|Year : 2018 | Volume
| Issue : 1 | Page : 6-11
α-tocopherol attenuates acetaminophen-induced testicular dysfunction in adult male rats
Kehinde Samuel Olaniyi1, Toluwani Bosede Agunbiade2
1 Department of Physiology, College of Medicine and Health Sciences, Afe Babalola University, Ado-Ekiti, Nigeria
2 Department of Medical Microbiology and Parasitology, College of Medicine and Health Sciences, Afe Babalola University, Ado-Ekiti, Nigeria
|Date of Web Publication||1-Mar-2018|
Dr. Kehinde Samuel Olaniyi
Department of Physiology, College of Medicine and Health Sciences, Afe Babalola University, P.M.B. 5454, Ado-Ekiti
Source of Support: None, Conflict of Interest: None
BACKGROUND: Acetaminophen (Paracetamol) is a widely used over-the-counter analgesic and an antipyretic drug known to inhibit cyclooxygenase II enzyme paramount in the prostaglandin synthesis and essential for normal male reproductive function. α-tocopherol (Vitamin E), an antioxidant has been used in the management of a number of conditions including infertility. However, there is a dearth of information on the role of α-tocopherol in the management of testicular dysfunction. This study was designed to investigate the ameliorative effect of α-tocopherol in acetaminophen-induced testicular dysfunction.
MATERIALS AND METHODS: Adult male Wistar rats were randomly allotted into groups; Control 1 (vehicle 1; received 0.2 ml of olive oil), Control 2 (vehicle 2; received 0.2 ml of distilled water), acetaminophen- treated (ACE-treated; received 500 mg/kg b. w), α-tocopherol-treated (AT-treated; received 100 mg/kg b. w), and α-tocopherol + acetaminophen-treated (AT + ACE-treated). The treatment lasted for 14 days, and the administration was given orally. The body weight change was monitored using animal weighing balance (Olympia SCL66110 model, Kent Scientific Corporation, Torrington, CT06790, USA), semen analysis and biochemical assay were performed.
RESULTS: The results showed a significant increase in body weight gain and significant alteration of spermatozoa integrity in the acetaminophen-treated group when compared to the vehicle-treated groups. These alterations were associated with decreased hypophyseal-gonadotropic hormones, testosterone, and increased testicular tissue oxidative redox status. Concomitant administration of α-tocopherol during treatment with acetaminophen ameliorated the alterations.
CONCLUSION: The present study demonstrates that administration of α-tocopherol during treatment with acetaminophen preserves testicular function. This might be due to its antioxidative effect and enhancement of hypophyseal-gonadotropic hormone secretion.
Keywords: Acetaminophen, antioxidant, cyclooxygenase-II, redox status, spermatozoa, α-tocopherol
|How to cite this article:|
Olaniyi KS, Agunbiade TB. α-tocopherol attenuates acetaminophen-induced testicular dysfunction in adult male rats. Int J Health Allied Sci 2018;7:6-11
|How to cite this URL:|
Olaniyi KS, Agunbiade TB. α-tocopherol attenuates acetaminophen-induced testicular dysfunction in adult male rats. Int J Health Allied Sci [serial online] 2018 [cited 2018 Dec 16];7:6-11. Available from: http://www.ijhas.in/text.asp?2018/7/1/6/226250
| Introduction|| |
Acetaminophen, commonly known as paracetamol and chemically named N-acetyl-p-aminophenol, is classified as a mild analgesic. It is commonly used for the relief of headaches, other minor aches and pains and is a major ingredient in numerous cold and flu remedies. In combination with opioid analgesics, paracetamol can also be used in the management of more severe pain such as postsurgical pain and providing palliative care in advanced cancer patients. Although paracetamol is used to treat inflammatory pain, it is not generally classified as a nonsteroidal anti-inflammatory drug (NSAID) because it exhibits only weak anti-inflammatory activity. Acetaminophen is thought to act primarily in the CNS, increasing the pain threshold by inhibiting isoforms of cyclooxygenase (COX), COX-1, COX-2, and COX-3 enzymes involved in prostaglandin synthesis, unlike NSAIDs that inhibits COX in peripheral tissues and thus has no peripheral anti-inflammatory effects. An earlier study has shown shrinkage of the seminiferous tubules and disturbance of regular arrangement of the spermatogenic cells in the testis of paracetamol-treated mice, i.e., the spermatogenic cells were not arranged in regular layer as in the case of the normal. Acetaminophen toxicity may result from a single toxic dose, from ingestion of large doses of acetaminophen (e.g., 7.5–10 g daily for 1–2 days)., High doses (500–1000 mg/kg) have been reported to impair fertility in male rats. These observations raise the possibility that the use of paracetamol interrupts normal reproductive function due to its antigonadotrophic activity. It has been recognized since 1940s that α-tocopherol (Vitamin E) is a powerful antioxidant that is absolutely vital for the maintenance of mammalian spermatogenesis. The effect of supplementation of Vitamin E on sperm characteristics, lipid peroxidation, and seminal plasma enzymes of mature male rabbits has been analyzed and the results indicated that supplementation of Vitamin E reduced production of free radicals and improved semen quality. People consume paracetamol uncontrollably the effect of paracetamol on male reproductive function cannot be ignored and it is highly imperative to search for protective measures to minimize its harmful effects. Hence, this study is designed to elucidate the possible ameliorative effect of α-tocopherol on acetaminophen-induced testicular alterations in adult male rats.
