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).
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.
Conflicts of interest
There is no conflict of interest to declare.
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Keywords:© 2012 The Egyptian Journal of Histology
cyclophosphamide; melatonin; testis