Introduction

Acrylamide (ACR, CH2 = CH-CONH2), classified as a group 2 A human carcinogen, is extensively used in the production of paper, plastics, tobacco, and wastewater treatment, including sewage [1, 2]. The Swedish National Food Administration (SNFA) states that significant amounts of ACR are released when certain foods like cereal products and potatoes are cooked above 120 °C [3]. Human exposure to ACR can occur through skin absorption, inhalation, or ingestion [4]. Upon consumption, ACR is primarily absorbed in the gastrointestinal tract and distributed to peripheral tissues via the bloodstream [5]. Additionally, ACR can undergo conjugation with N-acetyl-S-(3-amino-3-oxopropyl)-cysteine by glutathione S-transferase (GST) [6], and the cytochrome P450 enzyme complex (CYP450) converts ACR to glycidamide (GA) [7]. ACR exposure has been associated with neurotoxicity, genotoxicity, carcinogenicity, hepatotoxicity, immunotoxicity, and reproductive toxicity [8,9,10,11,12,13,14].

Neurotoxicity is the most prominent consequence of ACR exposure [15], characterized by symptoms such as skeletal muscle weakness, ataxia, and axon degeneration in both the central and peripheral nervous systems, even crossing the blood-brain barrier (BBB) [16, 17]. ACR can traverse the blood-placental barrier in the human placenta and the blood-breast milk barrier in nursing mothers [18]. It has been observed that ACR can breach the blood-testis barrier (BTB) within 1 h of oral administration in mice [19].

In rodents, a single dose of ACR ranging between 100 and 200 mg/kg is considered lethal, while repeated low-concentration exposures of 10 to 50 mg/kg can induce neuropathy [20]. ACR can induce liver and kidney toxicity through the PI3K/AKT/mTOR pathway and inflammatory factors like nuclear factor kappa B (NF-κB) and tumor necrosis factor-alpha (TNF-α) [21, 22]. ACR-induced hepatotoxicity may be indicated by increased levels of liver enzymes (AST, ALT, and ALK) [23]. In mouse bone marrow cells, ACR and its major metabolite, glycidamide, have been found to cause DNA damage, chromosomal aberrations, and micronuclei [24].

The metabolism of ACR by CYP450 E1 can generate reactive oxygen species (ROS) in reproductive organs, including the testis and ovary, leading to lipid peroxidation and cell death [20, 25,26,27,28]. However, research on the effects of ACR on the reproductive system has mainly concentrated on male subjects, resulting in a relative scarcity of data regarding its potential impacts on the female reproductive system [8]. Previous studies have indicated no significant increase in cancer mortality among industrial workers with prolonged exposure to high levels of ACR [29, 30], but a positive association has been observed between ACR exposure and reproductive cancers, such as endometrial and ovarian tumors, as well as estrogen receptor-positive breast cancer in postmenopausal women [31, 32]. Exposure to ACR has been linked to alterations in levels of various hormones involved in reproductive processes [33]. In one study conducted by Nagata and colleagues, which examined preschool-aged children exposed to ACR at an estimated intake level of 1 µg/kg/day, increased urinary levels of androstenediol, dehydroepiandrosterone (DHEA), testosterone (T), estrone (E1), and estradiol (E2) were observed. A positive correlation was found between urinary levels of T and androstenediol in boys, while in girls, no significant associations between ACR intake and hormone levels were found [34]. In another study, premenopausal Japanese women with the highest acrylamide intake showed significantly lower levels of total and free E2 (18.2% and 19.3% lower, respectively) and higher levels of follicle-stimulating hormone (FSH) (23.5% higher). However, there were no notable changes in plasma levels of dehydroepiandrosterone sulfate (DHEAS), luteinizing hormone (LH), prolactin, and sex hormone-binding globulin (SHBG) [35].

Interestingly, male animal studies have yielded complex and contradictory findings regarding the effects of ACR on reproductive hormones. For instance, one study demonstrated that a three-week administration of ACR (5 mg/kg) increased T levels in rats without affecting FSH or LH levels [36]. In contrast, another study found that ACR (10 mg/kg, administered for 20 days during pregnancy) decreased serum T, FSH, and LH levels in adult male rats [37]. Although the understanding of ACR’s impact on human spermatozoa is limited, the majority of animal studies consistently report detrimental effects of ACR on sperm parameters, including count, motility, morphology, and quality [36, 38,39,40,41,42,43,44,45]. This review primarily focuses on the effects of ACR on testicular and sperm function in animal models and explores the potential benefits of using antioxidants to mitigate ACR-induced damage.

