Ameliorative Potential of Ginger ( Zingiber officinale ) following Repeated Coexposure with Fluoride and Dimethoate in Blood and Brain of Wistar Rats

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Introduction
Te organophosphorus (OP) compounds have been rampantly used as insecticides to improve productivity in agriculture, horticulture, and allied industries and have also found applicability in the feld of medicine [1][2][3].Nonetheless, the residues of these agrochemicals bioaccumulate in the soil causing environmental pollution and contaminating grains, vegetables, and fruits and ultimately entering the human food chain [4][5][6].OP poisoning causes multisystemic dysfunctions, particularly neurotoxicity which is a global community health problem.Manifestations of chronic lowdose exposure include electroencephalographic changes and neurodevelopmental disorders such as autism, attention defcit hyperactivity disorder, dementia, and Parkinson's disease [7,8].However, acute neurotoxicity can result in acute cholinergic syndrome, OP-induced intermediate syndrome, and delayed neuropathy [9][10][11][12].Dimethoate (O, O-dimethyl-S-methyl carbamoyl methyl phosphorothioate) (DM), an OP compound, is widely used for the control of agricultural and household pests [13,14].Te WHO has enlisted DM as a class II (moderately hazardous) agent and a putative neurotoxin due to its ability to inhibit acetylcholinesterase (AChE) activity as a result of which a buildup of ACh occurs at nerve synapses, causing overstimulation of muscles which eventually leads to seizures, paralysis, exhaustion, and death [1].Additionally, continuous low-level exposure to DM incurs damage to antioxidant enzymes, and the ensuing oxidative stress has been incriminated as a main risk factor for neurological disorders (Alzheimer's disease, Huntington's disease, and Parkinson's disease) [15,16].Furthermore, DM also causes nephrotoxicity, hepatotoxicity, cardiotoxicity, genotoxicity, and hematotoxicity [4,[17][18][19].
Fluoride (F) is another neurotoxin that is a highly abundant, reactive, and electronegative element and often exists as a complex with other minerals in rocks and soil from where it readily leaches into groundwater due to natural weathering and erosion.In addition, volcanic emissions, industrial efuents or byproducts, and anthropogenic activities, such as coal burning and mining, pollute natural ecosystems with F. Drinking water containing geogenic F constitutes the largest source of F ingestion for humans and animals.Besides, the consumption of tea, tomatoes, spinach, grapes, fuoridated salt and the use of cosmetics, dental gels, and toothpaste also potentiates F intake [20,21].Endemic fuorosis is common worldwide, and Indian states (Jammu and Kashmir, Gujarat, Andhra Pradesh, Rajasthan, and West Bengal) are at higher risk of fuorosis due to heavy contamination of groundwater [22][23][24].
Te European Food Safety Authority (EFSA) recommendation limits the F intake to 50-70 μg/kg/day which is safe and prevents the development of dental caries; however, due to its narrow safety margin, an intake of >0.1 mg/kg/day predisposes to the development of dental fuorosis [21,25,26].Te WHO recommended upper limit for F in drinking water is 1.5 ppm.Skeletal fuorosis can occur after an intake of 6-10 mg of F per day for at least 10 days [20].Apart from skeletal deformities, such as arthritis and osteoporosis, fuorosis also predisposes to neurological developmental disabilities and heightens the risk of formation of certain brain tumors as it can readily permeate the bloodbrain barrier [27][28][29].Intellectual impediments in humans, such as impaired memory, slow learning ability, and low intelligence quotients, have been reported in areas where the groundwater carries high F content [30].Research on the role of F as a neurotoxicant is ongoing, and despite the controversies, several past and recent in vitro studies have revealed its toxic nature to neuronal cells and human embryonic stem cells [21,31,32].F-mediated oxidative damage to erythrocytes impedes their oxygen-carrying capability through methemoglobin formation and induces derangements in erythrocytic energy and redox metabolism [33].Many OP compounds employed as broad-spectrum insecticides accumulate and inhibit plasmatic antioxidant machinery [34].ROS-mediated metabolic and antioxidant enzymatic alterations can impinge perfusion of nervous tissue and glucose uptake by the brain and can be a potential risk factor for neurodegenerative disorders [35].Terefore, a concoction of environmental toxicants may interact to exacerbate direct neuronal damage inficted by individual toxicant exposure.Besides, existing brain damage can be further potentiated by a hindrance to oxygen supply to the brain after oxidative damage to red blood cells (RBCs) by coexposure to extraneous poisons.In the current scenario, a high likelihood of simultaneous coexposure to F and DM exists in animals and humans; however, only a few studies have evaluated the consequences of their coexposure on erythrocytes and the brain, and need scientifc attention.
Ginger (Zingiber ofcinale Roscoe, Zingiberaceae), a perennial rhizome, is a popular spice that has been widely consumed in Chinese, Ayurvedic, and Unani herbal medicine systems for the treatment of a wide range of illnesses such as catarrh, sore throat, rheumatism, neurological disorders, asthma, gingivitis, toothache, stroke, constipation, dementia, and diabetes [36][37][38].In a recent study, ginger was found to provide protection against DM-induced hormonal disturbances [39].Bioactive components identifed in ginger include 6-gingerol, 8-gingerol, 10-gingerol, zingerone, 6shogoal, sesquiterpenes, phenols, quercetin, and curcumin, which collectively confer antioxidant, anticancer antimicrobial, antiulcerative, and anti-infammatory properties [18].It also inhibits vascular smooth muscle proliferation and may be useful for the treatment of vascular diseases [40].Terefore, the present study was designed to evaluate the efect of combined exposure of F-and DM-induced oxidative stressmediated hematotoxicity and neurotoxicity in Wistar rats and the ameliorative potential of ginger.

