Histopathological and biochemical effect of quercetin on monosodium glutamate supplementation-induced testicular toxicity

Despite the wide usage of monosodium glutamate (MSG) as a flavor enhancer in many types of food, it has been reported as a toxic agent to humans and experimental animals. It also adversely influences male fertility. Several research studies attributed detrimental effects of MSG on reproductive organs to oxidative stress. The current study investigated the effects of MSG on testis and the potential role of quercetin in attenuating them. MSG-treated rats showed a considerable elevation in lipid peroxidation level and reduction in glutathione concentration, superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities in the homogenate of testis tissues. Treatment with quercetin in combination with MSG provided significant protection. When QU was used, the toxic side effects were significantly reduced, with a considerable reduction in lipid peroxidation and an increase in SOD and GPx activities, and glutathione concentration. Quercetin may be used in combination with MSG to improve the histopathological, ultrastructure, oxidative stress, and biochemical parameters of testicular toxicity induced by MSG due to its antioxidant effects.


Background
Monosodium glutamate (MSG) is the sodium salt of glutamic acid [1]. Glutamate is one of the most often encountered amino acids in nature. [2]. MSG produces a flavor called umami savory'' [3]. Its use as a flavor in cooking has grown in popularity across the world in recent years [4]. MSG delivers umami flavor to food only when it is present in its free form, not when it is linked along with other amino acids in protein [5,6]. Free glutamate liberates during the fermentation of starch, sugar, beet, sugarcane or molasses, aging, ripening, and heat cooking process [6,7].
MSG has been reported as toxic to humans and experimental animals [8][9][10][11]. According to previous studies, MSG has the capacity to cause a wide range of harmful adverse effects on organs, including the liver [12,13] and ovary [14,15]. Furthermore, Pavlovic et al. [16] have indicated that administration high concentration of MSG induced oxidative stress in rat thymus, which could possibly mediate thymocyte apoptosis triggered by MSG.
Dysfunction of male reproductive is a case whereas malfunctioning of structures of the male reproductive system occurs as a result of various factors such as diet, reactive oxygen species (ROS), hormonal disorders, testicular inflammation, endocrinal disturbance, genital infection, chronic health problems, and genetic defects [11].
Many research studies have been conducted on the utilization of natural antioxidants and dietary supplements. Phenolic chemicals, such as flavonoids, not only extend Page 3 of 13 Abdul-Hamid et al. Beni-Suef Univ J Basic Appl Sci (2021) 10: 73 the shelf life of food but also function as antioxidants in a variety of biological systems [21][22][23][24]. Quercetin (QU) is a flavonoid found mostly in apples, tea, onions, almonds, cauliflower, cabbage, and a variety of other foods. It has anticarcinogenic [24,25], anti-inflammatory [26], antiviral, and antibacterial effects [27,28]. Quercetin has been reported to be a powerful antioxidant [25]. Its high antioxidant activity is due to the presence of aromatic hydroxyl groups in its structure [29]. There are several hypotheses to explain the antioxidant mechanisms by quercetin including oxygen radical scavenging and provides protection against lipid peroxidation [30], metal ion chelation, and modulation of cell antioxidant responses [31]. It also protects macrophages from oxidative stress by keeping glutathione in its reduced form [32].
Several studies [33,34] have demonstrated quercetin's protective properties against different toxic agents and generated oxidative stress in several organs. A study by Farombi and Onyema [35] has indicated that QU effectively attenuated MSG-induced lipid peroxidation and modulated altered antioxidant status and antioxidant capacities in liver, kidney and brain of the rat. Furthermore, a recent study revealed that QU alleviated MSGinduced excitotoxicity of the spinal cord tissue in aged rats by suppressing the induced oxidative stress and impeding the programmed cell death via targeting p38 MAPK pathway [36]. On the other hand, although, quercetin is used as a supplementary diet for various health benefits, there are safety concerns regarding its use especially in cancer patients [37] Thus, this study is designed to explore the protective benefits of quercetin against MSG-related testicular toxicity.

Chemicals
Monosodium glutamate (MSG) and quercetin (QU) were purchased from Sigma-Aldrich (Nasr City, Cairo, Egypt). The biochemical kits were purchased from Human Diagnostics Chemical Company, Germany.

Animals
Thirty white male albino rats (Rattus norvegicus) from VACSERA (the Egyptian Organization for Biological Vaccine Production breeding unit, Cairo, Egypt) weighting about 140-160 g. Animals were kept under observation for about 15 days before the onset of the experiment to exclude any intercurrent infections and were housed in stainless steel cages in the department of animal house and given enough food and water ad-libitum. The animals were weighed weekly during the experimental periods. At the end of experiment, the rats were sacrificed by anesthesia inhalation under light diethyl ether (5%). This study was carried out according to the guidelines by the Animal Ethics Committee (Approval number is BSU/FS/ 2015/9).