Oxidative stress in human spermatozoa and testicles

Oxidative stress, triggered by an excess of ROS and reactive nitrogen species (RNS), significantly impairs the functionality of the male reproductive system, leading to a condition known as Male Oxidative Stress Infertility (MOSI) [46]. The dysfunction of male reproductive organs is closely associated with oxidative/nitrosative stress, as indicated by the presence of biochemical markers of damage [47, 48]. While a certain concentration of endogenous ROS is necessary for normal spermatozoa function during events like sperm-oocyte fusion, acrosome reaction, and capacitation, an excessive accumulation of ROS is a common pathological occurrence in the semen of infertile men [49]. Prolonged exposure to ROS in semen samples irreversibly damages various macromolecules, including polyunsaturated fatty acids in cell membranes, proteins, and nucleic acids, thereby impairing the functionality of germ cells, which are highly sensitive to elevated levels of free radicals [47, 50].

Several factors contribute to the high production of ROS. These include the development and proliferation of abnormal and immature spermatozoa [51], invasion of white blood cells [52], and defects in the scavenger/antioxidant system of the seminal fluid [53]. In the presence of triggers such as inflammation and infection, leukocytes in the seminal fluid can become activated and produce large amounts of ROS, often reaching concentrations up to 100 times higher than those found in sperm [54]. Activation of macrophages and polymorphonuclear neutrophils (PMNs) increases the production of ROS through a burst of respiratory activity and the generation of NADPH via the hexose monophosphate shunt [55]. In vitro sperm processing techniques, such as freeze/thaw procedures, excessive centrifugation, and low antioxidant activity in culture media, can also induce ROS formation in sperm samples [48]. Elevated levels of ROS, including hydroxyl radicals, superoxide radicals, nitric oxide, hydrogen peroxide, and peroxynitrite, adversely affect regular sperm production and quality by interacting with membrane proteins and lipids, as well as with the mitochondrial and nuclear genomes [56]. Testicular oxidative stress can impact sperm quality by causing peroxidative damage to their DNA [57]. Recent research on male infertility has focused on DNA damage, including DNA breaks, base mutations, DNA strand breaks, and chromatin cross-links, which are often observed in sperm from infertile males. These DNA abnormalities are attributed to increased levels of ROS in sperm, as intact DNA is crucial for successful fertilization [58, 59]. Sperm, like other aerobic cells rich in mitochondria, are susceptible to oxidative damage due to the presence of mitochondrial DNA (mtDNA). Some infertile men exhibit multiple losses of mtDNA, possibly due to free radical damage to spermatogonia [60].

ROS-induced DNA damage increases the likelihood of apoptosis, a form of programmed cell death, in germ cells [58]. Apoptosis can be triggered by internal and/or external stimuli without the presence of inflammation. It can be initiated by cytokine/stress-induced kinases, leading to the overexpression of E-selectin in sperm vascular endothelium, which recruits neutrophils to the testicular tissue and further increases ROS levels, resulting in peroxidative damage to the cell membrane and induction of apoptosis in germ cells [61]. Under pathological conditions such as cryptorchidism, toxin exposure, or testicular torsion, apoptosis in germ cells is significantly elevated, leading to disruption of sperm functionality [62]. Apoptosis is a normal process in spermatogenesis for the elimination of immature spermatozoa [63]. Germ cell death has a profound impact on the testicular epithelium [64], and during acute induction, Sertoli cells engulf a large number of apoptotic germ cells, which can overwhelm normal Sertoli cell processes [65]. This biological process triggers the synthesis of pro-inflammatory mediators, including interleukin-1β (IL-1β), interleukin-6 (IL-6), and other pro-inflammatory molecules, through the activation of the NF-κB pathway. In summary, excessive production of ROS and RNS in the male reproductive system leads to oxidative stress, which impairs sperm functionality, causes DNA damage, induces apoptosis in germ cells, and disrupts normal spermatogenesis.

