|Year : 2020 | Volume
| Issue : 1 | Page : 16-21
Protective effect of N-acetylcysteine on changes in serum levels of Pituitary–Gonadal axis hormones and testicular tissue in acrylamide-treated adult rats
Elham Shahrzad1, Mehrdad Shariati2, Syrus Naimi3, Mohammad Amin Edalatmanesh4
1 Department of Biology, Fars Science and Research Branch, Islamic Azad University, Fars; Department of Biology, Shiraz Branch, Islamic Azad University, Shiraz, Iran
2 Department of Biology, Kazerun Branch, Islamic Azad University, Kazerun, Iran
3 Department of Genetics, Kazerun Branch, Islamic Azad University, Kazerun, Iran
4 Department of Biology, Shiraz Branch, Islamic Azad University, Shiraz, Iran
|Date of Submission||30-May-2019|
|Date of Decision||06-Aug-2019|
|Date of Acceptance||30-Sep-2019|
|Date of Web Publication||03-Jan-2020|
Department of Biology, Kazerun Branch, Islamic Azad University, Kazerun
Source of Support: None, Conflict of Interest: None
Background: Acrylamide (ACR) has cytotoxic effects on various tissues of the body, including the reproductive system. The purpose of this study was to evaluate the protective effects of N-acetylcysteine (NAC) on changes in serum levels of pituitary–gonadal axis hormones and testicular tissue in ACR-treated adult rats. Materials and Methods: Forty-two adult male Wistar rats were randomly divided into 7 equal groups including control group, sham group received only distilled water intraperitoneally, ACR group received 50 mg/kg ACR orally, NAC group received 40 mg/kg NAC intraperitoneally and ACR+NAC1, ACR+NAC2 and ACR+NAC3 groups received 10, 20 and 40 mg/kg NAC intraperitoneally, respectively, and then received 50 mg/kg ACR orally. After 28 days of treatment, serum levels of LH, FSH and testosterone were measured by ELISA method, and the testicular tissue was evaluated histopathologically. Results: Hormonal and histopathological analysis indicated that compared to the control, sham and NAC groups, the administration of ACR alone decreased FSH and testosterone levels while increased LH level, and also, it decreased spermatogenic and Leydig cells, but it had no effect on Sertoli cells. The administration of NAC alone had no influence on the level of hormones and spermatogenesis. Coadministration of ACR+NAC ameliorated the serum levels of FSH, LH and testosterone and increased the number of spermatogenic and Leydig cells and recovered spermatogenesis disrupts, in a dose-dependent manner compared to the ACR group. Conclusion: As a potent antioxidant, NAC could inhibit ACR-induced toxicity effects in a dose-dependent manner and ameliorate spermatogenesis in rats.
Keywords: Acrylamide, N-acetylcysteine, rat, testis, testosterone
|How to cite this article:|
Shahrzad E, Shariati M, Naimi S, Edalatmanesh MA. Protective effect of N-acetylcysteine on changes in serum levels of Pituitary–Gonadal axis hormones and testicular tissue in acrylamide-treated adult rats. Adv Hum Biol 2020;10:16-21
|How to cite this URL:|
Shahrzad E, Shariati M, Naimi S, Edalatmanesh MA. Protective effect of N-acetylcysteine on changes in serum levels of Pituitary–Gonadal axis hormones and testicular tissue in acrylamide-treated adult rats. Adv Hum Biol [serial online] 2020 [cited 2020 Apr 4];10:16-21. Available from: http://www.aihbonline.com/text.asp?2020/10/1/16/275087
| Introduction|| |
Acrylamide (ACR) with C3H5 NO molecular formula is a low molecular weight, colourless and odourless compound which is found in foods rich in carbohydrates cooked at high temperatures. Previous studies have shown that ACR has cytotoxic effects on various tissues of the body such as the liver, kidney, intestine, lung, smooth and skeletal muscles and the nervous and reproductive systems.,,, The main mechanism for the formation of ACR in the heated starch foods is by Maillard reaction. In this reaction, the addition of a carbonyl group reducing sugars to asparagine amino acid causes the formation of ACR. In human body, a large amount of ACR (approximately 85%) is conjugated with glutathione, while its remaining (approximately 15%) is converted to oxidised glycidamide during an enzyme reaction, in which cytochrome P450 2E1 is involved. The ACR mutagenic property in humans and mice is due to the ability of glycidamide to react with DNA. In addition, both ACR and glycidamide can rapidly react with haemoglobin and essential enzymes.,