Preparation of ZO Extract.
After being identifed by the University of Kashmir Taxonomists (dated 03/03/2020, voucher specimen No. 2921), the required quantity of Zingiber ofcinale (ZO) rhizomes was cleaned and dried and converted to a fne powder in the laboratory using an electric grinder.Te ethanol and distilled water in a ratio of 1 : 1 were used as a solvent for the extraction purpose in a Soxhlet apparatus while setting the temperature of the hot plate at 65-70 °C.Te extract from ZO was dried using a rotatory evaporator (15 rpm, 55-60 °C) and kept in a glass jar under desiccation and refrigeration.

Sample Collection and Analysis.
Te cervical dislocation method was employed to sacrifce the animals, and the brain samples were collected for histopathological examination and antioxidant biomarker studies using 10% formalin and ice-cold 0.5 M phosphate bufer (pH 7.4), respectively.Before the animals were sacrifced, blood samples (3-4 mL) were directly collected from the hearts of the animals in heparinized tubes.A Tefon-coated homogenizer was used (1000 rpm, 5-7 min, 4 °C) to prepare a tissue homogenate (10%).While reduced glutathione (GSH) levels were determined using whole blood, the erythrocyte sediment and plasma were separated from the blood samples by centrifugation method (3000 rpm, 15 min, 4 °C) for further analysis.Te erythrocyte sediment thus obtained was diluted in the ratio of 1 : 1 (v/v) using normal saline solution (NSS) and centrifuged (10 min) to obtain a bufy coat after discarding the supernatant.Tis was followed by the addition of NSS to the RBCs on a v/v basis.Te process was repeated three times.Te activities of diferent antioxidant enzymes were determined using 1% hemolysate prepared by mixing 100 μL of washed RBCs and 9.9 mL of 0.1 M PBS (pH 7.4).

Fluoride Estimation.
Te F level in the brain and plasma was measured (w/v wet basis) following a standard method of extraction [42,43].

Determination of Antioxidant Biomarkers in Brain and
Blood.Phenylacetate was used as a substrate to measure the activity (U/mL, where one unit equals μmol of phenol formed per min) of arylesterase (AE) [44].Reduced glutathione (GSH), total thiols (TTH), and total antioxidant status (TAS) were determined following well-known standard methods [45][46][47].Te glutathione peroxidase (GPx) and catalase (CAT) activity levels were determined as per the methods of Hafeman et al. [48] and Aebi [49], respectively.Te activities of glutathione reductase (GR) and superoxide dismutase (SOD) were evaluated following the methods elaborated elsewhere [50,51], respectively.Similarly, advanced oxidation protein product (AOPP) and malondialdehyde (MDA) levels in brain tissue were evaluated following standard methods [52,53].
2.7.Histopathology.Te samples from the cerebrum and cerebellum of animals from various groups were collected in formalin, and the parafn-embedded sections were stained with hematoxylin and eosin after proper processing (washed, dehydrated, and cleared) [54].Te sections were evaluated for various histomorphological alterations.

