MATERIALS AND METHODS

Materials: In this experiment, female adult Albino mice were used. It was obtained from the Animal House of the Faculty of Pharmacy, King Saud University, Riyadh. The animals were distributed in special cages equipped with drinking water in their ventilated rooms subjected to the appropriate natural factors of moisture, light and temperature between 25 and 35°C.

Feeding: The animals were provided with the appropriate food, the feed of animals (No. 648) was obtained from the General Establishment of Grain Silos and Flour Mills in Riyadh. The daily diets contain; raw protein 20% phosphorus 6.0%; raw fat 5.3%, vitamin A 20% International unit/g; Ash 6%, vitamin D 2.2% International unit/g; Calcium 1%, vitamin E 70% International unit/g. In addition to the rare mineral elements: Cobalt, copper, iodine, iron, manganese and zinc, water was left as needed. The experimental animals were selected with an average age of 12-15 weeks and an average body weight of 50-60 g.

Sodium glutamate uses: A single sodium glutamate was used and obtained from a commercial center for the sale of foodstuffs. It contained white powder packed in bags and was dissolved in drinking water. The water was taken daily for 1 month for female rats at a dose²⁹ of 360 mg kg^–1 b.wt.

Experimental design: Animals were divided into two groups:

Assessment of histological analysis: All samples of the liver and kidney were taken from pregnant mice and kept in a 10% neutral formalin solution for the preparation of histological sections using Hematoxylin and eosin (H and E) stains.

RESULTS

Morphological studies
Animal weight and relative changes in weights of organs (liver and spleen): The data showed significant increase (p<0.001) in the weight of mice treated with MSG compared to control group (19.7 vs. 28.5 g) (Table 1). In addition, significant decrease in the ratio of liver index (2.65 vs. 1.65) and increase in the ratio of spleen index (0.75 vs. 1.28) were reported in MSG treated rats (p = 0.001) compared to those of control rats as shown in Table 1.

Internal shape of the viscera: In the mono-sodium glutamate group, the amount of fatty tissue was very clearly observed around the viscera of the mice compared to control mice as shown in Fig. 1a-f.

Histological analysis
Histological changes in liver structure: Sections of mice livers were analyzed histologically by using Hematoxylin and eosin (H and E) stains. Normal liver sections showed normal liver architecture. The liver of mice consisted of several liver lobules confined by the connective tissue and the bovine region.

Fig. 1(a-f):	Shape of the internal organs of mice, (a) Mice of control group are free of any increase in fatty tissue around the bowel, (b) Ovary, (c-d) MSG treated mice showed an increase in the amount of fatty tissues around both the bowels (arrows) and (e-f) Ovary

Fig. 2(a-d):	(a-b) Normal and (c-d) Glutamate treated liver tissues
	1: Normal hepatic tissues showed normal hepatic lobes, 2: Each hepatic segment of a central vein, surrounded by ribbons of interstitial and branching hepatic cells. Each hepatic placenta consists of a central vein (CV) surrounded by ribbons of interstitial and covalent cells (c). In glutamate treated, central venous infiltration (1) narrowing of the blood pockets (arrow), the death of most hepatic cells and 3: Appearance of many fatty cavities within cells were significantly shown after treatment with glutamate

Table 1:	Relative changes in organ weights (liver and spleen) in rats with hepatic fibrosis induced by MSG-toxicity
Liver index: Liver weight/body weight×100, Spleen index: Spleen weight/body weight×100, *Student’s t-test, Control vs. MSG treated group (350 mg g–1 b.wt.)

Each hepatic placenta consists of a central vein surrounded by ribbons of interstitial and interconnected hepatic cells forming a complex network that encloses the hepatic sinusoids. Multi- polygon hepatic cells appeared with a granular cytoplasm containing a spherical nucleus which appeared with one or more nuclei as shown in Fig. 2a and b.

The effect of mono-sodium glutamate was evident on the hepatic tissue, where tissue degradation and change in the structure of liver cells showed signs of cellular death (necrosis). The concentration of nuclear chromatin and nucleic degradation was observed in most liver cells, endothelial lining rupture with congestion in the central vein, narrowing of most of the hepatic pockets, presence of hemolysis and the emergence of multiple cavities within the cells. This indicated the accumulation of lipid droplets, which indicated the onset of liver lipid, invasion of inflammatory cells in some areas around the central vein (Fig. 2c, d, Fig. 3a, b).

