Sodium Metabisulfite-Induced Hematotoxicity, Oxidative Stress, and Organ Damage Ameliorated by Standardized Ginkgo biloba in Mice

Sodium metabisulfite (SMB) is a biocide and antioxidant agent generally used as a preservative in food and beverage industries but can oxidize to harmful sulfite radicals. A standardized Ginkgo biloba (EGb-761) has demonstrated potent antioxidant and anti-inflammatory activities, which is beneficial for the treatment of diseases that exhibit oxidative stress and inflammation. The present study sought to investigate the putative ameliorative effects of EGb-761 against SMB-induced toxicity in mice. Thirty-two male Swiss white mice were randomized into control, SMB-treated, SMB + EGb-761-treated, and EGb-761-treated groups. EGb-761 (100 mg/kg/day) and SMB (98 mg/kg/day) were administered by gastric gavage for 40 days. Oral administration of EGb-761 restored SMB-induced decrease in body weight and prevented SMB-induced thrombocytopenia, leukocytosis, and anemia. Furthermore, EGb-761-treatment protected against SMB-induced liver and kidney injury depicted by decreased serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, bilirubin, creatinine, urea, uric acid, and albumin. Furthermore, EGb-761 treatment attenuated SMB-driven dyslipidemia and metabolic acidosis. Besides, EGb-761 supplementation abrogated SMB-driven oxidative stress as depicted by stabilized reduced glutathione (GSH) levels in the brain, liver, kidney, spleen, heart, and lungs. SMB induced a significant increase of tissue levels of malondialdehyde (MDA), serum nitric oxide (NO), interferon-gamma (IFN-γ) and tumor necrosis factor-α (TNF-α) which were abrogated by EGb-761 treatment. In conclusion, these results deepen our understanding of EGb-761 in light of various detrimental effects of SMB-driven toxicities. These findings provide a novel approach that can be optimized for preventing or treating exposure due to SMB toxicity.


Introduction
Sodium metabisulfte (SMB) is an inorganic chemical widely used as a preservative in the food, beverage, and pharmaceutical industries due to its ability to stop the growth of microorganisms and its antioxidant properties [1].It is a sulfating agent that reacts with water to release toxic sulfur dioxide (SO 2 ) when ingested or inhaled.Sulftes can be produced endogenously through the degradation of sulfur-containing amino acids such as cysteine and methionine or they can be obtained externally through food, drink, medicine, or from the environment by inhalation of polluted air [2].Te toxicity of sulftes is mitigated in vivo by sulfur oxidase that converts sulftes (SO ) [3].Te established and acceptable daily intake of ingested sulftes expressed as SO 2 equivalents is 0.7 mg/kg body weight [4].Te toxic efects associated with SMB include male infertility [5], pneumonitis [6], increased lipid peroxidation [7], neurotoxicity [8], alterations in immunological, biochemical and hematological parameters [2], cytotoxicity of cells, and damage to the heart [1,9].
Usage of SMB within the recommended concentrations is usually safe but it becomes toxic when used in excess or due to prolonged exposure.At higher concentrations, SMB acts as a prooxidant [7], and prolonged exposure may lead to deleterious efects on biological systems causing organ damage such as hepatotoxicity, nephrotoxicity, coronary artery disease, brain edema, and dementia [6].Te toxicity is caused by the generation of sulftes which are converted to sulfates by sulfur oxidase.Sulfates are oxidants that can be converted to reactive oxygen species and other SO 2 free radicals responsible for various adverse efects [10].From the literature review, it is evident that the concentration of SMB is not regulated in most countries and most fesh food vendors have the temptation to use copious amounts in order to extend the self-life of food.
Ginkgo biloba leaf extracts possess powerful antioxidant properties that neutralize oxygen free radicals, the major cause of neurodegenerative diseases and aging [18].In addition, EGb-761 can mitigate against lipid peroxidation by acting as a free radical scavenger and can reduce infammation in diseases such as arthritis, irritable bowel syndrome, cancer, and heart diseases [19].Tis is achieved via the reduction of infammation by inhibiting the transcription of genes responsible for infammatory responses and histamine release [18].Besides, accumulating evidence suggests that Ginkgo biloba works by inhibiting infammatory mediators such as NO, TNF-α, and inducible nitric oxide synthase (iNOS) [20].Furthermore, ameliorative efects of Ginkgo biloba against lead and fuorideinduced toxicity have been documented [21].Tese previous fndings informed our use of Ginkgo biloba in this study, given that organ damage, infammation, and oxidative stress are some of the features associated with SMB toxicity.Terefore, in the present study, the efects of EGb-761 treatment on various toxicities initiated by SMB exposure in mice were evaluated.

