Long-Term Effect of Aspartame on Male Reproductive System: Evidence for Testicular Histomorphometrics, Hsp70-2 Protein Expression and Biochemical Status

Aspartame was purchased from Sigma-Aldrich (St Louis, MO, USA, CAS No. 22839-47-0). Acridine orange was purchased from sigma chemical Co. (St. Louis, MO, USA). All other chemicals used were commercial products of analytical grade. The rabbit anti-mouse primary antibodies for Hsp70-2 (Cat NO. SKU: 407), were obtained from Biocare (Biocare, California, USA).

All experimental protocols were conducted on the basis of the proofed principles for laboratory animal care (7506025.6.24), approved by the Ethical Committee of the University of Tehran. For this study, a total number of 36 NMRI mature male mice (8-10 weeks of age), weighing 25-35 g were used. The animals were provided from the Laboratory Animal Sciences Center, Pasteur Institute of Iran, Karaj, Iran. Before initiation of the treatment period, the mice were maintained for two weeks in order to acclimatize. The mice were housed in special cages under well-ventilated conditions at normal temperature (22 ± 5°C) with 12:12-hour light-dark cycles and fed standard pellet diet (Tehran pellet, Iran).

In this experimental study, The European Food Safety Authority has confirmed acceptable daily intake (ADI) for 40 mg/kg bodyweight/day of aspartame. This ADI was approved by the food and drug administration (FDA) for the European countries (EFSA Journal 2013). After labeling the mice, they were randomly divided into four groups of nine mice. The treatment groups received aspartame for 90 days by gavage as follows:

1. The first group (control): The animals of this group received normal saline at the dosage of 0.5 ml.

2. The second group was called low dose aspartame and it received 40 mg/kg bodyweight/day of aspartame.

3. The third group was called medium dose aspartame and it received 80 mg/kg bodyweight/day of aspartame.

4. The forth group was called high dose aspartame and it received 160 mg/kg bodyweight/day of aspartame.

Thereafter, the animals were kept under standard conditions and monitored for 90 days. On the basis of the fact that the duration of the chronic dose of aspartame is ninety days to have probable pathogenicity, this period was chosen for this experiment. The dosages and duration of the treatment in the present study were chosen on the basis of earlier studies (13, 14) (Fig .S1, See Supplementary Online Information at www.ijfs.ir).

Following the 90-day period, all animals were anesthetized using a mixture of ketamine and xylazine cocktail (0.10 ml xylazine and 1 ml ketamine and 8.90 ml distilled water), with the dose of 0.1 ml/10 g BW (15). In order to obtain serum, 15 minutes after anesthesia induction, the blood samples were centrifuged at 3000 g for 10 minutes at room temperature (RT) and stored at -70°C for further analyses. The testicular specimens were removed and rinsed with chilled normal saline. One of the testes from each individual mouse was snap frozen in liquid nitrogen and then kept in -70°C until further biochemical analyses and the other testes were fixed in Bouin’s solution for histological examinations.

The testes were quickly dissected out, cleared of adhered connective tissue and weighed on a digital scale (with a minimum accuracy of 0.001 g). For Histomorphometrical study, Dino-Lite digital lens and Dino Capture 2 Software were used. Furthermore, histometrical structures of the testes, including testicular capsule thickness, germinal epithelium height and diameter of seminiferous tubules, as well as the number of Sertoli and Leydig cells were evaluated. In order to classify spermatogenesis, Johnsen’s criteria were used. This classification is based on graded scoring between 1-10 for each tubule cross-section, according to presence or absence of main cell types organized in the order of maturity:10, complete spermatogenesis exists and tubules are normal in arrangement; 9, there are many spermatozoa with disorganization in germinal epithelium; 8, only a few spermatozoa are observed; 7, lacking spermatozoa while many spermatids exist; 6, only a few spermatids are present; 5, absence of spermatozoa and spermatids but existence of many spermatocytes; 4, only a few spermatocytes exist; 3, only spermatogonia are observed; 2, presence of only Sertoli cells and the absence of germ cells, and 1, no germ cells or Sertoli cells are present. Tubule cross-sections with scores of 9 and 10 were considered mature tubules (15).

Paraffin blocks were sectioned at 5-6 μm and stained with Hematoxylin and Eosin (H&E), Periodic acid-Schiff (PAS) and Masson's trichrome. Masson’s trichrome staining was used to show the amount of collagen fibers and fibrosis in testicular tissue. In order to analyze carbohydrate ratio in testicular germinal epithelium, PAS was conducted on specimens. Also, for the purpose of histochemical evaluations, frozen sectioning method was carried out. The samples were embedded using optimal cutting temperature compound (OCT gel) and sections of testicular tissues were prepared at 15-20 μm levels at -40°C using cryostat (SLEE, Germany). Also, the Sudan black B (SB) staining was performed to evaluate the rate of lipid foci supplement in treatment and control animals and identify the Leydig cells cytoplasmic bio-steroid supplement. The alkaline phosphatase staining (ALP) was conducted to demonstrate the ratio of this enzyme as a biomarker for inflammation. The photomicrographs were taken by a SONY on-board camera (Zeiss, Cyber-Shot, Japan).

