Discussion
Acrylamide is one of the most important agents that attracted considerable attention of the scientific community and general public due to its extensive presence in food and variety of applications (Kuorwel, Lumori, & Andrew, 2018). Mammalian studies have provided a lot of evidence that acrylamide induced a range of reproductive effects in males including disruption of reproductive development, alteration of steroid hormone balance, testicular lesions and atrophy, disruption of spermatogenesis as well as infertility(Xie et al., 2017).
In the present study, repeated administration of acrylamide to adult rats caused reduction in the relative testicular weight and marked deterioration of the histological tissue architecture. As regards sperm morphology, acrylamide apparently decreased sperm count and motility meanwhile it increased significantly the incidence of sperm head and tail abnormalities. Though the mechanisms whereby acrylamide induced such toxic effects on male rat reproductive system, an array of mechanistic approaches have been postulated. Acrylamide effect on rodent reproductive performance was discussed earlier in a review byTyl and Friedman(Tyl & Friedman, 2003). It was concluded that acrylamide may induce such toxicity through its metabolite; glycidamide binding to spermatid protamines, causing dominant lethality and effects on sperm morphology; and acrylamide binding to motor proteins, causing distal axonopathy, including hindlimb weakness/paresis, and effects on mounting, sperm motility, and intromission (Aras, Cakar, Ozkavukcu, Can, & Cinar, 2017).
It is known that sperm motility is ultimately related to healthy mitochondria and therefore, mitochondrial damage might result in reduction of sperm movement. This mitochondrial inhibitory potential provides a possible explanation for poor sperm motility in rats exposed to acrylamide (Mu et al., 2017).
Indeed several recent studies indicated that flavonoids had the protective property against acrylamide-induced oxidative stress and cell apoptosis in vivo and in vitro(Zhang et al., 2017) and(He et al., 2017). However, the protective effects of functional food and nutraceuticals on acrylamide-induced toxicity merit further investigation. TQ administered to acrylamide-challenged rats improved sperm morphology as manifested by increased sperm count and motility. It also increased the relative testes weight and decreased sperm head and tail abnormalities. Histological alterations induced in testicular tissues were also abrogated by the polyphenolic compound. Similar results were reported for TQ following other toxic chemicals.
Generally speaking, it is important to say that many studies elucidated the protective effects of TQ against the reproductive toxicities induced by different agents; such as diesel exhaust particles (Tavakkoli et al., 2017) and streptozotocin (Atta et al., 2018). TQ was able to ameliorate the deleterious effects of cadmium chloride on sperm motility, count and abnormalities in rats (Sayed, Hassanein, & Senosy, 2014).Mabrouk and Ben Cheikh (Mabrouk & Ben Cheikh, 2016) reported that TQ improved spermatogenic function by increasing epididymal sperm count in rats exposed to lead acetate. TQ also significantly boosted motility, morphology, count, viability of sperm cells, germinal thickness in morphine-treated mice (Salahshoor, Haghjoo, Roshankhah, Makalani, & Jalili, 2018).
In our results acrylamide group co-treated with capsaicin shows increase in sperms count and motility and decrease in sperm head and tail abnormalities , this is paralyzed with a previous study which shows that in cocks fed with a diet containing 1% red hot pepper (10 g/kg diet), their body weight gain decreased, whereas the testes weight, length, width and wall thickness of tubules seminiferous contortus increased, and the completion of spermatogenic cell serial formation took place earlier when compared to control group (Özer, Zik, Erdost, & ÖZFİLİZ, 2006).
Capsaicin tested as a second nutraceutical in the present work has shown remarkable protective effects on both spermatogenic and steroidogeneic functions. It increased sperm count and motility and further decreased sperm head and tail abnormalities. Also, the histopathological alterations induced by acrylamide were mitigated. Similar results were previously reported. Park et al. (Park et al., 2017) documented that the pungent principle or red pepper; capsaicin was able to protect against testicular injuries induced by transient scrotal hyperthermia. Low testicular weight, severe vacuolization of seminiferous tubules followed by loss of spermatogenic cells, and appearance of multinucleated giant cells were all mitigated.
Acrylamide can affect sperm parameters as well as sperm chromatin condensation and DNA integrity in mice. These abnormalities may be related to the reduction in blood testosterone. (Pourentezari et al., 2014). Interestingly, acrylamide notably decreased the serum levels of sex hormones; testosterone, LH and FSH. This coping with many previous studies; that documented deterioration in the male sex hormones following exposure to the endocrine disruptor; acrylamide (Erdemli et al., 2019). FSH, LH, and testosterone are known to regulate and sustain testicular function. The main function of FSH is to regulate and promote the spermatogenesis in males. Needless to say that testosterone is the critical hormone which maintains spermatogenesis in the testis (Xiao, Nabi, Yang, Hao, & Wang, 2018). Evidence for the critical role of the LH-testosterone signaling pathway in initiating and maintaining spermatogenesis has been obtained from several animal models and experimental approaches (O’Donnell, Meachem, Stanton, & McLachlan, 2006). LH/testosterone and FSH are the pivotal endocrine factors controlling testicular functions and they are crucial for spermatogenesis (Ramaswamy & Weinbauer, 2014).
