Melatonin-Caffeine Combination Modulates Gamma Radiation-induced Sperm Malformations in C57BL/6 Male Mice at Sublethal Dose of Gamma Radiation.

AIMS: The aim of this study was to assess the protective effect of the melatonin-caffeine combination against γ radiation-induced alterations in the morphological characteristics of sperms. SETTINGS AND
DESIGN: C57BL/6 male mice (n = 30) were randomly divided into five groups: control, radiation (2 Gy), melatonin (100 mg/kg body wt.) + radiation (2 Gy), caffeine (30 mg/kg body wt.) + radiation (2 Gy), melatonin-caffeine (100-30 mg/kg body wt.) + radiation (2 Gy).
MATERIALS AND METHODS: All the mice were sacrificed 24 h postirradiation, and cauda epididymis was collected. In this study, sperm concentration along with any abnormality in their morphology (amorphous heads, pinheads, hookless, coiled tails, midpiece defect, and tail-less) was observed in the control and treatment group of animals.
RESULTS: Radiation exposure (2 Gy) considerably decreases the sperm count when compared with the control group. However, pretreatment with melatonin and melatonin-caffeine combination before gamma irradiation increased the sperm count (P < 0.05), but with caffeine alone could not produce a significant difference. The higher rate of abnormal sperms was observed in the γ-irradiated mice when compared with the control group (P < 0.05). Besides, the numbers of sperm that are hookless and coiled tails were significantly increased after irradiation. Melatonin significantly reduced the number of sperm with amorphous heads and coiled tails. Melatonin-caffeine combination further reduced sperm malformations when compared with the melatonin + 2 Gy radiation and caffeine + 2 Gy radiation group.
CONCLUSIONS: This study suggests that caffeine exerts a protective effect when given in combination with melatonin against gamma irradiation in sperms of C57BL/6 mice and could be a potent combination for the development of radioprotector. Copyright:

INTRODUCTION

Gamma radiation impacts the reproductive system profoundly.[1] Gamma radiation affects normal cells and, as per Bergonie and Tribondeau’s rule, rapidly dividing cells such as spermatocytes are most susceptible, rapidly depleting after irradiation. In contrast, other mature germ cells and a small subfraction of testicular stem cells thrive. Humans are exposed to artificial sources of ionizing radiation from nuclear power plants, atomic bomb test fallout, medical x-rays, and radioactive sources used for cancer treatments. Still, our knowledge about their possible early and severe adverse effects on the testis is limited. Men undergoing chemotherapy or radiotherapy for cancer treatment experience infertility due to killing of a spermatogonial sperm cell. Direct irradiation to the testis can affect germinal epithelium at lower doses, and gamma radiation can cause either permanent or reversible aspermia at higher doses.[1] Direct irradiation of the testis in lower doses will affect the germinal epithelium: doses of irradiation greater than 0.35 Gy cause aspermia which may be reversible. The time taken for recovery increases with larger doses; however, with doses over 2 Gy, aspermia may be permanent.[2]

Cell exposure to ionizing radiation results in oxidation either by direct radiation interactions with macromolecules or by the products of water radiolysis. To cope with changes in the redox environment, the system produces transient responses at chemical, cellular, and tissue levels to mitigate radiation toxic effects. As with other indications of sperm dysfunction, the importance of ROS, caused by either increased ROS generation or impaired antioxidant defense, as a primary instigator of sperm DNA damage is well established.[34] Sperms are particularly vulnerable to damage from ROS because of their high polyunsaturated fatty acid content and limited ability to repair the damage. Sperm from infertile men are often associated with high levels of ROS caused by either increased generation or impaired antioxidant defense.[56] Associations between oxidative stress and sperm DNA damage have been reported in numerous studies.[56] The oocyte can provide limited repair to damaged sperm DNA postfertilization.[78] However, if inadequately repaired, such damage can predispose to mutations in the developing embryo with the potential to induce disease in the offspring.[9] It is acknowledged that a greater proportion of inherited diseases have their origin in the paternal germline.[10] Furthermore, cancers arising from germ Cell mutations showed greater paternal contribution than the maternal contribution.[11] This fact is further illustrated by the finding of higher rates of hematological cancers (leukemia and lymphomas) in offspring of men who smoke with the suspected causal link being the increased level of oxidative sperm DNA damage.[12]