Epididymides were carefully refined from their surrounding tissues under 10X magnification provided by a Stereo Zoom Microscope (TL2, Olympus Co., Tokyo). The caudate part of the epididymis was trimmed and minced in 5 ml TCM199 medium for 30 minutes, with 5% CO₂, at 36.5°C in a CO₂- equipped incubator (LEEC Co., England). After centrifugation, the sperm pellet was re-suspended in 0.5 ml of TCM199 medium. A small aliquot (20 μl) of sperm suspension was glass-smeared. The slides were air-dried and then fixed overnight in Carnoy’s solution (methanol/acetic acid, 3:1). Next, they were stained for 5 minutes with a freshly-prepared acridine orange stain (AO). After washing and drying, the slides were examined using a fluorescent microscope (Leitz, Germany; excitation of 450-490 nm). On each slide, an average of 200 sperms were evaluated and two types of staining patterns were identified including yellow (single-stranded DNA) sperms and green (double-stranded DNA) (16).

The percentage of spermatozoa with single-stranded DNA was calculated from the ratio of spermatozoa with red, orange, or yellow fluorescence, to the total spermatozoa counted per sample.

Sperm count was assessed by a standard hemocytometer method (15). The motility of the sperm was evaluated according to the WHO (WHO, 2010) standard method for manual examination of sperm motility (17). Accordingly, the sperm samples were diluted 1:8 in TCM199 before the assessment. A 20-μl sample of the sperm was placed on a sperm test area and evaluated under 1,000X magnifications. Only the motile sperms with forward progression were counted within 10 boxes and recorded. Finally, motility was calculated based on the following equation: Motility (%) = [motile sperm/(motile+non-motile sperm)]×100.

The Eosin-nigrosin staining method was performed to assess the sperm viability. For this purpose, 50 μl of sperm was mixed with 20 μl of eosin in a sterile test tube. After 5 seconds, 50 μl of nigrosin was added and mixed thoroughly. Then, the mixture of the stained sperm was smeared on the slide and examined under a light microscope (1,000X magnification, Olympus, Germany). The colorless sperms were considered live and the yellow to pink stained sperms were marked as dead. The sperm count was performed according to the standard hemocytometric method previously described by Pant and Srivastava (16). The sperm viability and motility are reported in percentage and compared between groups.

Following 90 days, blood samples were obtained directly from the heart under light anesthesia (induced using diethyl ether). After 15 minutes, the samples were centrifuged at 3000×g for 10 minutes at RT to obtain serum. Serum concentration of testosterone was measured by enzyme-linked immunosorbent assay (ELISA) as described by the manufacturer (Demeditec Diagnostics GmbH, Germany). In brief, 100 μl of serum sample and control (from the kit) were dispensed into the ELISA wells, and 100 μl of Enzyme conjugate was added into the wells and thereafter, incubated 60 minutes at RT. Next, the content of the wells was discarded and rinsed 4 times with diluted Wash Solution (300 μl per well), and 200 μl of Substrate Solution was added to each ELISA well. The samples were thereafter incubated in the dark for 30 minutes. Finally, 50 μl of Stop Solution was added to each well and the absorbance of each sample was determined at 450 nm.

Some important detectable oxidative stress biomarkers, including total antioxidant capacity (TAC), and activities of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) and nitric oxide (NO) content were measured in the blood samples as described previously (15, 18).

Determination of GSH-Px activity was performed by GSH-Px detection kit (Ransel, RanDox Co, UK) based on manufacturer’s instructions. One unit of GSH-Px was defined as μM of oxidized NADPH per minute mg-1 of protein. A decrease in absorbance was recorded by spectrophotometry against blank, at 340 nm.

The SOD activity was evaluated at 505 nm using a standard curve. The SOD activity was determined by the SOD detection kit (RanSod, RanDox Co, UK) based on the manufacturer’s instructions. Serum NO level was measured according to the Griess reaction (17) and expressed as μM/l. CAT activity, on the basis of a previously described method, was evaluated. Here, the blood samples were homogenized in Triton X-100 1% (Merck, Germany) and then diluted using phosphate buffer (pH=7.0). For initiation of the reaction, hydrogen peroxide was added to the mixture and the level of enzyme activity on the basis of the competency of the CAT in decompensation of hydrogen peroxide, was determined. This was gained through scanning the decrease in absorbance at 240 nm against a blank containing phosphate buffer instead of the substrate. The value of log A1/ A2 of a measured interval was used for unit definition due to the initial reaction of the enzyme, where the value of A1 refers to the absorbance at 240, at time 0 seconds and A2 is the absorbance at 240, at second 15. These enzyme activities were expressed as U g-1 Hb in blood. Then the measurement of the protein level in supernatant took place using the colorimetric method of Lowry with bovine serum albumin (BSA) as standard (15).