| Materials and Methods|| |
The entire drugs used in the study were obtained from Apro Pharmaceutical Ltd, Ado-Ekiti, Ekiti State, Nigeria.
Animals, grouping and protocol
Thirty adult male Wistar rats weighing 180–200 g were obtained from the animal house, College of Medicine and Health Sciences, Afe Babalola University, Ado-Ekiti, Ekiti State, Nigeria. The rats were housed in wire mesh cages and maintained in a well-ventilated room at 25°C ± 2°C, on a 12-h light/12-h dark cycle. Rats had unrestricted access to standard rat chow and tap water. After acclimatized for 2 weeks, the rats were randomly allotted into groups (n = 6 each); Control 1 (vehicle 1; received 0.2 ml of distilled water), Control 2 (vehicle 2; received 0.2 ml of olive oil), acetaminophen- treated (ACE-treated; received 500 mg/kg b. w), α-tocopherol-treated (AT-treated; received 100 mg/kg b. w) and α-tocopherol + acetaminophen-treated (AT + ACE-treated). The treatment lasted for 14 days, and the administration was given orally. The investigation was conducted in accordance with the National Institutes of Health Guide for the Care, and Use of Laboratory Animals and ethical approval was obtained from the Institutional Review ethical committee of Afe Babalola University, and every effort was made to minimize both the number of animals used and their suffering. Initial and final body weights were monitored using animal weighing balance (Olympia SCL66110 model, Kent Scientific Corporation, Torrington, CT06790, USA) and the body weight gain was estimated.
Sample preparation and biochemical analysis
At the end of treatment, the rats were anesthetized with pentobarbital sodium (50 mg/kg, i. p). Blood sample was collected from the apex of the heart into heparinized bottle and centrifuged at 3000 rpm for 15 min using a bench centrifuge and the plasma was stored frozen until it was needed for biochemical assay. Biochemical analysis of plasma malondialdehyde (MDA) and superoxide dismutase (SOD) and hypophyseal-gonadotropic hormones (Follicle stimulating hormone [FSH], luteinizing hormone [LH], and testosterone [TT]) were measured using standardized enzymatic colorimetric methods using assay kit obtained from Randox Laboratory Ltd.(Co. Antrim, UK), and enzyme-linked immunoabsorbent assay kits from Fortress diagnostics, respectively.
The testes were excised, cleared of adhering connective tissues, blotted, and weighed. After weighing, 500 mg of tissue was carefully removed and homogenized with a glass homogenizer. The homogenate was used for measurement of testicular oxidative stress markers (MDA and glutathione).
The epididymis was minced with scissors in 5 ml of normal saline, placed in a rocker for 10 min and allowed to incubate at room temperature for 2 min. After incubation, the supernatant was diluted at 1:100 with a solution containing sodium bicarbonate and 1 ml formalin (35%). The new improved Neuber's counting chamber (hematocytometer) was used in counting the total number of spermatozoa. About 10 μl of the diluted sperm suspension was transferred to each counting chamber of the hematocytometer and observed under a binocular light microscope.