Experimental Design.
Te present study employed one control (naïve) group of mice and three treatment groups.In brief, thirty-two healthy male Swiss albino mice (5-6 weeks old) were randomly allotted into the four groups; each group containing 8 mice.Group 1 served as control and received distilled water and mice pellets.Group II mice received 98 mg/kg/day of sodium metabisulfte (SMB).Group III mice received 100 mg/kg/day of standardized Ginkgo biloba (EGb-76) and 98 mg/kg/day of SMB.EGb-76 is a wellcharacterized and standardized extract of Ginkgo biloba leaves that contain 24% favone glycosides (primarily quercetin, kaempferol, and isorhamnetin) and 6% terpene lactones (2.8-3.4% ginkgolides A, B, and C and 2.6-3.2%bilobalide).Notably, ginkgolide B and bilobalide constitute approximately 0.8% and 3% of the total extract, respectively [22].Group IV mice received 100 mg/kg/day of EGb-761 only.Te mice were exposed to the treatments through oral administration using gastric gavage for 40 days.Te animals were housed in sterile plastic cages under a controlled room temperature of 23-25 °C and a 12 hour light/dark cycle and allowed to acclimatize for one week before the start of the experiments.Te mice were fed on pellets (Unga feeds, Kenya) and had access to clean water ad libitum.

Preparation of Sodium Metabisulfte and Ginkgo biloba.
Sodium metabisulfte 98 mg/kg/day (Sigma Aldrich, St Louis, MO) and standardized Ginkgo biloba extract (EGb 761) (eCRATER USA) were prepared fresh daily by dissolving them in sterile distilled water.Te choice of 100 mg/ kg/day dosage of EGb-761 was based on previous studies that showed potentiation of protective efects against leadinduced toxicity [23,24].

Determination of Body Weight.
Te live body weights of animals from each experimental group were measured every three days throughout the experimental period.Te body weight measurements were done using an analytical electronic balance (Mettler PM34, DoltaRange ® ).

Euthanization of Mice and Sample Collection and
Preparation.After 40 days post-treatment, mice were sacrifced through euthanization with ketamine (50 mg/ml) and xylazine (100 mg/ml) (Merck KGaA, Darmstadt, Germany) in a ratio of 4 : 1 through intramuscular injection.Blood samples were collected intracardially from individual mice and placed in heparinized tubes for complete hemogram analysis and for biochemical analysis; blood was collected in sterile Eppendorf tubes.To obtain serum, blood in the Eppendorf tubes was left to settle for one hour at normal room temperature and centrifuged at 10,000 rpm at 4 °C for 5 min (Centurion Scientifc Ltd., K240R, UK).Mice were perfused with sterile PBS bufer after which the spleen, kidney, liver, lungs, heart, and brain were harvested and placed in Eppendorf tubes that were under dry ice.Te snapfrozen whole brain, kidney, heart, lungs, spleen, and liver 2 Journal of Toxicology were homogenized on ice-cold water (4 °C) in 0.5 ml of 0.25 M sucrose, 5 mM HEPES-Tris, pH 7.4, with protease inhibitor cocktail to a fnal concentration of 10% (w/v).