The MDA level as an indicator of lipid peroxidation in serum was determined according to the procedure described by Buege and Aust. Here, 100 μl of serum specimens using a glass homogenizer was homogenized in 0.15 M/l KCl at a ratio of 1 to 9 ml. One volume of homogenate was blended thoroughly with two volumes of a stock solution of 15% w/v trichloroacetic acid, 0.375% w/v thiobarbituric acid, and 0.25 M/l hydrochloric acid. After heating and cooling cycles, the solution was clarified by centrifugation at 1000 ×g for 10 minutes. The absorbance of the clear solution was read at 535 nm and MDA content was figured out using 1.56 ×10⁵ M-1 cm-1 as molar absorbance coefficient. MDA levels are presented as mM per ml protein (15).

Evaluation of TAC was carried out on the basis of the manual of the kit (TAS test kit, Randox Laboratories Ltd, GB).

Immunohistochemical staining was done in order to analyze Hsp70-2 positive cells distribution. For this, before beginning the staining process, 5-μm tissue sections were heated at 60°C for approximately 25 minutes in a hot-air oven (Venticell, MMM, Einrichtungen, Germany). After deparaffinization in two changes of xylene, the sections were rehydrated using an alcohol gradient (96, 90, 80, 70, and 50%). The antigen retrieval process was performed in 10 mM sodium citrate buffer (pH=7.2). Immunohistochemical staining was conducted according to the manufacturer’s protocol (Biocare, USA). In brief, endogenous peroxidase was blocked in a peroxidase blocking solution (0.03% hydrogen peroxide containing sodium azide) for 5 minutes. Washing the sections was done with phosphate-buffered saline (PBS, DNAbiotech, Iran, pH=7) and subsequently incubation was performed with Hsp70-2 (1:600) biotinylated primary antibodies (Biocare, USA) at 4°C in humidified chamber overnight. After rinsing with PBS, the sections were incubated with streptavidin–HRP (streptavidin conjugated to horseradish PBS containing an anti-microbial agent) for 20 minutes. Followed by rinsing in washing buffer and adding a 3,3' Diaminobenzidine (DAB) chromogen, they were incubated for 10 minutes and counter stained with hematoxylin for 10 seconds. Then, the sections were dipped in ammonia (0.037 Ml), rinsed with distilled water, and cover slipped. Positive immunohistochemical staining could be observed as brown stains under a light microscope (8).

For RNA extraction, the collected testicles and those previously stored at -70°C, were used; RNA extraction was performed on the basis of the standard TRIzol method (8). For this, 20-30 mg of testicular tissue from an individual animal of each group was homogenized in 1 ml of TRIZOL. Then, in order to avoid genomic DNA contamination, the colorless aqueous phase was collected carefully. The quantitative assessment of RNA was performed using a nanodrop spectrophotometer (260 nm and A260/280=1.8-2.0), followed by storage of the samples at -70°C. For reverse transcriptionpolymerase chain reaction (RT-PCR), the cDNA was synthesized in a 20-μl reaction mixture containing 1 μl of oligo (dT) primer, 1 μl of RNAse inhibitor, 4 μl of 5X reaction buffer, 1 μg of RNA, 1 μl of M-MuLV reverse transcriptase and 2 μl of a 10 mM dNTP mix, on the basis of the manufacturer’s protocol (Fermentas, GmbH, Germany). The cycling protocol for 20 μl reaction mix was 5 minutes at 65°C, followed by 60 minutes at 42°C, and 5 minutes at 70°C to finalize the reaction. For evaluating the PCR reaction, a total volume of 27 μl containing primers pair's sequences (each 1 μl), 13 μl of PCR master mix and cDNA as a template (1.5 μl) and 10.5 μl of nuclease free water, were used. The following PCR conditions were considered; general denaturation at 95°C for 3 minutes for 1 cycle, followed by 35 cycles of 95°C for 20 seconds; annealing temperature (62°C for Hsp70-2, and 58°C for GAPDH) for 60 which participate in antioxidant defense system (6). Hsp70 functions as a cell supportive factor against many stresses that induce the production of reactive oxygen species(ROS), which in turn affect cellular molecules including DNA, proteins and lipids. Also, Hsp70 protein is known to be seconds; elongation: 72°C for 1 minute and 72°C for 5 minutes. Final PCR products were analyzed on 1.5% agarose gel electrophoresis and densitometric analysis of the bands were done by using PCR Gel analyzing software (ATP, Iran). The control was set at 100% and experimental samples were compared to the control. Specific primers were designed and manufactured by Sinaclon (Sinaclon Co., Iran). The primers pair's sequences and product size for primers used in RT-PCR are presented in Table 1.

