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STUDY THE HARMFUL EFFECTS OF SODIUM METABISULFITE ON LIVER, TESTEs, AND LIPID PEROXIDATION IN MALE MICE, (MUS MUSCULUS)

Purpose The present study was designed to evaluate the toxic effect of Sodium metabisulfite (SMB) on testes and liver of 8 weekold mouse, which were grouped as control (C) and dose group, SM (500 mg/kg B.W SMB). The Experimental group was injected 0.1ml SM (500 mg/g B.W + 10 ml distilled water) for twenty-one days, while a control group was given 0.1 ml water for the same durations. Recovered testes were subjected to morphological, morphometric, and histological analysis. A Hormone evaluation was also conducted. Methods Dose group SM (500 mg/kg B.W SMB) showed morphological defects like wrinkles on the surface, shrinkage, small size, whereas the testes of control group appeared normal just like control (C). Morphometric analysis of dose group SM (500 mg/g B.W SMB) showed significant decrease in length, width and weight of testis as compared to control group. Blood analysis showed significantly levels of testosterone and glutathione reductions in dose group SM (500 mg/g B.W SMB) as compared to the control group. While the level of Melanodialdehyde (MDA) increased significantly in dose group SM (500 mg/g B.W SMB) as compare to the control group. Histological defects were observed in testes of dose group SM (500 mg/g B.W SMB) like deshaped somniferous tubule, degenerated spermatids, vacuolization, rupturing of germinal epithelium, absence of sperms in the lumen, degenerated peritubular myoid cells, hyalinization, cluster of degenerated spermatozoa in lumen and absence of leydig cells. Histology of the liver showed various defects like mitotic cells, vacuolization, degeneration of hepatic cells and cell shrinkage, broad suicidal spacing, absence of bile canaliculus cells, regenerated kuppfer cell, and interrupted sheet of hepatocytes. Conclusion The harmful impact of sodium metabisulfite on the liver and reproductive system of male mice. A Sharp decrease in the level of testosterone was formed to be a key factor for studied deformation in morphology, morphometry, and histology oftestes. Sodium metabisulfite-induced deformities were also indicated in the liver tissue due to a significant decrease in glutathione and an increase in MDA (Malondialdehyde) level. Hence it is strongly recommended to reduce the use of sodium metabisulfite in our daily use products as it’s not only harmful to mice but also for men.


INTRODUCTION
Sodium metabisulfite (SM) is a white translucent or powder strong substance with a slight sulfur scent and this compound turns into a destructive corrosive when they are blended in with water. As they become a destructive corrosive, SM can cause hack and wheezing when breathed in. SM happens because of the impacts of sulfur dioxide, which influences the tangible nerves and causes arbiter discharge. Sodium metabisulfite is a broadly utilized biocide, usually as an additive in food handling and customer items. rolls, chocolate, jam, brew, wine, and so forth and in prescriptions, like parenteral amino corrosive arrangements. Sulfite salts are part of different chemicals like sodium metabisulfite(SMB), potassium metabisulfite(KMB), sodium bisulfate, potassium sulfite, and sodium sulfite and are perceived as "sulfating specialists" since they discharge sulfur dioxide (1) . Sulfites impacts poisonous impacts on the regenerative framework, the respiratory framework, and the sensory system and can likewise prompt hypersensitive responses. Sodium metabisulfite (SM) is a white glasslike or powder strong concoction with a slight sulfur scent and this compound turns into a destructive corrosive when they are blended in with water. As they become a destructive corrosive, SM can cause hack and wheezing when breathed in. (3) Sodium metabisulfite reduces testosterone, an abatement of 68%.Testosterone is an essential germ cell perseverance element since it elimination prompts germ cell apoptosis and it is fundamental keeping up spermatogenesis and male readiness. (2) Sodiummetabisulfite are the quantitative auxiliary parts of the gonad. Besides, it intends to present a defensive operator that could be effectively overcome with the least antagonistic impacts. In the body, lipid emulsions are cleared from the blood dependent on their physicochemical properties and the physiological reaction of the reticuloendothelial framework. (4) Sulfite is essentially scattered to all body tissues, including the central system (CNS), by being invested in the stomach. (3) The liver and kidney show high range of sulfite, while gonad shows low sulfite oxidase exercises. (5) Theimpacts of sulfating specialists of these structures on the brain, while hardly any investigations have been distributed on these consequences for the gonad. (4)