Hematological Determination and Biochemical Analysis.
Analysis of individual blood samples from diferent experimental groups was done using an automated Beckman Coulter Counter (Benchman, Indianapolis, USA) to obtain full hemogram parameters.Serum levels of liver enzyme markers: alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gammaglutamyl transferase (GGT), direct and total bilirubin, creatinine, urea, uric acid, albumin, total cholesterol, HDL, and triglycerides were assayed using an automatic analyzer (Integra 400 plus analyzer, Roche Diagnostics).
2.6.Cytokine ELISA.Serum levels of proinfammatory cytokines TNF-α and IFN-c and IL-10 anti-infammatory cytokine were measured by sandwich enzyme-linked immunosorbent assay (ELISA) (Termo Fisher Scientifc Inc., California, USA).Te ELISA kits were used according to the manufacturer's detailed protocol.Te ELISA optical reader (Multiskan EX-355, Termo Electron Corporation, Waltham, Massachusetts, USA) was used to measure the absorbance that was set at 450 nm.

Reduced Glutathione (GSH)
Assay.Reduced GSH content was determined by employing the method of Grifth [25] with some modifcations.In brief, the brain, liver, kidney, heart, lungs, and spleen homogenates were mixed with a solution containing sulfosalicylic acid (4.31% (w/v)) and 0.25 mM EDTA.Te GSH in the homogenates was determined chemically by reacting to GSH with Ellman's reagent (DTNB) and measuring the absorbance of the reaction product at 412 nm using a multidetection microplate reader (Biotek Synergy HT).

Nitric Oxide and Malondialdehyde
Assay.Serum levels of nitric oxide (NO) were measured by the Griess assay kit (Sigma-Aldrich, St Louis, MO), which was used according to the manufacturer's instructions.NO production was quantifed by measuring color change at 540 nm using a spectrometer (SpectraMax 340PC384, Molecular Devices, Sunnyvale, USA).Malondialdehyde (MDA) levels were measured by assays of thiobarbituric acid reactive species (TBARS) [26].Te quantifcation of the thiobarbituric acid reactive species (TBARS) was quantifed by a spectrometer set at 535 nm.
2.9.Statistical Analysis.Statistical analysis was done using the GraphPad Prism software package (version 5.0).Oneway ANOVA was done to compare the treatment groups with controls.For internal comparisons, Turkey's post-hoc test was used.Te results were given as a ± SEM with signifcance set at p < 0.05.

Efects of SMB and EGb-761 on Body Weight.
Tere was a progressive increase in the live mean weight across all the groups of mice up to 18 days posttreatment.However, an intermediary decrease in body weight was observed in mice orally administered with SMB relative to other groups of mice (Figure 1).Notably, this decrease in general body weight was reversed by EGb-761 administration.

Efects of SMB and Ginkgo biloba on White Blood Cells
and Teir Subtypes.Exposure to SMB resulted in a significant increase in the levels of total white blood cell count (WBC) relative to those in the control group (Figure 3(a)).
Remarkably, the administration of EGb-761 signifcantly restored SMB-driven leukocytosis.Te results of WBC subtypes further confrmed that exposure to SMB resulted in a signifcant reduction in neutrophils (Figure 3

Efects of Sodium Metabisulfte and Ginkgo biloba on
Platelets and Teir Indices.Exposure of mice to SMB led to a signifcant decrease in platelet levels when compared to the control group (Figure 4   SMB-treated mice when compared to control.Evidently, treatment with EGb-761 stabilized lipid levels across the board (Figure 5(c)).

Efects of SMB and Ginkgo biloba on Liver Function.
Serum activities of ALT, AST, and ALP were signifcantly increased in the SMB-treated group compared to the control (Figures 6(a)-6(c)), indicative of active liver injury.Intriguingly, administration with EGb-761 protected mice against SMB-induced liver damage.In addition, our results indicated the serum levels of direct bilirubin and total bilirubin activities were signifcantly increased in mice exposed to SMB, which were reduced in the EGb-761-treated group (Figures 6(d)-and 6(e)).On the contrary, hepatic gamma-glutamyltransferase (GGT) was comparable in all the treated groups (Figure 6(f)).

Te Impact of SMB and Ginkgo biloba Kidney Function.
Exposure of mice to SMB caused a signifcant increase in the serum levels of creatinine, urea, and uric acid in comparison to the controls (Figures 7(a)-7(c)).Tese heightened levels of creatinine, urea, and uric acid were reduced by treatment with EGb-761.Conversely, SMB caused a signifcant decrease in serum albumin levels; such changes were nullifed by EGb-761 (Figure 7(d)).