Melatonin, an antioxidant that is secreted by the pineal gland, is an extremely powerful antioxidant, and it is known that melatonin affects reproduction via its action on the hypothalamus[1314] and hypophysis,[15] and that melatonin may alter the secretion of gonadotropins. Melatonin treatment changes the number of atretic follicles in mice.[16] Pretreatment of melatonin before the whole-body irradiation with 8 Gy of gamma radiation modified the quantitative features of seminiferous tubules and reduced the ill effects produced by irradiation. It has also been reported that melatonin can significantly reduce the damaging effects of radiation on Leydig cells.[1718]

Human peripheral blood lymphocytes that were pretreated with melatonin exhibited a lower incidence of radiation-induced chromosomal damage as compared to irradiated cells without melatonin treatment.[1920] In another study, the rate of survival of the irradiated mice increased considerably with the treatment of melatonin before irradiation, and high doses of melatonin were found effective in protecting mice from lethal effects of acute whole-body gamma irradiation.[21]

Several observations support the fact that melatonin has a radioprotective effect against gamma irradiation,[2021222324252627282930] but only a few studies have reported the protective mechanism of melatonin on mouse epididymal sperm parameters in whole-body irradiated mice.[31]

Caffeine, 1,3,7-trimethylxanthine, a major component of coffee, offers radioprotection against oxygen-dependent radiation-induced damage. It is a radioprotector in a variety of systems.[323334353637]

Several studies have recommended the use of melatonin as a radioprotector in case of planned or unplanned gamma radiation exposure, but could not be used practically due to its behavioral toxicity specifically hindered alertness. On the other hand caffeine a potent radioprotector, has a stimulatory action on the central nervous system, elevates mood, and underlies alertness by blocking adenosine’s inhibition of cells in the brain. Although melatonin and caffeine have shown potential radioprotective efficacy individually, they have never been tested in combination against gamma radiation-induced damage in sperms. In the present study, the radioprotective effect of melatonin and caffeine combination has been studied on the mice sperms.

MATERIALS AND METHODS

Chemical and reagents

Melatonin, caffeine, and soybean oil were procured from Sigma Chemical Co. (St. Louis, MO, USA). DMSO and DPX were procured from Himedia Laboratories Pvt. Ltd. (India). Eosin Y was procured from Merck Ltd., India.

Animals

Male C57BL/6 mice of 8–10 weeks old were obtained from the experimental animal facility of the Institute of Nuclear Medicine and Allied Sciences (INMAS), Delhi, India, and the animal ethics committee approval number is INM/IAEC/2014/06/009. Mice were housed in cages under optimum conditions of temperature (25°C ±2°C), humidity (50–60%), and light (12 h of light and 12 h of dark), and provided with standard food and water ad libitum. After acclimatization (5–6 days), mice were weighed, and the average body weight was 23.0 ± 2 g. The protocols were approved by the institutional animal ethics committee.

Preparation of drug

The melatonin solution was freshly prepared in soybean oil to obtain a final dose of 100 mg/kg (200 μL) for each animal. The required quantity of melatonin was dissolved in 2% DMSO and made up to the final volume with soybean oil and mixed thoroughly for a minute. Caffeine was dissolved in water to obtain a final dose of 10, 20, 30, 50, 70, 100 mg/kg, 500 μL for each mouse, and administered 1 h before γ irradiation.

Drug administration

Mice were administered melatonin intraperitoneally 30 min before γ irradiation.[38] Caffeine was administered orally 1 h before radiation.

Optimization of drug dose

Various combinations of melatonin and caffeine (100–10 mg, 100–20 mg, 100–30 mg, 100–50 mg, 100–70, and 100–100 mg) were prepared, and mice were administered vehicle and melatonin–caffeine 30 min and 1 h prior to 2 Gy of gamma radiation respectively. The sperm counts were measured 24 h post-irradiation. The optimized dose of melatonin–caffeine combination was used in the following study.

