الفهرس 
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Abstract
Globally, cancer is considered as a public health problem and a leading cause of death. Thus, chemotherapy has been presented to be useful in slowing the progression of different sorts of cancers. Chemotherapy includes cytotoxic drugs that are derived from natural products and considered to be toxic for both the cancer cells and also the healthy normal proliferating cells.
As an efficient chemotherapeutic agent, TXL, a member of taxane family and a classic microtubule-targeting agent encourages tubulin polymerization and interposes with depolymerization of microtubules which is crucial in the division of cells. However, TXL has been successfully used against various types of cancers, it causes cell death that results in severe side effects reducing its prominent efficiency.
On the other side, MLT a natural derivative of the amino acid tryptophan which was extracted from the bovine pineal gland, has been known with its powerful antioxidant, free-radical scavenging, and cell-modulating properties. Also, MLT has been shown to inhibit the progression of tumor cell growth in various types of cancers.
Therefore, the present investigation was planned to show, assess and evaluate the deleterious consequences evoked by TXL on the hepatic and testicular tissues of adult rats and the probable ameliorative effect of MLT supplementation against such toxic effects using histological, histomorphometrical, immunohistochemical and ultrastructural approaches.
Fifty male Wistar rats (Rattus norvegicus) were equally allocated into five groups of ten rats each as follows:
• Control group: rats were injected daily intraperitoenally (i.p) with 1 ml physiological saline solution for 30 consecutive days.
• Sham group: rats were daily i.p injected for 30 days with physiological saline plus absolute ethanol with a final concentration below 0.1%.
• TXL-treated group: rats received i.p 7.5 mg/kg b.w. injections once a week (days 0, 7, 14, 21, and 28).
• MLT-treated group: rats received a daily i.p injection of 10 mg/kg b.w. for 30 days.
• MLT+TXL-treated group: rats were concomitantly treated with MLT (10 mg/kg b.w., daily) and TXL (7.5 mg/kg b.w., once a week) in the same manner.
At the end of the experimental period, the control and treated rats were anesthetized using diethyl ether, dissected, and their livers and testes were carefully excised, washed with physiological saline solution, and processed for histological, histomorphometrical, immunohistochemical and ultrastructural investigations.
For histological preparation:
Samples of the liver and testis of all animals’ groups were cut into small segments and fixed for 24 h in aqueous Bouin’s fixative. Then, they were prepared using the routine protocols of paraffin sectioning. 4-6 µm thick sections were stained with Ehrlich’s hematoxylin and counterstained with eosin (H&E).
For histomorphometrical measurements:
Five random fields from H&E-stained hepatic and testicular cross-sections of all animals’ groups were selected and analyzed. For the liver, the numbers of normal hepatocytes, necrotic hepatocytes, deteriorated Kupffer cells, infiltrated inflammatory cells in the both central and portal zones were counted. Also, the numbers of dilated/congested hepatic sinusoids, central and portal veins were estimated.
For the testis, the thickness of the tunica albuginea, diameter of seminiferous tubules, and height of germinal epithelia were measured, as well as the numbers of seminiferous tubules without sperm, apoptotic germinal epithelia, and apoptotic Leydig cells were counted.
For immunohistochemical preparations:
Buffered neutral formalin-fixed paraffin-embedded hepatic and testicular tissue sections from all experimental rats were prepared and immunostained for detection of Bcl-2, P53, Cas3 and CD163 reactive proteins following the standard Avidin Biotin Complex (ABC) method. Finally, sections were counterstained with Mayer’s hematoxylin, rinsed with tap water, dehydrated, cleared, and covered by cover slips. In each case, negative control sections were prepared.
For quantification of immunohistochemical parameters:
Image analysis was used to quantify the immunoreactivities of Bcl-2, P53, Cas3 and CD163 reactive proteins. The immunostained sections were first examined and photographed by a compound light microscope equipped with a computed image analysis system to assess the number of positive cases which estimated by the area percentage of the positive immunostained cells in relation to the total number of cells examined at X200 magnification in 5 selected fields using the computed image analysis system.
For ultrastructural preparation:
Small segments of the freshly excised livers and testes were fixed for 24 h in cold 4F1G, then post-fixed for 2–4 h in 1% phosphate buffered osmium tetroxide (pH 7.3). After fixation, specimens were processed for semithin sectioning. Then, areas of interest were selected and the blocks were trimmed. Ultrathin sections were cut, mounted on cupper grids and double stained with uranyl acetate and lead citrate for examination and photographing by transmission electron microscopy (TEM).
Statistical analysis:
Data are reported as means ± SEM. Differences in animals’ groups were compared by one-way analysis of variance (ANOVA) using SPSS/17.0 software. Statistical significances among groups were assessed by Tukey’s multiple comparison post hoc test and A P value ≤ 0.05 was considered statistically significant.
Results:
The results obtained from the current study are summarized as follows:
The Liver:
Hepatic tissues obtained from TXL-treated group revealed variable histological and histomorphometrical alterations in both the centrilobular and periportal zones including:
• Damaged architecture of the parenchymal tissues.
• Increased number of dilated and congested central and portal veins.
• Damaged and eroded endothelial linings of blood vasculature.
• Elevation in the number of accumulated infiltrated inflammatory cells in the centrilobular and periportal zones.
• Abundance of dilated and congested blood sinusoids with activated Kupffer cells and eroded endothelial linings.
• Hepatocytes showed clear signs of necrosis manifested by coarsely granulated and vacuolated cytoplasm, cloudy swelling and their nuclei underwent devastative symptoms of pyknosis, karyorrhexis and karyolysis.