TQ did not only improve the spermatogenic function but also boosted the steroidogenic sex hormones. It almost restored testosterone, FSH and LH serum levels to baseline. Similar findings were documented for TQ in an array of testicular toxicity models. TQ significantly attenuated cadmium- and lead-induced decreases in serum testosterone in rats (Fouad, Albuali, & Jresat, 2014) and(Mabrouk & Ben Cheikh, 2016).TQ ameliorated testicular tissue inflammation and restored the normal balance of sex hormones; testosterone, LH and FSH induced by sodium nitrite both in vivo and in vitro(Alyoussef & Al-Gayyar, 2016).Aithal et al.(Aithal, Haseena, Das, & Saheb, 2016) also showed that TQ increased the serum testosterone levels in arsenic-induced and sterptozotocine-induced testicular toxicity in male rats.
Capsaicin significantly increased the serum levels of testosterone, FSH and LH. Capsaicin appears to enhance testicular cell proliferation and can affect the release of ghrelin and testosterone directly or indirectly. It was also suggested that capsaicin-sensitive nerves contribute both to the regulation of blood content of dehydroepiandrosterone; testosterone precursor under normal and fructose-induced metabolic syndrome (Spiridonov, Tolochko, Ovsyukova, Kostina, & Obut, 2017).
Also our results showed increase in FSH and LH levels in capsaicin (10mg/kg) co- treated acrylamide groups compared to acrylamide alone (35mg/kg) trteated groups and this agreed with the results ofErdost et al. (Erdost, Ozer, Yakisik, Ozfiliz, & Zik, 2006) who pointed out that the number of FSH and LH immune-reactive cells increased in the unit area of the hypophysis when red hot pepper was added to chicken diets.
Acrylamide induced marked decrease in the testicular activity of LHD-X. LDH-X, a pachytene spermatocyte marker enzyme in the testis, is widely present in sertoli and spermatogenic cells, and plays an important role in testicular energy production and can be used as a marker in evaluating the function of spermatogenic cells. Odet et al. (Odet et al., 2011) hypothesized that in addition to its role in glycolysis, LDH-X is part of a complex involved in ATP homeostasis that is disrupted in sperms lacking LDH-X. LDH-X is a special enzyme produced at the phase of primary spermatogenic cells (Wu et al., 2017). The inhibition of LDH and LDH-X activities may induce denaturalization of spermatogenic cells (Ahmed, 2015). LDH-X has been suggested to be an index of testicular toxicity following exposure to different testicular toxicants (Adedara et al., 2017).
The correlation between disrupted LDH-X activity and sperm motility has been earlier studied by Odet et al.(Odet et al., 2011).Abd-Ellah et al.(Abd-Ellah, Aly, Mokhlis, & Abdel-Aziz, 2016) reported a positive correlation between LDH-X activity and sperm count. In addition, the study clarified that the decrease in testicular LDH-X activity in rat may be due to greater loss of germ cells from the testis, followed by their passage into epididymis. Decreased LDH-X level in the rats from Day 15 and Day 19 groups may be a consequence of enhanced lipid peroxidation after exposure to acrylamide, which may be due to fragmentation of the mitochondrial membrane ultra-structure that in turn affects the membrane bound LDH-X function.
In our recent experiment; pre-administration of TQ (15mg/kg) before acrylamide (35 mg/kg) attenuated the acrylamide-induced decrease in the testicular LDH-X activity, Which come in parallel with Mabrouk et al . (Mabrouk, Salah, Chaieb, & Cheikh, 2016) findings. Also our results show that co-administration of capsaicin (10mg/kg) with acrylamide (35mg/kg) attenuated acrylamide-induced decrease in the testicular LDH-X activity.
The finding in the current study that acrylamide induced oxidant stress in testicular tissue by increasing MDA and reducing GSH levels as well as the decreases in SOD and CAT activities may be one of the forerunners of such sperm morphological defects. The finding by Sun et al. (Sun, Wang, Gupta, & Rosen, 2018)that exposure to acrylamide and its metabolite; glycidamide increased ROS level and decreased mitochondrial membrane potential, might lend support to this issue. Consistent with that was the finding byShi et al. (Shi et al., 2018) that the liberated lipid peroxides destroy the structure of lipid matrix in the membranes of spermatozoa leading to loss of motility and impairment of spermatogenesis and decreased sperm count.