Te Impact of Ginkgo biloba on SMB-Driven Electrolyte
Imbalance.Exposure to SMB resulted in a signifcant decrease in the serum levels of potassium, sodium, and chloride ions (Figures 8(a)-8(c), respectively), indicative of SMB-driven active metabolic acidosis.In the presence of EGb-761, this phenomenon was alleviated.

Te Impact of Sodium Metabisulfte and Ginkgo biloba on
Malondialdehyde Levels.Exposure to SMB resulted in a signifcant increase in the levels of malondialdehyde (MDA) in the liver, brain, spleen, lungs, kidney, and serum relative to the control (Figures 9(a)-9(f ), respectively), depicting SMB-driven lipid peroxidation.Treatment with EGb-761 was able to alleviate this SMB-driven augmentation of MDA levels.In stark contrast, MDA levels in the heart were comparable for the normal control and mice that were administered with SMB and EGb-761 (Figure 9(g)).

Efects of Sodium Metabisulfte and Ginkgo biloba on
Nitric Oxide Levels.Te levels of NO were signifcantly increased upon exposure of mice to SMB when compared to the control (Figure 11).Administration of EGb-761 decreased the SMB-induced increase of serum NO levels.

Efects of Sodium Metabisulfte and Ginkgo biloba on
Cytokine Levels.Exposure to SMB caused a signifcant elevation of the proinfammatory cytokine tumor necrotic factor-alpha (TNF-α) and interferon-gamma (IFN-c) (Figures 12(a) and 12(b)), indicative of infammatory responses.Notably, this increase in proinfammatory cytokines was diminished in the presence of EGb-761.Te serum levels of the anti-infammatory cytokine interleukin-10 (IL-10) were comparable in all treated and control groups of mice (Figure 12(c)).An analysis of the ratios of the proinfammatory cytokines versus the antiinfammatory cytokines TNF-α: IL-10 and IFN-c-IL-10 revealed that the ratios were signifcantly higher in the SMB-exposed group of mice, which were reduced in the presence of EGb-761 (Figure 12(d) and 12(e)).