Statistical Analysis.
Te results expressed as mean-± standard errors are presented in tables and fgures and were obtained by analyzing the data using ANOVA (p ≤ 0.05) and Duncan's multiple range tests (SPSS 21.0).

Alterations in Antioxidant
Status of Blood.Alterations in the activities of AChE, AE, nonenzymatic (TAS, TTH, and GSH), and enzymatic (CAT, SOD, GPx, and GR) components of antioxidant system and cellular damage indicators (oxidation of protein and lipids) in erythrocytes of Wistar rats are presented in Tables 2 and 3.

Levels of GSH, TAS, and TTH.
When given alone, DM and F did not afect TAS but caused a signifcant (p < 0.05) reduction in GSH and TTH levels; however, their simultaneous administration caused a signifcant (p < 0.05) fall in GSH, TTH, and TAS when compared to control levels.ZO restored decreased levels of TTH in all the intoxicated groups and also corrected the fall in TAS values in dual intoxicated rats.Similarly, quercetin also restored both TAS and TTH levels in the combined toxicity group (Table 2).

Activities of AE and AChE.
Even though all groups treated with toxicants underwent a signifcant (p < 0.05) decline in AE values as compared to the control values, dual toxicant exposure created a signifcant reduction (p < 0.05) in AE as compared to the respective single toxicant exposure.AChE values were found to be signifcantly afected in the case of dual toxicant exposure as well as F exposure.ZO remediated depletion in AE and AChE after single toxicant exposure.ZO and quercetin caused a complete recovery of AChE but only a partial amelioration of AE levels.Te mean values along with the standard error are presented in Table 2.

Activities of CAT and SOD.
Both DM and F alone signifcantly (p < 0.05) reduced SOD but not CAT levels when compared to control.Teir concomitant exposure, however, caused signifcant (p < 0.05) reductions in both CAT and SOD levels.Supplementation of quercetin and ZO was efective in complete recovery of CAT levels in coexposed rats, and on the other hand, SOD levels in these rats showed signifcant improvement upon ZO supplementation only (Tables 2 and 3).

Activities of GPx and GR.
All types of toxicant exposure caused a signifcant (p < 0.05) decrease in activities of GPx and GR, but the decline was steepest in the case of dual intoxication.ZO and quercetin showed comparable efectiveness in the complete resurrection of the levels of GPx and GR reduced upon coexposure to F and DM (Table 3).

Levels of AOPP and MDA.
Te levels of AOPP were signifcantly (p < 0.05) elevated only after F or combined exposure whereas MDA levels were signifcantly increased by each toxicant when administered individually.F along with DM yielded signifcantly (p < 0.05) higher MDA concentration as compared to these toxicants when given alone.ZO and  4 Journal of Food Biochemistry quercetin manifested similar protective abilities by providing a complete safeguard to AOPP and MDA activities in the face of dual toxicant administrations (Table 3).

Alterations in Antioxidant Status of the Brain.
Alterations in the activities of AChE, antioxidant system (TAS, TTH, AE, CAT, SOD, GPx, and GR), and cellular damage indicators (protein and lipids) in the brain tissue of Wistar rats were accessed to determine the extent of damage and its amelioration with ginger and quercetin.

Efects on Activities of AChE and AE in the Brain.
Administration of toxicants (groups II, III, and IV) led to a signifcant (p < 0.05) decrease in values of AChE and AE.
ZO extract brought about a complete amelioration of the levels of both enzymes in all toxicant-administered groups including the combination group (Table 4).In contrast, quercetin only signifcantly (p < 0.05) improved AChE levels in the combined toxicant-administered rats with no signifcant impact on the reduced AE activities.