The data was analyzed using SPSS program version 19.0 (SPSS Inc, Chicago, IL, USA). All results are presented as mean ± SD. Differences between quantitative histological and biochemical data were analyzed by oneway ANOVA, followed by Tukey test, using Graph Pad Prism, 4.00. The P<0.05 were considered statistically significant.

The results of histomorphometric studies showed that the thickness of testicular capsule in the high-dose group of aspartame, had a significant increase compared to the control group, whereas, the number of Sertoli and Leydig cells showed a significant decrease (P<0.05) in this group. Also, in medium- and high-dose aspartame-treated groups, a significant decrease (P<0.05) was observed in the diameter of the seminiferous tubules, the height of the germinal epithelium and the Johnsen’s score (Table 2).

Our histological observations revealed that aspartame, in a dose-dependent manner, could increase disarrangement and produce severe edema in connective tissue. An increase in germinal epithelium dissociation (GED) and tubular depletion in medium- and high-dose aspartametreated groups, was observed. Especially in the highdose group, aspartame could induce drastic morphologic changes in the testes. There were some atrophied seminiferous tubules indicating severe reduction in the number of germ cells and intensive immune cells infiltration, edematous fluid accumulation and intertubular space widening in interstitial connective tissue. Moreover, Sertoli cells lost their junction with germ cells and looked amorphous with irregular and smaller nuclei (Fig .1).

Also, concerning the histochemical features observed following Masson’s trichrome staining, it was found that the groups do not differ in the density of collagen fibers. Histochemical analyses of the PAS-stained specimens elucidated that the cells in three first layers of spermatogenesis cell series, Sertoli and Leydig cells faintly reacted with PAS in medium- and high-dose aspartame-treated groups and the carbohydrate ratio was severely decreased in their cytoplasm. In Sudan black B staining, in seminiferous tubules, brown to black particles which contain lipid were clearly seen inside the cytoplasm of the cells close to the lumina of seminiferous tubules and Leydig cells. No cytoplasmic lipids in Leydig cells and spermatogenesis series cells in the control group, were observed. Animals in the aspartame receiving groups showed high lipid-stained sites in the cytoplasm of the Leydig cells and spermatogenesis series cells. In testicular tissue section, alkaline phosphates staining indicated the highest rate of small brown particles in the cytoplasm of Leydig cells and spermatogenesis cells in the high-dose group, compared to the other groups. In addition, it should be noted that the level of alkaline phosphatase reaction in the groups treated with aspartame was dose-dependently increased (Fig .1).

Observations showed that aspartame in a dose-dependent manner, significantly (P<0.05) reduced the sperm count. Survival rate and sperm motility in medium- and high-doses aspartame-treated groups were significantly decreased (P<0.05) compared to the control group. Also, the average percentage of abnormal sperms as well as the percentage of sperms with damaged DNA, in medium- and high-dose aspartame-treated groups was significantly increased (P<0.05, Fig .2, Table 2).

Aspartame effects on various parameters of oxidative stress biomarkers in serum and blood samples are shown in Figure 3. As can be seen, aspartame administration resulted in a significant increase (P<0.05) in MDA levels in the high-dose group as well as NO in medium- and high-dose aspartame-treated groups compared to the control group. Also, our observations showed that aspartame could induce a significant decrease (P<0.05) in TAC and CAT activity in the highdose group and consequently led to a significant decrease (P<0.05) in the level of GSH-Px and SOD in both medium- and high-dose aspartame-treated groups compared to the control group.

The mRNA and protein levels of Hsp70-2 were analyzed. In order to clarify Hsp70-2 expression in different cellular layers of germinal epithelium, immunohistochemical analyses were done. Our finding revealed that, biosynthesis of Hsp70-2 increased in low-dose aspartame-treated group (especially at spermatocytes and spermatids cell lineages) versus the control group. However, it was significantly decreased in medium- and high-dose aspartame-treated groups. The immunohistochemical results were confirmed by the semiquantitative RT-PCR analysis. A significant (P<0.05) increase in the mRNA level of Hsp70-2 was observed in the animals treated with low-dose aspartame. However, the mRNA levels of Hsp70-2 decreased in medium- and high-dose aspartame-treated groups (Fig .4).