Oxidative stress occurs due to the imbalance in the ratio of biological oxidants and antioxidants. ACR influences by inducing oxidative stress and activating oxygen reactive species (ROS), which reduces the glutathione storage and increases the lipids' peroxidation. It has been shown that ACR can reduce sperm motility. Furthermore, testicular and seminal vesicles atrophy and decreased mating ability in experimental animals have been reported due to testicular ACR poisoning. ACR can be combined with proteins containing sulfhydryl group located at the tail and the nucleus of sperm (such as protamine) and thereby affects fertility., Some studies have shown that the use of antioxidant compounds can modify the harmful effects of ACR on various tissues of the body. Antioxidants will purify the free radicals and protect the cells from harmful oxidative reactions.,, N-acetylcysteine (NAC) is a sulfidryl compound derived from the amino acid L-cysteine and is usually used as an antidote for the treatment of acetaminophen poisoning. The researches have shown that NAC, as an antioxidant and precursor of glutathione, can reduce the effects of oxidative stress and neutralises the intracellular damages of free radicals., By increasing glutathione, inhibiting inflammatory processes and preventing the expression of pre-apoptotic genes, NAC can act as a potent antioxidant. Studies have shown that administrating NAC does not cause severe side effects so that its long-term use at high doses was uncomplicated. Therefore, the present study aimed to investigate the protective effect of NAC as an antioxidant on changes in serum levels of pituitary–gonadal axis hormones and testicular tissue in ACR-treated adult rats.
| Materials and Methods|| |
The present study was an experimental research with the statistical population of 42 adult male Wistar rats weighing 220 ± 20 g. The animals were provided from the animals' house at Islamic Azad University of Kazerun and maintained at the same place. Before starting the study, the animals were kept together for 2 weeks to adapt to the new conditions. During the experiment, all animals were kept in the same standard conditions at 22°C ± 2°C, 12 h of daylight and 12 h of darkness and 70% humidity in polycarbonate cages, and they had free access to enough water and food ad libitum. The protocol of this study was approved by the Ethics Committee of Islamic Azad University of Kazerun, Iran, in relation to working with laboratory animal care (Ethical code No. IR.IAU.SHIRAZ.16330525951004).
Animals were randomly divided into 7 equal groups (n = 6 in each group) of control, sham, ACR, NAC, ACR+NAC1, ACR+NAC2 and ACR+NAC3. The control group did not receive any treatment. In this study, distilled water was used as the medications' solvent; therefore, the animals in the sham group received distilled water intraperitoneally. The animals in the ACR group received 50 mg/kg dose of ACR (Merck, Germany) orally. The rats in the NAC group received 40 mg/kg of NAC (Merck, Germany) intraperitoneally. The animals in ACR+NAC1, ACR+NAC2 and ACR+NAC3 groups received 10, 20 and 40 mg/kg NAC intraperitoneally for each day at 9 A.M., respectively, and then received 50 mg/kg of ACR orally at 5 P.M. The duration of treatment in all groups was 28 days. Based on the previous studies, the dose of NAC and ACR was selected., One day after the end of treatment, animals were anaesthetised using ether (Merck, Germany), and blood sampling was performed directly from the heart. Then, for histopathological evaluations, the left and right testes of all rats were removed.
Blood sampling was performed directly from the heart using a 5cc syringe. Blood samples were placed in the laboratory for 20 min at 37°C until agglutination was performed, and they were then centrifuged at 3000 rpm for 5 min to separate the serum. Before measuring serum levels of FSH, LH and testosterone hormones, the serum was stored at −20°C. FSH, LH and testosterone serum levels were measured using ELISA kits (FSH and LH: BT Lab, China; Testosterone: IBL, Germany).