Efects on the Levels of TAS and TTH in the Brain.
Te groups II, III, and IV which were intoxicated with DM, F, and their combination, respectively, exhibited signifcantly (p < 0.05) decreased levels of TAS and TTH as compared to control.Supplementation with ZO completely restored changes in TAS and TTH values in the dual intoxicated rats while quercetin could only signifcantly (p < 0.05) circumvent TTH levels.Te mean values along with the standard error are presented in Table 4.

Efects on Activities of CAT and SOD in the Brain.
A signifcant decline in SOD and CAT values was seen after the administration of toxicants.In addition, dual administration of toxicants caused a signifcantly steeper depreciation in the values of both these enzymes when compared to their respective single toxicant administration.ZO administration successfully upgraded SOD and CAT levels which fell in response to F or DM alone.Meanwhile, quercetin and ZO caused complete amelioration in the levels of these antioxidants in dual intoxicated groups (Tables 4 and 5).

Efects on Activities of GPx and GR in the Brain.
Toxicants singly or in combination signifcantly (p < 0.05) lowered GPx and GR contents in comparison to the control levels.Dual toxicant insult triggered a signifcantly higher depreciation in levels of the GPx and GR as compared to the respective values of individual toxicant-treated rats.ZO administration in all toxicant groups caused complete restoration of GPx and GR levels including the group receiving both the toxicants simultaneously.Quercetin also restored GPx levels completely to the levels of control.While signifcant improvements in GR levels in comparison to the rats treated with both the toxicants were observed,the GR values in the quercetin group were signifcantly lower than those in the control group (Table 5).

Efects on the Levels of AOPP and MDA in the Brain.
A signifcant (p < 0.05) elevation in AOPP and MDA levels was recorded after toxicant administration (groups II, III, and IV) as compared to the control group.Simultaneous administration of F and DM caused a further signifcant (p < 0.05) rise in their respective levels as compared to their corresponding values after a single toxicant administration.ZO treatment enabled signifcant (p < 0.05) and complete amendment in AOPP and MDA levels in all groups treated either with single or combined toxicants.On the other hand, quercetin fully corrected only AOPP levels while MDA levels could only be partially repaired upon its supplementation (Table 5).

Fluoride Levels in Plasma and Brain Tissue.
Alterations in F levels in plasma and brain induced by F and DM alone as well as in combination and efect of hydroalcoholic extract of ZO in Wistar rats are presented in Table 6.Rats subjected to only DM treatment did not show altered F levels in plasma or blood whereas animals given Fonly treatment caused a signifcant (p < 0.05) increment in F levels in plasma as well as in brain tissue.Interestingly, DM administration along with F manifested signifcantly (p < 0.05) higher F levels in plasma as well as in brain as compared to control; however, their levels were signifcantly reduced as compared to F-only treatment.ZO supplementation signifcantly (p < 0.05) reduced increased F levels in brain as well as in plasma of NaF-only intoxicated rats.Quercetin completely restored plasma and brain F contents to baseline levels; in contrast, ZO could completely restore F levels in brain but not in plasma in rats with combined administrations of toxicants.

Histopathological Alterations in Brain (Cerebrum and Cerebellum)
3.3.1.Cerebrum.Cerebral sections in group I did not show any pathological changes.Neuronal morphology was normal, and glial cells were fnely distributed throughout the neuropil and meningeal lining also showed no pathological abnormalities (Figure 1(a)).In group II rats, mild perineuronal edema as well as perivascular edema, hemorrhage, and spongiosis were noticed besides mild neuronal degeneration (Figure 1(b)).Group III rats revealed moderately severe spongiosis along with neuronal degeneration, pyknosis, and perineuronal as well as perivascular edema (Figure 1(c)).In group IV rats, the cerebrum had pathological changes of the most severe degree.Te presence of randomly distributed multifocal glial aggregates was recorded apart from neuronal degeneration and neuronophagia (Figure 1(d)).Also, perivascular edema, mild difuse gliosis with severe vacuolation in neuropil leading to formation of microcavitation, was present in group IV (Figure 1(e)).Congestion of meningeal vessels along with hemorrhage and lymphocytic infltration in the meninges was also recorded in some rats of group IV.Group V rats did not have any signifcant changes in their cerebral cortex and their architecture mirrored that of group I rats (Figure 1(f )).In group VIII, rats had less severe changes when compared to those seen in group IV.Focal glial aggregation was absent.However, mild congestion and fbrinoid necrosis of the vascular wall leading to perivascular edema and hemorrhage were recorded.Overall, spongiosis was also subdued (Figure 2(c)).However, in comparison to group VIII, group IX rats had more severe lesions consisting of multifocal small aggregates of glial cells which were randomly distributed in the neuropil (Figure 2(d)).Moreover, spongiosis and neuronal degeneration were also evident (Figure 2(d)).Lesions in group IX were, however, less intense when compared to the changes observed in group IV.