Discussion
Food preservatives are widely employed to circumvent food contamination due to microbial growth or undesirable chemical variations in packaged and stored food [27].In the face of the well-known application of these preservatives in the beverage and food industry, the extent of their detrimental and toxic impact requires scrutiny.Sodium metabisulfte (SMB) is commonly used as a preservative in food processing and consumer products to combat the growth of microorganisms [1].Excessive consumption of SMB, either through higher dosages or prolonged usage, causes many undesirable toxic and adverse efects [28].
It is increasingly evident that overuse of food additives as preservatives can signifcantly increase the development of human diseases [29].Ginkgo biloba leaf extract is a potent antioxidant and anti-infammatory agent.Besides this, it has been shown to have immunomodulatory efects as well as ofer protection against various drug-induced organ pathologies.In this study, the role of standardized Gingko biloba leaf extract (EGb-761) in ameliorating sodium metabisulfte (SMB)-induced toxicities was evaluated.From our In this study, exposure to SMB-induced weight loss in mice relative to the control.Similar fndings have been evident before in rats fed on high doses of SMB [2,30], as well as pigs and rabbits [31,32].SMB-induced changes in feeding behavior contributed to the loss of weight.Furthermore, the weight loss following SMB exposure could be due to the toxicological efects of SMB [9].Administration of signifcantly attenuated the SMB-induced weight loss.It is plausible to assume that the lipolysis-inducing property of EGb-761 may have contributed to this outcome as shown in a previous study [33].
Interference with the production of blood cells by chemical toxins is a common phenomenon with serious health implications.Tere was clear evidence of an SMBdriven decrease in the levels of RBCs, HGB, PCV, and the red cell indices, suggestive of anemia.Tese results corroborate a recently conducted study by Aslam [30], which showed that sulftes provoked a signifcant production of ROS that resulted in oxidative damage to the RBC membrane.In addition, RBC damage can be linked to lysis or feasible shrinkage of erythrocytes in blood [34].Te decline in the frequency of PCV may be associated with the reduction in the size of RBCs and the drop in the rate of synthesis of hemoglobin, which in turn controls the development and maturation of RBCs.Notably, MCH levels provide an indication of the actual content of hemoglobin in the RBC cytoplasm.Hence, MCV and MCHC levels are dependent upon the content of RBCs [35].Possibly, the decrease in MCHC levels could be associated with the toxic efects of SMB in the bone marrow impairing its ability to produce hemoglobin at a requisite rate.Such efects on the hematopoiesis would afect the synthesis and production of all blood cells, consequently afecting the transport of oxygen and immune functions.
In the current study, it was demonstrated that EGb-761 reversed the SMB-induced anemia, indicative of a benefcial modulatory efect of EGb-761 on the hematopoietic system.Tese outcomes may be ascribed to the suppressive efect of SMB on the host hematopoiesis system.Ginkgo biloba has demonstrated a robust capacity to impede lipid peroxidation of RBC membranes, glutathione depletion, and methemoglobin development [36].It is, therefore, plausible that these efects of EGb-761 may have played a fundamental role and perhaps protected the RBC from SMB-induced oxidantdriven damage.White blood cells, also known as leukocytes, are highly versatile and play a critical role in coordinating and shaping the immune response.Any chemical-driven changes in WBCs would have a detrimental impact on immunity.In this study, exposure to SMB signifcantly increased the levels of WBCs, lymphocytes, basophils, and monocytes.Such fndings had previously been shown by El-Kadi et al. [2].From this study, SMB induced leukocytosis, perhaps due to stimulation of the lymphoproliferative responses by sulftes.We noted a remarkable SMB-driven depletion of neutrophils.Such suppression of neutrophils has the potential to predispose individuals to bacterial infections.Moreover, monocytosis and a signifcant increase in levels of basophils that was observed in mice exposed to SMB in the present study may predispose to infammation-related ailments, given that efector basophils monocytes are implicated in infammatory responses [37].Remarkably, our fndings demonstrated that treatment with EGb-761 can alleviate these detrimental efects due to its ability to stabilize WBC levels in the presence of SMB.Te mechanism by which Ginkgo biloba ameliorates SMB-driven derangement of WBC and its subtypes could be multifactorial perhaps due to its antioxidant and anti-infammatory activities [20].