The fluid from the caudal epididymis was diluted with Tris buffer solution  0.5 ml. An aliquot of the solution was then observed under the light microscope. The mean motility estimation was reported as the final motility score for each sample.
The morphology of the spermatozoa was determined using the original dilution for motility, diluted 1:20 with 10% neutral buffered formalin. The sperm cells were categorized based on the presence of one or more abnormal features such as tail defects (short, irregular coiled, or multiple tail), neck and middle piece defects (distended, irregular, bent, or abnormally thin middle piece), and head defects (small, large, double, or detached head). Findings were expressed as a percentage of morphologically normal sperm.
All data were expressed as mean ± standard error of the mean statistical group analysis was performed with SPSS, version 22 of statistical software. One-way analysis of variance was used to compare the mean values of variables among the groups. Bonferroni's test was used to identify the significance of pairwise comparison of mean values among the groups. Statistically significant differences were accepted at P < 0.05.
| Results|| |
α-tocopherol improves body weight gain and testicular weight in male rats treated with acetaminophen
Treatment with acetaminophen significantly reduced body weight gain and testicular weight when compared to the vehicle-treated groups (P < 0.05). However, administration of α-tocopherol significantly improved the body weight gain [Figure 1] and testicular weight [Table 1].
|Figure 1: Effect of α-tocopherol and acetaminophen treatment on body weight of male Wistar rats. Data are expressed as mean ± standard error of the mean n = 6. Data were analysed by one-way analysis of variance followed by Bonferroni post hoc test. (*P < 0.05 vs Vehicle1, $P < 0.05 vs. Vehicle 2, #P < 0.05 vs. ACE)|
Click here to view
|Table 1: Effect of a-tocopherol and acetaminophen treatment on testicular weight of male Wistar rats|
Click here to view
α-tocopherol preserves spermatozoa integrity in male rats treated with acetaminophen
As indicated in [Figure 2], treatment with acetaminophen significantly decreased sperm count (a), % sperm motility (b), % normal morphology (c), and life/death ratio (d) when compared with the vehicle-treated groups. While concomitant treatment with α-tocopherol significantly preserved spermatozoa integrity.
|Figure 2: Effect of α-tocopherol and acetaminophen treatment on sperm count (a), percentage motility count (b), percentage normal morphology (c) and life: Death ratio (d) of male Wistar rats. Data are expressed as mean ± standard error of the mean n = 6. Data were analysed by one-way analysis of variance followed by Bonferroni post hoc test. (*P < 0.05 vs. Vehicle1, $P < 0.05 vs. Vehicle2, #P < 0.05 vs. ACE)|
Click here to view
α-tocopherol elicits antioxidant effect in male rats
Treatment with acetaminophen did not significantly alter plasma oxidative stress status, whereas treatment with α-tocopherol significantly decreased MDA concentration and increased SOD concentration when compared to the vehicle-treated groups [Figure 3]. Furthermore, concomitant treatment with α-tocopherol during acetaminophen treatment did not alter the plasma oxidative stress when compared to ACE-treated group.
|Figure 3: Effect of α-tocopherol and acetaminophen treatment on plasma oxidative stress biomarkers; plasma malondialdehyde concentration (a) and plasma SOD concentration of (b) male Wistar rats. Data are expressed as mean ± standard error of the mean n = 6. Data were analysed by one-way analysis of variance followed by Bonferroni post hoc test. (*P < 0.05 vs. Vehicle1, $P < 0.05 vs. Vehicle 2, #P < 0.05 vs. ACE)|
Click here to view
α-tocopherol reduces testicular oxidative stress in male rats treated with acetaminophen
MDA and glutathione are potent oxidative stress markers. Treatment with acetaminophen significantly increased testicular level of MDA and reduced glutathione level when compared with the vehicle-treated groups. In addition, co-treatment with α-tocopherol significantly reduced MDA and increased glutathione level when compared with ACE-treated group [Figure 4].