Histological changes in kidney structure: In normal group, histological sections showed that the kidney is made up of two cortex cortices and appears granular because it contains renal pellets, renal tubules, medulla, the universal tubules and henal hoops. The kidney is made up of the Bowman's capsule, which consists of an outer mantle layer and an inner mantle that surrounds a mass of blood capillaries known as glomerulus. The renal tubules are characterized by proximal close-edged tubules with a narrow cavity lined with low epithelial cells based on a basement membrane. The brush edge, a spherical core nucleus and an acid cytoplasm and distant spores have a large cavity and are characterized by cubic epithelial cells based on a basal membrane with acidic cytoplasm and a large spherical nucleus (Fig. 3c, d).

In glutamate treated group, the effect of glutamate mono-sodium was evident on the kidney tissue where a change was observed in the histological structure where the glomeruli had a narrowing of the cavity with some of them, swelling in the tubules, narrowness of the cavity and congestion in the capillaries, as well as the appearance of cell death and necrosis of most of them.

Fig. 3(a-d):

Liver and kidney tissues treated with glutamate, (a-b) Tissues of liver showed severe congestion and bleeding in the central (1), decomposition and death of most hepatic cells (2) and the emergence of many intracellular fatty cavities (3) attribution of inflammatory cells around the central vein (Arrow) and (c-d) In kidney tissues, showed normal kidney structures, which consists of cortex (1) and marrow (2) and convoluted tubules and the far-flung tubes (3)

Cells, in addition to the concentration of nuclear chromatin in others and shrinkage and disappearance in the renal pellets. Shrinkage of renal glomeruli and the emergence of bleeding and also observed erosion in the walls of renal tubules and blood congestion, resulting in the emergence of large areas of destruction and bleeding in some areas of renal tissue alive, only some debris cells remain (Fig. 4a-d).

DISCUSSION

The effect of mono-sodium glutamate on health and structure of body organs, especially liver and kidney was investigated in this study. There was a significant increase in body weight (28.5 g vs. 19.7 g for control) and accumulations of fatty tissues around the viscera were observed in rats treated with mono-sodium glutamate compared to normal control group. Histological examinations showed significant alteration in the normal structure of both liver and kidney in rats treated with mono-sodium glutamate compared with those of control rats. It was observed that the toxic effects of mono-sodium glutamate occurs via inflammatory and apoptotic inducing pathways. Cellular cell death was observed in all sections of both kidney and liver such as the concentration of nuclear chromatin, DNA fragmentation and finally shrinkage and nucleic lysis was observed in most liver and kidney cells which confirmed necrotic cell death. The whole cellular architectures of both liver and kidney tissue were significantly changed up on treatment with mono-sodium glutamate. Our results were in accordance with previously reported studies which showed that an increase in the levels of glutamate leads to over stimulation and cellular apoptosis^22-24.

Because glutamate is an important neurotransmitter in the human brain, which is a key element in learning and memory, there is a continuous study by neuroscientists about the possible side effects of MSG in food as its increase leads to over stimulation and apoptosis. Studies have shown that mono-sodium glutamate MSG for 10 days reduced the number of neurons in the cochlea, affecting neonatal hearing function²³. The intake of MSG with a dose of 10, 20, 30, 40 and 80 mg kg^–1 b.wt., for oral male rats for 28 days led to increase brain weight with damage to brain neurons decreased in level of Catalase with an increase in nitric oxide level²².

Fig. 4(a-d):	Structures kidney tissues treated with glutamate, (a-b) Kidney tissues showed, blood haemorrhage, dissolution and disappearance of some renal pellets in the tissues (1), death, narrowness in narrow glomerular cavity (1, 2) and (c-d) Decomposition of most of the nephropathic cells (2)

The MSG also weakens brain function and causes oxidative damage. This is what scientists proved when treating experimental animals at a dose of 2 g kg^–1 b.wt., for 7 days²⁴. It also affected the kinetic coordination and a number of neurons in the cerebellum if a dose of 2 g g^–1 b.wt., was given for 10 days orally². It was found that the administration of MSG for Wister mice at a dose of 10 mg kg^–1 b.wt., for 30 days showed an imbalance in the antioxidant balance and an increase in the level of choline in the brain²⁵. The administration of male white glutamate (MSG) with a dose of 8 mg kg^–1 b.wt., in drinking water for one month resulted in a defect in the level of neurotransmitters and oxidative stress indicators in the brain tissue¹⁶.