Experimental design
Male albino rats were grouped as the following (six animals each): • Group (1): Normal control group was administered distilled water.

Blood sampling
After coagulation at room temperature, blood samples was centrifuged at 3000 r. p. m for 15 min, the serum samples were removed and kept at − 20 °C until for testosterone estimation.

Tissues sampling and homogenate preparation
Rats in each group were sacrificed under moderate anesthesia at the end of the experiment. Around 0.5 g of testes tissues were removed quickly, ice-cooled and homogenized in 5 mL of 0.9% NaCl (1 percent w/v) using a Teflon homogenizer (Glas-Col, Terr Haute, IN, USA). The homogenate was centrifuged at 3000g for 15 min at 4° C. The supernatant was collected and preserved at − 20 °C until use. Lipid peroxidation, glutathione content (GSH), glutathione peroxidase (GPx), and superoxide dismutase (SOD) activity are all measured as part of the antioxidant defence system. Furthermore, for histological investigations, portions of testes tissues were preserved in neutral buffered formalin.

Antioxidant-capacity assay 2.6.1 Determination of lipid peroxidation level
Lipid peroxidation in testes homogenate was assessed according to Pressus et al. [40]. Malondialdehyde (MDA) formed from the breakdown of polyunsaturated fatty acids serves as a convenient index for determining the extent of lipid peroxidation. Lipid peroxidation products are quantified by their reaction with thiobarbituric acid.

Determination of glutathione content
Glutathione content (GSH) level was measured according to Beutler et al. [41]. The method based on the reduction of 5,5′dithiobis (2-nitrobenzoic acid) (DTNB) with glutathione (GSH) to produce a yellow compound. The reduced chromogen directly proportional to GSH concentration and its absorbance can be measured at 405 nm.

Determination of glutathione peroxidase activity
Glutathione peroxidase activity (GPx) activity was determined according to Paglia and Valentine [42]. The assay is an indirect measure of the activity of c-GPx. Oxidized glutathione (GSSG), produced upon reduction of an organic peroxide by c-GPx, is recycled to its reduced state by the enzyme glutathione reductase. The oxidation of NADPH to NADP + is accompanied by a decrease in absorbance at 340 nm (A340) providing a spectrophotometric means for monitoring GPx enzyme activity.

Determination of superoxide dismutase activity
Finally, according to Nishikimi et al., [43], this assay relies on the ability of the purified SOD to inhibit the initial rate of photo activated phenazine methosulphate-mediated reduction of O2° to O2 which then reduced nitroblue tetrazolium dye. 1.5 U/assay of the purified enzyme produced 80% inhibition.

Determination of testosterone level
Serum testosterone was determined by ELISA, based on the principle of competitive binding, according to Huang et al. [44] Darney [45] and Sakuma et al. [46]. The testosterone (an unknown amount) presents in the sample and a defined amount of testosterone conjugated to horseradish peroxidase compete for the binding sites of testosterone antiserum coated to the wells of a microplate, incubated for one hour on a shaker and then the microplate is washed four times. After addition of the substrate solution, the concentration of testosterone is inversely proportional to the optical density measured.

Light microscopic study
Paraffin sections prepared according to Bancroft et al. [47]. Testicular tissue samples were fixed in 10% neutral buffered formalin (pH 6.8). Then, tissue samples were dehydrated before being embedded in paraffin wax, sectioned at 4-5 µm, and stained with hematoxylin and eosin for histological analysis.

Transmission electron microscope (TEM) preparation
TEM thin sections were processed as reported by Bozzola and Russell [48]. 1-3 mm segments of testis were promptly fixed in 3% glutaraldehyde (freshly prepared), washed in phosphate buffer (pH 7.4) and then postfixed in osmium tetroxide (1%). Then, the samples were embedded in fresh pure resin at room temperature overnight before being transferred to capsules containing fresh resin. Semi-thin sections of testis tissues were cut and stained with toluidine blue. Ultrathin sections were prepared and stained to be examined on a TEM (model CX 100, JEOL, Japan) operated at 60 kV.

Statistical analysis
Data were analyzed using one way analysis of variance (PC-STAT software program) followed by least significant degree (LSD) analysis to evaluate multiple comparisons between different groups [49]. The data are presented as means ± Standard error, and values of P < 0.01 and P < 0.001 are significantly different.