Method

C57BL/6 male mice (n = 24) were randomly divided into five groups. Mice of all groups were sacrificed 24 h after various treatments. The description of each group is mentioned as follows:

Group 1: Control: sham irradiated

Group 2: Radiation (2 Gy): received 2 Gy of gamma radiation

Group 3: Melatonin + radiation (2 Gy): received 100 mg/kg body wt. of melatonin i.p 30 min prior to 2 Gy of gamma irradiation

Group 4: Caffeine + radiation (2Gy): received 30 mg/kg body wt. of melatonin orally 1 h before 2 Gy of gamma irradiation

Group 5: Melatonin–Caffeine + Radiation (2Gy): Received 100 mg/kg body wt. of melatonin (i.p) and 30 mg of Caffeine orally 30 min and 1 hr respectively before 2Gy of gamma irradiation

Gamma irradiation

Mice were placed in well-ventilated Perspex boxes (23.5 × 23.5 × 3.5 cm) and exposed to 2 Gy whole-body radiation using Cobalt Teletherapy Unit, Bhabhatron-II (Panacea Biotech India) at a dose rate of 0.780 Gy/min, source-to-surface distance (SSD) of 100 cm, and field size of 20 × 20 cm2. The radiation facility was provided by the Institute of Nuclear Medicine and Allied Sciences, Delhi, India.

Sperm collection, count, and assessment of morphology

Mice from each group were killed by cervical dislocation, and their cauda epididymis was removed. The cauda was weighed and macerated in 5 mL of phosphate-buffered saline (pH 7.2) in a petri dish using a scalpel. The sperm counts and motility were observed in Neubauer’s hemocytometer using an Olympus microscope (CH20i) with 100 × 10 magnifications. Sperms were then stained with 1% Eosin Y and incubated for 30 min. Thirty minutes after staining, smears were prepared, allowed to dry in the air, and mounted under a coverslip with DPX mounting medium. For each suspension, 1000 × 3 sperms were examined for assessment of morphology. Various abnormalities were observed. Amorphous heads were identified as the sperms that lack the usual hook and is deformed, hookless as the sperms without hook, pinhead as oblong shaped and smaller in size, coiled tailed as the sperms with the tails that form a coil, tail-less sperms as the sperms without the tail, and headless as the sperms without the head [Figure 1].

Figure 1

Representative images of various sperm abnormalities: a) Normal sperm, b) Amorphous head, c) Pinhead, d) Coiled tail, e) Headless, f) Hookless, g) Tail-less

Statistical analysis

Data are presented as mean ± standard deviation (SD) of all treatment in the experiment. The sperm counts and percent normal sperms of different treatment groups were compared using Student’s t-test, and P ≤ 0.05 (calculated using SPSS, version 16.0) was considered as significant.

RESULTS

The prepared melatonin drug was a clear and viscous solution. The drug was stable for 6 months when kept at 4oC in the airtight vial, covered with a dark paper. Caffeine solution was clear in appearance and was stable for a month when kept at 4oC in the airtight vial.

Optimization of formulation

This study assessed the protective effect of melatonin and melatonin–caffeine combination on γ-radiation-induced sperm malformation in C57BL/6 male mice. The melatonin dose was optimized based on our previous experiments[38] and melatonin–caffeine dose was optimized based on the measurement of sperm count 24 h postirradiation. The maximum number of sperm count was observed in melatonin–caffeine combination at the dose of 100–30 mg (0.80 ± 0.02), and hence this dose has been used for further studies [Figure 2].

Figure 2

Optimization of caffeine–melatonin dose based on sperm counts/mg of epididymis after 2 Gy of gamma radiation

Sperm count

First, the epididymal sperm concentration was compared between the negative control group of animals that were sham-irradiated and the group of animals that were irradiated with 2 Gy of gamma radiation [Figure 3]. The results indicate that the irradiation with 2 Gy of gamma radiation (0.52 ± 0.07 × 106 sperms/mg) significantly reduced the sperm count when compared with control (0.78 ± 0.09 × 106 sperms/mg). Melatonin treatment 30 min before 2 Gy of gamma radiation increased the sperm concentration to 0.69 ± 0.06 × 106 sperms/mg, while caffeine treatment an hour before gamma radiation increased it to 0.60 + 0.03 and melatonin and caffeine when given in combination 30 min and 60 min, respectively, before 2 Gy of gamma radiation increased the sperm concentration to 0.72 ± 0.08 compared with the radiation alone group (0.52 ± 0.07 × 106 sperms/mg) which is a significant difference compared to melatonin or caffeine-alone treatment group.