The MSG was classified as a cause of headache in the International Classification of Headaches and Disorders, where 45 patients with migraines were followed and analyzes were conducted during the occurrence of migraine headaches. Glutamate levels were significantly increased compared to control group³. The MSG was the study of obesity and type 2 diabetes. A study found that ingestion of MSG for pregnant mice at a dose of 60 mg/day from the 5th day of pregnancy to the last day before birth showed a sharp increase in fat and cholesterol levels for mothers. For pregnant women and maternal obesity during pregnancy⁵. The administration of pregnant women with MSG at a dose of 4 mg g^–1 b.wt., on 2, 4, 6, 8 and 10 days of pregnancy and the killing of animals was observed that the mice were obesed with high levels of triglycerides and total cholesterol with increased proportion of deformed embryos with significant obesity of the survived offsprings²⁶.

Animal studies indicated that chronic treatment with MSG leads to renal failure by its effect on the renal oxidation system²⁷ and ingestion of mono-sodium glutamate MSG at a dose of 4 g kg^–1 for 180 days to change the system of oxidation in the kidney as well as changes in the tissue in the kidney where the glomeruli crowded and the occurrence of swelling in the tubules and congestion in the capillaries as well as the emergence of death of cells and necrosis for most of them²⁸. The exposure to the substance of mono-sodium glutamate MSG In a dose of 3 and 6 g kg^–1 oral for one month to the occurrence of a defect in the thyroid gland where there was an increase in the layer of epithelial cells lining the glands with a decrease in the diameter of the vesicles and irregularity of the form with the enlargement of some of the vesicles and the density of the nuclei and the emergence of cells multi-nuclei²⁹. Testicular rats was affected by exposure to the substance of mono-sodium glutamate MSG, which led to the inhibition of testosterone as the weight and size of testes and diameter of sperm and sperm cells, as a result of obesity caused by the use of mono-sodium glutamate MSG, which led to the rise of leptin hormone³⁰.

In contrast, MSG was found to affect the ovary, where female newborns were given a dose of 2 mg g^–1 b.wt., for 75 days in drinking water. Histological sections of the ovary showed an increase in the number of cells primary ovaries without an increase in the number of follicular vesicles, with a multitude of pathological changes in the cells such as the nucleus with the appearance of some fibrous tissue in the ovarian stomas^30-32. This was due to the effect of MSG on the work of the pituitary and adrenal gland. In a study, 10 female Sprague dawley were given a dose of 0.08 mg kg^–1 b.wt., of MSG orally for 14 days and mice were dissected and the fallopian tubes were extracted and investigated with microscopy. The data showed a rise in the epithelial layer of the fallopian tube and atrophy and necrosis in the cells, due to the increase in the amount of nutrients consumed by rats and increase the level of estrogen in the blood, which may lead to infertility in women³².

In a study, aimed at evaluating liver function in the model of obesity caused by the use of MSG during the first 10 days after birth at a dose of 4 g kg^–1 by subcutaneous injection, the results showed a defect in the liver was evident in the incidence of ADP accompanied by a defect in the oxidation system due to malfunction in the mitochondria which was (MSG). The treatment with MSG after birth and for 29-32 weeks led to an increase in body mass index and blood sugar and the detection of oral glucose tolerance test of type 2 diabetes mellitus, (Cirrhosis of the liver and inflammation of the liver and inflammation of the liver with changes). Satisfactorily in hepatic tissue if given with a dose (4 g kg^–1) under the skin, where liver lipid and inflammation of the liver cells and inflammation of fatty cells and fatty lipids were significantly observed in the liver tissue, confirming non-alcoholic fatty liver disease (NASH)³⁵.

Previous research study, showed that exposure to 6 concentrations of mono-sodium glutamate (250, 500, 2000, 4000 and 8000 μg mL^–1) resulted in DNA damage in isolated human lymphocytes at all concentrations³⁶.

CONCLUSION

The present study concluded that mono-sodium glutamate produces more toxic effects on liver and kidney and considered as a major caustic effective agent against human health. It was recommended that the addition of monosodium glutamate to children's food should be avoided. Also, children should be avoided from having fast foods containing the mono-sodium glutamate, which has been shown to affect children directly.

ACKNOWLEDGMENT

The authors thank the Princess Nourah Bint Abdulrahman University (Riyadh, Saudi Arabia) for supporting this study.

SIGNIFICANCE STATEMENT

This study confirmed that exposure to mono-sodium glutamate at doses of 360 mg kg^–1 b.wt., produces more toxic effects on liver and kidney and considered as a major caustic effective agent against both animal and human health when it received in drinking water for prolonged periods. The study contributes to the effective monitoring of studies mono-sodium glutamate consumption and the cellular risk effects that may lead to cancer or severe complications.