Although several studies on toxic effect of aspartame metabolite have been studied, controversial reports over the use of aspartame owing to the fact that it releases methanol as one of its metabolite during metabolism exist. This present study is proposed to investigate whether aspartame (40 mg kg−1 b.wt) administration for 90 days could induce oxidative stress and alter antioxidant status of epididymal sperm in Wistar strain male albino rats. To mimic the human methanol metabolism, methotrexate (MTX)-treated rats were included to study the effects of aspartame. Oral intubations of FDA approved 40 mg kg−1 b.wt aspartame were given daily for 90 days to Wistar strain male albino rats and studied along with controls and MTX-treated controls. Sperm count, viability, morphology, morphometry and motility were assessed. A significant decrease in sperm function of aspartame treated animals was observed when compared with the control and MTX control. The free radical generation were observed in epididymal sperm by assessing the scavenging enzymes, enzymatic and non-enzymatic antioxidants. Result suggest that there was a significant increase glutathione-s-transferase (GST), with a significant decrease in reduced glutathione (GSH), superoxide dismutase activity (SOD), glutathione peroxidase levels (GPx), catalase activity (CAT) and glutathione reductase concentration. The increase in free radicals generation could have ultimately caused the lipid peroxidation mediated damages on the testis. Aspartame treated animals also revealed the reduced space in seminiferous tubules, which resulted in reduced Leydig cells when compared with control in histopathology. These findings demonstrate that aspartame metabolites could be a contributing factor for development of oxidative stress in the epididymal sperm.
Increased production of free radicals or decrease in the anti-oxidative capacity of cells causes imbalance on the redox status of the cell leading to membrane dysfunction, DNA damage and inactivation of protein if not counteracted. In this modern world, people are accustomed to carbonated beverages along with food intake containing artificial sweetener aspartame. A commonly used low calorie artificial sweetener Aspartame, (l-aspartyl-l-phenylalanine methyl ester) discovered in the year 1965 by James Schlatter of the G.D. Searle Company. The increased market of dietary products and the progress of novel synthetic sweetening compounds have not been sufficiently explored. Approximately 50% of the aspartame metabolite is phenylalanine, 40% is aspartic acid and 10% is methanol. It has been reported that not only the metabolites of methanol but methanol itself is toxic to the brain.1 The principal metabolic outcome of methanol is due to the direct oxidation of formaldehyde into formate. Recently, an experiment confirmed that aspartame is a multipotential carcinogenic agent when given at a daily dose of 20 mg kg−1 body weight, a quantity well beneath the acceptable daily dose of 40 mg kg−1 body weight.2 Formate is metabolized twice as fast in the rat as in the monkey.3 The rodents do not develop metabolic acidosis during methanol poisoning due to their elevated liver folate content. In order to create similar results in human beings, only folate-deficient rodents can be used as a model to accumulate formate in order to develop acidosis.4 Henceforth, in order to imitate the human condition, a folate deficiency status is used as a model and it is induced by administering MTX. Relatively small amount of aspartame can significantly increase methanol levels in blood.5, 6 Parthasarathy et al.,7 reported that methanol is primarily metabolized to formaldehyde and then to formate, escorted by the formation of superoxide anion and hydrogen peroxide. Ashok et al.,5, 6, 8, 9 have also reported that chronic methanol exposure a by-product of aspartame, leads to a disturbed free radical-scavenging system. Recent studies on aspartame have been carried out to understand the mechanisms of neurotoxicity.10, 11 It is also appropriate to point out that methanol released by aspartame may enhance an effect in the brain which could be a contributing factor for the observed change in the locomotor and anxiety levels.12 Oxidative stress has a significant role in sperm function. Thus, using aspartame as a chemical stressor, this hypothesis is aimed at evaluating the functional antioxidant markers and the morphometric analysis of the testis.
Materials and methods
Wistar strain male albino rats (200–220 gm) were maintained under standard laboratory conditions with water and food. For the folate-deficient group, folate-deficient diet was provided for 45 days before the experiment and methotrexate (MTX) was administered for a week before the oral intubation of aspartame. The folate deficiency was confirmed by monitoring the formiminoglutamic acid (FIGLU) level in the urine, after this confirmation the aspartame oral intubation was started. The animals were handled according to the principles of laboratory care framed by the committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. Prior to the experimentation, proper approval was obtained from the Institutional Animal Ethical Committee (No: 01/032/2010/Aug-11).
