Sorafenib Induced Oxidative Stress in Testicular Tissue of Male Swiss Albino Mice

 

Surekha D. Shetty¹, Laxminarayana Bairy K.², Ashwini Aithal P.¹*

¹Department of Anatomy, Melaka Manipal Medical College (Manipal Campus),

Manipal Academy of Higher Education (MAHE) Manipal, Karnataka, India.

²Department of Pharmacology, RAK College of Medical Sciences,

RAK Medical and Health Sciences University, Ras Al-Khaimah, UAE.

 

ABSTRACT:

Objective: Male gonadal toxicity is a common complication of modern anti-cancer treatments. Oxidative stress can lead to damage to the structure of testis and germ cells. Oxidative stress develops in association with an imbalance between reactive oxygen radicals and the antioxidant reserve system. Antioxidants are compound that protect cell against the damaging effects of reactive oxygen species. The aim of this study was to investigate the effect of sorafenib on antioxidative enzyme superoxide dismutase in testicular tissue of male Swiss albino mice. Materials and Methods: The animals were segregated into control, positive control, and treatment groups (n=6 in each group). Treatment group received 25, 50 and 100mg/kg body weight of sorafenib orally for seven consecutive days at intervals of 24 hours between two administrations. Positive control group received 100 mg/kg body weight of imatinib. The animals were sacrificed at the end of 1^st, 2^nd, 4^th, 5^th, 7^th and 10^th week after the last exposure to sorafenib. The testis were removed, weighed, and processed for superoxide dismutase activity assay. Results: The superoxide dismutase activity was reduced significantly (P<0.05) during the 1^st, 2^nd, 4^th, 5^th and 7^th week sampling time in mice treated with all the doses of sorafenib. Superoxide dismutase activity returned closer to control group in 10^th week sampling time. Conclusion: The administration of sorafenib decreases the superoxide dismutase activity in testicular tissue. It lead to imbalance between antioxidant system and reactive oxygen species generation, which produced the oxidative stress.

 

 

 

INTRODUCTION:

Chemotherapy is associated with male infertility. Chemotherapy and radiation therapy are linked with changes in the male genital tract. In chemotherapy, drugs with alkalizing properties have the highest dose-dependent adverse drug reactions on testicles¹. Oxidative stress also leads to damage to the structure of testis and germ cells². Oxidative stress develops in association with an imbalance between reactive oxygen radicals (ROS) and the antioxidant reserve system. ROS are the product of normal cellular metabolism. Sperm is one type of cell that manufactures free oxygen radicals.

 

With reactive oxygen radical production at low levels, sperm cell capacitation, acrosome reaction, and sperm binding to the zona pellucida take place³. Uncontrolled ROS production leads to sperm abnormalities and infertility.

 

Antioxidant defence mechanisms in the testis are important in the protection of sperm against ROS. Superoxide dismutase (SOD) is one of the major antioxidant enzymes that protect the male reproductive organs against the harmful effects of ROS^4,5. The SOD plays an important role in testicular development and spermatogenesis. Changes that take place in this enzyme may lead to impaired testicular functions and cessation of sperm development. Oxidative stress is an important mediator of apoptosis. Mitochondria play a significant role in the apoptotic process^6,7.

 

Oxidative stress is a major factor in the a etiology of male infertility. At the level of the isolated spermatozoon, ROS attack can induce lipid peroxidation and deoxyribonucleic acid (DNA) fragmentation disrupting both the motility of these cells and their ability to support normal embryonic development⁸. At the level of the testes, oxidative stress is capable of disrupting the steroidgenic capacity of Leydig cells as well as the capacity of the germinal epithelium to differentiate into normal spermatozoa⁹.

 

MATERIALS AND METHODS:

Animals:

Male Swiss albino mice (9-12 weeks old) were used for the study. The animals were segregated into control, positive control, and treatment groups (n=6 in each group). Treatment group received 25, 50 and 100mg/kg body weight of sorafenib orally for seven consecutive days at intervals of 24 hours between two administrations. Positive control group received 100 mg/kg body weight of imatinib. Control group remained in home cage for the same duration to match their corresponding treatment groups. The animals were sacrificed at the end of 1^st, 2^nd, 4^th, 5^th, 7^th and 10^th weeks after the last exposure to the drug.

 

Preparation of tissue homogenate:

Testes were removed and weighted. The testes were then minced in phosphate buffer solution at a ratio of 1:10 and homogenized. The tissue homogenate obtained was cold centrifuged. The supernatant was taken for estimation of SOD activity by EpiQuik™ Superoxide Dismutase Activity Assay Kit.

 

Statistical analysis:

The data generated are analysed by one-way Analysis of Variance (ANOVA) followed by Bonferroni’s post hoc test. Values of P<0.05 were considered statistically significant.

 

RESULTS:

Effect of Sorafenib on superoxide dismutase activities:

The SOD activity reduced in all the treated group of mice. For the mice treated with 25 mg/kg of sorafenib, SOD activity was significantly reduced during the 4^th and 5^th week sampling time. In 50 mg/kg of sorafenib, SOD activity was significantly reduced during the 4^th 5^th and 7^th week sampling time. In higher dose (100 mg/kg) group SOD activity was significantly reduced in all the sampling weeks, least during 5^th week (Table. 1). The time response graph shows that sorafenib affects the SOD activity in a time dependent manner (Figure. 1). Positive control had a significant effect on the SOD activity in 1^st, 2^nd, 4^th, 5^th, and 7^th week sampling time. SOD activity returned closer to control group in 10^th week sampling time. SOD activity was significantly reduced in both positive control and 100 mg/kg of sorafenib in all the sampling week except 10^th week.

