About 50% of the infertility conditions are attributed to male factors (Wu et al., 2020). The main cause of male infertility is still unknown but about 30-80% of the cases were attributed to the release of a high amount of reactive oxygen species (ROS) (Tremellen, 2008). Many drugs are implicated in infertility disorders due to the induction of oxidative stress (Smith et al., 2006). The testis is considered a susceptible organ to lipid peroxidation due to its high lipid content and high O2 consumption (Ilbey et al., 2009)(Hassan et al., 2019). This oxidative stress occurs due to an imbalance between ROS and the endogenous antioxidant defense system. Excessive ROS levels lead to detrimental deviations, that can abolish lipids, proteins, nucleic acids, and other cellular compounds, followed by cellular death (Konuk et al., 2012). Consequently, elevated ROS levels in testicular tissue might cause infertility (Ebokaiwe et al., 2015).
In the current study, our findings revealed that intraperitoneal injection of tramadol (20 mg/kg) to male rats for 2 months increased the level of oxidative stress evidenced by elevated MDA and NO and on the other hand a significant decrease in GSH and SOD levels in testicular tissue. Accordingly, we concluded that oxidative stress is well-thought to be a principal factor in the induction and progression of tramadol-induced reproductive toxicity (Ahmed & Kurkar, 2014). Our findings were in agreement with the prior studies reported by Abdel-Latif Ibrahim et al (M. A. L. Ibrahim & Salah-Eldin, 2019), and Ghoneim et al (Ghoneim et al., 2014), Who reported that MDA levels were significantly elevated after tramadol exposure in some tissues. In the current research, intraperitoneal injection of tramadol (20 mg/kg) significantly decreased the intracellular GSH content in testicular tissue, compared to the control group. Same findings recorded by, Ghoneim et al (Ghoneim et al., 2014) and Sheweita et al (Sheweita et al., 2018). Deficiency of the antioxidant system was evidenced by depletion of GSH levels in testicular tissue which in turn leads to a further increase in MDA levels. This condition can be followed by necrosis and apoptosis due to oxidation of the thiol group in the proteins of the mitochondrial membrane (Kandemir et al., 2017). Our results also revealed a significant decrease in SOD activity, which agreed with some previous studies (Ahmed & Kurkar, 2014) (M. A. L. Ibrahim & Salah-Eldin, 2019). The depletion of GSH and SOD indicates suppression of ROS scavenging capability. Our findings also showed NO levels in testicular tissue were increased, when compared to the control group. In parallel with our results, previous findings suggested that tramadol might induce testicular dysfunction due to the augmented production of NO and oxidative stress (Bodera et al., 2013). Our results also revealed that intraperitoneal injection of boldenone (5 mg/kg) induced oxidative stress in testicular tissue which was indicated by significant increase in MDA and NO and on the other hand a significant lowering of GSH and SOD in testiculat tissue. In a previous study (Behairy et al., 2020), authors verified an elevation of MDA and No as well as a decreament of SOD and GSH in rabbits due to injection with boldenone. Furthermore, a significant induction of lipid peroxidation, as well as complete inhibition of antioxidant capacity in testicular tissues in rats (S. S. Ibrahim & Said, 2019).
Physiologically, testis undergoes programmed cell death (apoptosis) to get rid of damaged cells (Turner & Lysiak, 2008). On the other hand, it was reported that there is a significant relationship between apoptosis and male infertility (Chen et al., 2006). Two important molecules are implicated in cell death; Bax (proapoptotic) and Bcl2 (antiapoptotic) and their ratio decides if the cell will experience apoptosis and in addition, caspase3 represents the intrinsic pathway of apoptosis (Arisha et al., 2019). Bcl2 can as well regulate the activity of caspase indirectly through heterodimerization of Bcl2 with Bax to prevent apoptosis (M. A. L. Ibrahim & Salah-Eldin, 2019), so cellular apoptosis is attributed to dysregulation of Bcl2 or Bax (Atici et al., 2004). In the current study, our findings reported that injection of tramadol for 2 months resulted in a significant increase in Bax and a significant decrease in Bcl2 leading to an increase in Bax/Bcl2 ratio. Additionally, tramadol induced caspase3 activity. All these findings indicate the presence of cellular apoptosis in testicular cells. Our results were in agreement with (Koohsari et al., 2020) who recorded an upregulation of Bax and caspase3 and a downregulation of Bcl2. Similar findings were recorded in rat cerebral cortex and lung tissues (Awadalla & Salah-Eldin, 2016). Similarly, the injection of boldenone for 2 months showed a significant dysregulation of apoptotic biomarkers; Bax, Bcl2 and caspase3 as recorded in tramadol injection, indicating the existence of apoptosis. Previous study conducted on rabbits, results revealed that boldenone leads to activation of apoptosis in liver parenchyma and upregulation of caspases activity (Mayada et al., 2015).
The reproductive efficiency of male and female is attributed maily to hypothalamic-pituitary-gonadal axis so any interference in this axis might lead to reduced or complete infertility (Faccio et al., 2013). Exposure of testes to heavy metals or any toxicants can cause damage of the mitochondria and downregulate expression of steroidogenic acute regulatory protein (StAR) and then inhibiting secretion of steroids. In the current study, injection of tramadol for 2 months had significantly reduced the serum levels of FSH, LH and testosterone. Previous study conducted on tramadol showed similar results (Abdellatief et al., 2015). These findings may be due the interference of opioids with the normal release of GnRH at the level of the hypothalamusleading to the decrease of FSH, LH and testerone (Aloisi et al., 2009). Injection of boldenone also for 2 months revealed similar results as those obtaine in groups injected with tramadol showing a significant reduction in FSH, LH and testosterone levels in serum. These results are in agreement with those of Behairy et al (2020) (Behairy et al., 2020). We also reported a significant downregulation of StaR and HSD17B3 expression after injection of both tramadol and boldenone. Reduction of steroid levels as well as downregulation of StaR and HSD17B3 may be attributed to elevated levels of ROS and reduction of antioxidant capacity as SOD, glutathione peroxidase (Yan et al., 2019) and finally leading to down regulation of StaR and HSD17B3 (Arisha et al., 2019). Mixture of tramadol and boldenone had been injected in rats for the first time to scout their additive effect because nowadays both drugs are being concurrently abused specially between youth. We found that the mixture showed significant detrimental effect in most of the parameters as compared to the grous administered the drugs alone.