Background
Azathioprine (AZA) is an immunomodulatory and cytotoxic drug often used to treat inflammatory bowel disease, autoimmune disorders, organ transplant rejection, and cancer [
1]. It functions via multifaceted pathways; it inhibits purine metabolism, which leads to DNA damage [
2], and at high chemotherapeutic doses inhibits DNA synthesis. By contrast, its anti-inflammatory effect is mainly mediated via inhibition of the small GTPase Rac1, leading to apoptosis of activated T-lymphocytes [
3]. AZA also increases oxidative stress; upon its administration, as it is rapidly metabolized into several toxic and non-toxic metabolic compounds, including the active 6-mercaptopurine (6-MP) that is formed through a conjugation reaction with glutathione (GSH). This leads to the depletion of GSH [
4] and a surge in reactive oxygen species (ROS). 6-MP is further metabolized by xanthine oxidase (XO) to thiouric acid, and this reaction also creates ROS [
5].
Despite their effectiveness, antimetabolic drugs may cause drug-induced toxicity with increased risk of death, even when used at standard doses [
6]. As expected for immunosuppressive drugs, common side effects of AZA treatment in both animals and humans are bone marrow suppression and lymphocyte depletion. However, its active metabolite 6-mercaptopurine (6-MP) damages rapidly dividing cells, such as those in the bone marrow, intestinal epithelium, and reproductive organs of adults [
7,
8]. One of these major drug–related disorders is testicular atrophy and infertility, with genetic polymorphisms of the thiopurine methyltransferase enzyme, which is responsible for thiopurine metabolism, possibly contributing to its mechanism. Population studies have shown that patients with low enzymatic activity have a high risk for severe, potentially fatal toxicities [
9].
The usage of nutraceuticals, amino acids and vitamins as adjunctive therapy to chemotherapeutic agents and drugs with reported toxicity is effective in improving drug safety and reducing toxicities and side effects [
10]. One promising nutraceutical that is commonly used as an adjuvant for chemotherapy is taurine (TAU), which is a sulfur-containing amino acid that does not contribute to protein synthesis and that is traditionally considered as an inert molecule without any reactive groups. It can be obtained either exogenously through dietary source as poultry, beef, pork, seafood, and processed meats or endogenously through biosynthesis from methionine and cysteine precursors. Both sources are important to maintain the physiologic levels of taurine, and either can help to compensate the other in cases of deficiency. Taurine supplementation has been proposed to have beneficial effects in the treatment of epilepsy, heart failure and cystic fibrosis [
11].
Interestingly, TAU has been detected in the testes of humans and has been identified as the major free amino acid of sperm cells and seminal fluid [
12]. It has been localized to Leydig cells of the testes, the cellular source of testosterone in males, and to the cremaster muscle, efferent ducts, and peritubular myoid cells surrounding seminiferous tubules [
13]. The major function of taurine in leukocytes, neutophile and any inflammed cell is to trap chlorinated oxidants (HOCl) and convert them into less toxic taurine chloramine (TAU-Cl) and TAU-Cl production is found to result in decreased NO production [
14].
It has become increasingly apparent that oxidative stress plays a major role in a broad range of human diseases and in many diseases including the destruction of male rat reproductive system. By virtue of its antioxidant activity, TAU-Cl also plays a crucial role as a cytoprotectant and attenuates apoptosis in several inflammatory and chronic diseases [
15]. There is growing consensus that the beneficial effects of TAU-Cl are due to its antioxidant properties, aided by its ability to improve mitochondrial function by stabilizing the electron transport chain and inhibiting ROS generation [
16].
Taurine levels in spermatozoa are also correlated with sperm quality, presumably due to its antioxidant activity that protects against lipid peroxidation and its effect on spermatozoa maturation by facilitating the capacitation, motility, and acrosomal reaction of sperm [
17,
18]. To this end, this study aimed to elucidate the toxic effect of AZA on the testicular functions in male rats and unravel the potential protective effects of TAU-Cl combination with AZA, focusing on the induced inflammation, oxidative perturbations and apoptosis.
Discussion
The clinical use of AZA as an immunosuppressant and chemotherapeutic agent has been associated with various organ toxicities, however, reports on its safety regarding testicular functions are scarce. The current study highlights the gonadotoxicity of AZA, accompanied by the induction of inflammation, oxidative stress and apoptosis, and the efficacy of TAU-CL in providing testicular protection.
