is accepted that the increased lipid peroxidation is one of the toxic manifestations of CP administration in testis. It has been reported that CP treatment results in elevated MDA levels because of the excessive generation of free radicals, and reduced GSH levels and GSH-Px, CAT and SOD activities in testis [2,4,5,29]. While alone CP administration significantly (p < 0.05) increased the MDA levels and SOD activities, it significantly decreased the CAT activities when compared with the control group. CP treatment did not significantly alter the GSH levels and GSH-Px activities. Antioxidant enzyme activities such as SOD and CAT in lipid peroxidation may sometimes decrease [34] or increase [35]. Increment in MDA levels can be attributed to the CP-induced excessive production of free radicals and consequently elevated lipid peroxidation. Reduction in CAT activities may be attributed to excessive utilization of this antioxidant to scavenge the free radicals.

In vitro experiments have designated LC as one of the most efficient antioxidants (1O2) quencher [36]. Structurally, it is an acyclic carotene with 11 conjugated double bonds, normally in the all-trans configuration. The double bonds attribute to its powerful antioxidant actions [37]. EA inhibits generation of O2˙ and ˙OH in both enzymatic and non-enzymatic systems by its metal-chelating property, thus providing protection against lipid peroxidation [38,39]. In our earlier studies, we found that LC and EA-protected testes and spermatozoa from toxicity induced by some chemotherapeutics such as cisplatin and adriamycin [40,41]. In the present study, administrations of LC and EA to CP-treated rats resulted in statistically significant decreases in tail abnormality and insignificant decreases in total abnormality compared with the alone CP group. While EA administration to CP-treated rats provided a moderate improvement, LC caused a pivotal amelioration in testicular histological view compared with the alone CP group. Both LC and EA administrations improved the CP-induced decreased diameters of seminiferous tubules, GCLT and Johnsen’s Testicular Score. LC and EA administration to CP-treated rats significantly reduced the increased MDA levels in comparison with the only CP group. A trend towards to the normalization in CAT and SOD activities was observed in both the CP + LC and CP + EA groups. These improvements in sperm morphology and testicular tissue and oxidant–antioxidant balance after LC or EA administrations may be attributed to potent-free radical scavenger effects of these antioxidants.

In conclusion, this study suggests that LC and EA protect morphological structure of sperms and testicular tissue against CP toxicity. These protective actions of LC and EA seem to be closely involved with the suppressing of plasma lipid peroxidation and increasing of antioxidant enzyme activities in erythrocytes. Therefore, LC or EA may be used combined with CP in cancer patients, transplantation and autoimmune diseases to improve CP-induced injuries in sperm morphology and blood oxidative stress parameters.

Acknowledgements

The authors acknowledge for financial support from The Scientific and Technological Research Council of Turkey (TÜBİTAK); Project number: 106O123.

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