• Coagulative necrosis was also observed.
Immunohistochemically, TXL induced changes in the immunohistochemical expression of Bcl-2, P53, Cas3 and CD163 reactive proteins represented as;
• A significant decrease (P ≤ 0.05) in the immunoexpression of Bcl-2 reactive protein.
• A significant increase (P ≤ 0.05) in the levels of P53, Cas3 and CD163 reactive proteins.
Ultrastructurally, TXL displayed various hazardous ultrastructural changes in almost all organelles manifested by:
• Rough endoplasmic reticulum fragmented into small bars, detached from the nuclei, lost its attached ribosomes and appeared hypertrophied.
• Smooth endoplasmic reticulum appeared proliferated, hypertrophied and lysed.
• Mitochondria suffered from degenerated cristae, lack of cristae and matrices, ruptured limiting membranes, and appeared as electron dense bodies.
• Increased number of lysosomes.
• Nuclei appeared mostly with destructive nuclear envelopes and showing prominent degree of chromatolysis; Pyknosis, karyorrhexis and karyolysis.
On the other side, hepatic tissues obtained from MLT+TXL-treated rats displayed marked improvement in their histological, histomorphometrical and ultrastructural architectures of both the centrilobular and periportal zones presented by:
• Normal intact radiating hepatic cords.
• Normal configuration of central veins with intact endothelial linings.
• Narrow hepatic sinusoids which lined with intact endothelial and Kupffer cells.
• Intact portal triads having well-organized hepatic portal veins, hepatic portal arteries, lymphatic vessels and bile ductules.
• Almost normal structures of all cytoplasmic organelles including; the rough endoplasmic reticulum, smooth endoplasmic reticulum, mitochondria and lysosomes, besides regular nuclei.
Immunohistochemically, MLT upregulated the immuno-expression of Bcl-2 reactive protein and downregulated the levels of immunostainability for P53, Cas3 and CD163 reactive proteins.
The Testis:
TXL-treated rats revealed severe histological and histomorphometrical alterations as follows:
• Tunica albuginea appeared thickened and irregular.
• Seminiferous tubules exhibited irregular with ruptured surrounding basal laminae.
• Reduction in the thickness of germinal epithelia.
• Increased numbers of atrophied seminiferous tubules which have necrotic spermatogenic cells and appeared almost devoid of spermatozoa.
• Reduction in the size and number of elongated spermatids, mostly appeared with abnormal residual cytoplasm and accordingly loss of spermatozoa.
• Formation of multinucleated giant cells and sloughing of germ cells into the tubule lumens.
• Dilated intercellular spaces between such destructed germ cells.
• Malformed Sertoli cells which appeared frequently separated from the underneath basal laminae.
• Congested interstitial tissues with decreased number of Leydig cells which appeared mostly degenerated.
Immunohistochemically, TXL induced changes in the immunoexpression of Bcl-2, P53, Cas3 and CD163 reactive proteins represented as;
• A significant decrease (P ≤ 0.05) in the immunoexpression of Bcl-2 reactive protein.
• A significant increase (P ≤ 0.05) in the immunoexpression of P53, Cas3 and CD163 reactive proteins.
Ultrastructurally, testicular tissues obtained from TXL-treated rats illustrated marked ultrastructural alterations of the tunica albuginea, seminiferous tubules and interstitial tissues as follows:
• Thickened and irregular tunica albuginea and basal laminae.
• Deformed spermatogonia appeared small in size and had few electron-dense mitochondria, rough endoplasmic reticulum and cytoplasmic vacuoles.
• Atrophied primary spermatocytes had electron-dense mitochondria, fragmented rough endoplasmic reticulum and dilated smooth endoplasmic reticulum, besides the nuclei exhibiting chromatolysis.
• Ruptured rounded spermatids with karyorrhectic nuclei, few mitochondria, rough endoplasmic reticulum and increased cytoplasmic vacuoles.
• Atrophied elongated spermatids possessed electron-dense acrosomal head caps, pyknotic nuclei and short flagella with disarranged and electron-dense mitochondria.
• Detached Sertoli cells with irregular nuclei, electron-dense mitochondria, dilated smooth endoplasmic reticulum, phagosomal bodies and cytoplasmic vacuoles.
• Markedly dilated intracellular spaces.
• Deteriorated Leydig cells had pyknotic nuclei, deformed mitochondria, fragmented rough endoplasmic reticulum, reduced smooth endoplasmic reticulum and few lipid droplets.
Whereas, the testes obtained from MLT+TXL-treated rats revealed an improvement in the histological, histomorphometrical and ultrastructural architecture of the testicular tissues, represented with:
• Regular tunica albuginea.
• Nearly integral seminiferous tubules covered by intact thin basal laminae and had normal diameters and endothelial height.
• Normal numbers and structures of spermatogenic cells including spermatogonia, primary spermatocytes, rounded spermatids, elongated spermatids, spermatozoa and Sertoli cells.
• Regular interstitial tissues enclosing intact clusters of Leydig cells.
In addition, the immunohistochemical results revealed that MLT enhanced the immunoexpression of Bcl-2 reactive protein and decreased the levels of immunoexpression of P53, Cas3 and CD163 reactive proteins.
In conclusion: The current experimental study proved that MLT supplementation ameliorated the hepatic and testicular tissues of rats exposed to TXL from its deleterious and noxious impacts through the antioxidant and anti-apoptotic properties of MLT. Furthermore, this investigation revealed that MLT can be used as a protective agent during chemotherapeutic treatment to prevent the damage evoked in the hepatic and testicular tissues.