Melatonin mitigates methamphetamine-induced testicular oxidative stress, hormonal imbalance and seminiferous tubule degeneration in rats

Abstract

Background

Drug abuse has become a significant public health concern due to its effects on socioeconomic and mental health challenges. Methamphetamine (MAP) is a common psychostimmulant drug consume widely by youth. it excites central nervous system, alters neurotransmiters and affects body tissues including testis. This study examined the effects of melatonin on methamphetamine-induced testicular toxicity in adult rats.

Methods

Thirty-two (32) adult male Wistar rats (130 ± 2.70 g) were used were randomly assigned into four (4) groups of 8 animals each. Group 1 was given 0.5 ml/kg of phosphate buffered saline (PBS); group II received 20 mg/kg of MAP; group III was administered 10 mg/kg of MAP; and group IV was given 20 mg/kg of MAP plus 10 mg/kg of MAP. All the administration was carried out orally using oral cannula and lasted for 21 days. After the administration, oxidative stress level, Hormonal concentration, seminal fluid parameters were assayed. Histological analysis of the testis was done using H & E stain.

Results

Melatonin was able to improved antioxidant capacity of Superoxide Dismutase (SOD) and Glutathione Peroxidase (GPx), and reduced Malondialdehyde (MDA) concentration. It also regulate the hormonal concentration of LH, FSH and testosterone and enhanced the caudal epididymal sperm quality and restored testicular morphology.

Conclusion

Melatonin showed an antioxidant effects against methamphetanine-induced testicular toxicity and improve testicular functions in rat. Therefore melatonin has a therapeutic benefit against methamphetamine toxicity.

Introduction

Methamphetamine (METH), a potent stimulant within the amphetamine class, is recognized for its extensive systemic toxicity. Research indicates that METH consumption leads to significant pathological damage across multiple physiological frameworks, most notably the central nervous system, hepatic tissues, cardiovascular system, and reproductive structures [1–3]. Current estimates suggest that approximately 36 million people worldwide misuse amphetamine-type stimulants [4]. Posing particular concern is the rising frequency of METH abuse among young adults of reproductive age, posing significant long-term societal and biological risks [5, 6] Extensive research underscores the deleterious effects of methamphetamine (METH) on the male reproductive system, primarily characterized by structural testicular impairment and diminished semen quality [7, 8]. These toxicological outcomes are often rooted in endocrine disruption; specifically, METH exposure has been linked to systemic sex hormone imbalances and the down regulated expression of progesterone and estrogen receptors within testicular tissues [8, 9]. METH interferes with these processes by modulating the genes and proteins associated with calcium transport, leading to disrupted intracellular signaling [10, 11]. Data derived from rodent models provide further evidence of the biological mechanisms at play. These studies indicate that METH impairs fertility potential through oxidative stress, genotoxicity, induction of significant DNA fragmentation within germ cells, Spermatogenic failure which Interferes with the normal progression of sperm development [12–13].

Majority of earlier studies focused on the deleterious effects of methamphetamine on male reproductive system with little being explore on potential therapeutic agent against its methamphetamine-induced male reproductive toxicity. This study evaluated melatonin effects on methamphetamine-induced testicular toxicity in rats.