Figure 3

Sperm count per mg of tissue from the epididymis expressed as a mean ± standard deviation. The significance analysis was performed between the groups

Sperm morphology

Results of normal sperm morphology and frequency distribution of sperm malformations are shown in Figure 4 and Table 1, respectively. The statistically significant difference (P ≤ 0.05) was observed in percent normal sperm when the control and sham-irradiated groups (98.33 ± 1.15) were compared with the radiation group (84.80 ± 3.61). Pretreatment with melatonin 30 min before radiation increased the percent normal sperm (92.40 ± 2.00) when compared with the radiation group and the difference was found to be statistically significant at P ≤ 0.05. However, caffeine alone (87.03 ± 7.8) when given an hour before radiation did not produce a significant difference when compared with the radiation group. Melatonin, when given in combination with caffeine (95.47 ± 1.50), ameliorates the percentage of normal sperms when compared with the melatonin + radiation group.

Figure 4

Normal sperm morphology expressed as mean± standard deviation. The significance analysis was performed between the groups. (n = 3000 sperms examined)

Table 1

Comparison of the distribution of sperm malformations between the negative control, radiation and the animals treated with melatonin and melatonin–caffeine before radiation

Group	Sperm counted	Frequency of sperm malformations						Abnormal	Normal
		Amorphous	Hookless	Pin head	Coiled tail	Tail less	Head less
Control	3000	26	9	2	12	1	0	50	2950
Radiation	3000	127	109	51	101	49	19	456	2544
Rad + Mel	3000	72	67	20	46	18	5	228	2772
Rad + Caf	3000	103	95	23	69	68	31	389	2611
Mel-Caf + Rad	3000	52	43	8	23	9	1	136	2864

The frequency distribution of sperm malformations was compared between the control group of animals that were sham-irradiated and the group of animals that were irradiated with 2 Gy of gamma radiation. There was a significant difference between the radiation group and the control group. The analysis of the results in the radiation group showed that the 2Gy of gamma radiation significantly increased the rate of sperm malformations (amorphous, hookless, coiled tail, headless, and tail-less). The amorphous sperm concentration increased to 127 as compared to 26 in control. Hookless sperm concentration increased to 109 as compared to 9 in the control group.

In contrast, the rates of all the sperm malformation were significantly reduced in the animals exposed to the same dose of γ radiation (2Gy) and pretreated with 100 mg/kg melatonin. In case of caffeine-only pretreatment, the reduction was seen in various abnormalities like a pinhead and coiled tail but not a significant reduction in amorphous head and hookless. The pretreatment with caffeine and melatonin before 2 Gy of gamma radiation significantly reduced the frequency of all the abnormalities when compared to either melatonin or caffeine-alone group.

DISCUSSION

Spermatogenesis plays a crucial role in the process of reproduction and hence supports continuity of life. Sperm formation is a complex process and involves several hormones and cascades of cellular events. The initial stages of spermatogenesis occur within the testis while the maturation in the epididymis until final ejaculation. It is known that no proliferation takes place once Sertoli or Leydig cells are irradiated.[39] However, testis has its own antioxidant defense system of antioxidant enzymes and free radical scavengers for the maintenance of spermatogenic[40] and Leyding cells steroidogenic functions.[40] Radiation is known to affect the defense system and induce male infertility.[4142] Therefore, a safe radioprotector is needed to counter the radiation-induced male infertility in case of planned or unplanned radiation exposure.

At dose as low as 1 Gy, the number of spermatogonia decreases while their daughter spermatocytes are severely reduced.[43] At doses of 2 to 3 Gy, spermatocytes are damaged and spermatid numbers decrease.[44] Therefore, a dose of 2 Gy was used in the experiment because, at this dose, a previous study has reported a significant decrease in sperm motility and the number of sperm in the caudal epididymis.[45] The low sperm count observed in the radiation group 24 h post 2Gy of gamma radiation corresponds to an effect on sperms when transiting through the epididymis [Figure 3]. The decrease in sperm concentration corresponds to the fact that oxidative damage occurred in the epididymis.