Aspartame and MTX were purchased from Sigma-Aldrich (St Louis, MO, USA). Other molecular grade chemicals were purchased from Merck (Bangalore, India). All other chemicals were of Analar grade obtained from Sisco Research Laboratory (Bombay, India).
The European Food Safety Authority recently confirmed its daily acceptable intake (ADI) for aspartame of 40 mg kg−1 b.wt./day. This ADI is approved by FDA for the European countries (EFSA Journal 2013). Aspartame mixed in sterile saline was administered orally (40 mg kg−1 body weight) for 90 days and this dosage was based on the FDA approved Daily acceptable intake (ADI) limit.
The rats were divided into three groups, namely, saline control, MTX-treated control and MTX-treated aspartame administered groups. Each group consisted of six animals. MTX in sterile saline was administered (0.2 mg kg−1 per day) subcutaneously for 7 days to induce folate deficiency in MTX controls as well as to MTX+ aspartame treated groups.13 One week after treatment with MTX, folate deficiency was confirmed by estimating the urinary excretion of formaminoglutamic acid (FIGLU).14 From the eighth day, the MTX-treated aspartame group received the oral intubation of aspartame, whereas the other two groups received equivalent volumes of saline as an oral dose and all animals were handled similarly. The chronic dose of aspartame was given for 90 days and all the animals were fed folate-deficient diet except the control animals till 90 days.
The animals were sacrificed using higher dose of long acting Pentothal sodium (100 mg kg−1 b.wt). The blood samples and isolation of epididymal sperm was performed between 0800 hours and 1000 hours to avoid circadian rhythm induced changes. The epididymis was immediately removed and washed with ice-cold phosphate-buffered saline (PBS). Further dissection was made on ice-cold glass plate. The homogenate (10% wt/vol) of the epididymal sperm was prepared in a Teflon-glass tissue homogenizer, using ice-cold PBS (100 mm, pH 7.4) buffer and centrifuged separately in refrigerated centrifuge at 3000 r.p.m. for 15 min. The supernatant was used for analyzing the parameters in this study.
Plasma methanol level using HPLC
100 μl plasma was deproteinized with equal volume of acetonitrile and centrifuged for 7 min at 4 °C. The supernatant (20 μl) was analyzed for blood methanol and formate using an HPLC refractive index detector system (Shimadzu RID, Japan) (equipped with Rezex ROA-organic acid column 300 × 7.5 mm 2 I.D., Phenomenex) with the security guard cartridge (AJO 4490 Phe- nomenex). Column oven was used to maintain the temperature at 60 °C. The mobile phase was 0.026 N sulfuric acid. By using methanol as an external standard, the recovery of methanol (HPLC grade) from blood was found to be 92–96%. Linearity for methanol was found to be 5–500 mg per 100 μl. The detector sensitivity for methanol was found to be 5 mg per 100 μl and reproducibility was >93%.
Free radical scavenging enzymes
Interference of free radicals in auto oxidation of pyrogallol is used as a convenient assay for Superoxide dismutase (SOD) (EC.126.96.36.199) and expressed as units/min/mg protein.15 For catalase (EC.188.8.131.52) assay, standard hydrogen peroxide (0.2 m) was used as substrate and the catalase activity was terminated at intervals of 0, 15, 30 and 60 s by addition of potassium dichromate-acetic acid reagent and is expressed as units/min/mg protein.16 Glutathione peroxidase (EC.184.108.40.206) (GPx) activity was assayed by its ability to utilize the standard glutathione in the presence of specific amount of hydrogen peroxide (1 mM) and is expressed as units/min/mg protein.17 Glutathione-s-transferase (GST) was estimated by the method of Habig et al.18 The activity of GST in tissues is expressed as μmoles of 1-chloro-2,4-dinitrobenzene (CDNB) utilized/min/mg of protein. The activity of Glutathione Reductase was estimated by the method of Charles and Robert.19 The activity of GR in tissues is expressed as nanomoles of NADPH oxidized/min/mg protein. The vitamin c was estimated by the method of Omaye et al.20 The vitamin C content is expressed as mg per gm wet tissue.