ACR changes sperm function in animals through ROS

Mitochondria in spermatozoa play a crucial role in ATP production, which is essential for sperm motility and fertilization, and the regulation of mitochondrial ATP production occurs through oxidative phosphorylation. Elevated levels of reactive oxygen species (ROS) in the cell can lead to disruptions in mitochondrial function, including a decrease in mitochondrial membrane potential (ΔΨm) and activation of inner mitochondrial membrane (IMM) enzymes involved in apoptosis, such as Caspase-3 and Caspase-9 (Fig. 1) [66]. Exposure to ACR at different doses (0.5–2 mM) has been shown to result in a decrease in ΔΨm in human spermatozoa [67]. A reduction in sperm motility is associated with increased ROS levels, decreased ΔΨm, and the formation of electrophilic aldehyde lipids (EALs) [68]. These EALs can hinder sperm motility by interfering with the mitochondrial electron transport chain and binding to proteins like dynein and AKAP-3/4, which are involved in regulating sperm motility [68]. Under oxidative stress conditions, BH3 protein, located in the outer mitochondrial membrane (OMM), becomes activated, leading to increased OMM permeability [69]. This leads to electron loss and the generation of free radicals, such as superoxide anion radical (O2−.), through their binding to O2 [69].

Fig. 1
figure 1

Possible mechanisms of mitochondrial dysfunction in testicular tissues and spermatozoa related to ACR administration and the defense mechanism against oxygen free radicals. ACR exposure leads to mitochondrial dysfunction due to the alteration of mitochondrial permeability transition pore (mPTP) activity, loss of ΔΨm, and decrease in ATP content. In addition, the overproduction of ROS in mitochondria leads to the release of apoptotic factors. Moreover, oxidative stress can activate several signaling pathways, such as inflammation and autophagy, via the activation of NF-κB and blocking of PI3K/AKT/mTOR. Therefore, such events may lead to DNA damage and apoptosis in spermatozoa, spermatogonia, and Leydig cells. Any damage to Leydig cells leads to lower production of testosterone, which in turn alters sperm parameters. On the other hand, antioxidant sources can counteract the production of ROS, and therefore intracellular antioxidant defenses such as SOD, CAT, GPx, and GSH can readily convert free radicals into H2O and O2

Full size image

ACR may also affect ΔΨm by disrupting the function of adenine nucleotide transporters located in the IMM, causing apoptotic substances to move from the membrane space into the cytosol [70]. Increased sensitivity of sperm to free radicals may be due to the unsaturated fatty acids that are abundant in the sperm membrane. Some types of ROS, such as H2O2, can enter sperm cells inhibiting the activity of the enzyme glucose-6-phosphate dehydrogenase (G6PD), which is involved in the control of glucose influx and the intracellular presence of NADPH. Therefore, inhibition of G6PD/NADPH metabolism results in decreased glutathione levels and accumulation of glutathione oxidase in sperm. It is considered that the overproduction of ROS leads to a decrease in both nonenzymatic and enzymatic antioxidants in Leydig cells, which in turn impairs spermatogenesis and decreases sperm count (Fig. 1) [71]. ACR administration decreases SOD and TAC as well as some parameters in human spermatozoa, such as progressive/total motility and viability, while it can increase MDA levels [72]. Incubation of spermatozoa with ACR also resulted in decreased activity of GST, an enzyme involved in mitigating oxidative stress, and a decline in mitochondrial membrane potential when cellular ROS levels are elevated.

According to a study conducted by Haleem and colleagues in 2022, exposure to ACR was found to result in a decrease in the activity of LDH-X in testicular tissue [73], while Rajeh and Khayyat in 2017 found that the administration of ACR (45 mg/kg) for five consecutive days did not lead to changes in serum lactate dehydrogenase (LDH) concentration [74]. LDH-X, also known as testis-specific LDH, is a unique isoform of the enzyme LDH primarily found in spermatogenic cells (pachytene spermatocytes and Sertoli cells) within the testicular tissue, and its activity is considered an indicator that could reflect overall tissue damage. LDH-X is involved in energy metabolism in the testes, particularly in the conversion of lactate to pyruvate, which generates NADH, a reducing equivalent that contributes to the production of ATP through oxidative phosphorylation in mitochondria. The ATP generated through LDH-X-mediated energy metabolism is crucial for various processes involved in spermatogenesis, including DNA replication, chromatin remodeling, and sperm motility. Sperm motility, in particular, requires a significant amount of ATP to power the flagellar movement necessary for sperm to reach the egg for fertilization [75]. It has also been suggested that the overproduction of ROS following ACR exposure could cause mitochondrial membrane fragmentation and subsequent alteration of membrane-bound LDH-X activity [73]. Therefore, reduced LDH-X can lead to a decrease in ATP production, resulting in diminished energy availability for sperm motility.