Beyond its well-established role in regulating circadian rhythms via pineal gland secretion, melatonin has emerged as a significant subject of clinical interest regarding its therapeutic potential in managing oncological, cardiovascular, and metabolic pathologies [14]. The presence of melatonin membrane receptors, specifically MT1 and MT2, within Leydig, Sertoli, and germ cells suggests that this hormone is integral to maintaining homeostatic reproductive function [15–17]. Clinical observations indicate that gonadotoxic interventions, such as chemotherapy, often result in a marked reduction in systemic melatonin concentrations and the down regulation of its associated receptors [18–20]. Researches have highlighted melatonin as a promising adjuvant therapy to mitigate the multi-organ toxicity affecting the renal, hepatic, cardiac, and neurological systems frequently induced by ionizing radiation and chemotherapeutic agents [21]. This cytoprotective efficacy is attributed to a multifaceted pharmacological profile, which includes oxidative stress mitigation, potent antioxidant and anti-nitrosative properties, anti-apoptotic signaling and modulation of immune responses it also regulates genetics via enhancement of gene expressions related to endogenous antioxidant defense mechanisms [22, 23]. A growing body of evidence specifically underscores melatonin’s ability to shield the male reproductive system from the deleterious effects of cytotoxic treatments, thereby potentially preserving fertility following cancer therapy [24, 25]. Melatonin exerted cytoprotective capacity by bolstering both enzymatic and non-enzymatic antioxidant defenses within testicular tissue [26]. By upregulating the catalytic activities of essential antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) and elevating reduced glutathione (GSH) levels, melatonin effectively mitigates oxidative damage triggered by radiotherapy and chemotherapy [22]. Previous research has demonstrated melatonin’s efficacy in countering testicular injuries resulting from metabolic dysregulation and various physical or chemical insults [23]. Furthermore, its ability to attenuate oxidative stress induced by microwave and radiofrequency electromagnetic radiation has been previously established [24–25]. Melatonin exerts a synergistic influence on the testicular antioxidant environment, protecting the tissue through both direct sequestration of free radicals and the indirect regulation of the biological defense network [26–28].

Materials and methods

Laboratory animals and care

Thirty-two (32) adult male Wistar rats (120–150) g were used for the study. The rats were purchased from the University of Ilorin Central Research Laboratory animal breeding facility and housed in the Animal House of the Faculty of Basic Medical Sciences, University of Ilorin, Nigeria. The animals were allowed to acclimatize for two weeks before the commencement of the study, The animals were maintained under normal day-night cycles, and allowed free access to standard chow diet water.

Animal treatment

Methamphetamine crystal was donated for the study by the Nigerian Drug Law Enforcement Agency (NDLEA) through the Kwara State Command. Melatonin was obtained from the a local pharmacy in Ilorin Kwara State Nigeria.

The animals were randomly assigned into four (4) groups of 8 animals each.

Group 1 was given 0.5 ml of phosphate buffered saline (PBS); group 2 received 20 mg/kg of MAP; group 3 was administered 10 mg/kg of MEL; and group 4 was given 20 mg/kg of MAP plus 10 mg/kg of MEL. All the administration was done orally and lasted for 21 days.

Animal euthanasia

On the 22nd day of the experiment, the rats were euthanized using 20 mg/ kg ketamine hydrochloride intramuscularly in order to make the rats unconscious. The right testis of rats were removed, weighed and immersed in cold sucrose solution, homogenised and used for biochemical assessment, The left testes were fixed in Bouin’s fluid, then processed for hematoxylin and eosin staining.

Assay for biochemical parameters

superoxide dismutase, glutathione peroxidase and malondialdehyde were assay. Assay kits for superoxide dimustase (SOD; KT-60703), glutathione peroxidase (GPx; MBS744364) and malondialdehyde (MDA; MBS9389391) were used to assess the testicular osxidative state of rats using spectrophotometric technique. All reagents and samples were placed at room temperature before starting the procedures. Testis were homogenized in ice cold 30% sucrose solution with an automated homogenizer at 4 °C. The homogenate was scooped and poured into a 5 ml plain specimen bottle and placed in a centrifuging tube containing ice. The homogenate was centrifuged for 15 min at 3,000 rpm. The supernatants were aspirated into plain labeled glass cuvettes placed in ice. The supernatant was used to assayed for the level of SOD, GPx and MDA and the procedure was carried out according to the manufacturer’s instruction on the assay pack.

Blood sample was taking and centrifuged for 15 min at 3000 rpm, blood serum was taking and used to assay for level of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (TT) using Accu-bind ELISA Microwells from Cal biotech, with CAT Nos: FSH46F, LH231F and TE187S and procedure was performed according to the manufacturer’s instruction.

Estimation of epididymal sperm characteristics

The bilateral caudal epididymis’s was minced into small fragments in 1 ml of PBS (pH 7.2) in order to release the sperm and subsequently incubated at 37◦C for 5 min [29]. The total sperm concentration was evaluated using a Neubauer hemocytometer. Sperm motility was identify based on progressive motility, non-progressive motilit and immotility (IM). Spermatozoa with progressive and non-progressive were classified as motile sperm [30, 31]. The sperm smear was stained with eosin, and sperm morphology and viability which was observed at 400× magnification, and the ratio of normally shaped sperm with pin head and normal tail [32] were calculated.