Also, the administration of acrylamide resulted in significant elevation in testicular and epididymal MDA and significant reduction in the level of GSH and the activities of glutathione-S-transferase (GST), glutathione peroxidase (GPX) and glutathione reductase (GR) (Lebda, Gad, & Gaafar, 2014). In a recent study conducted by Erdemli et al.(Erdemli et al., 2019), offspring male rats previously exposed in utero to acrylamide exhibited marked testicular oxidative stress as shown from the deterioration in the activities of CAT and SOD and the contents of GSH and MDA.
The protective effects of TQ on both spermatogenesis and steroidogenesis may reside at least in part on its antioxidant effects observed in the present work and elsewhere in a plethora of previous studies. In the current study, TQ significantly increased testicular GSH content and CAT and SOD activities while it reduced lipid peroxidation in testicular tissue. TQ significantly attenuated cadmium-induced decreases in serum testosterone, and testicular GSH and SOD activity and significantly decreased the elevation in testicular MDA (Fouad et al., 2014). TQ was also found to increase total anti-oxidant capacity with concomitant reduction in testicular lipid peroxidation following testis reperfusion injury in rats (Erol et al., 2017).
Javdan et al. (Javdan, Ayatollahi, Iqbal Choudhary, Al‐Hasani, & Pazoki‐Toroudi, 2018)studied the role of capsaicin in tissue damage after testicular torsion. Testicular torsion-related oxidative stress causes a sequential chain of DNA damage, lipid peroxidation and cell death that leads to the derangement in the sperm functions and infertility. Capsaicin improved testicular morphology and decreased apoptosis in testes by targeting Forkhead Box O1 (FOXO1) gene and apoptotic pathways. Capsaicin attenuated spermatogenic cell death induced by scrotal hyperthermia through its antioxidative effects as shown by the diminished MDA level in testicular tissue (Park et al., 2017). Very recently, it was reported that combined treatment of capsaicin and curcumin improved significantly the oxidant/anti-oxidant status in male Sprague-Dawley rats fed a high fat diet. The combo increased significantly the activities of glutathione transferase, Cu-Zn SOD, glutathione peroxidase and CAT, but decreased TBARS and ROS levels in liver and testicular tissues (Tanrıkulu-Küçük et al., 2019).
Immunohistochemical localization of NF-κB/p65 in testicular tissues after challenging animals with acrylamide revealed marked immunoreactivity for the protein denoting its marked translation in testicular tissue. This is the first finding to date that acrylamide would dramatically activate the NF-κB/p65 pathway in testicular tissue. Nuclear factor-kappa B is a family of transcription factors implicated in numerous stress responses including apoptosis within male testicular cells (Baldwin Jr, 1996).Pentikainen et al. (Pentikäinen et al., 2002) demonstrated that under serum free conditions, an excessive amount of apoptotic activity was seen in human seminiferous tubules, concomitant with increased amounts of NF-κB activity.
Another plausible mechanism for the toxic effects of acrylamide on spermatogenesis could be explained by virtue of its endocrine effects. As we know, FSH plays an important role in the process of spermatogenesis. Binding of FSH to its receptors on Sertoli cells leads to activation of adenylyl cyclase and subsequently the production of cAMP. Through this pathway, FSH indirectly leads to the activation of protein kinase A (PKA), which turns out to be a regulator of NF-κB (KANGASNIEMI et al., 1990). Increased level of PKA causes an increase in NF-κB binding activity (Delfino & Walker, 1998); the way in which PKA controls NF-κB is through phosphorylation of IκB, which leads to its degradation (Ghosh & Baltimore, 1990). So, by decreasing FSH production, acrylamide would probably inhibit the PKA-mediated degradation of NF-κB, and this would ultimately lead to its superfluous localization in testicular tissue observed in the present work.
Interestingly, TQ notably decreased the expression of testicular NF-κB/p65. Similar previous results were reported in other testicular toxicities. Fouad et al.(Fouad et al., 2014) have speculated that the protective effect of the TQ in arsenic-induced testicular injury in rats is ascribed to its modulatory effect on NF-κB. The downregulatory effects of TQ on NF-κB/p65 were also reported in an array of other toxidromes including cisplatin-induced nephropathy (Al-Malki & Sayed, 2014),experimental diabetes (Usta & Dede, 2017) and Freund’s Complete Adjuvant-induced arthritis in rats (Arjumand, Shahzad, Shabbir, & Yousaf, 2019).
