Cisplatin (CPT) compound has shown important biological activities such as antitumor, antituberculous and antimicrobial. However, several researchers have reported the adverse effects of this drug especially on male reproductive system following its administration. Therefore, this study explores the possible ameliorative effect of dexrazoxane against cisplatin-induced testicular tissue using mouse model. Swiss albino mice were randomly selected into three groups (n=5). Group I served as control while group II received 5mg/kg cisplatin and mice in group III were co-treated with 5mg/kg/bwt cisplatin +10mg/kg/bwt dexrazoxane via intraperitoneal injection respectively. Experimental animals from each group were sacrificed at the intervals of 6 hr, 12 hr and 24 hr. The estimation of biochemical and hormonal analyzes by enzyme immunosorbent assay (ELISA) were carried out. Sperm quality and role of apoptosis were also measured in this study. Data were analyzed by Duncan ANOVA at p<0.05. It was observed that CPT leads to significant decrease in level of all the antioxidants measured. CPT leads to significant decrease in the testicular testosterone level and all the antioxidant enzymes measured. Cisplatin leads to depletion in the number of spermatozoa cells and increased aberrant spermatozoa cells structure. However, recovery was seen in the group co-treated with dexrazoxane. This study gives insight on the adverse effects of cisplatin compound on male reproductive system also shows that melatonin remarkably improved the deleterious effect seen in the all parameters tested.

Cisplatin is a platinum based chemotherapy agent. It has been used for treatment of numerous human cancers including testicular cancers. Cisplatin mode of action has been linked to its ability to crosslink with the purine bases on the DNA; interfering with the DNA repair mechanisms, causing DNA damage, and subsequently inducing apoptosis in cancer cells [1-3]. However, due to numerous undesirable side effects especially in younger patients and fetus, its use was restricted but combination therapies of cisplatin with other drugs have been highly encouraged to overcome drug-resistance and reduce toxicity. Dexrazoxane hydrochloride is a cardioprotective agent used to protect the heart against the cardiotoxic side effects of chemotherapeutic drugs such as anthracyclines [4], daunorubicin or doxorubicin or other chemotherapeutic agents [5]. Based on the available data legally binding the decision to implement dexrazoxane in order to allow children to receive dexrazoxane as primary cardioprotection against anthracyclineinduced cardiotoxicity without reducing anthracycline activity and without enhancing secondary malignancies [6], It was therefore speculated that dexrazoxane could be used for further investigation to synthesize new drugs [7]. Hence, this study, explore dexrazoxane use in the ameliorating the deleterious effect of cisplatin in testicular tissue dysfunction. 

2.0. Methods 

2.1 Ethnical Approval

The studies were conducted in accordance with the standards and permission established by The Ethics Committee of Animal, Ekiti State University Ado-Ekiti, Nigeria. Male Swiss-albino mice were housed in room at 22 ± 2°C with 40% relative humidity and with a 12-hr light ± dark cycle. They were fed with a standard rat chow and tap water ad libitum.

 2.2 Experimental Procedure 

Male Swiss-albino mice used for this study were weighed. The animals were randomly divided into three group I-III and treated as shown in the table below. 

Dosing 

2.3 Testicular Testosterone (T) and Luteinizing hormone (LH) concentrations. 

The testicular testosterone and luteinizing hormone levels in three mice from each group were measured. Briefly, testicular proteins were extracted with phosphate buffer (50 mM, pH 7.4) and centrifuged at 10,000 g for 20 min. The supernatant was used to estimate T and LH levels using ELISA, and were expressed in ng/ml. 

2.5 Biochemical Estimations study

Testicular tissues from each mouse were stored at -20oC for different biochemical assays Protein quantity was estimated according to Lowry’s method. 10% tissue homogenates (w/v) were prepared in chilled 100 mMTris-HCL buffer (pH 7.4). The values were expressed per mg of protein.

2.6 GSH Determination

Tetsis tissues were homogenized in 10 ml TCA (trichloroacetic acid) which is at the rate of 10%, and then centrifuged at +4 °C for 15 minutes. Afterwards, 0,5 ml of supernatant was taken, and mixed with 0,3 M 2 ml Na2HPO4. The mixture was thoroughly vortexed. This mixture was vortexed by the addition of 0,2 ml DTBN (Dithiobisnitrobenzene: prepared by dissolving in 1% sodium citrate). Absorbance was measured at 412 nm.

2.7. Measurement of reactive oxygen species (ROS) level 

The ROS assay was performed by the method of Hayashi., et al. (2017). In brief, 50 µl of testicular tissue homogenate and 1400 µl sodium acetate buffer was transferred to a cuvette. After then, 1000 ul of reagent mixture (N, N-diethyl paraphenylenediamine 6 mg/ml with 4.37 µM of ferrous sulfate dissolved in 0.1M sodium acetate buffer pH- 4.8) was added at 37°C for 5 minutes. The absorbance was measured at 505 nm using spectrophotometer [8].

2.8.          Measurement of MDA 

Testes tissues were homogenized in 10 ml TCA (trichloroacetic acid) which is at the rate of 10%, and then centrifuged at +4°C for 15 minutes. 750μl of the supernatant which was obtained was mixed with 0.67% TBA (thiobarbituric acid) in a ratio of 1:1. Afterwards, the solution was left in the water bath for 15 minutes. Finally, the absorbance was measured spectrophotometrically at 535 nm.

2.9. Superoxide dismutase (SOD) activity 

Superoxide dismutase (SOD) activity was assayed by a spectrophotometric method. Assay mixture containing sodium pyrophosphate buffer (pH 8.3, 0.052M), phenazine methosulfate (186 µM), nitroblue tetrazolium (300 µM) and NADH (780 µM) were diluted with appropriate enzyme in total volume of 3 ml. The mixture was incubated at 37oC for 90 sec and reaction was stopped by addition of glacial acetic acid. The reaction mixture was mixed vigorously by adding n-butanol and allowed to stand for 10 min before the collection of butanol layer. The intensity of chromogen in butanol was measured at 520 nm.

2.10. Sperm Parameters 

Caudal epididymidis was removed from each mouse and cleaned off from the epididymal fat pad, and minced in a pre-warmed Petri dish containing 500 µl phosphate buffer saline solutions (PBS, pH 7.4) at 37oC. Sperm motility was estimated and expressed as percentage incidence [4]. For sperm count, an aliquot of this suspension was charged into the Neubauer’s counting chamber and the spermatozoa were counted under light microscope. Total sperm count was calculated as the average of the spermatozoa count (N) in each chamber X multiplication factor (106) X dilution factor and was expressed in millions/ml. The sperm morphology was also evaluated [9].

2.11. Capases Estimation

Briefly, 1ml of assay buffer (20mM HEPES, 10% glycerol, 1M DTT, and 14ml of n-acetyl-DEVD-AMC/ml of buffer), and 50ml of sample were added to a microcentrifuge tube and protected from the light. Samples were incubated at 37°C for 60 mins after which fluorescence was measured on a spectrofluorometer with an excitation wavelength of 380nm and an emission wavelength of 440nm.

2.12. Statistical Analysis 

All data were expressed as mean ±standard error of the mean (SEM) and analyzed by one-way ANOVA followed by Duncan’s multiple comparison test using SPSS software version 22 (SPSS Inc., Chicago, Illinois). p<0> were considered statically significant.