Spermatogenesis is highly susceptible to testicular inflammation, which causes damage to the seminiferous epithelium and increases apoptosis of spermatogenic cells. The effect of inflammation on spermatogenesis is evidenced by the histological results in this study, suggesting a direct association between AZA treatment and testicular inflammation, as shown by elevated testicular IL1-ß and TNF-α levels. This association is supported by the study of Ramonda et al. (2014) [
29], who reported an increase in semen TNF-α levels associated with reduced sperm count, reduced motility, and altered morphology. These disturbances were successfully abrogated by TAU, as shown by Ahmed (2015) [
30], who attributed these corrective effects to the anti-inflammatory, anti-apoptotic (through the intrinsic apoptosis pathway), and steroidogenic effects of TAU. The anti-inflammatory action of TAU-CL, evidenced in the current study, confirms the previous findings of Latchoumycandane et al. (2015) [
31], who reported that TAU is effective as an anti-inflammatory supplement when given to alleviate the inflammation induced in the kidney by chronic ethanol ingestion.
Increased inflammation is associated with increased oxidative stress, which itself impairs sperm function [
32]. Indeed, inflammatory damage to the male genital tract leads to the increased generation of ROS. Superoxide, hydroxyl, and hydrogen hydroxide radicals are the major ROS present in seminal plasma [
33], and we chose to study the levels of the main free radical scavengers- SOD,CAT, and GST in this study.
We found that AZA treatment for 4 weeks significantly increased MDA levels and reduced GSH, SOD, and CAT levels; these decreases were corrected by TAU-CL pre-treatment. There is a positive correlation between abnormal and immature sperm and oxidative stress [
34]. Consistent with these findings, we showed that the percentage of abnormal, immature, immotile, and dead sperm was significantly increased in the AZA group, where levels of SOD and CAT were reduced [
35]. As ROS are known to promote apoptosis [
36], it could be that this is the mechanism resulting in decreased sperm count and viability.
In keeping with the reported ability of TAU to scavenge ROS, and attenuate lipid peroxidation [
15,
37], we found that pre-treatment with TAU-CL protected against the effects of AZA: restoring levels of oxidative stress markers and free radical scavengers to normal levels, and preventing the damage caused to sperm count and motility. The antioxidant effects observed with TAU-CL pre-treatment in this study may be associated with its sulfur moiety, and the modulation of GSH, SOD, and GSH levels by TAU is critical in the cellular defense against oxidative stress. As reported by Kim and Cha (2014) [
15], taurine undergoes halogenation in phagocytes upon inflammation, and is converted to taurine bromamine and taurine chloramine (TAU-CL). The latter is released from activated apoptotic neutrophils and suppresses inflammatory mediators such as, superoxide anion, NO, TNF-α, and interleukins, and prostaglandins in inflammatory cells. Moreover, TAU-CL stimulates the expressions of antioxidant proteins (heme oxygenase 1, peroxiredoxin, thioredoxin), and enzymes (glutathione peroxidase, and catalase) in macrophages. Therefore, a vital anti-inflammatory and cytoprotective role is exerted by TAU-CL is exerted to protect macrophages and surrounding tissues from the toxic deleterious effect of overproduced reactive oxygen metabolites during inflammation [
15]. Consistent with the antioxidant properties of TAU-CL observed in the present study, Zhang et al. (2014) [
38] also found a protective role of TAU in hepatocytes subjected to iron overload as an inducer of oxidative stress.
In the present study, we decided to measure caspase-9, a protease upstream of caspase-3 and a downstream effector of the Bcl-2-mediated mitochondrial apoptosis pathway, indicating an increase in apoptotic activity in testicular tissue. Bcl-2 is a multidomain, prosurvival protein that regulates apoptosis by preventing the release of proapoptogenic factors from the mitochondria (e.g., cytochrome c) and subsequent caspase activation. In our study, AZA treatment for 4 weeks induced the activation of caspase-9 and significantly reduced levels of the anti-apoptotic protein Bcl-2. This dysregulation of the fine-tuned apoptosis pathway is considered one of the mechanisms of AZA-induced damage to testicular function and might be a reason for decreased sperm viability and mobility.