Histopathological analysis of testicular tissue
After blood sampling, the left and right testes of all animals were removed from the abdominal cavity and fixed in the 10% formalin buffer solution. After performing standard tissue processing, the samples were blocked in paraffin (Aisan, China), and then, using a rotary microtome (Leitz, Germany), 5 sections of 5-μ thickness were provided from each testis and were stained with haematoxylin–eosin (Merck, Germany). Under a light microscope (Nikon, Tokyo, Japan) and using a × 10 Lattice lens, the number of spermatogonia, spermatocyte and spermatid cells were counted in five different areas at the level of one square millimetre in the wall of seminiferous tubules randomly in each section. Sertoli and Leydig cells were then counted with the magnification of ×40.
Data were analysed statistically using SPSS software version 20 (SPSS Inc., Chicago, IL, USA). The normal distribution of the data was confirmed using nonparametric Kolmogorov–Smirnov test, and then, the data were studied statistically among groups at P < 0.05 level using one-way ANOVA and post hoc by LSD test. The results were expressed as mean ± standard error, and the graphs were designed using GraphPad Prism software version 6 (GraphPad Prism, Inc., San Diego, CA, USA).
| Results|| |
In [Figure 1], the effect of ACR, NAC and ACR + NAC on the serum levels of FSH, LH and testosterone in different groups has been shown. The serum levels of FSH, LH and testosterone among control, sham, NAC and ACR + NAC3 groups were not significantly different (P > 0.05) [Figure 1]a, [Figure 1]b, [Figure 1]c. The serum levels of FSH and testosterone in ACR and ACR + NAC1 groups were significantly decreased in comparison to control, sham, NAC, and ACR + NAC2 and ACR + NAC3 groups (P < 0.05) [Figure 1]a and [Figure 1]c; but, the serum level of LH in both groups was significantly increased compared to control, sham, NAC, ACR + NAC2 and ACR + NAC3 groups (P < 0.05) [Figure 1]b. The serum levels of FSH and testosterone in ACR + NAC1, ACR + NAC2 and ACR + NAC3 groups in a dose-dependent manner increased compared to ACR group, and this increase was significant in ACR + NAC2 and ACR + NAC3 groups (P < 0.05) [Figure 1]a and [Figure 1]c. The serum level of LH in ACR + NAC1, ACR + NAC2 and ACR + NAC3 groups in a dose-dependent manner significantly decreased compared to ACR group (P < 0.05) [Figure 1]b.
|Figure 1: Comparison of mean and standard error of the serum levels of FSH (a), LH (b) and testosterone (c) hormones in control, shame, ACR-treated rats in ACR group, NAC-treated rats in NAC group and ACR + NAC-treated rats in ACR + NAC1, ACR + NAC2 and ACR + NAC3 groups. Superscript letters (a-e): P<0.05, as compared with control, sham, ACR, NAC and ACR + NAC1 groups, respectively. ACR: Acrylamide, NAC: N-acetylcysteine.|
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In [Figure 2], the effect of ACR, NAC and ACR + NAC on the number of spermatogonia, spermatocyte, spermatid, Sertoli and Leydig cells in different groups has been shown. There was not a significant difference in the number of spermatogonia and spermatocyte cells among control, sham, NAC and ACR + NAC3 groups (P > 0.05) [Figure 2]a and [Figure 2]b. Furthermore, there was no significant difference in the number of spermatid and Leydig cells among control, sham, NAC, ACR + NAC2 and ACR+NAC3 groups (P > 0.05) [Figure 2]c and [Figure 2]d. There was no significant difference in the number of Sertoli cells in the studied groups (P > 0.05) [Figure 2]e. The number of spermatogonia and spermatocyte cells in the ACR group decreased significantly compared to control, sham, NAC, ACR + NAC1, ACR + NAC2 and ACR + NAC3 groups (P < 0.05) [Figure 2]a and [Figure 2]b. The number of spermatid and Leydig cells in the ACR and ACR + NAC1 groups decreased significantly compared to control, sham, NAC, ACR + NAC2 and ACR+NAC3 groups (P < 0.05) [Figure 2]c and [Figure 2]d. The number of spermatogonia and spermatocyte cells in ACR + NAC1, ACR + NAC2 and ACR + NAC3 groups significantly increased in a dose-dependent manner compared to the ACR group (P < 0.05), but only ACR + NAC3 groups had no significant differences compared to control, sham and NAC groups (P > 0.05) [Figure 2]a and [Figure 2]b. The number of spermatid and Leydig cells in the ACR + NAC1, ACR + NAC2 and ACR + NAC3 groups increased in a dose-dependent manner compared to the ACR group, which was significant in the ACR + NAC2 and ACR + NAC3 groups (P < 0.05) while it had no significant differences compared to control, sham and NAC groups (P > 0.05) [Figure 2]c and [Figure 2]d.
