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)