Studies have shown that melatonin produced an increase in sperm concentration, possibly by reducing the apoptosis of the germinal epithelium by scavenging ROS.[46] It has also been reported that melatonin-reduced sperm malformations in the rats exposed to cisplatin.[47] In another study, pretreatment of melatonin (10 mg/kg) before irradiation to testicular tissue reversed the MDA, end product of lipid peroxidation, which indicates a reduction of myeloperoxidase activity.[48] Since melatonin is lipophilic in nature and highly soluble in lipids, it protects macromolecules in all cellular components by its antioxidant property and its stimulatory action on antioxidant enzymes. High lipid content of testis and Leydig cell mitochondria and microsome makes it more sensitive to lipid peroxidation and melatonin reverses lipid peroxidation as reported in several studies.[3145] Melatonin scavenges free radicals directly or indirectly, hence protecting cells from the degenerative damage to them by gamma radiation.[495051] A study reported that pretreatment of melatonin before 5 Gy of gamma radiation inhibited radiation-induced expression of ATM-dependent p53 pro-apoptotic markers, ATM, p53, p21, Bax, cytochrome C, active caspases-3, and caspases-9.[31] This decreased expression of pro-apoptotic proteins was associated with the increase of anti-apoptotic Bcl-x protein leading to a balanced Bax/Bcl-x ratio in melatonin pretreated mice which is of great significance for cell survival.[52] Our study is in accordance with reported study. Melatonin exerted protective effects on the sperm concentration and percent normal sperms, as seen in the melatonin treated group. In our study, we have found that melatonin and melatonin–caffeine combination neither reduce sperm concentration nor cause sperm malformations [Figures 3 and 4].

However, caffeine alone did not produce a significant protective effect against the damaging effect of radiation. It resulted in various abnormalities, but when given in combination with melatonin not only could protect the sperms from apoptosis but also fewer abnormalities were observed [Table 1]. Caffeine has shown radioprotection in various models.[323334353637] It has also been shown to potentiate radioprotection for the induction of chromosomal aberrations in the bone marrow of mice given pre as well as post-irradiation treatment.[53] Radioprotection efficacy of caffeine pretreatment as suggested in most of the studies was due to its ability to scavenge reactive oxygen species generated by radiation based on its antioxidant property. However, caffeine, when given post-irradiation, has been shown to potentiate radiation-induced micronuclei and chromosomal aberrations in human lymphocytes.[5455] However, caffeine has been associated with various effects ranging from no effect to protective effect and radiosensitization. Our study demonstrated that caffeine ameliorated the protective effect of melatonin on sperm concentration and structure by significantly reducing the sperm abnormalities such as amorphous head, hookless, pinhead, coiled tail, and headless sperms when compared with melatonin-alone-treated group before 2 Gy of gamma radiation. The most probable cause of this protective effect is that coadministration of caffeine increased the Cmax of melatonin by 137% and AUC by 120%, thus increasing more scavenging of ROS by melatonin as explained by Hartter et al.[56] So, caffeine increased the oral bioavailability of melatonin probably due to an inhibition of the CYP1A2 catalyzed the first-pass metabolism of melatonin, hence resulting in increasing the efficacy of melatonin.[56]

To the best of our knowledge, this study is the first to report the radioprotective efficacy of the combination of melatonin and caffeine on the sperms of gamma-irradiated mice. Our data suggest that melatonin and caffeine when given in combination exert protection against DNA damage to the sperms.

Research on chemical radioprotectors needs to be expanded to include studies on drugs that not only provide protection against radiation but also prevent radiation-induced behavioral disruption and decrease in performance, as well as studies on agents that will modify the behavioral toxicity of radioprotectors. Behavioral effects were observed in mice and rats after administration of melatonin at doses of 400 mg/kg by weight, vasodilatation of the extremities indicated by a reddening of the ears and feet, and piloerection and ptosis were common. Most importantly, a marked lack of motor activity, muscle relaxation, and ataxia were observed.[57] The central nervous system stimulant caffeine can mitigate these effects produced by melatonin. The data and results on radioprotection by combinations of caffeine and melatonin on the whole body will be published in detail elsewhere. Caffeine administration does not have an adverse effect on the radioprotective efficacy of melatonin. The results on combinations of caffeine and melatonin indicate that the toxicities of major radioprotective compounds can be ameliorated and is a promising approach for maximizing radioprotection. However, this is the preliminary study; more studies are needed to establish the use of the combination of melatonin and caffeine as a radioprotector.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.