Platelets in tandem with coagulation factors are indispensable during the thrombosis and hemostasis processes.Furthermore, platelets are involved in the infammatory response and wound healing [38].Terefore, a change in the content of platelet levels will critically interfere with these vital physiological and biochemical processes and present a higher threat for patients on blood thinning drugs [39].To this end, exposure to SMB signifcantly suppressed platelet levels as well as MPV, P-LCR, and PDW, a clear indication of thrombocytopenia.From our study, treatment with EGb 761 signifcantly attenuated SMB-driven thrombocytopenia.Tese fndings suggest that EGb-761 may have a modulatory role in the thrombocytosis and hemostasis processes.Tis phenomenon warrants further inquiry.Collectively, the results demonstrate that SMB negatively afected the hematopoietic processes.Importantly, EGb-761 supplementation reversed the SMB-induced hematotoxicity.
Lipid metabolism plays a critical role as an important source of macromolecular structures for the cell as well as a source of cellular energy.Consequently, alterations in lipids play a signifcant part in several pathophysiological disorders [40].In the current study, exposure to SMB resulted in a signifcant elevation of total cholesterol and triglycerides with a concomitant decrease in the levels of high-density lipoproteins.Tis implies that people sufering from metabolic disorders and who are constantly exposed to SMB may aggravate the development of severe forms of the disease.In the presence of EGb-761, lipids metabolism was stabilized, demonstrating a possible modulatory role of Ginkgo biloba in lipid metabolism [33].In a previous study, exposure to SMB was shown to infuence lipid metabolism through increased release of free fatty acids (FFA) into the plasma [7], usually accompanied by inhibition of the enzyme lipase resulting in severe hypertriglyceridemia and hypercholesterolemia [7].
Sodium metabisulfte has been directly linked with severe liver damage in several studies [30].In this study, liver enzymes that are important metric indexes of liver injury were measured to evaluate whether exposure to SMB affected liver function, and if administration of EGb-761 protected from SMB-driven liver damage.We report, that exposure to SMB signifcantly increased serum AST, ALT, and ALP, denoting liver damage.SMB-driven liver injury has been demonstrated in a prior study [30].Te levels of these enzymes are elevated and released into the plasma under hepatocellular membrane stress, depicting liver injury [2].It is noteworthy that in the presence of EGb-761, the SMB-driven elevation of liver enzymes was abrogated.
Bilirubin is a byproduct released following the prompt destruction of the RBC.Heightened levels of bilirubin and its buildup in the hepatocellular environment result in infammation and organ injury [41].SMB induced signifcant elevation of bilirubin.Notably, SMB-driven elevation of bilirubin was attenuated by oral administration of EGb-761.Since bilirubin is a product of RBC breakdown, the results point to a possible novel protective efect on the liver and RBCs.
Additional investigations determined the integrity of renal function in the presence of SMB and EGb-761.Creatinine and urea are important markers of kidney function, and their upsurge or reduction mirrors a dysfunction of the kidney [42].Indeed, the breakdown of liver protein compounds has been implicated in the intensifcation of urea and creatinine levels in animal models [2].Tese increased levels of urea and creatinine may be linked to the kidney injury that was observed in this study as revealed by a signifcant increase of serum creatinine and urea levels in mice exposed to SMB.Te observed upsurge in serum levels of uric acid may be attributed to the reduction in urinary excretion of the metabolites.Herein, we report that SMB-driven kidney injury was attenuated by administration of EGb-761, inferring protection against nephrotoxicity.Tis result is in harmony with the previous work where Ginkgo biloba was observed to have a renoprotective efect against cisplatin-induced nephrotoxicity and renal damage due to ischemic reperfusion [43].
Uric acid is the end product of purine degradation [44].Hyperuricemia has been implicated as a driving force behind cognitive impairment, cardiovascular maladies, and oxidative stress [45].In the present study, mice exposed to SMB had signifcantly high uric acid levels, which is in agreement with prior studies [30].Augmented levels of uric acid are associated with increased production of proinfammatory cytokines such as TNF-α, IL-1β, and IL-6 [46].Tus, SMBdriven elevation of uric acids may in part contribute to infammation of the kidney and liver, which will directly exacerbate the pathophysiology of these organs.We report that administration of EGb-761 signifcantly reduced these elevated levels of uric acid.Tis modulation of uric acid by EGb-761 may attenuate SMB-driven toxicity and infammation of the kidney and liver.

