Effects of monosodium glutamate on testicular structural and functional alterations induced by quinine therapy in rat: An experimental study

Abstract Background Quinine (QU) as an anti-malarial drug induces alterations in testicular tissue. Toxic effects of monosodium glutamate (MSG) on the male reproductive system have been recognized. Objective To investigate the impact of MSG administration on the intensity of gonadotoxicity of QU. Materials and Methods Sixty eight-wk old Wistar rats weighing 180-200 gr were divided into six groups (n = 10/each): the first group as a control; the second and third groups received low and high doses of MSG (2 & 4 gr/kg i.p.), respectively, for 28 days; the fourth group received QU for seven days (25 mg/kg); and in the fifth and sixth groups, QU was gavaged following the MSG administration (MSG + QU) from day 22 to day 28. Serum testosterone and malondialdehyde (MDA) levels were measured. Testes samples were prepared for tissue MDA levels, histomorphometry, and immunohistochemistry of p53. Sperm analysis was performed on cauda epididymis. Results Serum and tissue MDA levels were increased in treated groups compared to the control group. This increment was higher in the MSG + QU groups. The testosterone levels were reduced significantly (p < 0.0001) in all treated groups. In addition, histomorphometric indices and tubular epithelium population were reduced significantly (p < 0.0001) in QU, MSG + QU, and consequently in high-dose MSG, QU, MSG + QU groups. All spermatogenic indices were reduced in the treated groups, particularly in the MSG + QU groups. Sperm motility and viability indices were reduced significantly (p = 0.003) in the MSG + QU groups. Finally, the overexpression of p53 was observed in the MSG + QU groups. Conclusion The administration of MSG before and during QU therapy may intensify testicular tissue alterations.


Introduction
Quinine (QU) is one of the most common medications used for the treatment of malaria disease due to Plasmodium falciparum. QU treatment is associated with side effects such as irreversible deafness, amblyopia, and at high doses can result in cinchonism syndrome (1).
Some studies have reported QU-induced toxicity on the male reproductive system. Histological alterations of the seminiferous tubules (STD), reduction in testosterone levels, decrease in sperm population and motility, and changes in lipid peroxidation indices have been observed following the administration of QU in experimental animal studies (2,3).
Monosodium glutamate (MSG), a sodium salt of L-glutamic acid, is the most common food additives that act as a preservative or improves the meal palatability (4). Changing lifestyles and increased access to processed food have led to increased MSG consumption. In this regard, it It has been reported that the average daily intake of MSG through processed foods is about 1 gr in some European countries, 4 gr in Asia, and 10 gr in Germany (5). Some studies have identified the negative toxic effects of MSG on the male reproductive system (6)(7)(8). It has been documented that MSG-induced testicular toxicity is associated with increased oxidative stress (4). Oxidative stress can induce the overproduction of reactive oxygen radicals and hydrogen peroxide leading to oxidative DNA damage and peroxidation of cell membranes and consequently cell death (6).
QU as a common antimalarial medication has various side effects on the male reproductive system and MSG is in abundance in many processed foods; these factors can increase the possibility of the simultaneous consumption of these cytotoxic compounds. Accordingly, this study was designed to investigate the potential role of MSG administration prior to QU therapy on the intensity of the QU-induced structural and functional alterations of testicular tissue through the experimental animal study.

Animals and exposure
In this experimental study, Sixty eight-wk old Wistar rats weighing 180-200 gr were used. The animals were placed in standard cages under a 12-hr light/dark cycle. During the period of the experiment, the standard laboratory chow and water were available ad libitum to the animals.
QU hydrochloride was solubilized in distilled water (20 mg/ml as stock treatment solution) and administered orally (gavage) at a dose of 25 mg/kg once a day for a period of seven days (2). MSG was solubilized in distilled water (concentration: 1 g/ml) and administered intraperitoneally at low-and high doses (2 and 4 gr/kg consequently), respectively, once a day for 28 days (9, 10). The animals were randomly divided into six experimental groups (10 rats/group): In group 5 and 6, QU was gavaged following the MSG administration from day 22 to day 28 ( Figure  1).

Biochemical analysis
Twenty-four hours after the final treatment, the animals were anesthetized with xylazine hydrochloride (10 mg/kg i.p.) and ketamine hydrochloride (100 mg/kg i.p.). The blood samples were collected through cardiac puncture. The assessment of the serum testosterone levels was carried out through the standard ELISA method with a commercial assay kit (Monobind Inc. USA).

Sampling and preparation
The animals were euthanized through sodium thiopental (100 mg/kg i.p.). The left and right testicles were separated and dissected from their epididymis and weighed as total testes weight. Moreover, the organ relative weight (i.e., the organ weight/body weight × 100) was recorded. The left testicles were used for histologic studies and the right testicles were prepared for tissue lipid peroxidation measurement and immunohistochemistry (IHC). The sperm analysis was prepared on the left epididymides.

Serum and tissue lipid peroxidation levels
The quantification of the serum and tissue lipid peroxidation was completed by the determination of thiobarbituric acid levels (11). Testicles samples were homogenized in 50 mM Tris/HCl, pH 7.5 (1/10, w/v) and centrifuged at 3000 g for 10 min. An aliquot of serum or tissue samples were incubated (95°C) for 2 hr with thiobarbituric acid. The sample-coated microplates were analyzed using a microplate reader and the absorbance was measured at 532 nm.

Histomorphometry
Testicular samples were fixed immediately in 10% neutral buffered formalin solution, dehydrated, and paraffin-embedded. The sections were stained with hematoxylin and eosin (H&E) and analyzed with an optical microscope (Olympus CX22, Tokyo, Japan).
The measurement of the height of germinal epithelium (GEH) and the diameter of STD was performed on the images obtained via Am Scope digital camera (Am Scope MD500). The images were processed by the image analysis software (Am Scope, Version ×86, 3.7.7934).

