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The effect of melatonin on the testes of rats treated with cyclophosphamide: histological and immunohistochemical study

Edrees, Zakaria Abd-El hameeda; kader, Hanaa Abd-Ela; Embaby, Azza S.b; hameed, Eman Abd-Elb

doi: 10.1097/01.EHX.0000419785.84206.d2
Original articles

Background Cyclophosphamide (CP) is used extensively as a chemotherapeutic and an immunosuppressive agent during organ transplantation. However, its clinical utility is limited by adverse actions on the human reproductive system. Melatonin is detected in the human and animal reproductive system, hence assumed to play a useful role in the reproductive cells.

Aim of work To examine the effect of melatonin on the histological and immunohistochemical changes that appear in the cells of the testes of albino rats treated with CP.

Materials and methods Forty-two adult male albino rats were classified into group I (control), subgroups IIa and IIIa (CP by an intraperitoneal injection for 2 and 5 weeks, respectively), subgroups IIb and IIIb (CP and melatonin by an intraperitoneal injection for 2 and 5 weeks, respectively), and subgroups IIc and IIIc (CP for 2 and 5 weeks and left for 2 and 5 weeks for recovery, respectively). The rats were weighed, sacrificed, and the right testes were weighed and processed for paraffin sections. A histological study was carried out using H&E and Masson’s trichrome. Immunohistochemical study using the BCL2 antiapoptotic onchoprotein and a morphometric study were carried out.

Results A significant decrease was found in the mean body weight in subgroups IIa, IIIa, and IIIc and in the mean testicular weight in group III. There were distortions in some seminiferous tubules, degeneration of spermatogenic cells, perivascular and intertubular fibrosis, and negative immunoexpression of BCL2. These changes were more marked in group III than in group II. The use of melatonin in subgroups (IIb, IIIb) conferred partial protection against all of the above-mentioned changes.

Conclusion Prolonged administration of CP to rats can induce testicular lesions. Withdrawal of the drug does not ameliorate this effect. The concomitant administration of melatonin with CP led to a partial improvement in testicular lesions induced by CP.

aDepartment of Histology, Faculty of Medicine, Cairo University, Cairo

bDepartment of Histology, Faculty of Medicine, Beni-Suef University, Beni-Suef, Egypt

Correspondence to Azza S. Embaby, Department of Histology, Faculty of Medicine, Beni-Suef University, Beni-Suef, Egypt, Tel: +20 111 238 2889; fax 0020235381183 e-mail: azza_embaby2010@yahoo.com

Received March 22, 2012

Accepted June 28, 2012

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Introduction

Most of the chemotherapeutic drugs used in the treatment of neoplastic cells induce various kinds of damage to normal cells. Cyclophosphamide (CP) is used in the treatment of certain malignant tumors such as lymphomas, leukemias, and in some non-neoplastic autoimmune diseases such as lupus erythematosus and glomerulonephritis 1.

CP treatment is associated with oligospermia and azoospermia as well as biochemical and histological alterations in the testes and epididymis of rats and humans. Moreover, disturbances in gonadotropin secretion, testicular damage, and decreased plasma testosterone levels are found in patients treated with CP 2.

Melatonin is a natural compound found in humans, animals, plants, and microbes. In animals, it is synthesized in various organs such as the pineal gland, retina, intestine, bone marrow cells, and skin. The circulating levels of the hormone melatonin vary in a daily cycle 3.

Melatonin acts as a powerful antioxidant and as a free radical scavenger of hydroxyl and peroxyl radicals. Indeed, melatonin was shown to be twice as potent as vitamin E in eliminating peroxyl radicals and it is more effective in scavenging hydroxyl radicals than glutathione and mannitol. As melatonin-binding sites have been detected in the reproductive systems of different species, it seems reasonable to assume that melatonin exerts its actions through a direct interaction with the steroidogenic cells of the reproductive organs 4.

The antioxidant function of melatonin has been associated with its capacity to scavenge reactive oxygen/nitrogen species (ROS/RNS). In addition, melatonin reduces free radical levels by stimulating the activities of antioxidative enzymes 5,6.

This study aims to examine the histological and immunohistochemical changes that may appear in testicular cells of albino rats treated previously with CP alone or in combination with melatonin.

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Materials and methods

Drugs

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Cyclophosphamide

In the form of an endoxan ampoule of 200▒mg (AstaMedica, Germany) a vial was dissolved in 10▒ml saline. Two doses were calculated. The first dose was in 100▒mg/kg and the second was in 20▒mg/kg/day 7.

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Melatonin

Melatonin was used in the form of tablets, 3▒mg each (Natures Bounty, USA); each tablet was dissolved in 5% ethanol. The total dose of melatonin was calculated at a dose of 10▒mg/kg/day and divided into two equal doses. Half of the total dose was injected 2▒h before the administration of CP and the other half was injected 2▒h after the administration of CP 4.