Several studies have shown that ACR can decrease progressive/total sperm motility, sperm abnormalities, sperm concentration, chromatin condensation, and sperm DNA integrity, and increase sperm protamine deficiency and sperm DNA damage [36, 38, 76,77,78,79,80] (Table 1). Chapman and Michael in 2003 reported that the effects of ACR on sperm chromatin condensation may be due to a reduction in serum T levels [81]. Also, DNA damage induction in germ cells and spermatozoa could be due to the activation of cytochrome P450 2E1 (CYP2E1) [82, 83]. CYP2E1 is involved in the conversion of ACR to the highly reactive metabolite glycidamide and in dominant lethality. Indeed, the function of CYP2E1 depends on its activation by O2, so the destruction of the oxygen-enriched P450 complex releases the O2−., resulting in DNA damage and cell death (Fig. 2) [84]. One study showed for the first time that the major site where sperm DNA damage occurs is the epididymis because CYP2E1 is expressed there [85]. Also, the liver, spleen, brain, spermatocytes, and the midpiece of mature spermatozoa are the other sites where CYP2E1 is expressed [85,86,87].

Fig. 2
figure 2

Schematic representation of acrylamide (ACR)-induced testicular damage. ACR could be involved in the activation of inflammation (by increasing TNF-α, IL-1, IL-1β, IL-6, COX-2, NF-κB, and p38α MAPK levels), oxidative stress (by increasing MDA, NO, LPO, 8-OHdG or decreasing CAT, GPx, SOD, GSH, and GSSG activities) autophagy (by upregulating Beclin-1, LC3A, and LC3B expression), apoptosis (by upregulating Bax, Caspase-3, Cytochrome-C [Cyt-C], p53, and ERK and downregulating Bcl-2, Nrf2, and PI3K/AKT/mTOR signaling expression), and DNA damage (by upregulating the expression of CYP2E1) in sperm and testicular cells such as germ cells and Leydig cells (res arrows), which in turn could lead to the formation of abnormal spermatozoa. However, antioxidant therapy could alleviate these side effects (green arrow) and play an effective role in maintaining low levels of oxidative stress, thereby enabling normal cell signaling cascades and spermatogenic functions and preventing ROS-induced cell damage

Full size image

ACR exposure during the prepubertal period could also change sperm production and function. For instance, prepubertal exposure to ACR (from postnatal day 23 (PND) to PND60) decreased total sperm production in a dose-response manner, reduced mitochondrial activity, decreased mRNA expression of early growth factor 2 (EGR2), RHCG (a transcriptional marker for spermatocytes), and LRRC34 (a transcriptional marker for spermatids), and increased androgen receptor (AR) expression at the transcriptional level in rats [88]. EGR2 is responsible for the proliferation and maintenance of spermatogonia A and B, and its expression also decreased during the differentiation of spermatogonia into pre-leptotene spermatocytes [89, 90]. Therefore, it appears that ACR can have negative effects on spermatogonial proliferation, leading to a decrease in sperm production, whereas higher expression of AR may be an attempt to restore spermatogonial proliferation and a compensatory mechanism against downregulated EGR2 expression.

It has been documented that the level of carboxymethyl lysine increases as a form of advanced glycation following the incubation of sperm with ACR [72]. Advanced glycation refers to a process in which sugars react with proteins, lipids, or nucleic acids, resulting in the formation of advanced glycation end products (AGEs) [91]. These AGEs can accumulate in various tissues, including the reproductive system. In the case of sperm, the accumulation of AGEs, particularly in the acrosomal region of the sperm head, can induce oxidative stress and inflammation, leading to impaired sperm function and reduced sperm quality [91]. Furthermore, the disruption of the BTB by AGEs can further compromise sperm DNA integrity and contribute to DNA damage [91].

Therefore, the available studies provide substantial evidence that exposure to ACR has detrimental effects on sperm health and function by interfering with mitochondrial function, reducing ΔΨm, and activating CYP2E1. ACR also promotes the formation of AGEs in sperm, compromising DNA integrity. These findings highlight the adverse effects of ACR on sperm parameters such as progressive/total sperm motility and reduced sperm concentration, underscoring the importance of maintaining mitochondrial health and oxidative balance for optimal sperm function.