The population of spermatogenic cells lineage
The number of Sertoli cells, the spermatogonia, the spermatocyte, and the round spermatids were counted in 20 cross-sectioned STD and reported as the mean of the cellular population (total counted cells/20 tubules) for every type of cells (12).

Microscopic indices of spermatogenesis
The quantitative investigation of spermatogenesis in testicular tissue was completed by the measurement of three indices: tubular differentiation index (TDI, the number of STD with more than three layers of germinal cells derived from type-A spermatogonia); spermiogenesis index (SPI, the ratio of STD with spermatozoids to the empty tubules); and repopulation index (RI, the ratio of active spermatogonia to inactive cells) (13).

IHC of p53
Paraffin-embedded tissue sections were prepared for immunohistochemical study (14). Briefly, tissue section samples were deparaffinized in xylene and rehydrated through alcohol gradients (90%, 80%, 70%, and 50%). Antigen retrieval was carried out on deparaffinized and rehydrated slides kept in 10 mM sodium citrate solution (pH 6.0) at a temperature of 95°C in a water bath for 40 min. Immunohistochemical staining was conducted according to the manufacturer's protocol (St John's Laboratory Ltd., UK). Endogenous peroxidase activity was blocked with 0.3% H 2 O 2 . Tissue slides were washed with PBS (pH 7.2) and incubated with primary antibody (1:500) at 4°C overnight. Sections were treated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (as secondary antibody) (Agrisera Antibodies, SE-911 21 Vännäs, SWEDEN) at 37°C-incubator humidified chamber container with a wet paper towel for 1 hr. Diaminobenzidine (DAB) chromogen was added to tissue sections and incubated for 5 min. Tissue slides were dehydrated and coverslipped after hematoxylin counterstaining.

Epididymal sperm analysis
Sperm analysis was conducted on the left epididymides. For sperm counting, the cauda epididymis was cut into small pieces. The contents of epididymis were diluted using the HTF medium (1:200 v:v). After shaking once, 10 µl of the specimen were transferred to a Neubauer hemocytometer and placed for 5 min in the humidified chamber. The number of counted cells in five squares with a light microscope under a 20× microscope objective was expressed as the number of sperm/ ml (15).
For the evaluation of sperm motility percentage, one drop of the specimen was placed on an incubated glass slide (37°C) and covered with a lamella. The percentage of motile cells was recorded in 10 different microscopic fields under a 20× microscope objective.
The evaluation of sperm viability was carried out by adding 10 µl of 0.50% eosin Y and nigrosin staining solution into an equal volume of the specimen. The examination was done on the slides incubated for 2 min at room temperature. The head of dead sperm cells was stained with pink while the head of live cells appeared pale. One hundred randomly chosen spermatozoa were evaluated under a 100× microscope objective.

Statistical analysis
Statistical analyses of evaluated parameters were performed using one-way ANOVA followed by Tukey's test. All data are expressed as mean ± SD. Statistical analyses were performed using the GraphPad Prism software (version 5.04; Graph Pad Inc., CA, USA). P < 0.05 was considered as statistically significant.

Weight of testicles
The mean testicular weight was decreased in all treated groups compared to the control group (Table I). This reduction was significant between the control and the HD-MSG + QU groups (p = 0.0119). No significant difference was seen in the organ relative weight between the control and treated groups. (p = 0.344)

Serum testosterone levels
The blood concentration of testosterone was reduced significantly (p < 0.0001) in all treated groups in comparison to the control group (Table I)

Lipid peroxidation
The levels of malondialdehyde in the serum and testicular tissue were increased in all treated groups compared to the control group (Table I)

Testicular tissue morphometry
The mean diameter of the STD was reduced in all treated groups compared to the control group (Table I)

Microscopic indices of spermatogenesis
The mean of all indices of spermatogenesis was reduced non-significantly in MSG-treated groups in comparison to the control group (Table   II). The administration of the QU or the LD-MSG + QU led to a significant decrease in the TDI and RI and a non-significant decrease in the SPI compared to the control group.
Moreover, the administration of the QU following the higher dose of MSG led to a significant decrease in all indices of spermatogenesis in comparison to the control and the LD-MSGreceiving groups.

Sperm analysis
All indices of sperm analysis were reduced non-significantly in the MSG-receiving groups in comparison to the control group (Table   II)

Histology of testicular tissue
The histologic study showed various alterations in testicular tissue of treated groups in comparison to the control group ( Figure   2). The atrophy of STD and loss of tubular architecture, a decrease in the germ cells population, impaired cellular arrangement, and the increase in the interstitial connective tissue were the most prominent alterations observed in the treated groups. These changes were observed in higher degrees in the MSG + QU groups.

IHC of p53
Immunostaining

Discussion
In immune responses (17). Some MSG-related side effects such as brain neurotoxicity, obesity and metabolic defects, Chinese restaurant syndrome, and the negative effects on reproductive organs have been discussed more (18).
In this study, the mean testicular weight was This study demonstrated that the administration of the MSG or the QU led to a significant reduction in the blood testosterone levels. This reduction was seen more once the QU was gavaged to rats after MSG consumption. The reduction of testosterone levels has been reported in the QU-treated rats (2,21). Also, some studies have pointed out the effects of MSG on testosterone reduction (7,22).

Conclusion
This study showed that in adult rats, the administration of MSG prior to QU therapy increases the gonadotoxic effects of this antimalarial drug. This study indicates a potential role of MSG (as a common dietary supplement with cytotoxic and organotoxic characteristics) in increasing the QU-induced alterations in the male reproductive system. However, further studies are required to explain the mechanisms involved in the incidence of the aforementioned changes.