Toxic effect of cyclophosphamide on sperm morphology, testicular histology and blood oxidant-antioxidant balance, and protective roles of lycopene and ellagic acid

Toxic Effect of Cyclophosphamide on Sperm Morphology,

Testicular Histology and Blood Oxidant-Antioxidant Balance,

and Protective Roles of Lycopene and Ellagic Acid

1_{Department of Pathology, Faculty of Veterinary Medicine, Fırat University, Elazıg˘, Turkey,}2_{Department of Reproduction and Artificial}

Insemination, Faculty of Veterinary Medicine, Fırat University, Elazıg˘, Turkey,3_{Department of Pharmacology and Toxicology, Faculty of}

Veterinary Medicine, Dicle University, Diyarbakır, Turkey, and4_{Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine,}

Fırat University, Elazıg˘, Turkey

(Received 17 November 2009; Accepted 26 January 2010)

Abstract: In this study, the toxic effect of cyclophosphamide (CP) on sperm morphology, testicular histology and blood oxi-dant–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 admin-istration 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 dam-ages 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

liv-ing cells. One of these drugs is cyclophosphamide (CP;

C

H

Cl

N

O

P; MW: 279.10 g⁄ mol;

N-bis(2-chloroethyl)-1-oxo-6-oxa-2-aza-1k

_{-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

treat-ment 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,

composi-tion of sperm head basic proteins and increases abnormal

sperm rate, and manifest biochemical and histological

altera-tions 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

sus-ceptible 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,

mitochon-dria 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; C

H

;

MW:

536.87;

w,w-Carotene,

2,6,10,14,19,23,27,31-Octa-methyl-dotriaconta-2,6,8,10,12,14,16,18,20,22,24,26,30-tridec

aene), 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 (

O

)-quenching

ability and to act as a potent antioxidant, preventing the

oxi-dative damage of critical biomolecules including lipids,

pro-teins and DNA [11]. Ellagic acid (EA; C

H

O

; MW:

302.20; 3,7,8-tetrahydroxy[1]-benzopyrano[5,4,3-cde]

[1]benz-opyran-5,10-dione) has potent radical scavenging and

chemopreventive properties [12,13]. Raspberries,

strawber-ries, walnuts, longan seed, mango kernel [14,15] and

pome-granate [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

Author for correspondence: Gaffari Trk, Department of Reproduc-tion and Artificial InseminaReproduc-tion, Faculty of Veterinary Medicine, Fırat University, 23119 Elazıg˘, Turkey (fax +90 424 238 81 73, e-mail gturk@firat.edu.tr; gaffariturk@hotmail.com).

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 Eczacıbas¸ı-Baxter (_Istanbul, Turkey). LC 10% FS (Redivivo TM, Code 7803) was obtained from DSM Nutritional Products (_Istanbul, Turkey). EA was supplied from Fluka (Steinheim, Ger-many) 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 ani-mals were obtained from Fırat University, Experimental Research Centre (Elazıg˘, Turkey) and were housed under standard laboratory conditions (temperature 24 € 3C, humidity 40–60%, a 12-hr light : dark cycle). A commercial pellet diet (Elazıg˘ Food Company, Elazıg˘, 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 adminis-tered 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 (0.01NNaOH; 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 solu-tion + 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 4C; then the plasma was removed immediately and stored at )20C 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 phos-phate buffer solution was added and the erythrocytes were centri-fuged, and the upper buffer part removed by Pasteur pipette. The erythrocytes were diluted 10 times with ice-cold water, vortexed and stored at)20C until used.

Assessment of morphologically abnormal sperm. To determine the per cent of morphologically abnormal spermatozoa in left cauda

epidid-ymis, the slides stained with eosin–nigrosin (1.67% eosin, 10% nigro-sin and 0.1M 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 spermato-zoa were expressed as per cent [21].

Biochemical analyses. Plasma malondialdehyde (MDA) concentra-tion, the end-product of lipid peroxidaconcentra-tion, 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 dismu-tase (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 con-verts 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 H2O2into

H2O and 1⁄ 2 O2in 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)ˇˇ.) into H2O2and

molec-ular oxygen, is one of the most important antioxidative enzymes. SOD activity determination was based on SOD’s inhibition of the reaction of O2)ˇˇ., from xanthine by xanthine oxidase and the

reduc-tion 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 lm and were stained with haematoxylin and eosin [26]. Light microscopy with ocular micrometer was used to measure the diameters of semi-niferous 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. Dif-ferences were considered to be significant at p < 0.05. One-way ANO-VAand post hoc Tukey-HSD test were used to determine differences

between the groups. TheSPSS⁄PCprogram (Version 10.0; SPSS,

Chi-cago, 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

por-tion of the spermatozoa, only CP administrapor-tion caused

sta-tistically 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

abnormal-ity were brought near values to control by LC or EA

admin-istrations to CP-treated rats, these adminadmin-istrations could not

improve significantly this parameter when compared with

the alone CP group.