Table 1 Effects of ACR on sperm function
Full size table

ACR through inflammation, apoptosis, and autophagy alters testicular structure and function

ACR administration could result in severe damage to testicular tissues, such as degeneration and necrosis of spermatogonia, including primary spermatocytes and spermatids, vacuolization of Leydig cells and primary spermatocytes, enlargement of interstitial areas (edema) via induction of inflammatory cell infiltration and fibrosis, pyknosis of Leydig cells, focal hemorrhage, formation of giant cells in the spermatids, and debris from spermatozoa in the lumen of testicular tubules (Table 2) (Fig. 2) [28, 40, 73, 98,99,100,101,102,103]. It also appears that a reduction in testicular weight and reproductive accessory organs such as the epididymis may have resulted from ACR-induced tissue damage [92, 104, 105]. On the other hand, connective tissue deformities, multinucleated giant cells, reduced mean Johnson’s score, reduced testicular tubule diameter, and atrophic tubules were observed in the testes of the male offspring of rats whose mothers received ACR during pregnancy (Table 2) [37, 106]. Multinucleated giant cells observed in the testis are atypically large cells that contain multiple nuclei within a single cell. These cells are a specific type of degenerating germ cell, thought to derive from secondary spermatocytes and spermatids [107]. After toxic exposures in animals, their presence is frequently observed within the lumen of seminiferous tubules, indicating an underlying inflammatory process [108]. Also, Johnsen’s score is a commonly utilized system for evaluating testicular damage. It provides a comprehensive grading system using scores ranging from 10 to 1, which correspond to the level of histological findings [109]. A decrease in the number of cells involved in spermatogenesis within the seminiferous tubules is associated with lower scores.

Research suggests that ACR administration increases the expression of genes and the level of factors involved in testicular inflammation, such as NF-κB/p65, TNF-α, p38α mitogen-activated protein kinase (p38α MAPK), prostaglandin-endoperoxide synthase 2 (COX-2), IL-6, IL-1, and IL-1β [39, 42, 43, 73, 110]. The transcription of pro-inflammatory mediators such as interleukins is regulated by NF-κB, while p38α MAPK could regulate the inflammatory response in cells. Inflammation can impair BTB function in animals [111]. For example, NF-κB/p65 activation had negative feedback on the expression of proteins involved in the tight junction of BTB [73]. In 2001, Wachtel et al. demonstrated that the activation of NF-κB/p65 and TNF-α receptors can suppress occludin expression in astrocytes [112]. Therefore, the activation of NF-κB and p38α MAPK by ACR could induce changes in the inflammatory response in testicular cells, probably resulting in BTB damage and cell death. In the rat testes, a number of studies found that an administration of ACR decreased GSH levels and the activity of antioxidant enzymes such as SOD, GPx, and CAT while increasing DNA damage and levels of apoptotic parameters such as Bax, cytochrome-C (Cyt-C), p53, and Caspase-3, and autophagic parameters such as Beclin-1, LC3A, and LC3B [28, 41, 42, 82, 96, 110, 113, 114]. Among all types of spermatogenic cells, spermatogonia and spermatocytes responded more positively to Bax when rats were treated with ACR [115]. Kucukler et al. (2020) demonstrated that ACR can inhibit PI3K/AKT/mTOR signaling pathways [110]. PI3K, a key signaling molecule, regulates numerous cellular processes, such as apoptosis and cell growth. Furthermore, PI3K is responsible for activating AKT, and the downstream mTOR pathway, which is positively regulated by AKT, controls essential processes, including cell growth and autophagy. In addition, Beclin-1 has been shown to be a scaffolding protein for the formation of the PI3K complex as an autophagic protein [116]. Upon oxidative damage, LC3B, a type of autophagosome, is activated [116]; hence, autophagic or apoptotic testicular death induced by ACR may be due to inhibition of PI3K/AKT/mTOR signaling pathways [110]. In mice, ACR was unable to alter AKT levels in both Leydig cells and testes, whereas ACR exposure activated phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK1/2) in TM3 Leydig cells and mouse testicular tissue [98]. ERK is a signaling pathway that plays a crucial role in various cellular processes, including apoptosis. In the testis, ERK signaling has been implicated in regulating apoptotic pathways by regulating pro-apoptotic proteins and downregulating anti-apoptotic proteins [117]. ERK signaling could also interact with the PI3K/AKT pathway, activating caspase cascades in testicular germ cells [117]. Studies suggest that ACR can induce cell apoptosis by triggering the Caspase-3 and ERK pathways [118]. Therefore, ACR exposure leads to oxidative stress, decreased antioxidant activity, increased apoptotic and autophagic parameters, inhibition of the PI3K/AKT/mTOR signaling pathways, and activation of the ERK signaling, suggesting mechanisms underlying ACR-induced testicular cell injury and death.