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Animals

Forty-two adult male albino rats, each weighing about 150–200▒g, were used in this study after obtaining consent from the ethical committee. The animals were fed ad libitum and allowed free access to water during the entire experiment, with a 12-h light–dark cycle. The animals were classified into the following groups, and each group was kept in separate cages. All rats were weighed at the beginning and at the end of all experiments. Before sacrification, the rats were physically examined. The right testes of animals used were weighed after sacrification.

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Group I

The control group included 12 rats; they were injected with the same vehicle used as a solvent for each drug. At the end of each experiment, two control rats were sacrificed with the injected rats of each experimental group.

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Group II

Group II included 15 rats, which were subdivided into three subgroups as follows:

Subgroup IIa: Five rats were injected intraperitoneally twice 1 week apart with CP (100▒mg/kg/body weight dissolved in saline) 7.

Subgroup IIb: Five rats were injected (intraperitoneally) twice 1 week apart with both CP (100▒mg/kg/body weight) and melatonin (10▒mg/kg body weight dissolved in 5% ethanol) daily for 2 weeks 4.

Subgroup IIc: Five rats were injected (intraperitoneally) twice 1 week apart with CP (100▒mg/kg/body weight dissolved in saline) and then left for another 2 weeks without any drug treatment.

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Group III

Group III included 15 rats, which were subdivided into three subgroups as follows:

Subgroup IIIa: Five rats were injected (intraperitoneally) daily for 5 weeks with CP (20▒mg /kg/body weight dissolved in saline) 8.

Subgroup IIIb: Five rats were injected (intraperitoneally) daily for 5 weeks with CP (20▒mg/kg/body weight) as well as with melatonin 10▒mg/kg body weight daily for 5 weeks 9.

Subgroup IIIc: Five rats were injected (intraperitoneally) daily for 5 weeks with CP (20▒mg/kg/body weight) dissolved in saline and then left for another 5 weeks without any drug treatment.

At the end of each experiment, the rats were anesthetized (ketamine 200▒mg/kg body weight, intraperitoneally), weighed, and sacrificed. The abdomen was reached with an abdominal midline incision was done. midline incision; the right testes for all animal groups were dissected, weighed, fixed in Bouin’s solution, and processed for paraffin sections 5▒µm in thickness.

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The prepared histological slides were stained with the following:

  1. H&E for observation of histological changes 10.
  2. Masson’s trichrome stain for observation of collagen fibers 11.
  3. Immunohistochemical studies with (BCL2 onchoprotein) to examine the apoptotic cellular changes 12.
  4. Morphometric study using the image analyzer computer system with Leica Qwin 500 software (Cambridge, England) to determine the following:
    1. Area%of the stained collagen fibers around seminiferous tubules per high-power field.
    2. Area% for the distribution of the immunohistochemical stains in the cells of the seminiferous tubules.
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Results

Body weight

There was a significant decrease (P<0.01) in the mean body weight in subgroups (IIa, IIIa, IIIc) in comparison with the control group (Histogram 1 and Table 1).

Histogram 1

Histogram 1

Table 1

Table 1

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Testicular weight

In group III, a significant decrease in the mean testicular weight was observed (P<0.01) (Histogram 2 and Table 1).

Histogram 2

Histogram 2

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Histological and immunohistochemical results

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Group I

The seminiferous tubules were separated by interstitial tissue. The tubules were lined by stratified epithelium of spermatogenic cells. The interstitial cells of Leydig appeared rounded or polygonal in shape, with an acidophilic cytoplasm and large rounded nuclei (Fig. 1). The spermatogenic cells were spermatogonia, primary spermatocytes, and spermatozoa filling the lumen of the tubules (Fig. 2). Masson’s trichrome-stained sections of control rats showed fine collagen fibers around intertubular blood vessels (Fig. 3). Positive BCL2 immunoexpression was found in the cytoplasm of the spermatogenic and Leydig cells (Fig. 4).

Figure 1

Figure 1

Figure 2

Figure 2

Figure 3

Figure 3

Figure 4

Figure 4

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Group II

Subgroup IIa: The separation of spermatogenic cells from their underlying basement membrane was observed in most seminiferous tubules (Fig. 5). Some dilated congested blood vessels were observed between the seminiferous tubules (Figs. 5 and 6). Sertoli cells showed partial cytoplasmic vacuolation (Fig. 7). Masson’s trichrome-stained sections showed increased perivascular and peritubular collagen fibers (Fig. 8). A decreased immunoexpression was observed in the cytoplasm of all spermatogenic and Leydig cells compared with group I (Fig. 9).