Reduced glutathione (GSH)
Reduced GSH was measured by its reaction with 5.5-dithiobis-2-nitro benzoic acid (DTNB), to form a compound that absorbs at 412 nm.21 The level of GSH is expressed as μg of GSH per mg of protein.
Lipid peroxidation was measured by estimating malondialdehyde (MDA), an intermediary product of lipid peroxidation, using thiobarbuteric acid and is expressed as nanomoles of malondialdehyde (an intermediary product of lipid peroxidation) per mg protein.22
Sperm count, viability and motility
Sperm count was carried out according to the procedure described by Atessahin et al.23 Briefly, spermatozoa were collected from entire portion of the epididymis, by mincing epididymis with anatomical scissors in 5 ml of pre-warmed (35 °C) physiological saline, placed in a rocker for 10 min and incubated at room temperature for 2 min. The supernatant fluid was diluted 1: 100 with a solution containing 5 gm sodium bicarbonate, 1 ml formalin (35%) and 25 mg eosin per 100 ml/H2O. Total sperm number was determined with a haemocytometer. Approximately, 1:100 of diluted sperm suspension was transferred to each counting chamber and was allowed to stand for 5 min and counting under a light microscope at 40 × 10 magnifications. About 1: 50 of sperm suspension was mixed with an equal volume of 0.05% eosin-Y and nigrosin. After 2-min incubation at room temperature, slides were viewed by bright-field microscope under 40 × 10 magnifications (Nikon Corporation, Tokyo, Japan). Dead sperm appeared pink and live sperm were not stained.24 Two hundred sperm were counted for each sample and viability percentages were calculated. Percentage of motile sperm was assessed using graded semi-quantitative scale of 0–5, and the spermatozoa were evaluated for the rate of forward movement and graded accordingly, taht is, 0=no movement; 1=sluggish or tail movement alone; 2=intermittent sluggish movement; 3–4=fair and good movement; 5=maximum movement in forward direction.
Morphology and morphometry
The fixed sperm were smeared on a glass slide and stained with phosphate-buffered saline solution of Giemsa (Merck, Darmstadt, Germany).25 Sperm analysis for head and tail defects was performed as per WHO Laboratory Manual26 for semen analysis. The length of the head and flagellum was evaluated in 100 spermatozoa (Intact sperm) per animal under the light microscope by using ocular micrometer scale at 40 × 10 magnifications.
Animals were deeply anesthetized with ketamine hydrochloride. Rats were then perfused transcardially with PBS, followed by buffered 10% formalin. The testis, was removed, and preserved in formalin until processed for histology. Then kept on running water to remove formalin pigments and dehydrated with ascending grades of alcohol. After impregnation with paraffin wax, the paraffin blocks were made. They were processed and sections were cut with 10 μm in thickness using Spencer Lens, rotatory microtome (No: 820, Newyork, USA) and then stained with hematoxylin and eosin stain as follows for testis.
Statistical analysis was carried out using the SPSS statistical package version 17.0 (IBM Corporation, Armonk, NY, USA). The results are expressed as mean±s.d. and the data were analyzed by the one-way analysis of variance (ANOVA) followed by Turkey’s multiple comparison tests when there is a significant ‘F’ test ratio. The level of significance was fixed at P0.05.
The data from various groups for the individual parameters are presented as bar diagram with mean±s.d.
Blood methanol level
The data from various groups are presented as table with mean±s.d. (Table 1). The blood methanol level significantly increased in aspartame treated animals when compared with control and MTX-treated control. However, the control did not deviate from the MTX-treated control animals.
Free radical scavenging enzymes
The data from various groups are presented as bar diagram with mean±s.d. (Table 1). Activity of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and vitamin C was significantly reduced in aspartame treated animals when compared with control and MTX control. Activity of GST was significantly increased in aspartame treated animals when compared with control and MTX control. However, the control did not deviate from the MTX-treated control animals.
The data from various groups are presented as bar diagram with mean±s.d. (Figure 1). The level of lipid peroxidation was increased markedly in aspartame treated animals compared with control and MTX-treated control. The control as well as MTX-treated controls showed similar lipid peroxidation level.