Table 2 Effects of ACR and antioxidant therapy against testicular structure
Full size table

ACR changes hormone production and steroidogenesis in male animals

Testicular physiology is affected by several hormones, including T, LH, and FSH. However, the effects of ACR on the production of T, LH, and FSH are controversial (Tables 1 and 2, and 3) [92, 124]. In mouse Sertoli and Leydig cell lines (TM4 and TM3, respectively) treated with ACR or glycidamide, the activity of 3β-hydroxysteroid dehydrogenase (3β-HSD, in Leydig cells) and 17β-hydroxysteroid (17β-HSD, in Sertoli cells) decreased (Table 3) [125]. Of the two cells mentioned, Leydig cells were more sensitive to the treatments compared with Sertoli cells [125]. These results suggest that ACR or GA may affect T biosynthesis in Leydig cells by suppressing steroidogenic enzyme activities. Additionally, ACR can decrease T levels by inducing abnormalities in the cytoskeleton of Leydig cells, which is involved in cholesterol uptake [124, 126]. Yildizbayrak and Erkan showed that administration of ACR (2 mmol/L, for one day) resulted in damage to TM3 and decreased T levels, probably via downregulating steroidogenic acute regulatory protein (StAR) and luteinizing hormone/choriogonadotropin receptor (LHCGR) expression, and upregulating CYP11A1 expression (Table 3) [127]. LHCGR is a crucial protein expressed on Leydig cells that, when bound to LH, stimulates T production. StAR plays a critical role in transporting cholesterol from the outer to the inner mitochondrial membrane of Leydig cells, where cholesterol is converted into steroid hormones, initiating steroidogenesis [128]. Thus, reduced expression of LHCGR and StAR can impact T production and alter its synthesis [99]. Hence, the increased activity of CYP11A1, involved in converting cholesterol to pregnenolone [129], in response to ACR treatment, maybe a compensatory mechanism of Leydig cells to maintain androgen formation balance, including T. Furthermore, ACR can disrupt the integrity of the basement membrane of testicular tubules by downregulating occludin, a crucial protein involved in the formation and maintenance of tight junctions expressed in Sertoli cells, leading to damage to the BTB (Fig. 2) [73, 123, 130]. A positive correlation has been found between lower expression of junctional proteins such as occludin and lower serum T levels [73]. ACR can also lead to the death of Leydig cells through the induction of oxidative stress and apoptosis [37], leading to decreased T production.

It is demonstrated that ACR specifically affects T biosynthesis in Leydig cells by suppressing the activities of key proteins involved in steroidogenesis and disrupting the cytoskeleton responsible for cholesterol uptake. However, elevated serum T levels may be due to Leydig cell hyperplasia observed in studies where ACR upregulated the expression of Ki-67, a cell proliferation marker, in hyperplastic Leydig cells [36, 119]. In rats, one month of ACR administration decreased serum levels of FSH and T due to damage to spermatogenic cells and Leydig cells, while LH production increased, possibly because the number of Sertoli cells was unaffected [105]. Administration of ACR for 10 days (40 mg/kg) decreased FSH and increased LH levels [93], while its administration for 21 days (5 mg/kg) failed to alter the levels of LH and FSH in rats [36]. It seems the duration and dose of ACR treatment are indeed important factors that can influence the alternation of FSH and LH production. On the other hand, any disruption of hormone production of LH and FSH may be due to the effects of ACR on the pituitary gland (responsible for LH and FSH production). In this regard, a separate study conducted by Nadhim and AL-Derawi (2022) on rats demonstrated that the administration of ACR for a duration of six weeks (at a dose of 4 mg/kg) induced significant infiltration of inflammatory cells and degenerative changes in both the anterior and posterior regions of the pituitary gland [131]. Collectively, it is evident that ACR-induced testicular damage can occur through alterations in LH and FSH production. In Sertoli cells, the binding of FSH to its receptors (FSHR) expressed on these cells plays a crucial role in promoting spermatogenesis and T production. This binding activates adenylyl cyclase, leading to the formation of cyclic adenosine monophosphate (cAMP). Alongside T, cAMP is involved in regulating spermatogenesis. LH, on the other hand, binds to its receptors (LHR) located on Leydig cells, stimulating and regulating T synthesis. Therefore, any disruptions in T synthesis or spermatogenesis caused by ACR can have an impact on the normal histology of seminiferous tubules, potentially leading to structural changes.