II. STUDY DESIGN AND METHODOLOGY:
This study is an experimental study.
A group of 30 male mice (Mus musculus) were placed under the diverse controlled conditions, e.g., 12hour day/night cycle, 27±2°C temperatures with 40-50 ٪humidity rates. These were kept in iron cages having a size of 24 inches in length and 12 inches wide. The most available feed named Chick feed No.14, richly supplied with minerals, proteins, and multivitamins were given to mice. The Control group was labeled as (C), while the experimental group was labeled as SM. The Dose was prepared on 500 mg/kg B.W of SMB was dissolved in 10ml of distilled water in such a way that every 0.1 ml contained the desired (500 mg/kg) concentration of the required dose. The Dose was given intraperitoneally to mice with the help of an insulin syringe.

III. HORMONE MEASUREMENT:
Blood samples were directly collected from the hearts of male mice to estimate various analyses in the body. 1) Testosterone: A Radio immune assay (RIA) was used to measure the blood testosterone level.

3) Malondialdehyde (MDA):
MDA esteem was also evaluated by following the protocol of Ohkawa et al. (1979). The tissue test was 200 ul, and 8.1% sodium dodecyl sulfate (SDS), 1.5 mL of 20% acidic damaging game plan (pH3.5). The mix was made up to 4.0 mL with refined water and warmed in a water shower at 90°C for 60 min. Subsequent to cooling with fixture water 1.0mL of refined water and 0.5 mL of n-butanol were added and shaken enthusiastically and centrifuged at 4000 rpm for 10 min. The upper butanol layer was taken and its absorbance at 532 nm was analyzed.

IV.
SURGICAL INCISION / TISSUE SAMPLING: After the extraction of blood, testicles were removed for additional analysis. Mice were given anesthesia. By giving careful incision, testicles and liver were removed from the body. These organs were moved to saline for additional procedure. Tissue Sampling and Processing, after dismemberment, balls of each creature were set in Petri dish containing 0.9% saline. The balls were then situated in Bouin's fixative for preservation. Testes were seen under a binocular stereoscopic magnifying lens and morphological abnormalities were noted. By then these bent gonads were small caught with the assistance of advanced camera.

V. MORPHOMETRIC ANALYSIS:
Computerized Vernier caliper was used to choose the length and width of testes of control and experimental groups. Advanced weight assessing balance was used to decide the heaps of each group Analysis of the testicles include in each group. There were the various steps, which included lack of hydration by adopting particular categories of liquor, clearing and embedding in paraffin wax. The embedded tissues were transitionally segmented at 6-7μm at microtome. The consecutive segments were strained out on neat slides which covered with egg whites. At that point slides were recolored withHematoxylin. Histological slides after micro photographed were handled in adobe photograph examine for the change of sharpness and difference in shading alongside resizing utilizing advanced trimming, snaps making with arrows and star just as background change.

VII. STATISTICS:
Information was inspected through the unpaired Ttest. A probability level of p <0.001 was assessed vast. VIII.

RESULTS:
The morphological assessment of control and the trial bunches were broke down. The morphological deformities initiated by sodium metabisulfite. The control group demonstrated typical type of the testicles. Testicles outer looked sparkly and smooth and gave off an impression of being a bigger and heavier. Their tones were smooth in appearance.

Control group:
In the histological examination of control group, histopathologically irregularities were not found. The Control group of testes exhibited the ordinary appearance and plan of seminiferous tubules like, round formed seminiferous tubules were seen, which obviously showed sertoli cells, spermatogonia and create spermatozoa. A layer of peritubular myoid cells (smooth muscle) was found around the seminiferous tubules, reserved by connective tissues and interstitial Leydig cells (Fig: 2.1).