Histological analysis

Testis tissues were fixed in Bouin’s fluid for 24 h before embedding. An extablished protocol of [33]. A 5 μm paraffin sections testes was cut and stained with hematoxylin and eosin (H&E). This staining was used to evaluate the cytoarchitecture of seminiferous tubules, focusing specifically on the morphology and number of both interstitial cells and spermatogenic cells. Images were viewed under a light microscope and images were taken with amscope digital camera.

Statistical analysis

All quantitative data from this study were analyzed using analysis of variance ANOVA and Tukey post hoc test for multiple comparison with the GraphPad Prism version 10.0.2 and expressed as mean ± standard error of the mean (M ± SEM) and p < 0.05 is considered statistically significant.

Results

The body weight of the experimental rats increase across the groups, but the changes in body weight was lowest significantly in MAP group compared to other groups at p < 0.05. Table 1. the testicular weight and testiculosomatic index were highest in MAP group compared to other group Table 1.

Table 1.

Body weighy, weight changes, testicular weight

Groups	Initial Weight	Final Weight (g)	Weigh Changes (g)	Testicular Weight (g)	Testis/somatic Index
PBS	124.00±1.90	177.00±2.85	54.80±3.48	1.81±0.15	1.09±0.10
MAP	151.80±2.71	172.80±3.76	21.60±3.31a	1.97±0.16	1.12±0.07
MEL	106.00±1.48	154.60±9.21	47.60±8.38b	1.32±0.18	1.00±0.09
MAP + MEL	152.00±5.39	179.20±7.41	27.60±5.96a	1.39±0.30	0.78±0.19

PBS (phosphate buffered saline; MAP (Methamphetamine); MEL (Melatonin); MAP + MEL (Methamphetamin followed Melatonin)

^{a, b, c} show statistical significant difference from PBS, MAP and MEL groups respectively at p < 0.05

Changes in hormonal profiles following methamphetamine and melatonin exposure in adult male Wistar rats

There was a non-significant difference in serum level of LH across the experimental groups MAP, MEL and MAP + MEL compared to the PBS group at (P < 0.05). This study also found a non significant difference in serum level of FSH of PBS when compared with MAP, MEL and MAP + MEL groups at (P < 0.05). there was a Significant difference in the testosterone level of the MAP and MEL groupS when compared with MAP + MEL group at (P < 0.05), and a non significant difference when compared with PBS at (P < 0.05) (Table 2).

Table 2.

Concentration of luteinizing hormone, follicle stimulating hormone and testosterone in rats exposed to methamphetamine and melatonin

Groups	LH (mlU/ml)	FSH (mlU/ml)	TT (ng/ml)
PBS	4.81±2.13	7.59±1.71	2.51±0.31
MAP	7.49±0.37	2.53±0.52	0.41±0.07
MEL	4.69±1.38	1.80±0.50	0.30±0.06
MAP + MEL	9.95±1.32	5.24±1.05	6.03±0.48bc

PBS (phosphate buffered saline; MAP (Methamphetamine); MEL (Melatonin); MAP + MEL (Methamphetamin followed Melatonin). LH (Luteinizing Hormone); FSH (Follicle Stimulating Hormone); TT (Testosterone)

^{a, b, c} show statistical significant difference from PBS, MAP and MEL groups respectively at p < 0.05

Serum level of luteinizing hormone (LH), follicle stimulating hormone (FSH), and testosterone (TT) in male rat.

Alteration in antioxidant capacity and oxidative damage in rats administered methamphetamine and melatonin

Significance difference was found in SOD activities of PBS group when compared with MAP group at (P < 0.05). Also, there was significant increase in SOD level of MAP + MEL compared to MAP and MEL groups at (P < 0.05). GPx activities of the MAP group was non significantly lower compared to that of the PBS group but significantly lower than MEL and MAP + MEL groups at (p < 0.05). The concentration of MDA in MAP group is significantly high compared to PBS and MEL groups but no significant different in MDA level of MAP compared to MAP + MEL group at p < 0.05. Table 3.

Table 3.