Figure 5

Figure 5

Figure 6

Figure 6

Figure 7

Figure 7

Figure 8

Figure 8

Figure 9

Figure 9

Subgroup IIb: Some seminiferous tubules appeared lined with nearly normal spermatogenic cells. Some spermatogonia contained small dark nuclei (Fig. 10). Masson’s trichrome-stained sections showed fine collagen fibers around dilated congested venules and around tubules (Fig. 11). A comparable immunoexpression of BCL2 to group I was observed in the cytoplasm of the spermatogenic cells and Leydig cells (Fig. 12).

Figure 10

Figure 10

Figure 11

Figure 11

Figure 12

Figure 12

Subgroup IIc: Some seminiferous tubules showed disorganization of their lining epithelium (Fig. 13). Minimal collagen fibers were evident around dilated vessels (Fig. 14). Decreased immunoexpression was observed in the spermatogenic and Leydig cells compared with group I, and subgroups IIa and IIb (Fig. 15).

Figure 13

Figure 13

Figure 14

Figure 14

Figure 15

Figure 15

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Group III

Subgroup IIIa: Distortion of some seminiferous tubules was observed, with the appearance of thickened obliterated blood vessels (BVs) in the interstitial tissue. The spermatogenic cells were separated from the underlying basement membrane (Fig. 16).

Figure 16

Figure 16

Also, there was marked vacuolation of the cytoplasm of the spermatogenic cells (Fig. 17). Dilated congested blood vessels with dense perivascular collagen fibers were evident (Fig. 18). Negative immunoexpression in the cytoplasm of the spermatogenic cells as well as the interstitial cells of Leydig was observed (Fig. 19).

Figure 17

Figure 17

Figure 18

Figure 18

Figure 19

Figure 19

Subgroup IIIb: Some seminiferous tubules had an intact basement membrane and were lined by almost normal spermatogenic cells with clear primary spermatocytes and spermatids within their center (Fig. 20). Minimal intertubular collagen fiber was observed (Fig. 21). Increased immunoexpression was observed in the spermatogenic and Leydig cells (Fig. 22).

Figure 20

Figure 20

Figure 21

Figure 21

Figure 22

Figure 22

Subgroup IIIc: Seminiferous tubules with thinned-out spermatogenic cells, central separated spermatid cells, and marked peritubular fibrosis were observed (Fig. 23). There was marked vacuolation of the cytoplasm of the spermatogenic cells that were separated from the underlying basement membrane (Fig. 24). Acidophilic exudates appeared in interstitial tissue (Fig. 25). Decreased immunoexpression was observed in the cytoplasm of spermatogenic and Leydig cells compared with group I and other subgroups (Fig. 26).

Figure 23

Figure 23

Figure 24

Figure 24

Figure 25

Figure 25

Figure 26

Figure 26

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Morphometric results

The area% of BCL2 immunoexpression denoted a significant decrease in subgroups IIa, IIc, IIIa, IIIb and IIIc compared to control and subgroup IIb (Histogram 3 and Table 2). A significant increase in area % of connective tissue was recorded in subgroups IIa, IIc, IIIa, IIIb and IIIc compared to control and subgroup IIb (Histogram 4 and Table 2).

Histogram 3

Histogram 3

Table 2

Table 2

Histogram 4

Histogram 4

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Discussion

Many drugs used for cancer chemotherapy produce toxic side effects in multiple organs, including the testes. A wide range of adverse effects including reproductive toxicity have been found following CP treatment in humans and experimental animals. Adult male patients treated with CP have shown decreased sperm counts. Several studies on male rats have confirmed that the administration of CP resulted in oligospermia, azoospermia, and histological alterations in the testes 1,13,14.

A means to reduce the side effects of anticancer drugs with preservation of their chemotherapeutic efficacy is necessary; therefore, in this study, we combined the CP treatment with melatonin, which is normally detected in the cells of the reproductive system.

This study aimed to examine the abnormal histological and immunohistochemical changes in the testes of albino rats treated with CP. Also, another aim was to evaluate the possible protective role of using a melatonin drug in combination with CP treatment.

The subgroups of animals that were injected with CP alone showed general signs of deterioration such as piloerection, hair loss, lethargy, and a hunched posture, with a marked decrease in the entire body weight as well as in testicular weight. These results have been confirmed by previous studies 1,14, in which a significant reduction in the weight of the testes following CP treatment has been reported.

In the present study, CP-treated animals showed severe necrosis and reductions in the germinal cell thickness. On application of the immunohistochemical stain, there were clear apoptotic changes in the cytoplasm of the testicular cells.