Dose group SM (500 mg/kg B.W SMB):
In dose group SM (500 mg/kg B.W SMB) certain segments epithelium layer was split which is also noted. Degeneration of interstitial cells and tubular malformation of seminiferous tubules were noted in some section as well. Histological deformities were observed in the form of tubular shape seminiferous tubules and decay of interstitial cells. In the specific sections less number of a sperm and spermatids were seen. Degenerated connective tissue and Leydig cells were also originate around the tubules heaving vacuoles. Other defects observed were desquamation of germinal epithelium, desquamated ( Sulfite salts are added as additives to an assortment of nourishments in different types of food items e.g. bread rolls, chocolate, jam, brew, wine, and so forth and in prescriptions, like parenteral amino corrosive arrangements. Sulfite salts are important for various synthetics like sodium metabisulfite and sodium sulfite and are perceived as "sulfating specialists" since they discharge sulfur dioxide. Sulfites impacts poisonous impacts on the regenerative framework, the respiratory framework, and the sensory system and can likewise prompt hypersensitive responses. (14) Sulfite is essentially scattered to all body tissues, including the central system (CNS), by being invested in the stomach. Ingested sulfite salts respond with water and make bisulfate, sulfite, and sulfur dioxide which can bring about poisonousness. (6) Digestion of the amino acids, for instance, methionine and cysteine that contain sulfur typically produce extensive endogenous proportion of sulfites in the body. Hence, human body organs are presented to sulfites both endogenously and exogenously. (16) Sodium metabisulfite lessens testosterone, a diminishing of 68%. The morphometric alteration in epididymis found in the SMB treated social events might be a direct result of testosterone decline. In dose group SM seriously wreck the testicular tissues and impacts (18) spermatogenesis when contrasted with control gathering. Investigation demonstrated that SM has direct harmful impact on sperms morphology in bull semen. (17) .
Testicles acquired from dose group SM (500 mg/kg B.W) demonstrated imperfections like dim type of testicles just as wrinkles on their surfaces. These were additionally deshaped. (Fig 1.1, 1.2,1.3, 1.4,) and furthermore demonstrated deviations in the size, shape, appearance and surface of testicles and showed up not to be smooth and glossy to some reach. To the Leydig cells and Sertoli cells prompting weakened epididymis sperm development measure and decreased limit of sperms to enter oocytes. (20) The Morphometric examination demonstrated the after effect of the control and the experimental groups.
In control group testicles of mice uncover that length, width and weight of testicles stay unaffected ( Fig:  1.5, 1.6, 1.7, Table: 1). in experimental group length, width and weight of testicles is hurtfully influenced and decreased size of seminiferous tubules caused the decrease in weight of testicles by SM (Fig 1.8 .Treatment of SM to mice indicated huge decrease of glutathione hormone in experiment group when contrasted with control group. (15) High centralization of SM causes pressure diminishes the glutathione level in the cell. It was proposed SM change balance among union and usage of GSH (glutathione stress hormone). The high amount of SM lessens the measure of GST. (14) In the current investigation, the degree of Malondealdehyde (MDA) announced variety in male mice because of organization of dose.
In control group MDA level indicated typical reach while in experimental group it builds (Fig: 1 Current histological assessment revealed that round shape seminiferous tubules, sperm organized lumen of tubules, peritubular myoid cells, germinal epithelium and interstitial cells remains in normal condition in control group of mice. (Fig: 1.10 X. CONCLUSION: In conclusion this study provides convincing evidence that the harmful impact of sodium metabisulfite on the liver and reproductive system of male mice. Sharp decrease in the level of testosterone was formed to be key factor for studied deformation in morphology, morphometry and histology of testes. Sodium metabisulfite induced deformities were also indicated in liver tissue due to significant decrease in glutathione and increase in MDA (Malondialdehyde) level. Hence it is strongly recommended to reduce the use of sodium metabisulfite in our daily use products as its not only harmful for mice but also for man.