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

lev-els 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.

When the structure of testes was histopathologically

exam-ined; it was observed that histological appearances of

testicu-lar 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,

intersti-tial 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

adminis-tration to CP-treated rats provided a moderate improvement.

However, LC administration to CP-treated rats caused a

piv-otal 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).

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

mor-phology, histopathological status of testis, plasma lipid

per-oxidation 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, O

ˇˇ .)

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

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

Groups MDA (nmol⁄ ml) GSH (nmol⁄ gHb) GSH-Px (U⁄ gHb) CAT (k⁄ gHb) SOD (U⁄ gHb)

Control 5.80 € 0.31a 0.56 € 0.07a 44.57 € 10.54 41.4 € 4.8a 1.45 € 0.08b LC 5.53 € 0.63a 0.19 € 0.01b 42.35 € 3.58 24.7 € 2.7b 0.73 € 0.05a EA 4.62 € 0.50a _{0.64 € 0.11}a _{25.08 € 5.02 42.6 € 3.9}a _{1.98 € 0.25}bc CP 7.19 € 0.39b _{0.59 € 0.14}a _{41.82 € 8.36 27.4 € 3.63}b _{3.00 € 0.32}e CP + LC 5.74 € 0.56a _{0.6 € 0.15}a _{31.28 € 6.50 34.3 € 3.59}ab _{2.75 € 0.15}de CP + EA 4.96 € 0.06a _{0.79 € 0.12}a _{33.70 € 2.71 33.8 € 2.19}ab _{2.25 € 0.15}cd

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).

Table 1.

Mean ± S.E.M. values of abnormal sperm rate, DST, GCLT and Johnsen’s Testicular Score. Parameters

Abnormal sperm rate (%)

Groups Head Tail Total DST (lm) GCLT (lm)

Johnsen’s Testicular Score (0–10) Control 2.28 € 0.31 3.78 € 0.78A 6.06 € 2.01A 223.6 € 2.20a 76.40 € 0.98a 9.67 € 0.21ac LC 2.16 € 0.39 1.83 € 0.37A 3.99 € 1.67A 225.1 € 2.00a 75.67 € 1.19a 10.00 € 0.00a EA 1.89 € 0.41 2.55 € 0.31A 4.44 € 0.72A 224.5 € 1.90a 74.73 € 0.99a 10.00 € 0.00a CP 3.39 € 0.62 6.50 € 1.03B 9.89 € 3.28B 185.8 € 2.93b 55.20 € 0.72b 7.83 € 0.40b CP + LC 3.33 € 0.29 3.39 € 0.35A 6.72 € 0.72AB 216.0 € 2.14a 63.60 € 0.60d 9.17 € 0.31ac CP + EA 3.11 € 0.67 3.75 € 0.62A 6.86 € 1.90AB 217.6 € 11.28a 68.20 € 0.83c 9.00 € 0.26c 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).

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

associ-ated with rapid loss of intracellular ATP leading to axonemal

damage, decreased sperm viability and increased mid-piece

morphological defects, and even it completely inhibits

sper-matogenesis 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 _Ilbey 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

A B

C D

E F

G H

Fig. 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 Ser-toli 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·).

control group. Our findings are in agreement with the above

reports.

Lifestyle,

medical

conditions

⁄ treatments

and

environmental factors increase ROS production.

Mitochon-dria 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

toxic-ity 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

peroxida-tion 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,

disorgani-zation and reduction in germinal cells, atrophy in tubules,

vacuolization in Sertoli cells, multi-nucleated giant cell

for-mation, interstitial oedema and congestion, reduced

diame-ters 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

histo-logical 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

dis-rupting the structure and function of testis.

The degree of excessive ROS production-induced oxidative

stress in the organism can be determined with direct or

indi-rect measurement of free radicals and enzymatic

⁄

non-enzy-matic

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

antioxi-dant mechanisms, enzymatic or vitamin complexes to

pre-vent 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

activi-ties in testis [2,4,5,29]. While alone CP administration

signifi-cantly (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

sig-nificantly alter the GSH levels and GSH-Px activities.

Anti-oxidant 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

conse-quently elevated lipid peroxidation. Reduction in CAT

activi-ties 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 (

_O

2

) 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 O

ˇˇ . andˇˇ.OH in both enzymatic and

non-enzymatic systems by its metal-chelating property, thus

pro-viding 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

chemother-apeutics such as cisplatin and adriamycin [40,41]. In the

pres-ent study, administrations of LC and EA to CP-treated rats

resulted in statistically significant decreases in tail

abnormal-ity and insignificant decreases in total abnormalabnormal-ity 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

seminifer-ous 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

Table 3.

The existence of some pathological lesions in testicular tissues of different treatment groups (LC, lycopene; EA, ellagic acid; CP, cyclophosphamide).

Groups

Parameters Control LC EA CP CP + LC CP + 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 _{) ) )} + ₎ +

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

(TB_ITAK); Project number: 106O123.

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