Cerebellum.
Te cerebellum of control rats showed normal cerebellar architecture with healthy Purkinje cells, granule cell layer, and molecular layer (Figure 3(a)).In group II, mild degenerative changes in Purkinje cells and neurons of the granule cell layer were discernible along with spongiosis in the molecular layer (Figure 3(b)).Similarly, in group III, Purkinje cell degeneration, atrophy, and loss were seen but spongiosis was minimal (Figure 3(c)).Most severe changes in the cerebellar cortex and white matter were noted in group IV and included necrosis and loss of Purkinje cells as well as granule layer cells (Figure 3(d)).Moderately severe congestion, gliosis, and perivascular edema with severe spongiosis characterized by extensive dilatation of myelin sheaths could be appreciated in white matter and molecular layer (Figures 3(d) and 3(e)).Hemorrhage and gliosis were also observed in white matter in rats of this group.In contrast, no pathological alterations were appreciated in the cerebellum of group V animals (Figure 3(f )).However, mild Purkinje cell degeneration was observed in group VI rats (Figure 4(a)).Te cerebellum from group VII also showed mild degeneration of Purkinje cells and mild spongiosis in white matter tracts (Figure 4(b)).Group VIII cerebellum (Figure 4(c)) revealed only mild neuronal degeneration and congestion in comparison to the severe pathological lesions seen in the cerebellum of group IV rats.Te degree of spongiosis was also very mild in group VIII.Group IX had more severe lesions in their cerebellum (Figure 4(d)) as compared to those in group VIII including congestion, spongiosis in the white matter along with degeneration, and

