DILTIAZEM PREVENTS MONOSODIUM GLUTAMATE TOXICITY IN THE RAT TESTES

Increased exposure to food additives may exhibit harmful effect on the male reproductive system. Neonatal application of high doses of popular taste enhancer monosodium glutamate (MSG) to different kinds of animals cause lesions of the hypothalamic nuclei and the retina. Later in adulthood, animals exhibit a series of neuroendocrine disorders (stunted growth, obesity and decreased fertility). The mechanism of MSG action is not completely explained yet. We hypothesized that high concentration of MSG could alter permeability of neural membrane for calcium. The objective of our study was to find out whether the pretreatment with diltiazem, a calcium channel blocker, could prevent harmful effect of MSG in the rat testes. Male rat pups were treated with: 0.9% sodium chloride (C group), 4 mg/g BW of MSG (M group), 5 mg/g BW of diltiazem (D group) and diltiazem 5 mg/g BW with MSG (DM group) on 2 nd , 4 th , 6 th , 8 th , 10 th postnatal day. Animals were sacrificed at the age of six months. MSG treatment resulted in: stunted growth (decreased naso-anal length and tail length; p < 0.05), decreased relative testis weight (p < 0.05), and increased adipose tissue mass (Lee index; p < 0.05), testicular atrophy and decreased histomorphometric parameters (tubular area, tubular perimeter, Feret diameter, tubular diameter, epithelial height; p < 0.001). The rats of C, D and DM groups had normal testicular histology and morphometric parameters. Pretreatment with diltiazem has prevented the development of morphological disorders of testes. Our results suggest that calcium overloading may play an important role among mechanisms of MSG testicular toxicity. Acta Medica Medianae 2020;59(3):20-26.


Introduction
The decrease of male fertility has been reported in numerous countries over the past few decades. The meta-analyses data showed a decline in both quality and quantity of human sperm (1)(2)(3)(4)(5)(6). A modern life style carries increased exposition to different chemical, biological and physical etiologic factors. The harmful effects on male fertility are the most often attributed to: infections, the negative influence of alcohol, cigarette smoking habit, drug addiction, exposition to heat, excessive exercising, overweight or underweight, exposition to toxic products (lead, cadmium, etc.) and iatrogenic factors (7)(8)(9)(10).
A certain risk is hidden in increased use of food additives like monosodium glutamate (MSG). This popular taste enhancer is widely used in commercial and domestic food preparation. MSG is the salt of nonessential glutamic acid. It has a property to enhance the perception that flavors are well blended and full-bodied and disguise unwelcome tastes. This additive is present in almost all food products: dehydrated soups or sauces, canned and frozen foods and meals, fresh sausages, marinated meats, and stuffed or seasoned chicken, bottled soy or oriental sauces, manufactured meats, some hams, flavored tuna, vegetarian burgers and sausages, flavored chips and snacks (11)(12)(13)(14).
The way of MSG action is not completely explained yet. It is well known that MSG has high excitotoxic potential. We hypothesized that high concentration of MSG could alter permeability of neural membrane for calcium which could be involved in the mechanisms of MSG toxicity.
The objective of the present study was to examine whether the pretreatment with L-calcium channel blocker, diltiazem, may prevent toxic effect of MSG in Wistar rat testes.

Materials and methods
The study was carried out in 24 neonatal male Wistar rats. The pups were injected sub-cutaneously interscapulary on the 2 nd , 4 th , 6 th , 8 th and 10 th day of life with equal volume of: 0.9% of sodium chloride solution (C group), 4 mg/g BW of MSG (M group), 5 mg/g BW of diltiazem (D group) and 5 mg/g BW of diltiazem and 60 minutes later with 4 mg/g BW of MSG (DM group).The animals were housed in standard cages under controlled laboratory conditions. The room temperature was 23 ± 2 O C and air humidity 50 ± 5%. The rhythm of light and darkness was established (light phase from 6.00 a.m. to 6.00 p.m.). The pups were weaned at age of 28 days and subsequently had free access to tap water and to standard laboratory chow pellets ("Veterinarski zavod" Zemun). The animals were sacrificed at the age of six months under pentobarbital sodium anesthesia (40 mg/kg BW intraperitoneally). Biometric parameters were measured: body weight, naso-anal length, tail length, absolute and relative testis weight and Lee index. The Lee index was used for assessment of obesity. It was calculated by the formula: cube root of body weight (g) x10 3 /naso-anal length (mm) for each animal (45)(46)(47).
The testes were carefully removed, cleaned of the surrounding tissue and weighed. The relative testis weight was then calculated as: absolute testis weight (g)/[body weight of rat (g)] x100 for each animal (48). The testis tissue was prepared according to appropriate procedures then stained routinely with periodic acid-Schiff (PAS) and with haematoxylin-eosin (H-E methods), later analyzed and described. Serial sections stained with haematoxylin and eosin were subject of histomorphological examinations. Histomorphometric analysis of the testes was performed on Leica microscope equipped with Leica DC 180 and DC 480 camera using Image J v1.39d. programme. The tubular diameter was measured on at least 30 randomly chosen tubular profiles of round or nearly round shape for each of 3 sections per animal. Area, perimeter and diameter were measured at x20 magnification. The height of the seminal epithelium was measured at x40 magnification at randomly chosen tubular profiles of oval or round shape with at least 6 measurements per tubule, including the highest and the shortest part.
Results of statistical analysis are expressed as means ± standard deviation (SD). Statistical significance was determined with analysis of variance (ANOVA) test. The differences were considered significant at p < 0.05 or p < 0.001 level. All statistical analyses were performed using the SPSS statistical software (Version 15). All procedures on animals followed Guideline for Work on Experimental Animals approved by the Ethic Committee of Faculty of Medicine, University of Niš.