Table 3 Effects of ACR on leydig and sertoli cells
Full size table

Antioxidant therapy against ACR

The modern lifestyle is accompanied by an array of environmental pollutants and toxins, giving rise to concerns regarding their impact on human health. These hazardous substances have the ability to generate ROS, resulting in cellular damage to proteins, lipids, DNA, and overall cellular function. Consequently, the role of antioxidants as a potential defense mechanism against these harmful factors has garnered significant attention and research. Notably, antioxidants, including vitamins and various plant-derived compounds, play a crucial role in counteracting the detrimental effects of ROS by either neutralizing them or preventing their formation. Below, a compilation of studies demonstrating the ability of different types of antioxidants to counteract the deleterious effects of ACR is presented (Tables 1 and 2).

Capsaicin and thymoquinone treatment could have a protective effect against ACR-induced testicular injury and increase sperm motility and number [73, 95]. It has been shown that capsaicin plays a protective role against scrotal hyperthermia through its antiapoptotic and antioxidant properties [132]. Treatment with resveratrol, a phytoalexin found in a variety of plant species and used in traditional medicine [133], was able to attenuate DNA damage in both sperm and germ cells of mice treated with ACR over a long period of time [82]. Therefore, one of the mechanisms for resveratrol could be its inhibitory effect on CYP2E1 activity. One study showed that simultaneous supplementation of ACR-exposed rats with Portulaca oleracea seed extract was able to increase the expression of proliferating cell nuclear antigen (PCNA) in spermatogenic cells, decrease Caspase-3 expression in Leydig cells, and increase mRNA expression of two major genes involved in steroidogenesis, CYP11A1 and hydroxysteroid 17-beta dehydrogenase 3 (17β-HSD3), and increase testicular GSH and SOD [94]. Another study conducted by El-Beltagi et al. (2016) presented that oral administration of quercetin increased T levels and the activity of GSH and GST, whereas TNF-α levels decreased in testicular tissues of rats exposed to ACR [121].

In mice exposed to ACR for 35 days, treatment with vitamin E improved sperm chromatin quality [79]. Moreover, ACR exposure during pregnancy could lead to lifelong permanent damage, whereas daily intake of vitamin E could prevent testicular damage and hormonal changes in the offspring [37]. It was also demonstrated that ACR-induced damage to testicular tubules, characterized by shrinkage and atrophy, was mitigated when rats were treated with either vitamin E or 5-aminosalicylic acid [74]. Vitamin E, renowned for its antioxidative properties and capacity to scavenge free radicals, exhibits promising potential in ameliorating DNA integrity and sperm quality [134]. Furthermore, it has been observed to augment plasma antioxidant enzyme activity, elevate serum T levels, and enhance sperm motility and count in male rats [135]. Given its lipophilic nature, vitamin E readily accumulates within cell membranes, thereby effectively inhibiting lipid peroxidation and preserving membrane integrity. This protective mechanism may serve to shield cells from the injurious consequences of ACR exposure. Moreover, vitamin E has been postulated to enhance the body’s detoxification processes, encompassing the metabolism and elimination of ACR. Vitamin E holds promise in minimizing the adverse effects associated with their accumulation by facilitating the efficient clearance of ACR and its metabolites. Supplementation with garlic oil had a protective effect against ACR-induced testicular toxicity [102]. Garlic is a medicinal plant that has a wide range of biological activities due to its organosulfur compounds. The mechanism of action of garlic against reproductive toxicity induced by hazardous substances such as ACR may be due to its scavenging effect ROS, which increases GSH content in cells [136]. Cellular GSH depletion plays an important role in the genotoxicity of ACR [137]. Mechanistically, the initial phase of ACR biotransformation is due to the ability of ACR to form GSH-S conjugates by interacting with essential cellular nucleophiles that have an SH group and GSH [138].

Amino acids, such as L-cysteine can serve as an anti-oxidant and alleviate endocrine disrupting chemical-induced liver and reproductive cell injury [139]. The use of L-cysteine ameliorated testicular injury such as hyperplasia of Leydig cells induced by ACR in male albino rats by decreasing the production of NO and LPO via its activity against ROS and increasing the activity of antioxidant parameters including CAT, GSH, and SOD [120]. A treatment with L-cysteine could also decrease the number of multinucleated giant cells and protein expression of Bax and increase PCNA activity in a model of ACR-induced testicular toxicity [115]. An administration of N-acetylcysteine (derived from the amino acid L-cysteine, 10–40 mg/kg) was able to restore disorders of spermatogenesis by increasing the number of spermatogenic and Leydig cells and subsequently increasing serum levels of LH and testosterone in a dose-dependent manner [105]. Treatment with N-acetylcysteine was also able to suppress ACR-induced apoptosis in rat testes by reducing Fas mRNA expression (a marker of apoptosis) [97]. N-acetylcysteine possesses antioxidant properties, allowing it to counteract the harmful effects of free radicals and reduce oxidative stress in tissues. By mitigating oxidative stress, N-acetylcysteine has the potential to attenuate the inflammatory response and inhibit the excessive production of certain inflammatory molecules, including cytokines and chemokines. This ability to modulate inflammation may contribute to N-acetylcysteine’s protective effects against tissue injury induced by ACR.