The data from various groups are presented as bar diagram with mean±s.d. (Figure 2). The level of reduced glutathione was decreased in aspartame treated animals when compared with control and MTX-treated control. The control and the MTX-treated controls did not differ from each other.
Weight of testis and its size
The data from various groups are presented as table with mean±s.d. (Table 1). The control and the MTX-treated controls did not differ from each other. The weight and the diameter (oval) of the testis reduced from the normal size in the aspartame treated animals when compared with the controls. This change reveals the abnormality of the testis function.
Significant reduction in the percentage of sperm viability was observed in the aspartame treated rats when compared with control rats. Figure 4 shows the sperm motility in control and experimental groups; significant decline in sperm motility was observed in aspartame treated rats when compared with control rats. No alterations in motility were observed in the control and MTX control. The sperm concentrations and motility were markedly decreased in the aspartame treated animals (Figures 3 and 4).
Morphological analysis shows wide degree of abnormality in aspartame treated rat epididymal sperm, such as defects in the head (microcephalic, bicephalous, amorphous and acephalic) and tail. These morphological defects were significantly reduced in control groups and they did not deviate from the normal morphology when compared with the aspartame treated epididymal sperms (Table 1 and Figure 5).
The results are given in Figure 6. Effect of 90 days aspartame (40 mg kg−1 b.wt.) administration to Wistar albino rats on histopathological changes in testis. The systemic and well-differentiated seminiferous tubules and Leydig cells, with their regular orientation were noticed in control rat testes. Whereas aspartame treated animals revealed the reduced space in between seminiferous tubules, which resulted in minimized Leydig cells. The circular shape of seminiferous tubules and radial differentiation of their spermatogenic cells were also severely disturbed by the aspartame treatment. Instead of circular shape, the tubules were elongated and showed poorly differentiated spermatogenic cells which clearly show the defective architecture of seminiferous tubules within the testes of aspartame treated animals. The lumens of seminiferous tubules were also found to be reduced, indicating their compressed and bundled disorientation, whereas the same were radially oriented in control testes.
Oxidative stress has a major role in in the etiology of sperm function and quality. Antioxidants in the body delicately maintain the cellular balance and function, disturbance to this physiological processes any control outcomes in disease. The free radical generation in our study could be due to the methanol released during aspartame metabolism.5, 6, 8 Methanol is metabolized by three enzyme systems, alcohol dehydrogenase, catalase per-oxidative pathway and microsomal oxidizing systems. However the microsomal oxidizing system is known to be responsible for free radical generation.27 Antioxidants scavenging system are partially effective in combating the oxidative damage when the free radical generation overwhelms antioxidants.1, 9, 11, 12 GSH also acts as a cofactor which is needed for methanol detoxification.5
The testis has shown to be highly vulnerable to increased aspartic acid28 and methanol, as it crosses blood testes barrier and reduce spermatogenesis. The reduction in the testicular weight of aspartame treated rats may be due to reduced tubule size, spermatogenic arrest and inhibition of steroid biosynthesis in Leydig cells.29 Excessive reactive oxygen species (ROS) production that exceeds critical levels can overcome antioxidant defense system in the spermatozoa and seminal plasma causes oxidative stress.30 The controlled generation of highly ROS serves as a second messenger system in numerous different cell types. However, its unrestrained production is considered as an important factor in the tissue damage.31 ROS are regularly formed during the process of normal respiration. However, the production is retained at physiologically low intensities by intracellular free radical scavengers. It had demonstrated that the major sources of ROS in semen were resultant from the spermatozoa and infiltrating leukocytes.32
In the present study we are the first to report that FDA approved daily acceptable dosage (40 mg kg−1 b.wt) of aspartame showed the decreased epididymal sperm motility, concentration and abnormal morphometry. Increased lipid peroxidation results in sperm immobilization, reduced acrosomal reaction and membrane fluidity33 and DNA damages which also causes high frequencies of single and double DNA strand breaks.34 High levels of free radical generation disrupt the inner and outer mitochondrial membranes resulting in oxidative stress which affects the sperm motility by altering axoneme structure that hints to tail aberration in sperm35 and decrease in sperm motility.36 Increased LPO and altered membrane function can render defects in head (amorphous, microcephalic and acephalic), neck and tail of sperm.