12
Journal of Toxicology Serum albumin is a crucial protein with a vital physiological role and antioxidant activities [47].It is produced in the liver and can be used as a biomarker for early liver impairment and chronic liver diseases [48].In the current study, we assessed serum albumin levels in mice exposed to SMB and Ginkgo biloba.Exposure of mice to SMB resulted in a signifcant decrease in the serum levels of serum albumin, indicative of liver impairment.It was clear that EGb-761 treatment of SMB-exposed mice signifcantly increased the levels of serum albumin, demonstrating protection against liver injury.
Metabolic acidosis is a common condition characterized by a fall in pH by several toxins [49].In addition, metabolic acidosis is usually an indication of serious pathological states.Tus, understanding how exposure to toxicants such as SMB disrupts the physiological pH bufering system is important.A signifcant sodium metabisulfte-driven metabolic acidosis was noted in the present study as demonstrated by a signifcant decrease in serum levels of potassium, sodium, and chloride ions.Metabolic acidosis often arises, partly when there is the acceleration of movement of sodium ions into the cell in response to severe intracellular acidosis with the potential for cell dysfunction.Notably, SMB-driven metabolic acidosis was prevented by EGb-761 administration.Tis observation may be ascribed to its role in the maintenance of the membrane ultrastructure against lethal efects associated with the generation of free radicals as well as protection against modulation of enzymatic systems and ionic pumps [50].Nevertheless, the mechanisms by which EGb-761 regulates metabolic acidosis merit further investigations.
Oxidative is a phenomenon that is known to underlie or even aggravate the pathogenesis of several disease processes including but not limited to cancer, atherosclerosis, neurodegenerative diseases hypertension, diabetes mellitus, cardiovascular disease, atherosclerosis, reproductive system diseases, and aging [51].Moreover, elevated levels of lipid peroxides resulting from augmented production of free radicals may be important molecular mechanisms for sodium metabisulfte-associated deleterious efects [52].Te uncontrolled oxidation of sulfte into sulfte free radicals may trigger sulfte-driven lipid peroxidation [53].In addition, uncontrolled lipid peroxidation may drive the production of malondialdehyde (MDA).MDA is a critical marker of lipid peroxidation [54].In the current study, exposure of mice to SMB led to an increase in tissue and serum MDA, indicating the presence of lipid peroxidation.Remarkably, the administration of EGb-761 attenuated an SMB-driven increase in MDA levels, a protective efect that can be attributed to its antioxidant properties.Tese results are in line with published data [55], which demonstrated the ability of EGb-761 to scavenge free radicals with concomitant reduction in MDA associated with lipid peroxidation.
Indeed, oxidative stress is known to cause damage to important cellular biomolecules [56].Besides, accumulating evidence has shown that due to its sulftes and its derivatives, SMB can cause oxidative stress as a result of sulfte oxidation and DNA damage in vital organs such as the liver, brain, lung, and spleen [57].
In the physiological environment, cells cope with excessive ROS using highly versatile and potent endogenous antioxidant enzymes consisting of GSH, superoxide dismutase (SOD), glutathione peroxidase, and catalases.Depletion of these important antioxidant systems elicits elevation of lethal ROS, thus causing oxidative stress.Consequently, levels of antioxidant enzymes such as GSH are very good indicators of oxidative stress.In agreement with the earlier fndings [58], SMB administration signifcantly depleted GSH levels in the liver, brain, and heart with elevation of GSH being observed in the spleen, lungs, and kidney, indicative of oxidative stress.Depletion of GSH is a clear indication of overwhelming and lethal oxidative stress levels, whereas it is characteristically associated with an initial response to rising levels of oxidative stress [59][60][61].
Ginkgo biloba has been proposed as an antioxidant agent in numerous studies [62][63][64].Recently, it has been shown to exert its efect directly by scavenging ROS or elevating the expression of genes encoding antioxidant enzymes [65].Moreover, both in vitro and in vivo studies have shown that the antioxidant property of Ginkgo biloba is associated with its favonoid components, such as kaempferol and quercetin that suppress ROS [66].We also assessed the antioxidant activity of EGb-761 using GSH levels following exposure of mice to SMB.Remarkably, this is the frst study demonstrating that EGb-761 administration resulted in the assuaging of oxidative stress by SMB in vital organs such as brain, liver, kidney, lungs, spleen, and heart.
Te induction of nitric oxide synthase (iNOS) leads to the elevation of nitric oxide (NO), leading to the inhibition of the respiratory chain and a reduction in ATP formation [67].Besides, the excessive production of NO is the hallmark of diferent pathological disorders [68].Specifcally, NO facilitates the generation of lethal reactive metabolite, peroxynitrite (ONOO − ) [69], which nitrates vital lipids, nucleic acids, proteins, and/or enzymes in the physiological environment of vital organs, altering their structure and rendering them dysfunctional.Moreover, NO-mediated infammatory processes and oxidative stress events have also been outlined [70].Given that many toxic chemicals induce infammation and oxidative stress, the identifcation of novel compounds that are good candidates for the downregulation of infammatory mediators is of great signifcance.Herein, we found out that exposure of mice to SMB resulted in a signifcant increase in the serum levels of NO.Remarkably, EGb-761 nullifed SMB-induced elevation of NO.Overwhelming evidence has demonstrated that Ginkgo biloba protects cells from NO-induced neurotoxicity and several infammatory mediators [71].Tus, the protective ability of EGb-761 against SMB, noted in the current study, may be associated with its proven anti-infammatory and antioxidant activities [53].
Te body gets rid of detrimental stimuli such as toxic compounds and invading pathogens by mounting strong immune responses [72].Exposure to SMB has been demonstrated to enhance the pyroptosis process which ultimately results in increased amounts of proinfammatory cytokines IL-1β and IL-18 [73].Impairment of Journal of Toxicology these processes due to continuous exposure to sodium metabisulfte may cause chronic infammation.Our study revealed that exposure to SMB resulted in a signifcant increase of serum TNF-α and IFN-c, indicative of active SMB-induced infammation.In the presence of EGb-761, SMB-induced elevation of these proinfammatory cytokines was abrogated.It is well documented that a balance between anti-infammatory and proinfammatory cytokines defnes the infammatory state of the cellular environment.Tus, determining the ratio between the proinfammatory and anti-infammatory cytokines may help to determine the degree of infammatory status due to SMB exposure.Furthermore, we demonstrate a noticeable imbalance of proinfammatory and anti-infammatory cytokines in an SMB-administered group of mice that refects aggravated infammation.In addition, the antiinfammatory efects of EGb-761 treatment were also confrmed by a stable balance between proinfammatory and anti-infammatory cytokines, once again showing the anti-infammatory action of EGb-761.Te antiinfammatory properties of Ginkgo biloba have been proven in several studies [20,22,74].Accordingly, it has been shown that administration of Ginkgo biloba plays an important role in the resolution of infammation through the reduction of tumor necrosis factor (TNF-α) and interleukin 1β (IL-1β), while enhancing the level of antiinfammatory cytokine interleukin 10 (IL-10) [75].Its antiinfammatory properties are attributed to various favone glycosides and terpenoids contained in it.