|Figure 4: Effect of α-tocopherol and acetaminophen treatment on testicular tissue oxidative stress biomarkers; testicular malondialdehyde level (a) and testicular glutathione level of (b) male Wistar rats. Data are expressed as mean ± standard error of the mean n = 6. Data were analysed by one-way analysis of variance followed by Bonferroni post hoc test. (* P < 0.05 vs. Vehicle1, $P < 0.05 vs. Vehicle 2, #P < 0.05 vs. ACE)|
Click here to view
α-tocopherol improves hypophyseal-gonadotropic hormones and testosterone in male rats treated with acetaminophen
Plasma levels of gonadotropic hormones (FSH and LH) and TT significantly decreased in ACE-treated group when compared to vehicle-treated groups. However, treatment with acetaminophen significantly restored FSH, LH, and TT levels [Figure 5].
|Figure 5: Effect of α-tocopherol and acetaminophen treatment on circulating hypophyseal-gonadotropic hormones; follicle stimulating hormone concentration (a), luteinizing hormone concentration (b) and testosterone concentration of (c)male Wistar rats. Data are expressed as mean ± standard error of the mean n = 6. Data were analysed by one-way analysis of variance followed by Bonferroni post hoc test. (*P < 0.05 vs. Vehicle1, $P < 0.05 vs. Vehicle 2, #P < 0.05 vs. ACE)|
Click here to view
| Discussion|| |
The present study showed that administration of acetaminophen (500 mg/kg b. w) led to decrease in body weight gain, testicular weight, sperm count, % sperm motility, % normal morphology, and sperm viability when compared to vehicle-treated groups. This significant alteration in spermatozoa integrity was associated with increased testicular oxidative stress status (MDA and glutathione concentration) and decreased hypophyseal-gonadotropic hormones. However, treatment with α-tocopherol significantly restored body weight gain, testicular weight, and spermatozoa integrity. These effects were also associated with decreased testicular oxidative stress status and increased hypophyseal-gonadotropic hormones.
The present observation that acetaminophen significantly reduced the body weight gain when compared with vehicle-treated groups is consistent with an earlier study which reported that administration of 500 mg/kg of acetaminophen showed a significant loss in body weight. In addition, our study revealed that treatment with acetaminophen significantly decreased testicular weight. This is also inconsonance with the previous study., However, concomitant treatment with α-tocopherol significantly attenuated acetaminophen-induced body weight or testicular weight loss.
Acetaminophen is principally metabolized in the liver through two major hepatic routes: Glucuronidation and sulfation, the latter route can be rapidly saturated at doses above therapeutic level, then elimination by dose–dependent formation of Glucurono- and sulfo-conjugate derivatives,, and this may be the reason why not all organs are affected by the treatment of low toxic dose of acetaminophen like testis, where they need more time to be exposed to the tested dose before the elimination process take place. The present observation that treatment with acetaminophen significantly impaired spermatozoa integrity is consistent with earlier studies that acetaminophen could either interfere with spermatogenic processes in the seminiferous tubules or epididymal functions which may result in alteration of spermatogenesis. This suggests that acetaminophen could permeate the blood-testis barrier with resultant alteration in the microenvironment of the seminiferous tubules, since it has been reported that the decrease in sperm motility caused by chemical agents was due to their ability to permeate the blood-testis barrier  and thus, creating a different microenvironment in the inner part of the wall of the seminiferous tubules.
Furthermore, the current data revealed that the alteration of testicular spermatogenic and endocrine (TT) function during treatment with acetaminophen is associated with increased testicular oxidative stress and hypophyseal gonadotropic hormones (FSH and LH). This provides additional information to earlier observations that testicular dysfunction is associated with altered plasma oxidative stress., Hence, an intervention that could preserve and enhance testicular function will be of great importance, especially during treatment with acetaminophen.
From the result of the present study, it was found that the group that received α-tocopherol during treatment with acetaminophen revealed the attenuation of testicular toxicity induced by acetaminophen, which were assessed using seminal analysis and blood testosterone level. They exhibited an increase in seminal quality in all parameters and showed a significant increase in blood testosterone when compared to the acetaminophen-treated group. According to Aitken and Roman (2008), α-tocopherol has been shown to improve fertility by enhancing testosterone secretion. Earlier studies have revealed the mechanism of action of α-tocopherol, member of fat-soluble vitamins, which inhibits the formation of reactive oxygen species. It scavenges peroxyl radicals and binds with them to form a tocopheryl radical which can then be reduced by a hydrogen donor to its reduced state. As α-tocopherol is fat soluble, it is incorporated into cell membranes and protects them from oxidative damage. Previous studies have shown that deficiency of α-tocopherol led to oxidative stress that disrupted both the spermatogenic and endocrine function of the testes. This was consistent with Lucoseli and Fraga's observation that treatment with Vitamin E suppressed lipid peroxidation in testicular microsomes and mitochondria to reverse the detrimental effects of oxidative stress on testicular function mediated by exposure to NSAIDs. The present results affirm previous observations that α-tocopherol elicits antioxidative effect and provide additional information that concomitant treatment of α-tocopherol with acetaminophen preserves testicular function.