In agreement with our findings, Xu et al. (2016) [
39] documented an increase in the activity of Bcl-2/Caspase-9 apoptosis pathway in the testis of asthmatic mice subjected to apoptotic inducers like ovalbumin. The protective role of TAU-CL was highlighted by the changes in caspase-9 activity and Bcl-2 levels toward the control values, which –based on our histopathological analysis – appears to have restored normal numbers of germ cells during spermatogenesis.
This putative anti-apoptotic effect of TAU-CL is supported by a report that suggests TAU inhibits apoptosis by preventing the formation of the Apaf-1/caspase-9 apoptosome [
40]. In addition, Zulli et al. (2009) [
41] reported an anti-apoptotic effect of TAU, which encourages the use of TAU as a dietary supplement in conditions where there are aberrant or inappropriate levels of apoptosis. A sensitive and indispensable method for revealing disturbances in spermatogenesis is histopathological examination. Our histological results revealed widening of the interstitial spaces and disruption and atrophy of many STs with reduced numbers of spermatogenic cells and spermatozoa in the AZA-treated group.
These results agree with those of Akinloye et al. (2011) [
42], Nouri et al. (2009) [
43] and Padmanabhan et al. (2009) [
44], who reported that methotrexate caused significant increase in interstitial tissue and capsular thickness and decrease of testicular and body weight. Moreover, it caused significant decline in seminiferous tubule diameter and epithelium thickness, while cytoplasmic vacuolations were also reported by Karawya and El-Nahas (2006) [
45].
The current study showed a decrease in the diameter of the STs, which was confirmed by morphometrical studies; this finding agrees with those of Shrestha et al. (2007) [
46] and Khayatnour et al. (2011) [
47] who reported that 60 days of methotrexate administration intraperitoneally (ip), decreased diameter of seminiferous tubules, increased interstial spaces as well as distortion of morphology of Leydig cells in experimental group. Many cells appeared shrunken with pyknotic nuclei and deeply acidophilic cytoplasm, which are hallmarks of apoptosis [
48,
49]. AZA-induced reproductive dysfunction was confirmed by histomorphometrical analyses of rat testicular tissue; AZA significantly decreased the diameter and epithelial height of due to cell loss from the epithelium, epithelial sloughing in some tubules, and Leydig cell atrophy. Moreover, the results of this study showed a decrease in RI, TDI, and SPI, as presented in (Fig.
3b). As testosterone is essential for spermatogenesis, sperm maturation and motility any disruption in testosterone biosynthesis can adversely affect the structure and function of the testis [
50].
Oral AZA administration in male rats caused a significant decrease in body and testicular weight and reduced serum testosterone, LH, and FSH levels, which we believe may be attributed to the oxidative stress [
51] induced by this drug or decrease in the anabolic effects of testosterone [
52,
53]. This hypothesis is consistent with the data reported by Duan et al. (2017) [
54], who found a marked decline in serum FSH and LH levels and increased. In another study of El-Sharaky et al. (2010) [
55], the use of a Leydig cell toxicant, Gossypol acetic acid (GAA), resulted in decreased testosterone levels in rats, resulting in increased germ cell apoptosis. In addition, testosterone can affect Sertoli cell function and germinal cell degeneration; thus, premature detachment of spermatids could occur due to Sertoli cell dysfunction and decreased testosterone levels, and low testosterone levels can enhance the premature detachment of epithelial cells [
48,
56].
Leydig cell atrophy can be responsible for the reduction in serum testosterone levels. Therefore, changes in the seminiferous tubules, which were observed in the current histopathological study, may result from hormonal alterations induced by AZA and may not be a direct effect of the drug. Considering that normal spermatogenesis is adversely affected by increased oxidative stress and is promoted by increased endocrine activity in Leydig and Sertoli cells, it was not surprising that TAU was shown to protect spermatogenesis, decrease tubular atrophy and improve testicular and body weights [
57]. This may be partly due to the reduction in oxidative stress indicators [
17,
18], apoptotic markers and improved testosterone levels observed in this study [
58].