Discussion
Te incidence of neurological disorders, such as Parkinson's disease, Alzheimer's disease, dementia, and brain tumors, among the general public is on the rise.Likewise, an unprecedented increase in the number of kids being diagnosed with developmental disorders, such as autism, dyslexia, and slow learning, has been witnessed during the last few decades.Evidence is emerging that rising levels of environmental degradation and omnipresent pollution are major incriminating factors for the signifcant spurt in cases of mental health diseases around the world [55].Since inherent high lipid concentration in nervous tissue makes it especially susceptible to oxidant damage, pollutants, such as F and OP pesticides such as DM which can cross the blood-brain barrier, may induce oxidative brain injury and neuroinfammation [29,56,57].Erythrocytes transport gases and maintain systemic redox equilibrium but are among the frst to bear the brunt of oxidative damage in the event of excessive generation of reactive oxygen species owing to exposure to environmental pollutants, which not only damages their structure and reduces their fnite lifespan but also can impinge systemic oxygen delivery causing anemia and hypoxia [58,59].Antioxidant machinery in erythrocytes is apt to counteract oxidative damage but injury occurs when an overload of free radical generation overpowers this antioxidant response system causing peroxidation of membrane phospholipids reducing erythrocytic deformability and expediting their premature removal from the general circulation [60].Reduction in levels of enzymatic and nonenzymatic antioxidants, such as SOD, CAT, GSH, GPX, GR, TTH, and TAS, is indicative of exhaustion incumbent upon increased catabolism of free radicals such as H 2 O 2 .
Membrane integrity of erythrocytes is also afected by AChE activity alterations, and the latter is a marker for assessment of anemia [61].Moreover, since NO is produced by endothelial cells in the presence of ACh, fuctuations in AChE in response to poisoning can afect vascular dilation and leukocytic adhesion during infammation [62].Tiols are sulfhydryl group-containing compounds with exuberant antioxidant activity which assist in maintaining a reducing environment inside cells.Examples include oxidative stress busters such as GSH, a tripeptide rich in cysteine, and glycine and glutamic acid, which apart from being a potent guard against free radical damage and a mediator of detoxifcation reactions, also serves as a cofactor for GP X .
Enzyme GR regenerates GSH from its oxidized form, so excess free radical generation can crash GR values.Low-dose long-term exposure to a mixture of toxicants can overwhelm the antioxidant capacity of erythrocytes due in part to acceleration in auto-oxidation of hemoglobin, accumulation of fuorescent heme degradation products, oxidative stress-induced premature eryptosis, and sequestration of injured nondeformable RBCs out of circulation leading to diminishment in oxygen transfer to vital organs such as brain [63,64].Since oxidative phosphorylation serves as the only means of energy generation for neurons, an adequate oxygen supply to brain is quintessential and toxicant-induced oxidative damage to erythrocytes can also indirectly impact brain functioning or exacerbate primary oxidative brain damage.
Te present work studied the impact of simultaneous F and DM exposure on brain and blood antioxidation apparatus.While DM is a well-researched cholinergic toxin [1,13], repeated exposure to F also induces neuron apoptosis and is suspected to contribute to developmental delays and impaired memory [65,66].Tus, a combined F and DM exposure may be associated with furtherance in neurobehavioral alterations, cognitive defcits, and structural alterations in brain histology.Scientifc literature has presented clear evidence that F induces oxidative injury and neurotoxicity [60,67,68] while DM poisoning can cause AChE inhibition and toxicity in a wide variety of species including aquatic fauna and birds [13].Here, we assessed the induction of neurotoxicity and hematotoxicity after their individual as well as combined exposure.F-only exposure was toxic to erythrocytic antioxidant machinery as it brought a signifcant drop in levels of SOD, GPx, GR, GSH, TTH, and AChE and signifcant augmentation in values of MDA and AOPP which validates the fndings of earlier workers who have also documented role of F in oxidative erythrocytic damage [58, 69, and 27].Results of the present work also showed oxidative damage-induced signifcant fall in levels of erythrocytic GSH, TTH, AE, SOD, GPx, and GR and a signifcant increment in MDA after DM-only administration which agreed with the previously published results [60,70].It was noteworthy that TAS levels in RBCs were signifcantly reduced only after the coexposure to the toxicants.In brain, individual F and DM treatment significantly reduced the levels of TAS, TTH, AE, AChE, CAT, SOD, GPx, and GR besides signifcantly stepping up the contents of MDA and AOPP.Notably, all animals exposed to DM and F in concomitance had a more signifcant decrease in GP X , GR, AE, CAT, and SOD alongside a higher signifcant increase in MDA and AOPP in brain.Increased MDA and AOPP levels suggested greater oxidative damage to polyunsaturated lipids and proteins, respectively, after combined toxicity [70,71].Signifcantly higher alterations in AE, CAT, and MDA concentrations in blood were also observed in the combination group as compared to those receiving any single toxicant exposure, which is similar to the fndings of Okediran et al. [72].Escalation of lipid and protein peroxidation after toxic interaction also caused far greater damage in brain parenchyma visible as severe neuronal degeneration, perivascular vacuolation in neuropil, gliosis, and multifocal glial aggregate formation alongside congestion and hemorrhage in brain.Existing literature concurs with the current research that subacute exposure to F or OP compounds alone or in combination with metalloids can efectuate signifcant devitalization of protective antioxidant enginery in brain with signifcant alterations in SOD, CAT, GPx, and MDA levels besides incurring pathological damage to brain [73][74][75].Earlier researchers showed that coexposure to deltamethrin and F inhibited AChE activity, increased hepatic oxidative stress, and altered biochemical parameters indicative of hepatic damage in rats [76,77].Additionally, Dec et al. [78] suggested pre-and postnatal exposure to F-induced morphological changes in rat liver and brain by impairment of antioxidant defense mechanism and modulation in cyclooxygenase expression.Unpremeditated exposure to a concoction of toxic agents, such as heavy metals and pesticides, is a widespread and common concern all over the world; therefore, researchers are trying to unearth inherent preventive properties in natural ingredients that can counteract ill efects of environmental pollutants on health of all organisms including humans.Ginger is commonly used in Indian ayurvedic and other traditional systems of medicine for the treatment of various disorders [16], and our research provided scientifc evidence that it could serve as an excellent antioxidant supplement to mitigate dual F and DM neuro and hematotoxicity.In the present study, ginger subverted oxidative injury after combined F and DM exposure by signifcantly raising and completely restoring the altered levels of TAS, AE, AChE, CAT, GPx SOD, GSH, TTH, and AOPP and MDA in brain in contrast to quercetin which did not bring about restoration of TAS, AE, and GR.In blood, ginger displayed similar ameliorative potential as that of quercetin since both could bring about complete amelioration in TAS, GSH, TTH, AChE, CAT, GPx, GR, AOPP, and MDA but only partial correction was seen in the levels of AE and SOD which is in accordance with the previously published data [18].Hydroalcoholic extract of ZO was found to contain phytochemicals, such as favonoids, quercetin, and curcumin [18], which explains its slightly better efectivity in countering F and DM toxicity than quercetin in our study.Amara et al. [58] also advocated the use of antioxidants, such as selenium and vitamin E, to ofset DM-induced oxidative stress-mediated erythrocytic damage.Umarani et al. [79] documented the protective properties of rutin against F-induced oxidative cardio and hematotoxicity.Sutalangka and Wattanathorn [80] propounded ZO as a neuroprotectant and cognitive ability enhancer when used in combination with C. rotundus.Boric acid manifested neuroprotective efects and engendered improvements in altered levels of MDA and SOD besides shielding from DNA damage caused by in vitro F toxicity on rat synaptosomes [81].In a similar manner, Wattanathorn et al. [82] reported that alcoholic extract of ZO efectively improved cognitive functioning and neuron density in the hippocampus of rats who developed focal ischemic infarct after clamping of their right medial cerebral artery.Likewise, Shanmugam et al. [83] recommended ginger may be used to assuage hyperglycemia-induced oxidative stress-mediated neurotoxicity in streptozotocin-induced diabetic rats since treatment with ginger successfully lowered raised MDA levels and replenished SOD, CAT, GPx, GP, and GSH.In the current investigation, ginger signifcantly reduced pathological alterations in brain in rats given F or DM since neuronal degeneration, necrosis, gliosis, and spongiosis were signifcantly diminished in dually intoxicated rats treated with ZO.Interestingly, ZO seemed to confer greater amelioration than quercetin in rats administered both toxicants simultaneously as overall lesions in the brain of the gingertreated group were less severe than those in quercetinadministered rats.Similarly, neurodegeneration and neuroinfammation in the cerebral cortex and cerebellum in streptozotocin-induced diabetic rats were inhibited by ginger, and the latter also reduced expression of TNF-α (tumor necrosis factor-α) and caspase-3 in the diabetic rats [84].