Results
Statistically significant difference of mean values between analyzed groups was present in all analyzed biometric parameters except in body weight (Table 1). Parameters: naso-anal length, tail length, and Lee index in rats of M group were significantly lower (p < 0.05) than in rats of the other groups (C, D and DM). Absolute testis weight was significantly lower in rats of M group than in rats of C and D group. Relative testis weight was sig-nificantly lower in M than in D group of rats.
Histological examination revealed testicular atrophy in MSG treated rats. The most prominent histological changes in testes of MSG treated rats were: reduced diameters of the seminiferous tubules with decreased numbers of germ cells and decreased spermatogenesis, edematous interstitium and not prominent Leydig cells (Figure 1. M). Normal testicular histology was found in animals of control group, D and DM group (normal semini-ferous epithelium rich with developing germ cells through continuous spermatogenesis and the semi-niferous tubules containing spermatozoa) (Figure 1. C, D and DM).
Histomorphometric parameters: tubular area, tubular perimeter, Feret's diameter, tubular diameter and epithelial height were determined ( Table 2). The lowest values of measured parameters were in group M and they were significantly lower than in groups D, DM and C (p < 0.001). The values of parameters in animals of group C were significantly higher than in groups DM and M. The highest values of all measured parameters (tubular area, tubular perimeter, Feret's diameter, tubular diameter and epithelial height) were found in animals of group D. These parameters were significantly higher in group D than in groups DM and M, but not significantly higher in group D than in group C. All parameters in animals of group DM were significantly higher than in group M and lower than in groups D and C.    C -control group treated with 0.9% NaCl; M -group treated with 4 mg/g of MSG; D -group treated with 5 mg/g of diltiazem; DM -group treated with 5 mg/g of diltiazem and 4 mg/g of MSG.
Results are expressed as means ± standard deviation. The values of measured parameters in group M were significantly lower than in groups D, DM and C (p < 0.001)
It is referred that administration of high doses of MSG to adult Wistar rats, after two weeks, induced degenerative and atrophic changes in testes (43,55). Also, increased apoptotic changes in the germinal epithelial cells and decreased germinal epithelial thickness were registered in adult Wistar rats treated with MSG (3 g/kg/BW/) by gavage for 30 days (55).
These findings could be explained by effect of high concentration of MSG on glutamate receptors in peripheral tissues as testes. Activation of glutamate receptors by sustainable high concentration of MSG could alter ionic permeability of neural membrane and induce persistent depolarization (26,56). Neural death can be induced by such excessive activation of glutamate receptors and overloading with intracellular calcium (38,57).
The toxic effect of MSG on endocrine (male reproductive system) has been explained for years as the late consequence of hypothalamic nuclei impairment and persisting disturbances of the hypothalamic-pituitary-gonadal axis. Necrosis of hypothalamic structures is the most clearly demonstrated from three to five hours after application of MSG in neonatal animals. It is demonstrated that 24 hours after MSG application, necrotic cells are already phagocytized and signs of edema have disappeared (21,27).
The presence of the central effect of MSG is well known, but its mechanism is not clear yet. In our experiment the effectiveness of the treatment with MSG is confirmed with the next findings: stunted growth (significantly shorter naso-anal and tail lengths) and obesity (p < 0.05). Although the MSG treated rats were not significantly heavier than the controls, they were significantly more obese, what is confirmed with a significantly increased Lee index (p < 0.05) (45,(58)(59)(60)(61)(62).
In MSG-treated rats, we registered a significant reduction in absolute weight of testes and gonadosomatic index (relative testis weight) compared with these parameters in rats of C, D and DM groups. Our results confirm findings of the studies which registered the decrease of gonadal weights in MSG treated animals (32)(33)(34)(35)(36)(37)(38)40).
Decreased organ weight is a sign of toxic injury. In our study, marked testicular atrophy is shown by light microscopy in rats neonatally treated with MSG. We confirmed alterations in testes structure reported by other authors (38, 41-43, 63, 64).
Normal testicular histology was present in other groups of rats (C, D and DM). These findings are strongly supported by the results of histomorphometric parameters analysis. We recorded highly significantly decreased values of histomorphometric parameters (tubular area, tubular perimeter, Feret's diameter, tubular diameter and epithelial height) (p < 0.001) in rats neonatally treated with MSG compared with parameters in rats of C, D and DM group.

Conclusion
The results of our study show that the pretreatment with diltiazem is efficient in prevention of MSG toxicity on testes in Wistar rats. MSG harmful effects: stunted growth, decreased relative testis weight, increased adipose tissue mass, testicular atrophy and decreased histomorphometric parameters are prevented in rats pretreated with diltiazem. We consider that excessive activation of glutamate receptors and overloading with calcium could be responsible for neurotoxic potential of MSG. Subsequent studies should be done to elucidate if pretreatment with slow calcium channel-blocking agents could prevent toxic effect of MSG on testes in adult animals.