Superoxide Dismutase (SOD) Activities, Gutathione (GSH) activity and Malondialdehyde (MDA) Concentrations in testis of adult male Wistar rat

Groups	SOD (U/L)	GPx (U/L)	MDA (µM)
PBS	0.97±0.06	38.26±2.39	0.75±0.05
MAP	0.65±0.06a	17.16±3.42	1.18±0.05a
MEL	0.49±0.10	50.36±4.63b	0.71±0.02b
MAP + MEL	1.32±0.07bc	21.94±4.07a	0.96±0.25

PBS (phosphate buffered saline; MAP (Methamphetamine); MEL (Melatonin); MAP + MEL (Methamphetamin followed Melatonin)

^{a, b, c} show statistical significant difference from PBS, MAP and MEL groups respectively at p < 0.05

Seminal fluid parameters of adult rats exposed to methamphetamine and melatonin

The result of this study showed a significant different in the sperm viability (SV) in PBS group compared to MAP group only. The MAP also had a significant difference in SV compared to MEL group at p < 0.05. the SV concentration of the MAP + MEL showed a significant different compared to PBS and MMEL groups but non significant compared to the MAP group at p < 0.05.The sperm concentration.

(SC) was no significantly lower in MAP compared to PBS and MEL at p < 0.05 but significantly lower compared to MAP + MEL at p < 0.05. The MAP group recorded a significant difference in the level of Sperm motility (SM) compared to MEL and MAP + MEL groups but non significant compared to PBS group at (p < 0.05). There was a non-significant different in sperm morphology (SMP) in PBS group compared to MAP. The MEL group had a significant higher SMP compared to MAP and MAP + MEL groups. Also, MAP + MEL had a significant increase in the level of SMP compared to MAP at p < 0.05 Table 4.

Table 4.

Sperm count (SC) sperm motility (SM), sperm morphology (SMP) and sperm viability (SV) of male Wistar rat

Groups	S.C (X10^6)	S.M (%)	SMP. (%)	S.V (%)
PBS	255.80±7.67	81.28±1.02	83.64±1.27	88.21±0.44
MAP	232.30±3.17	75.67±0.82	78.41±1.00	77.18±1.69a
MEL	273.50±14.64	84.48±0.34b	86.65±0.44b	90.05±0.62b
MAP + MEL	259.00±5.96b	81.34±0.29bc	83.74±0.19bc	85.71±0.25ac

PBS (phosphate buffered saline; MAP (Methamphetamine); MEL (Melatonin); MAP + MEL (Methamphetamin followed Melatonin). SC (Sperm count); SM (Sperm Motility); SMP (Sperm morphology), SV (Sperm Viability)

^{a, b, c} show statistical significant difference from PBS, MAP and MEL groups respectively at p< 0.05

Histological observation

Representative photomicrograph of testes the control (PBS) shows seminiferous tubules lined by stratified germinal epithelial at varying stages of development. The interstitium is cellular, showing leydig cells and numerous capillary sized vascular channels. MAP: presents germinal epithelial cells which are arranged haphazardly, the basement membrane of the tubules appeared thickened. The intersitium is scanty showing few leydig cells. MEL: shows scanty interstitium. For MAP + MEL, the seminiferous tubule appear normal. The interstitium is cellular showing leydig cells and vascular channels. Figs. 1 and 2.

Fig. 1.

Representative photomicrograph of Rat testis exposed to methamphetamine and melatonin. PBS (Phosphate Buffered Saline), MAP (Methamphetamine), MEL (Melatonin) MAP + MEL (methamphetamine followed by melatonin). (Mag. X100)

Fig. 2.

Representative photomicrograph of Rat testis exposed to methamphetamine and melatonin (Mag. X400). PBS (Phosphate Buffered Saline), MAP (Methamphetamine), MEL (Melatonin) MAP + MEL (methamphetamine followed by melatonin). White arrow: Leydig cells/ interstitium, Black arrow: germinal epithelium, Red arrow: thickened basement membrane