There was a significant decrease in the free radical scavenging enzymes namely superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and vitamin C in spermatozoa of aspartame treated animals. This was due to the redox status and imbalance in the oxidant and antioxidant.37 Sperm are more vulnerable to excess reactive oxygen species because of high amount of polyunsaturated fatty acid present in their plasma membrane.38
Superoxide dismutase, which is involved in conversion of the O2· to H2O2 and O2 was significantly decreased in aspartame treated rats. Decreased SOD activity indicates either reduced synthesis or elevated degradation or inactivation of the enzyme. Increased concentration of H2O2 and HO· radicals in the aspartame treated rat sperm may have inhibited the SOD activity.39 SOD protects spermatozoa against spontaneous O2· toxicity and LPO. SOD and catalase also eradicate O2· produced by NADPH oxidase in neutrophils, and may have an important role in decreasing LPO and protecting spermatozoa. Catalase scavenges H2O2 and neutralize into H2O and O2·. There was a significant reduction in the activity of catalase in aspartame treated rats, and this may be because of the reduced conversion of O2· radical to H2O2 by SOD that leads to the accumulation of O2· radical; which might have inhibited the activity of catalase.
Spermatozoa and seminal plasma have their own anti-oxidative mechanisms to protect against ROS induced cellular damage. GSH is a major thiol in living organisms has a central role in coordinating the body’s antioxidant defense processes. The reduced GSH not only serves as a sensitive marker of oxidative stress but also has an important role in maintaining integrity of the cell system. Conditions that disturb intracellular levels of glutathione have been revealed to result in significant alteration in cellular metabolism. The tissue glutathione concentration reflects its potential for detoxification and it is important in preserving the proper cellular redox balance and for its role as a cellular protectant.40 GSH has a likely role in sperm nucleus decondensation and spindle microtubule formation. Reduced GSH can scavenge peroxynitrite and HO· as well as convert H2O2 to water with the help of Glutathione Peroxidase.41 Our results showed that reduced GSH, glutathione reductase and GPx was significantly decreased in the aspartame treated rats. Glutathione reductase is alarmed with the conservation of cellular level of GSH by effecting fast reduction of oxidized glutathione (GSSG) to reduced form.42 GPx has a significant role in the peroxyl scavenging mechanism and in sustaining functional integration of the cell membranes, spermatogenesis, and sperm morphology and motility.43 Our results showed that GST was significantly increased in the aspartame treated rats. GST has an important role in adaptive defense mechanism and also essential in eliminating toxic compounds by conjugation.44
Oxidative stress in the testis is one of the major factors that induce germ cell dysfunction. The antioxidants protect germ cells against oxidative DNA damage and have important roles in spermatogenesis. Aspartame metabolite, methanol may be one of the reason for the excessive generation of free radicals induced oxidative stress in the testis. Alterations in the susceptibility of cells to aspartame toxicity may be explained by the imbalance in the activities of the oxidants and antioxidants enzymes. From the present study it can be concluded that aspartame metabolite, methanol might have been a contributory factor for increased oxidative stress and it can either it acts as a chemical stressor with increased free radical generation or diminishing the decreased antioxidant defense system. We are the first to report this abnormality on aspartame consumption which attenuates the sperm function and morphology. As aspartame consumption is on the rise among common people, it is essential to create awareness regarding the usage of this artificial sweetener aspartame. Additional studies are required to evaluate the effect of aspartame in depth in the future.
The author is grateful to the valuable suggestion offered by Dr NJ Parthasarathy and Co-authors. The author is grateful to the valuable help given by L. Sundareswaran, Wankupar Wankhar is acknowledged. We thank Mr. GS. Sravan for his language check on this manuscript. The financial assistance provided by the Indian Council of Medical Research (ICMR) File. No. 3 / 1 / 2 / 29 / Nut. / 2012 / Dated 29-09-2013 for Senior Research Fellow is gratefully acknowledged. I acknowledge University of Madras for providing the infrastructure to conduct the research.