|Figure 2: Comparison of mean and standard error of the number of Spermatogonia (a), spermatocyte (b), spermatid (c), Leydig (d) and Sertoli (e) cells in control, sham, ACR-treated rats in ACR group, NAC-treated rats in NAC group and ACR + NAC-treated rats in ACR + NAC1, ACR + NAC2 and ACR + NAC3 groups. Superscript letters (a-e): P<0.05, as compared with control, sham, ACR, NAC and ACR + NAC1 groups, respectively. ACR: Acrylamide, NAC: N-acetylcysteine.|
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Also, histological findings [Figure 3] indicated that in control [Figure 3]a, sham [Figure 3]b, NAC [Figure 3]d, ACR + NAC2 [Figure 3]f and ACR + NAC3 [Figure 3]g groups, spermatogenesis was done completely and different types of spermatogenic cells with Sertoli cells were observed in seminiferous tubules. In ACR [Figure 3]c and ACR + NAC1 [Figure 3]e groups, the thickness of the germinal epithelium decreased and a vacuolar space was observed in germinal epithelium. Also, a low number of spermatogonia, spermatocyte, spermatid and Leydig cells were observed, and the spermatogenesis was disrupted. However, ACR + NAC1 group demonstrated a relative improvement in spermatogenesis compared to ACR group.
|Figure 3: Photomicrograph of testicular tissue in control, shame, ACR-treated rats in ACR group, NAC-treated rats in NAC group and ACR + NAC-treated rats in ACR + NAC1, ACR + NAC2 and ACR + NAC3 groups. In control (a) and sham (b) groups, complete types of spermatogenic cells with normal spermatogenesis are seen. In the ACR group (c), vacuolar spaces are visible in the germinal epithelium (black arrows). Spermatogenic cells density has decreased, luminal space has expanded and spermatogenesis has been disrupted. In the NAC group (d), all types of spermatogenic cells are observed with complete spermatogenesis. In ACR + NAC1 group (e), spermatogenic cells were observed slightly, but relative improvement of spermatogenesis was observed. In the ACR + NAC2 (f) and the ACR + NAC3 (g) groups, complete types of spermatogenic cells were observed and spermatogenesis improved. H and E staining. White bars, 50 μm. ACR: Acrylamide, NAC: N-acetylcysteine.|
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| Discussion|| |
In this study, the effect of ACR, NAC and ACR + NAC on testicular tissue and the serum levels of FSH, LH and testosterone were investigated after 28 days. Our histopathologic results indicated that ACR decreased the number of spermatogonia, spermatocyte, spermatid and Leydig cells by disrupting spermatogenesis, but it has no effect on Sertoli cells. It has been shown that following ACR administration, its residues can be detected in the reproductive system of male rats including testis, epididymis, seminal vesicle and prostate; therefore, this suggests that ACR can infiltrate the blood–testis barrier and damage the male reproductive system. Some studies have shown that ACR damages spermatogenic cells by increasing apoptosis and decreases Leydig cells; therefore, it can disrupt spermatogenesis., Ma et al. has shown that by increasing the dose of ACR in rats, the count, motility, morphology and viability of sperms are decreased. The evidence suggests that ACR can inhibit the activity of some motor proteins of the cytoskeleton, such as Dynein and Kinesin. These motor proteins play essential roles in the integration of cytoskeleton elements such as microtubules, microfilaments and intermediate filaments into structural and functional units. It is clear that a defect in the cytoskeleton of the cell can affect cell–cell adhesions, cellular shape, intracellular communications, metabolism, synthesis and secretion of biochemical substances; therefore, this can be explained by the fact that ACR can damage the reproductive system by targeting the cell's cytoskeleton system. The hormonal results of this study showed that ACR at 50 mg/kg decreases the serum levels of FSH and testosterone and increases LH. In confirming our results, Camacho et al. showed that ACR can decrease the serum levels of FSH and testosterone and increase LH in a dose-dependent manner. Immunohistochemistry evidence suggests that ACR can decrease and increase the percentage of the staining areas of FSH and LH hormones in the pituitary gland, respectively. In this study, decreased serum level of testosterone appear to be due to decreased Leydig cells; however, decreased testosterone level can also be due to ACR-induced cytoskeleton inhibition which decreases the level of testosterone by decreasing cholesterol absorption. Also, abnormality in the cytoskeleton can be associated with inhibiting the synthesis or transferring LH membrane receptors in Leydig cells which indirectly decreases testosterone levels., In vitro studies show that Leydig cells are more sensitive to ACR than Sertoli cells and when exposed to small amounts of ACR, the activity of the enzymes involved in steroidogenic such as 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase decreases significantly and thereby reduces testosterone biosynthesis. The increase in LH level with the decrease in testosterone level due to ACR administration indicates that the pituitary–gonadal axis can provide an appropriate negative feedback to decreased testosterone levels.