Conclusion
In summary, the present study demonstrates for the frst time that oral administration of standardized Ginkgo biloba (EGb-761) attenuated SMB-induced alteration of hematological parameters, metabolic acidosis, infammatory responses, oxidative stress, and organ [76] damage.Arguably, because exposure to SMB results in varied detrimental effects, our fndings have signifcant and immediate clinical implications.

Figure 1 :Figure 2 3 . 5 .Figure 2 :
Figure 1: Te efects of Ginkgo biloba on sodium metabisulfte-driven change in the general body weight.Change in body weight was analyzed using one-way ANOVA with Tukey's test for group comparisons.

Figure 3 :
Figure 3: Efects of sodium metabisulfte and/or Ginkgo biloba administration on WBC and subtypes in mice.Mean comparison procedures were done with one-way ANOVA with Tukey's multiple comparison post-hoc test.Te results are expressed as ± SEM.Te indicated level of signifcance was at * p < 0.05, * * p < 0.01, and * * * p < 0.001.

Figure 4 :Figure 5 :Figure 6 :Figure 7 :Figure 8 :
Figure 4: Efects of sodium metabisulfte and/or Ginkgo biloba administration on platelets and platelet subtypes in mice.Mean comparison procedures were done with one-way ANOVA with Tukey's multiple comparison post-hoc test.Te results are expressed as ± SEM.Te indicated level of signifcance was at * p < 0.05 and * * p < 0.01.

Figure 9 :Figure 10 :Figure 11 :
Figure 9: Comparison of the efect of sodium metabisulfte and/or Ginkgo biloba administration on the levels of malondialdehyde.Mean comparison procedures were done with one-way ANOVA with Tukey's multiple comparison post-hoc test.Te results are expressed as ± SEM.Te indicated level of signifcance was at * p < 0.05 and * * * p < 0.001.

Figure 12 :
Figure 12: Comparison of the efect of sodium metabisulfte and/or Ginkgo biloba administration on the levels of the cytokines.Mean comparison procedures were done with one-way ANOVA with Tukey's multiple comparison post-hoc test.Te results are expressed as ± SEM.Te indicated level of signifcance was at * p < 0.05, * * p < 0.01, and * * * p < 0.001.