Antioxidant-capacity assay
The MSG-treated group displayed a significant increase in level of MDA level in comparison to their respective control group (Fig. 1). Also, MSG administration resulted in a significant reduction in GSH content tissue along with GPx and SOD activities (Figs. 2, 3, 4) respectively. However, simultaneous administration of QU along with MSG significantly (P < 0.0001) mitigated the altered level of GSH, GSH-Px, and SOD activities when compared with MSG-treated group. When compared with the control and corn oil groups, rats treated with QU only depicted nearly comparable outcomes. The observed changes in serum testosterone are shown in (Fig. 5). A significant decrease in serum testosterone level in MSG-treated rats compared to the control rats was observed. Administration of QU to MSG-treated rats revealed a significant (P < 0.0001) amelioration in the altered level of testosterone when compared with the MSG-treated group.

Histopatological results
Testes of control rats (Fig. 6a-d) and quercetin treated rats ( Fig. 6e and f ) showed a normal morphology for the seminiferous tubules and interstitial tissue containing Leydig cells. Each tubule possessed epithelial cells involving Sertoli cells and germ cells which showed the complete process of spermatogenesis (Fig. 1b, d, and f ). Sertoli cells contain irregular nuclei, which were usually found toward the basement membrane of the seminiferous tubule. Spermatogonia were oval in shape, resting upon the basal lamina of the seminiferous tubule, above them were spherical primary spermatocytes, recognized by their copious cytoplasm and large nuclei containing coarse clumps of chromatin (Fig. 6b, d, and f ). MSG-treated rats' testicular tissues showed numerous histological abnormalities (Fig. 7ad). Irregular, shrunken and disorganized seminiferous tubules were observed (Fig. 7a). Congested blood vessels are also seen in (Fig. 7a and b). Degenerated interstitial tissue, irregular variable-sized seminiferous tubules, and apparent reduction in spermatogenic cells and sperms numbers (Fig. 7b). Moreover, detachment of germ cells from the basal lamina and cytoplasmic vacuolation were also seen (Fig. 7c). Pyknotic nuclei, vacuolation, sloughing of spermatogonia cells into the lumen of seminiferous tubules, decrease in number of sperms and widening of intercellular spaces are seen in (Fig. 7d). Testes treated with MSG and quercetin ( Fig. 8a and b) revealed a significant amelioration in testis structure. Tubules of MSG rats treated with quercetin restored their normal shape, spermatogenic layers and sperms except some tubules still have a marked decrease in the number of sperms and vacuolation.

Ultrastructure evaluation
Electron microscopic examination of the testis of control rats showed each seminiferous tubule to be lined with spermatogonia that attached to the thin basal lamina, primary spermatocytes having mitochondria and large spherical nuclei ( Fig. 9a and b). Sertoli cells have a triangular nucleus (Fig. 9b). Round spermatids with spherical nucleus, Note the differentiated part of acrosome (Fig. 9c). Cross-sections at the midpiece of sperms' tails containing 9 mitochondria and microtubules are observed ( Fig. 9d and e). Leydig cells with a normal nucleus and a marked increase in lipid droplets are seen (Fig. 9f ). The MSG-treated group revealed ultrastructural alterations vacuolization in spermatogenic cells, autolysis in spermatognia, and primary spermatocytes with a damaged nucleus and fragmented chromatin (Fig. 10a). Also, Sertoli cell with vacuolation is observed (Fig. 10b). Separation of part of the round spermatids with a degenerated nucleus, cytoplasmic vacuoles, damaged mitochondria and lysosomes are seen (Fig. 10c). Spermatid with irregular membrane, increased intercellular spaces and marked decrease in the number of sperms in lumen of seminiferous tubule (Fig. 10d). Degenerated interstitial tissues, Lydeig cells with abnormal nuclei, dilated rough endoplasmic reticulum and rapture of the nuclear membrane are observed in (Fig. 10e).
The treatment of MSG rats with quercetin led to a significant improvement in testis tissues. The basement membrane was thin, spermatogonia and Sertoli cells looked normal with normal nucleus (Fig. 11a). The primary spermatocytes retained their normal organelles except for the presence of some vacuoles (Fig. 11b). The round and late spermatids retained their normal structure and the moderate number of sperms appeared in (Fig. 11c). Leydig cells with normal nucleus, mitochondria, and lipid droplets (Fig. 11d).