Discussion

This study investigated the protective potentials of melatonin in Methamphetamine-induced testicular toxicity. The hormonal profiles observed in MAP group were characterized by an increase in LH and FSH levels and a decrease in testosterone level. The decrease in testosterone may be linked with the necrosis of Leydig cells, regulation of the hypothalamic-pituitary axis, and steroidogenic enzymes mRNA as well as increase in aromatase level [33–35]. The increase in the LH and FSH level may the result of feedback mechanism which signal increase in the production of the two hormones as a result of low level of testosterone. Liu et al. [36], observed a decrease in testosterone level of mouse exposed to methamphetamine similar to our findings, but their result was accompanied by low level of LH and FSH which may be due to differences in route of administration. Melatonin administered alone led to reduced level of LH, FSH, and low level of testosterone. Melatonin is a natural anti-gonadotropins, the reduction in the level of the gonadotropins under normal physiological conditions results in low testosterone secretion due to less stimulation of the Leydig cells. Also, melatonin stimulates the expression of testicular melatonin receptors in Leydig cells causing downregulation of the expression of enzymes for steroid synthesis, inhibits androgen secretion. However, intervention of melatonin in methamphetamine exposed rats showed high levels of LH and FSH, with testosterone equally higher than that of the MAP rats. This results indicated that MEL administered may exerted some protective potentials on the testicular tissue which brings about the positive feedback response that led to the increase testosterone level observed in this animals. The increase in testosterone level observed can either be due to the antioxidant potential of melatonin which has been shown to protect against testicular injury or the capacity of melatonin to down regulates adrenocorticotropic hormone-releasing hormone which becomes activated in response to stress [37, 38]. Previous studies have revealed that melatonin can improve testosterone levels following testicular injury [39, 40]. Jian et al., [41] reported that melatonin was able to enhanced testicular functions in mice following 24 h light exposure, which indicate that melatonin despite being a natural anti-gonadotropins, its biological role as antioxidant and anti-corticosteroids may account for its potential role in countering drug induced testicular damage.

We examine oxidative marker to assess the potentials of melatonin in preserving testes against methamphetamine toxicity. In this study the superoxide dismutase (SOD) and glutathione peroxidase level were low in methamphetamine treated group with an elevated level of oxidative stress marker malondialdehyde (MDA) relative to the control. This reduction observed in these enzymes concentration and increase level of MDA may be associated with methamphetamine-induced oxidative stress. It has been suggested that MAP activate reactive oxygen species (ROS) which disrupt redox homeostasis in biological tissue [42]. The ROS when excessive can result to oxidative stress in the testicular tissue due to its high fatty acid content, affecting spermatogenesis, spermatocytes, and inhibits Leydig cells thereby interfering with testosterone production [43–45]. A previous study by Li et al. [36], reported that MAP exposure in mice resulted in rise in MDA and ROS level. The antioxidant enzymes SOD and CAT level were slightly higher but GSH was found to be significantly low which further suggests that MAP exposure potentially increase ROS and disrupt antioxidant enzyme profiles. In rats that were given melatonin with methamphetamine showed and elevated level of SOD and GPx with reduction in MDA concentration. This findings may be due to antioxidant capacity of melatonin. Melatonin has been implicated to have a rich antioxidant potential in various human and animal research a function that is ascribed to its amphilic nature. Melatonin has been widely reported to exert antioxidant activities by increasing antioxidant enzyme levels, reduce oxidative stress marker MDA level, and enhance expression of antioxidant enzyme mRNA level [41–43]. previous studies have shown that melatonin can enhanced male fertility by improving antioxidant enzyme levels of SOD, CAT, and reduced MDA level [42, 45]

Epididymal sperm development is a vital part of male fertility, therefore we evaluated semen parameters and found that MAP exposed rats had low semen parameters including low sperm count, reduced sperm motility, morphology and viability. These observations may be as a result of MAP detrimental effects on the oxidative equilibrium which is manifested in the reduced antioxidant enzyme levels and increase in the marker of lipid peroxidation MDA. Oxidative stress can affect sperm qualities by increasing levels of DNA and protein oxidation, and lipid peroxidation and causes reduction in spermatogenesis in rats model of Methamphetamine addiction, resulting in low sperm quality [46]. Also, Liu et al. [47], showed in their study that methamphetmine exposure reduced sperm motility, morphology and concentration in mice. Melatonin treatment to the rats given methamphetamine showed increase sperm qualities, they have increased sperm count, viability level of motility and morphology compared to MAP alone rats. As reported earlier, the intervention of melatonin resulted in an elevated level of SOD and GPx and reduction in MDA concentration, which could be a factor in the increase level of the observed testosterone in the co-treated rat. These observed antioxidant potential couple with increased testosterone level could be a contributory factor to the enhanced sperm qualities observed in the sperm motility, concentration, morphology and viability in this study.