NAC is a potent antioxidant that has been clinically used for treating many different diseases for decades. This antioxidant plays an important role against oxidative stress and protects against cellular damages. In this study, NAC in healthy rats did not influence spermatogenic cells and the serum levels of FSH, LH and testosterone. In a study by Malmir et al., the administration of NAC did not influence the number of spermatogonia, spermatocyte, spermatid, Leydig and Sertoli cells, which is according to the results of this study. Also, Kunle-Alabi et al. showed that NAC administration in male rats did not influence the serum levels of FSH, LH and testosterone compared to the control group, but it can increase the count, motility and rate of sperm viability. It seems that NAC in the damaged testis can decrease the level of Malondialdehyde and increase the levels of glutathione and total protein, thus it can improve the spermatogenesis.
According to the results of this study, the administration of NAC in ACR-treated rats in a dose-dependent manner increases the number of spermatogonia, spermatocyte, spermatid and Leydig cells, therefore can improve spermatogenesis compared to the ACR group. Our study also showed that the serum levels of FSH, LH and testosterone following the administration of NAC at a dose of 40 mg/kg in ACR-treated rats did not differ significantly with control, sham and NAC groups. Malmir et al., indicated that in mice treated with Paranonylphenol, the administration of NAC can improve the spermatogenetic indices and level of testosterone compared to the control group. El-Kirdasy et al., also showed that following the administration of NAC in rats treated with Titanium Dioxide, the amount of apoptosis in spermatogenic and Sertoli cells decreases. In normal testes, apoptosis of germ cells play an important role in spermatogenesis and can maintain the balance between germ and Sertoli cells. It has been represented that spermatogonia and spermatocyte cells are more sensitive to apoptosis factors in comparison with Sertoli and Leydig cells. ACR can induce Caspase-3 and FAS genes that are associated with apoptosis and also increase apoptosis/cell proliferation in the testis. However, it seems that NAC with its antiapoptotic, antioxidant and anti-inflammatory effects, which is confirmed in various studies, can decrease the toxic effects of ACR on testicular function and thus improve and normalise sex hormones and spermatogenesis.,,
The limitations of this study include the short duration of treatment and not studying sperm parameters such as count, morphology, motility, vitality and sperm DNA fragmentation index. Therefore, to obtain better results, longer duration of treatment should be selected in the future studies and different sperm parameters should be evaluated.
| Conclusion|| |
Our results represented that ACR at a dose of 50 mg/kg has toxic effects on testes and could disrupt the spermatogenesis as well as the serum levels of pituitary–testicular axis hormones in the rats. The spermatogenesis and serum levels of pituitary–testicular axis hormones were not influenced in the case of administrating NAC in intact rats. Administrating NAC at various doses in ACR-treated rats does not have a similar effect on spermatogenesis and serum levels of pituitary–testicular axis hormones. The least effect of NAC was detected at 10 and 20 mg/kg, respectively, while 40 mg/kg NAC had the greatest effect on the spermatogenesis and serum levels of pituitary–testicular axis hormones in ACR-treated rats. Therefore, spermatogenesis and serum levels of pituitary–testicular axis hormones could be recovered by administrating NAC in ACR-treated rats in a dose-dependent manner. Hence, in those people at high risk for ACR toxicity, the NAC supplement can be useful in improving fertility.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]