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Abstract Chemotherapy is the main treatment of cancer. Cyclophosphamide is a nitrogen mustard alkylating agent, an anticancer, anti-neoplastic agents and immunosuppressive agent widely used in cytotoxic chemotherapy, which disturbs DNA synthesis and cell division. Medical plants are widely used against various diseases. Fennel oil is the essential oil obtained from the dried ripe fruits of foeniculum vulgare. The chief component of fennel, anethole, had different therapeutic effects. The present study examined the histological, histochemical, immunohistochemical and biochemical effects of cyclophosphamide on liver and testis of rat and the ameliorative effect of fennel oil. Animal groups: Animals were divided into four groups The first group: Animals of this group (12 rats) were kept as control groups. They were given standard diet and tap water only. The second group: Animals of this group (12 rats) were orally given fennel oil (1ml/kg body weight) once a week for six weeks. The third group: Animals of this group (12 rats) were orally given cyclophosphamide (15mg/kg body weight) once a week for six weeks. The fourth group: Animals of this group (12 rats) were orally given cyclophosphamide (15mg/kg body weight) followed by fennel oil (1ml/kg body weight) once a week for six weeks. After three and six weeks, six animals from each group were sacrificed. Liver and testis were immediately removed from rats after dissection and fixed in alcoholic Bouin’s fluid for histopathological study and general carbohydrates and in 10% neutral formalin for histochemical demonistration of total proteins and for immunohistochemical demonstration of caspase-3 and Ki-67 staining cells. Paraffin sections (5 microns thickness) were prepared and stained with Ehrlich’s hematoxylin and counter stained with eosin for histological examination. For histochemical purposes, total polysaccharides were demonstrated by periodic acid schiff’s (PAS) technique, total proteins were demonstrated by mercury bromophenol blue method. For immunohistochemical study Avidin biotin method was used for detection of expression of caspase-3 and Ki-67 staining cell in liver and testicular tissue. For biochemical analysis, blood samples were collected in clean centrifuge tubes and left to clot in room temperature and then serum separated by centrifugation at 3000 rpm for 20 minutes. The collected serum stored at - 20°C until analysis, The activities of ALT, AST, CAT and SOD were determined. Also the level of MDA and GSH were measured. Testosterone and luteinizing hormone were also determined. The results of this study showed that: Animals treated with fennel oil showed no changes in histological, histochemical, immunohistochemical and biochemical investigations in both liver and testis. I. Liver: The histological observations of rats treated with cyclophosphamide showed less in the normal structure of hepatic lobules. Congestion of portal vein, leucocytic infiltration, cytoplasmic vaculation and fatty infiltration were detected. While, the light microscopic examination in groups treated with cyclophosphamide followed by fennel oil showed marked improvement in the histological structure of liver. The histochemical investigations involved the determination of total polysaccharides and proteins content in liver. Gradual depletion in total polysaccharides and proteins contents were observed in liver of rats treated with cyclophosphamide. On the other hand, the restoration of total polysaccharides and proteins contents were observed in hepatic tissues of rats which treated with cyclophosphamide followed by fennel oil. The immunohistochemical investigations include the expression of Proliferating marker Ki-67 and caspase-3. The expression of Ki-67 and caspase- 3 were strong in liver of rats treated with cyclophosphamide only. While the administration of cyclophosphamide followed by fennel oil showed slight or weak expression of Ki-67 and caspase-3 which were nearly similar to control group. II. Testis Animals treated with cyclophosphamide showed more pronounced histological change during different periods. Exfoliated germ cells, vaculation, degeneration and congestion were observed. On the other hand, an improvement observed in testis sections of rats treated with cyclophosphamide followed by fennel oil. The content of total polysaccharides and proteins were determined by many histochemical techniques. Treating animals with cyclophosphamide only showed gradual depletion in total polysaccharides and proteins in testis. On the other hand, rats treated with cyclophosphamide followed by fennel oil showed restoration of total polysaccharides and proteins contents in testis. The immunohistochemical investigations involved the expression of proliferating marker Ki-67 and caspase-3. A strong expression of caspase-3 was observed but weak expression of Ki-67 was observed in testis of rats treated with cyclophosphamide only. While rats treated with cyclophosphamide followed by fennel oil showed a slight expression of caspase-3 and strong expression of Ki-67 similar to control group. III. Biochemical analysis In the present work, the administration of cyclophosphamide included elevation in the activities of ALT, AST and MDA and reduction in the activities of antioxidant enzymes CAT and SOD and decrease in the level of testosterone and LH hormones and non-enzymatic antioxidant GSH in sera of treated rats. On the other hand, the biochemical results of rats treated with cyclophosphamide followed by fennel oil showed significant reduction in the activities of ALT, AST and MDA, while recorded an increase in the activities of SOD, CAT, levels of testosterone and LH hormones and GSH when compared with animals treated with cyclophosphamide only. In conclusion, fennel oil has ameliorative effect against cyclophosphamide toxicity in both liver and testis of rats. |