Discussion
Despite its widespread usage as a flavor enhancer in cooking, monosodium glutamate (MSG) is reported as a toxic agent that affects the structure and function of the male reproductive system in both humans and experimental animals [50]. The current study investigated the biochemical, histopathological, and ultrastructure alternations resulted from MSG-induced toxicity in the testis of adult rats. This study also assessed the amelioration effects of QU against MSG-induced testicular toxicity in rats.
MSG-induced testicular toxicity could be attributed to ROS-induced oxidative stress and lipid peroxidation. MSG has been reported to cause oxidative damage in testis [17,34,51,52].
Our data revealed that MSG administration resulted in an elevation in the level of lipid peroxidation in form of (MDA) in testis tissue of MSG-treated rats. The increased level of lipid peroxidation resulted in oxidative damage to the development of spermatozoa, which alters membrane functions according to [53,54]. In the current study, the elevation in MDA is also accompanied by depletion in the activity of antioxidant enzymes like, GSH, GPx and SOD which is considered as markers of cell damage according to Armand et al. [55], who mentioned that the high Our findings support the argument which suggested that monosodium glutamate (MSG) exposure induces lipid peroxidation and oxidative damage in [51,52,56] which may result in a decrease of ascorbic acid level [57]. Ascorbic acid is an endogenous antioxidant that presents in the testes to protect it from oxidative damage. When monosodium glutamate (MSG) induces oxidative damage to testis, the cell used ascorbic acid as a ROS scavenger which explains the depletion in its level [19,34,58].
Moreover, the current biochemical study revealed a decrease of serum testosterone levels of MSG-treated rats in comparison to the control group. These results are inconsistent with the findings [59,60].
The reduction in blood testosterone levels observed in the current investigation might be due to a direct harmful impact of Leydig cells which are responsible for the synthesis of testosterone hormone [61]. In another study, the researcher suggested that MSG administration destroys neurons of the hypothalamus which can lead to disturbance of the hypothalamic-pituitary-testis regulatory axis, which regulates testosterone synthesis by testicular Leydig [62,63]. This disturbance might explain the reduction in serum testosterone levels seen in the current study.
In harmony with prior research findings [15,18,20,64], the present investigation of histopathological results of MSG-treated rats represented by alterations of the seminiferous tubules which included atrophied seminiferous tubules, decrease in bundles of sperms, pyknotic nuclei, dilated and congested intertubular blood vessels, and cytoplasmic vacuolation.
Cytoplasmic vacuolation is considered as defense mechanism against harmful oxidative stress effects and vacuoles may collect these damaging substances preventing them from interfering with the biological activities of these cells [65,66]. Therefore, current study postulated that cytoplasmic vacuolation that appeared in testis might be a result of testicular toxicity and cell degeneration triggered by MSG according to [2,65]. Pyknosis of cell nuclei may clear the loss of functional efficiency of the cells [67]. Balasubramanian et al. [68] revealed that congestion of blood vessels observed in testicular tissue of MSG-treated rats is related to the suppression of prostaglandin production, which is known to be involved in the control of testicular circulation.
The current ultrastructure investigation of the testis of MSG-treated rats shows degeneration of Sertoli cells and spermatogenic cells, primary spermatocyte and Leydig cells with numerous cytoplasmic vacuoles and damaged mitochondria [69,70] explained these vacuoles by degeneration of abnormal germ cells resulting in wide gaps between nearby Sertoli cells. The current work also recorded enlarged intercellular spaces between spermatid, shrunken nuclei, irregular distribution of mitochondria and lysosomes in accordance with [71].
The histopathological and ultrastructure damage of testis may be explained by different mechanisms. First, MSG can directly influence testicular epithelial cells via functioning glutamate transporters and receptors in rat testes, inhibiting spermatogenesis [64,72,73]. The second mechanism suggested that MSG-induced oxidative damage may destroy most of the testicular germ cells and result in a decrease in testicular weight [57].
In the present investigation quercetin remarkably diminished the testicular histological and morphometric alterations produced by numerous toxic substances Uygur et al. [74].
As one of the strong antioxidants, quercetin can impact the redox state of cells by boosting the expression of c-glutamylcysteine synthetase, the rate-limiting enzyme in the production of GSH. [75,76]. Amália et al. [77] have also reported that quercetin acts as a promoter enhanced SOD and GPx levels.
Quercetin substantially reduced lipid peroxidation and boosted the antioxidant activity of SOD, GPx, and GSH concentrations in the current research. These results are in accordance with [78][79][80].
Our data also revealed that QU treatment enhanced testosterone levels in MSG-treated animals. These findings are in agreement with Khaki et al. [81] who reported that testosterone levels in diabetic animals enhanced after the treatment with QU.
So, the present data showed that many histological alterations and ultrastructure changes as well as the change in serum testosterone level induced by MSG in testis of rats were significantly improved after treatment with quercetin. The presence of aromatic hydroxyl groups in quercetin's structure, which serve as ROS scavengers, is responsible for its high antioxidant effects. [29,82].

Conclusions
Our study concluded that quercetin can efficiently modulate the MSG-induced toxicity in the rat testis and this action appears to rely mainly on QU's antioxidant properties. Our study showed that quercetin supplementation ameliorated the MSG-induced oxidative damage which was marked by lipid peroxidation, a decrease in enzymatic antioxidant (GPx and SOD), and non-enzymatic antioxidant (GSH), and low testosterone levels as well as severe histopathological and ultrastructure changes in the testis.