Results of our study demonstrated significant increase of inducible nitric oxide synthase (iNOS) and nuclear factor-ƘB-p65 (NF-ƘB-p65) expression in AZA-treated group. In parallel to the present data Ilbey et al.(2009) [
59] study has shown that cytotoxic chemotherapy species activate the nuclear factor-kB (NF-kB) family of transcription factors mediating inflammation and testicular damage.
The transcription factor NF-ƘB helps to control the expression of numerous genes activated during inflammation (i.e. cytokines, chemokines, growth factors, immune receptors, cellular ligands, inducible nitric oxide synthase [iNOS] and adhesion molecules). iNOS; is a protein that produces high amounts of nitric oxide (NO), and NO is highly reactive with other free radicals. Nitric oxide reacts with superoxide (O
−) producing peroxynitrite, which in turn leads to protein nitration, DNA damage, and poly (ADP-ri- bose) polymerase activation. NF-ƘB can also be activated by oxidative stress [
60].
Interestingly, we observed that TAU-CL-pretreatment exerted an inhibitory and downregulating effect on iNOS and NF-ƘB- p65 expression, thereby moderating the consequences of inflammation. In consistence with our findings,
Aydos et al.
(2014) [
61] demonstrated the downregulating effect of taurine treatment on NO level and nitric oxide synthase expression in ischemia/reperfusion-induced testicular injury in a rat.
TAU-CL has shown to provide cytoprotection against AZA-induced testecular injury by inhibiting the overproduction of inflammatory mediators by the virtue of its anti-oxidative properties. As a direct antioxidant, taurine would quench and detoxify some reactive intermediates such as hypochlorous acid generated by myeloperoxidase. As an indirect antioxidant, taurine may protect cells via intercalating into the membrane and stabilizing it. Molecular gonadal protective effect of TAU was also documented by controlling the regulation of testicular-ERK1/2, -p38, -AKT and NF-ƘB [
62], thereby moderating the consequences of inflammation.
Furthermore, the current study highlights that RT-PCR mRNA expression levels of Nrf2 and HO-1 were markedly decreased in AZA-treated group. Importantly, our results showed that pretreatment with taurine-chloramine significantly attenuated AZA-induced downregulation of Nrf2 and HO-1 expression. These findings are consistent with Yang et al. (2017) [
63] who demonstrated the modulatory effect of taurine on Nrf2 and HO-1 expression levels against irradiation induced testicular damage in mice.
A plethora of studies suggest that in mammalian cells Nrf2 plays an important role to maintain normal cellular physiological conditions under exogenous oxidative insult by regulating the minimal and induced expression of several antioxidants molecules, enzymes including HO-1 and xenobiotic transporters [
64], upregulation of HO-1expression through stimulation of Nrf2, conferring protection against oxidative damage and organ dysfunction [
65]. Taurine-chloramine could therefore protect testes from Azathioprine (Imuran)-induced testicular damage and such process was relevant with Nrf2/HO-1 antioxidant pathway activation. These results were supported by the histopathological findings of a remarkably larger seminiferous tubules diameter and greater germinal epithelium height in testes from the TAU/AZA group compared to the AZA group.
Taurine-chloramine improved not only the morphological and histomorphometrical damage but also the apoptotic cell number and morphology which confirms the ability of TAU-CL to protect against the toxic effects of AZA in rats. Previous studies have reported that TAU treatment significantly prevented histomorphological damage and decreased the number of apoptotic cells in a rat model of diabetes-induced testicular dysfunction by suppressing the increase in oxidative stress [
57,
59,
60].
As for the translation to medical care in oncologic diseases, the implication of TAU in clinical settings has proved effectiveness in reducing chemotherapy –induced toxicities. By virtue of its antioxidant impact, several studies pointed out that TAU co-administration decreases the risk of chemotherapy –induced hepatotoxicity, nephrotoxicity and also increases WBC count in patients with acute lymphocytic leukemia during their maintenance therapy [
66]. It can also attenuate chemotherapy complications; e.g. nausea and vomiting, taste and smell impairment [
67] as well as febrile episodes [
68] and leads to a more tolerable chemotherapy with lower incidence of adverse drug events for the patients. In the field of chemotherapy supportive care, our results add to these reported benefits of TAU a promising protective effect against AZA induced male testicular atrophy, which can be experimented in a pilot study with a focus on testicular functions.