These changes were in agreement with those reported by some authors 14 who found that CP induced drastic morphologic changes in the testis. Atrophied seminiferous tubules with severe hypocellularity (reduction in number of germ cells) and intraepithelial vacuolization were observed, along with vascular congestion, inflammatory cell infiltration, and edematous fluid accumulation in the interstitial space. They reported the precise mechanism by which CP causes testicular toxicity: CP can disrupt the redox balance of tissues, leading to oxidative stress. They attributed these changes to oxidative DNA damage caused by the hydrogen peroxide derivative of CP through the generation of H2O2. In addition, acrolein (a major CP metabolite) is produced, which interferes with the tissue antioxidant defense system and produces highly reactive oxygen free-radicals that are mutagenic to mammalian cells 14.

Moreover, the investigators 15,16 added that the plasma membranes of spermatozoa contained large quantities of polyunsaturated fatty acids and their cytoplasm contained low concentrations of scavenging enzymes; thus, they were susceptible to the damage induced by excessive ROS production induced by CP.

Other researchers have reported that these ROS can attack the unsaturated bonds of the membrane lipids in an autocatalytic process, with the generation of peroxides, alcohol, and lipidic aldehydes as byproducts of the reaction. Thus, the increase in free radicals in cells can induce lipid peroxidation by oxidative breakdown of polyunsaturated fatty acids in membranes of cells 17.

Furthermore, the authors 18 reported that the main highly ROS that have potential implications in reproductive biology were the superoxide anion, the hydroxyl radical, and hydrogen peroxide. Normally, the balance between ROS produced by pro-oxidants and that scavenged by antioxidants is maintained, and cellular damage arises when this equilibrium is disturbed.

Previous studies have reported that spermatogenic inhibition may also be because of decreased plasma testosterone levels in CP-treated rats through downregulation of steroidgenesis and the impairment of Leydig cells 19.

In this study, degeneration in the cells of the seminiferous tubules, vacuolation in their cytoplasm, and reduced seminiferous epithelial layers, perivascular fibrosis, edema, and hyalinization of intertubular tissue were observed. The same results have been reported previously by other investigators 14,20,21, who described the appearance of necrosis, degeneration, desquamation, disorganization, reduction in germinal cells, vacuolization in Sertoli cells, multinucleated giant cell formation, interstitial edema, and congestion in the intertubular tissue.

Similar results 22 have been reported: a beneficial action of melatonin (20▒mg/kg) in inhibiting apoptosis and liver damage resulting from the oxidative stress in malaria, which could be a novel approach in the treatment of this disease.

In the present study, the concomitant administration of melatonin with CP to subgroups (IIb, IIIb) led to a marked improvement in the histopathological changes. A significant reduction in the germ cell apoptosis and in the degenerative changes in the cells of the seminiferous tubules was observed.

These changes were in agreement with those observed by some researchers 6,23,24, who reported that melatonin may alleviate cadmium-induced cellular stress and germ cell apoptosis in testes.

However, other authors 4 have claimed that coadministration of melatonin with chemotherapeutic agents such as CP led to relatively normal tubules, with the usual arrangement of cells.

Moreover, the investigators 25 evaluated the protective effect of melatonin against irradiation-induced damage to rat testes that included amelioration of germ-cell depletion and apoptotic changes. The same results were reported at the ultrastructural level by other researchers 26 who observed the disappearance of the characteristics of apoptosis (condensation of the nuclei, vacuolization of the cytoplasm, increased cytoplasmic density, and apoptotic bodies) when the irradiated animals were pretreated with melatonin.

Many studies have explained the possible mechanism of melatonin protection as being dependent on its antioxidative action. Lena and Subramanian 27 reported that melatonin has the ability to scavenge up to four or more reactive species, which makes melatonin a potent antioxidant and a free radical scavenger. They concluded that melatonin could control the oxidative abuse by (i) directly scavenging a variety of radicals and ROS, (ii) inducing antioxidative enzymes which reduce the steady-state levels of ROS, (iii) inhibiting nitric oxide synthase, which generates nitric oxide, and (iv) stabilizing cell membranes that aid them in reducing oxidative damage.

Similar results 28 have been reported; melatonin improved prosurvival signals and reduced prodeath signals. Zhang et al. 29 have suggested that melatonin may potentially attenuate testicular damage by improving histopathological changes and reducing germ cell apoptosis in hyperlipidemic mice.

Finally, in this study, it was found that melatonin could provide partial protection against CP-induced testicular atrophy. Several clinical trials should be conducted in the future to combine melatonin treatment with other chemotherapeutic and toxic drugs to reduce their apoptotic cellular changes and toxicity.

Figure

Figure

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Acknowledgements

Conflicts of interest

There is no conflict of interest to declare.

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Keywords:

cyclophosphamide; melatonin; testis

© 2012 The Egyptian Journal of Histology