Immunohistochemical localization of NF-κB revealed partial, albeit significant decrease in the expression of the inflammatory marker following capsaicin-acrylamide group compared to the animals that received acrylamide alone. Such finding is unique since no previous studies addressed this issue in testicular tissues before. Indeed, many other reports demonstrated the downregulatory effects of capsaicin on NF-κB in other biological systems. Capsaicin is a quinone that has been shown to regulate a wide variety of activities that require NF-kappa B activation. An earlier study by Singh et al. (Singh, Natarajan, & Aggarwal, 1996)examined the effect of capsaicin and its analogue, resiniferatoxin, on the activation of NF-kappa B induced by different agents including TNF. Capsaicin treatment of cells blocked the degradation of I kappa B alpha, and thus the nuclear translocation of the p65 subunit of NF-kappa B, which is essential for NF-kappa B activation.
Of major interest in the current study was the finding that acrylamide perturbed the basement membrane of seminiferous tubules by downregulating occludin; one of its major junctional proteins. Occludin expresses in Sertoli cells, together with claudins, serving as a key component of tight junctions in the blood testes barrier (Morrow, Mruk, Cheng, & Hess, 2010). It was found that the deletion or functional silencing of genes encoding tight junction proteins, to which belongs occludin, may disrupt the blood testes barrier (BTB), which may cause immunological or other damages to meiotic and postmeiotic cells and ultimately lead to spermatogenic arrest and infertility (Jiang et al., 2014).
Ablating occludin in vitro led to a quantitatively significant decrease in tight junctin function. Decreases in tight junction adhesiveness have also been observed when silencing occludin in keratinocytes (Rachow et al., 2013). Thus, recalling that acrylamide significantly decreased serum testosterone level might explain its downregulatory effect on the junctional protein in the basement membrane of seminiferous tubules.
The unique finding in the current study that TQ upregulated occludin in testicular tissue as shown from the increased immunofluorescent reactivity could possibly be explained by virtue of its downregulatory effect on NF-κB/p65. The earlier finding by Wachtel et al. (Wachtel et al., 2001) that the down-regulation of occludin expression in astrocytes by tumor necrosis factor (TNF) is mediated via TNF type-1 receptor and NF-κB/p65 activation might lend support to this view. This again would support the negative feedback of NF-κB/p65 on the expression of the tight junction protein.
One of the outstanding results of the current study was the ability of capsaicin to upregulate the expression of the tight junctional protein; occludin in testicular tissue; to date, no similar results were reported. In a study conducted by Janyou et al.(Janyou et al., 2017), the authors investigated the effect of dihydrocapsaicin (DHC) on cerebral and blood brain barrier (BBB) damage in cerebral ischemia and reperfusion (I/R) models. Capsaicin increased the expression of tight junction proteins and significantly decreased oxidative stress and inflammation via down-regulation of reactive oxygen species (ROS), NADPH oxidase and NF-κB/p65.
The histopathological results in the present work confirmed the gained biochemical findings. The testicular degeneration and inflammation associated with acrylamide treatment were greatly improved with TQ (15mg/kg) and capsaicin intake (10mg/kg) in comparison with acrylamide alone challenged group. acrylamide groups which are pretreated with TQ (15mg/kg) and capsaicin (10mg/kg) showed marked improvement in testicular tissue. Seminiferous tubules showed mild vacuolization in seminiferous epithelium and germ cell and slight inter-tubular edema and congestion compared to acrylamide alone (35mg/kg) treated group which shows massive edema ,necrosis , vacuolation , degeneration of spermatogonial cells and severly congested blood vessels of seminiferous tubules. Yang et al. (Yang et al., 2005) reported that acrylamide induced histopathological lesions, such as formation of multinucleated giant cells and vacuolation associated with numerous apoptotic cells in seminiferous tubules, and such lesions appeared to increase Leydig cell death and perturb gene expression levels, contributing to sperm defects. Likewise, epididymal sperm reserves decreased significantly following oral exposure to acrylamide of weaned male Sprague-Dawley rats suggesting partial depletion of germ cells. In addition, histopathologic lesions were also present in the testes of treated rats (Wang et al., 2010).Tüfek et al.(Tüfek, Altunkaynak, Altunkaynak, & Kaplan, 2015) demonstrated beneficial effects of TQ on mean volumes of testes and seminiferous tubules, the number of spermatogenic cells and also Leydig cells in rats following feeding a high-fat diet. Also, the improvement of histopathological abnormalities produced by pretreatment of acrylamide by capsaicin is in accordance with the results of Sarioglu‐Buke et al.(Sarioglu‐Buke, Erdem, Gedikoglu, Bingol‐Kologlu, & Tanyel, 2001)