Busulfan induces oxidative stress- and Bcl-2 family gene-related apoptosis in epididymal sperm and testis of adult male mice

Introduction: Busulfan as a chemotherapeutic agent causes testicular germinal epithelium depletion and cytotoxicity in germ cells. The aim of this study was to assess antioxidant status, reactive oxygen species (ROS) generation and apoptosis-related genetic markers of adult male mouse sperm following busulfan treatment. Materials and Methods: Forty adult NMRI mice (30 ± 5 g) were divided into two groups. Control and busulfan treated group were administered with 100 μL dimethyl sulfoxide and 3.2 mg/kg/day busulfan for 4 days, respectively. The superoxide dismutase and glutathione peroxidase assays were used for analyzing antioxidant status. Then, the levels of Bcl -2 family gene expression, lipid peroxidation and cytotoxicity were evaluated by Real-Time PCR, thiobarbituric and lactate dehydrogenase assays, respectively. Results: The results showed significant decrease on antioxidant status, increase on lipid peroxidation and lactate dehydrogenase in epididymal sperm and testis of busulfan treated mice in comparison with control (P< 0.05). Real Time PCR demonstrated significantly increased-Bax gene expression and decreased-Bcl-2 gene expression in epididymal sperm of treated group (P< 0.05). Conclusion: The high levels of lipid peroxidation and lactate dehydrogenase revealed increased-ROS and severe cytotoxicity in epididymal sperm and testis tissue following busulfan treatment at clinical dose. The oxidative stress and increased-ROS may induce Bcl-2 family gene expression-related apoptosis following busulfan therapy in normal cells. D ow nl oa de d fr om w w w .p hy ph a. ir at 2 0: 29 IR S T o n T hu rs da y O ct ob er 1 3t h 20 16 Busulfan-related oxidative stress in sperm and testis Physiol Pharmacol 19 (2015) 208-215 | 209 marrow hematopoietic cells and human diploid fibroblasts (WI38 cells) (Meng et al., 2003; Probin et al., 2006). Busulfan as a potent reproductive toxic agent induces DNA fragmentation in epididymal or ejaculated sperm that can result in activation of the p53 (a tumor suppressor gene) pathway (Evenson and Wixon, 2006). The p53 pathway is one of the common apoptosis-mediated pathways that is induced by DNA damage following irradiation and chemotherapy (Probin et al., 2007; Nasimi and Roohi, 2012; Vahdati et al., 2015). The reactions/interactions between DNA and Reactive Oxygen Species (ROS) causes oxidative stress which leads to DNA damage (Suriapraba et al., 2012). ROS can act as primary DNA damaging agents (Evenson and Wixon, 2006) and DNA damage is an early marker of apoptosis (Evenson and Wixon, 2006; Nasimi and Roohi, 2012). So, apoptotic events and ROS can increase DNA fragmentation (Evenson and Wixon, 2006). The air pollution is related to sperm DNA damage and poor semen quality that may be due to increased ROS (Evenson and Wixon, 2006). Levels of ROS are negatively associated with the quality of sperm, sperm viability and male fertility (Choudhary et al., 2010). In normal conditions, there are several antioxidant mechanisms in seminal plasma and inside the spermatozoa that are able to inhibit ROS activity, but, abnormal conditions such as smoking and air pollution can increase ROS generation and induce DNA damaged-related apoptosis in spermatozoa (Evenson and Wixon, 2006). Probin et al. (2007) presented busulfanrelated hematopoietic cell senescence via extracellular signalregulated kinase (Erk) and p38 mitogen-activated protein kinase (p38 MAPK). They showed that ROS can contribute to the induction of DNA damage and DNA damage induces senescence via p38 MAPK pathway in normal hematopoietic cell (Meng et al., 2003; Nasimi and Roohi, 2012). ROS are capable of activating various signal transduction pathways including the Erk-p38 MAPK cascade (Torres, 2003; Esposito et al., 2004). Also, the Erk-p38 MAPK cascade mediates senescence (Esposito et al., 2004) and apoptosis (Nasimi and Roohi, 2012). According to the recent studies busulfan can stimulate oxidative stress via GSH depletion and induces thioredoxin reductase inhibition in normal human diploid WI38 fibroblasts (Probin et al., 2006; Probin et al., 2007). Moreover, alterations on expression of Bcl-2 family members that is induced by anticancer drug treatment are potent factors for trigger or simply facilitate apoptosis in tumors (Probin et al., 2006; Nasimi and Roohi, 2012). Apoptosis as a main type of programmed cell death can exert itself via various proteins which are regulated by Bcl-2 family members (Probin et al., 2006; Nasimi and Roohi, 2012). The pro-apoptotic (Bax, Bad, Bid, and Bcl-xs) and anti-apoptotic (Bcl-2, Bcl-xl, and Bcl-w) members of Bcl-2 family can activate or inhibit the apoptotic pathways, respectively (Nasimi and Roohi, 2012; Kontos et al., 2013). On the other hand, the development of chemoprotection is not only for raising the effectiveness of cancer treatment but also for studying the underlying mechanisms of anticancer agentsinduced cytotoxicity (Suriapraba et al., 2012). There are different studies that indicate cytotoxicity and histopathology of chemotherapeutic agents in normal cells (Vahdati et al., 2015). Most of recnt studies used high single doses (including 20, 30 and 40 mg/kg) of these drugs (Mohammad-Ghasemi et al., 2008; MohammadGhasemi et al., 2009). Previous works on studying the adverse effects of busulfan have been done in doses higher than the ones that were clinically used. Based on our knowledge there is not enough information about the effects of busulfan on ROS production and apoptosis geneexpression alteration in sperm and testis of mammalian. Most of recent studies are about histological and cytotoxic effects of busulfan on reproductive tract of male (Mohammad-Ghasemi et al. 2008; Dehghani et al, 2013; Ahar et al., 2014). In the present study, the effect of busulfan on the levels of oxidative stress markers and ROS generation in sperm and testis, and apoptosis-related gene expression (Bcl2 family) in sperm of adult NMRI male mice were analyzed. Materials and methods