Administration of selenium was able to increase the synthesis of T hormone in the ACR-treated rats via upregulation of the expression of CYP17A1, HSD17β1, and StAR [93]. Selenium is an essential trace mineral that acts as a cofactor for several antioxidant enzymes, including glutathione peroxidases, which help neutralize harmful free radicals and reduce oxidative stress in tissues [140]. Therefore, enhancing the antioxidant defense system may help protect the testes from oxidative damage induced by ACR and mitigate testicular injury.

Plant extracts have been found to be effective in alleviating some environmental toxicant-induced health effects. Treatment of ACR-exposed rats with propolis extract (a honey-related product) ameliorated the deleterious effects of ACR on some reproductive parameters, such as sperm and hormones (LH, FSH, and T) [92]. ACR toxicity in the testes decreased when animals (rats) received 5-aminosalicylic acid [77], while eugenol (the major compound of clove oil) treatment enhanced sperm quality parameters, elevated levels of amino acids in seminal plasma, improved testicular oxidative/nitrosative stress biomarkers, and altered AMPK/p-AKT/mTOR signaling pathway in the rat exposed to ACR [123]. Administration of icariin (a flavonoid from Herba Epimedii) restored the number of some spermatogonial cells such as spermatocytes, spermatids, as well as Leydig and Sertoli cells in the testes of rats exposed to ACR [104]. A three-week administration of crocin (as a component of saffron) had a protective effect against ACR-induced testicular damage [103]. Crocin treatment improved testicular histopathological parameters in rats, such as testicular epithelial height, mean diameter of testicular tubules, and Johnson’s score, and increased hormone levels (T, FSH, LH) and levels of CAT, GSH, and SOD [103]. A 3-month treatment with green tea (Camellia sinensis) improved testicular histopathology and increased serum T levels due to its antioxidant properties [122]. Another experimental study found that administration of earthworm extract reduced the expression of Ki-67 in Leydig cells and the expression of p53 in spermatogenic cells of rats treated with ACR [36]; down-regulation of these two parameters could explain how earthworm extract could restore T production and increase sperm count in the ACR-treated group [36]. Plant extracts offer a multifaceted approach to combating testicular injuries caused by toxic agents. Acrylamide is a toxic compound known to induce oxidative stress and inflammation in testicular tissue. However, plant extracts abundant in antioxidants, such as polyphenols and flavonoids, can effectively scavenge the free radicals generated by ACR. This scavenging action reduces oxidative stress and safeguards the testes from damage. These extracts possess anti-inflammatory properties that assist in mitigating the inflammatory response triggered by ACR exposure, while some plant extracts may exhibit detoxifying effects, aiding in the elimination of ACR from the body and diminishing its harmful impact on the testes. Overall, incorporating plant antioxidants provides a natural and potentially effective approach to mitigate the adverse effects of ACR on reproductive health.

Conclusion

Due to the growing consumption of harmful compounds in daily life, the incidence of infertility within human populations is increasing. ACR is one of these compounds which can lead to male infertility. ACR has been shown to increase serum T levels as a result of Leydig cell hyperplasia. In contrast, numerous studies have explained that T production decreases in ACR models, as the overproduction of ROS subsequently results in damage to Leydig cells Furthermore, ACR can induce cellular autophagy by inhibiting the PI3K/AKT/mTOR signaling pathway and triggering apoptotic factors through the disruption of mitochondrial function. This leads to morphological damage in testicular tissues, sperm dysfunction, and ultimately DNA damage and cell death. This review shows that different concentrations of ACR have adverse effects on testicular tubule architecture (induction of vacuolization and hemorrhage), sperm parameters (viability and motility), sperm head and tail integrity, production of hormones (LH, FSH, and T), mitochondrial membrane potential, and antioxidant levels (SOD, CAT, GPx, and GSH) through increasing oxidative stress and production of ROS, while the use of antioxidants could act against ACR and attenuate the above adverse effects of ACR.