In the cytoarchitecture of the testes, our findings revealed the MAP group showed a disorganized germinal epithelium with haphazardly arranged germinal epithelial cells, thickened basement membrane and few and poorly vascularied interstial cells. The observed seminiferous tubule structure can be linked to the reduced testosterone level due to the scanty Leydig cells observed in the interstitium. Also, the disorganized cytoarchitecture observed may not be unconnected to the increased MDA level which is an indication of possible oxidative stress damage i.e. lipid peroxidation. Previous histological studies of the testes strengthened our findings, indicating the potential adverse roles of MAP on rats spermatocytes and spermatogenesis (Saberi et al. [48]).

Melatonin administration following MAP was able to restored the seminifeous tubule cytoarchitecture compared to the METH group. The seminiferous tubule of the MAP + MEL showed improve germinal epithelium and well vacularized interstitium and more Leydig cells. Imam et al. [49], reported that exogenous melatonin restored testicular integrity against sodium fluoride toxicity in rats. In a similar finding, Sabahi et al. [50], in their study showed that MEL enhance testicular seminiferous tubule integrity, protect Leydig cells and enhance spermatogenesis in mice treated with aflatoxin B1. These protective effects may be due to the direct antioxidant role of the MEL exerted on the testes.

Conclusion

This findings of this study shows that melatonin offers protective potential against the detrimental effects of methamphetamine on the cytoarchitecture of the rat testes, regulate hormonal balance and enhanced seminal fluid parameters by enhancing antioxidant activities of SOD and GPx, and lowers MDA concentration. Therefore melatonin may be a good agent due to its antioxidant potential in the management of methamphetamine-induced male infertility.

Abbreviations

MAP

Methamphetamine

MEL

Melatonin

LH

Luteinizing hormone

FSH

Follicle Stimulating Hormone

TT

Testosterone

SC

Sperm Count

SM

Sperm Motility

SMP

Sperm Morphology

SV

Sperm Viability

MDA

Malondialdehyde

SOD

Superoxide Dismutase

GPx

Glutathione Peroxidase

ELISA

Enzyme-linked immunosorbent assays

ANOVA

One-way analysis of variance

SEM

Standard error of the mean

UERC

University of Ilorin ethical review committee

IACUC

Institutional Animal Care and Use Committee

MDMA

Methylene deoxy-methamphetamine

GnRH

Gonadotropin-releasing hormone

DA

Dopamine

DAT

Dopamine Transporter

TH

Tyrosin Hydroxylase

OI

Organosomatic Index

Author contributions

Abubakar Lekan Imam: Concept and design of the study, definition of intellectual content, experimental studies, literature search, collection of data, analysis and interpretation of data, manuscript preparation, and submission of manuscript.Banmore Oyinlola Adebola: experimental studies, literature search, collection of data, manuscript drafting.Fatimo Ajoke Sulaimon, Kehinde Muibat Ibiyeye, Aliyu Ibrahim Adedo, Rukayat Jaji-Sulaimon, Olarewaju Ambali Danwahab, Babalola Abdussalam Abdulsalam, abriel Olaiya Omotoso, Moyosore Saliu Ajao: Concept and design of the study, experimental studies, collection of data, analysis and interpretation of datamanuscript editing and review and final approval of the version to be published.

Funding

Not applicable.

Data availability

The authors declare that the data supporting the findings of this study are available within the paper. Should raw data files be needed in another format, they are available from the corresponding author.

Declarations

Ethical approval and consent to participate

This research was approved by the University of Ilorin ethical review. committee (UERC) (UERC/ASN/2024/2978) in November 2024, following the. recommendation of the Faculty of Basic Medical Sciences ethical review committee, and consent to use the animals for the study was given by the university of Ilorin central research laboratory animal breeding facility after the university ethical review committee endorsement in compliance with the Institutional Animal Care and Use Committee (IACUC).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.