 

Table 1: Effect of sorafenib on superoxide dismutase activity (SOD). Each dose group from particular time represents mean ± SD from six animals.

Dose	Sampling Time
	1W	2W	4W	5W	7W	10W
NC	0.09±0.01	0.09±0.01	0.09±0.01	0.09±0.01	0.09±0.01	0.09±0.01
PC	0.05±0.01ϕ	0.05±0.01ϕϕ	0.04±0.01ϕ	0.04±0.01ϕ	0.05±0.01ϕ	0.07±0.01
S1	0.08±0.01##	0.08±0.01###	0.06±0.01***	0.06±0.01***	0.07±0.01###	0.08±0.01
S2	0.06±0.01	0.06±0.01	0.05±0.01*	0.05±0.01*	0.05±0.01*ааа	0.08±0.01
S3	0.04±0.01*Ƅ	0.04±0.01*ƄƄ	0.03±0.01*ƄƄ	0.03±0.01*ƄƄ	0.04±0.01*ƄƄ	0.06±0.01***

Significant values are NC (normal control) PC (positive control), ^ϕϕ < 0.01, ^ϕ < 0.001; NC versus treated, ^*** < 0.05, ^* < 0.001, PC versus treated, ^### < 0.05 ^## < 0.01; S1 (sorafenib 25mg/kg) versus S2 (sorafenib 50mg/kg), ^ааа < 0.05; S1 versus S3 (sorafenib 100mg/kg), ^bb < 0.01, ^b < 0.001. w = weeks.

Figure 1: Time response relationship for sorafenib induced changes in superoxide dismutase activity. Each time at particular dose represent mean ±SD from 6 animals. Significant values are NC (normal control) PC (positive control), ^ϕϕ < 0.01, ^ϕ < 0.001; NC versus treated, ^***< 0.05, ^* < 0.001, PC versus treated, ^###<0.05, ^##<0.01; S1 (sorafenib 25mg/kg) versus S2 (sorafenib 50mg/kg), ^ааа<0.05;

S1 versus S3 (sorafenib 100mg/kg), ^bb < 0.01, ^b < 0.001. w = weeks.

 

DISCUSSION:

Oxidation is a chemical reaction that transfers electrons or hydrogen from a substance to an oxidizing agent. Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions. When the chain reaction occurs in a cell, it can cause damage or death to the cell. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions¹⁰. Sterility is a major clinical issue, influencing larger part of the general population medicinally also, psychosocially. As of late, oxidative stress and free radical production has been involved in male infertility¹¹.

 

The present study demonstrated that administration of sorafenib decreases the SOD activity in testicular tissue. It leads imbalance between antioxidant system and reactive oxygen species generation, which produces the oxidative stress in testicular tissue. The SOD is considered as one of the most significant antioxidants that current researches specified as having a leading role on oxidative stress inhibition¹². Superoxide dismutases (SODs) are a class of closely related enzymes that catalyze the breakdown of the superoxide anion into oxygen and hydrogen peroxide. SOD enzymes are present in almost all aerobic cells and in extracellular fluid. Antioxidants enzymes such as SOD, CAT, and GSH dependently act in the metabolic pathways that involve free radicals¹³. In our study, SOD activity level is significantly reduced by toxicity of sorafenib when comparing with normal control. Declined SOD activity increases the ROS production that induced the oxidative stress. It causes the germ cells damage in the seminiferous tubule of testis. Oxidative stress has long-standing effect on male germ cell variation. It has been stated that oxidative stress prevents spermatogenesis, declines SOD activity, and induces DNA impairment as well as apoptosis¹⁴.

 

In our study sorafenib administration markedly decreased SOD activity in the mice testes. The SOD activity was decreased in significant manner from 4^th, 5^th and 7^th week sampling time in 25mg/kg, the 4^th, 5^th and 7^th week sampling in 50mg/kg, all week sampling time in 100mg/kg of sorafenib and 1^st to 7^th week sampling in positive control imatinib. Cytotoxicity and cell deaths were caused by oxidative stress¹⁵. Mitochondria are known to be major places of intracellular ROS generation, and they were easily damaged by oxidative stress¹⁶. Administration of drugs like methotrexate, busulfan and artesunate decrease the SOD activity in testicular tissue^17,18.

 

Numerous investigators denoted important decrease in antioxidant enzymes activities and testicular toxicity after cyclophosphamide administration to various animals^19,20. Male germ cells are more vulnerable to oxidative stress than other cells, since they have greater polyunsaturated fatty acids in their membranes than somatic cells. Oxidative stress shows a significant role in the etiology of imperfect sperm formation, function; sperm count and male infertility²¹. Cisplatin treatment caused a significant decrease in activities of antioxidant enzymes (SOD and catalase) with a rise in the levels of H₂O₂ and lipid peroxides in the testes and             epididymis^{22, 23}.

 

The ROS can attack sperm membrane lipids, DNA and proteins; change enzymatic systems; produce permanent alterations; cause cell death; and finally, lead to decline in the semen parameters related with male infertility^24,25. Chemotherapeutic drugs induce oxidative stress is associated with antioxidant defense mechanism and a general balance among pro-oxidants and antioxidant status of cells and results in producing oxygen free radicals and ROS^24,26.

 

CONCLUSION:

The present study proved that administration of sorafenib decreases the SOD activity in testicular tissue. It leads to imbalance between antioxidant system and reactive oxygen species generation, which produces the oxidative stress. Outcome of the study may help the clinicians to plan and address the fertility related issues in young patients of reproductive age who are treated with sorafenib for advanced renal cell carcinoma, hepatocellular carcinoma and differentiated thyroid carcinoma.

 

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Received on 22.05.2021           Modified on 28.09.2021

Accepted on 30.12.2021         © RJPT All right reserved