| Conclusion|| |
The present study demonstrates that administration of α-tocopherol during treatment with acetaminophen preserves testicular function. This might be due to its antioxidative effect and enhancement of hypophyseal gonadotropic hormone secretion.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Anderson BJ, Holford NH, Woollard GA, Chan PL. Paracetamol plasma and cerebrospinal fluid pharmacokinetics in children. Br J Clin Pharmacol 1998;46:237-43.
Ratnasooriya WD, Jayakody JR. Long-term administration of large doses of paracetamol impairs the reproductive competence of male rats. Asian J Androl 2000;2:247-55.
Hassan WB, Kalifa S, Kitta S. Effect of prolonged acetaminophen (panadol) ingestion on the mouse liver, kidney and testis histology. Saudia J Biol Sci 1999;6:168-78.
Goodman PB, Gilman AG. The Pharmacological basis of Therapeutic. 10th
ed. New York: McGraw-Hill; 2001. p. 704.
Evoy GK. American Hospital Formulary Service. AHFS Drug Information. Vol. 10. Bethesda MD: American Society of Health System Pharmacists; 2007. p. 2182.
Lucesoli F, Fraga CG. Oxidative stress in testes of rats subjected to chronic iron intoxication and alpha-tocopherol supplementation. Toxicology 1999;132:179-86.
Aitken RJ, Roman SD. Antioxidant systems and oxidative stress in the testes. Oxid Med Cell Longev 2008;1:15-24.
Yousef MI, Abdallah GA, Kamel KI. Effect of ascorbic acid and Vitamin E supplementation on semen quality and biochemical parameters of male rabbits. Anim Reprod Sci 2003;76:99-111.
Yokoi K, Myi ZK. Organ Apoptosis with cytotoxic drugs. Toxicology 2004;290:78-85.
Sönmez M, Türk G, Yüce A. The effect of ascorbic acid supplementation on sperm quality, lipid peroxidation and testosterone levels of male wistar rats. Theriogenology 2005;63:2063-72.
Oyewopo AO, Dare BJ, Leke JM, Olaniyan TO, Kadirs RE, Owolabi JO, et al
. Cottonseed extract and antifertility: Metabolic versus hormonal changes in rat model. World J Public Health Sci 2012;1:196-9.
Garba SH, Sambo N, Bala U. The effect of the aqueous extract of Kohautia grandiflora
on paracetamol-induced liver damage in albino rats. Niger J Physiol Sci 2009;24:17-23.
Yano CL, Dolder H. Rat testicular structure and ultrastructure after paracetamol treatment. Contraception 2002;66:463-7.
Luangpirom A, Kourchamp W, Junaimuang T. Attenuating effect of Allium ascalonicum
L. on paracetamol induced seminal quality impairment in mice. J Med Plants Res 2012;6:2655-9.
Bowman WC, Rand MJ. The reproductive system and drugs affecting the reproductive systems. Textbook of Pharmacology. 2nd
ed., Vol. 20. 1985. p. 1-8.
Baldessarini RJ. In drugs and the treatment of psychiatriod disorders. In: Goodman L.S, Gilman A, editors. The Pharmacological Basis of Therapeutics. Macmillan Publication Co., Inc.; 1980. p. 301-417.
Oyewopo AO, Olaniyi SK, Oyewopo CI. Tetracycline, amoxicillin and metronidazole elicits oxidative stress-induced testicular toxicity. World J Biomed Res 2016;3:32-6.
Tappel AL. Vitamin E as the biological lipid antioxidant. Vitam Horm 1962;20:493-510.
Makker K, Agarwal A, Sharma R. Oxidative stress and male infertility. Indian J Med Res 2009;129:357-67.
] [Full text]
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]