Conclusions
Present fndings have shed light on the fact that F in synergy with DM has the potential to signifcantly elevate oxidative stress in blood as well as in brain, and the combination can perpetrate appreciably greater damage in cerebral and cerebellar histoarchitecture than that inficted after exposure to any one toxicant.However, ginger extract supplementation not only signifcantly reversed the oxidative damage to the erythrocytic and brain antioxidant setup induced by 12 Journal of Food Biochemistry coexposure to F and DM but also alleviated dual exposureinduced pathological lesions in brain.Hence, ginger extract supplementation in diet can work as a prevention strategy to negate the occurrence of subacute neurotoxicity and hematotoxicity which can be an unwanted but unavoidable ramifcation of DM contamination in F endemic areas.

Figure 1 :Figure 2 :
Figure 1: (a-f ) Normal architecture of cerebrum of rats in group I with healthy neurons (arrow) and neuropil (arrow) (a), perivascular edema and hemorrhage (arrow), perineuronal edema, and neuronal degeneration (arrow) in group II (b), congestion, perivascular edema (black arrow), shrunken, and pyknotic neurons (green arrow) alongside spongiosis (star) in group III (c), large aggregates of glial cells (green arrows) in neuropil with severe neuronal degeneration (black arrow) (d) with severe spongiosis leading to microcavitation (arrow) in group IV (e), and no pathological lesions seen in the cerebrum of group V (f ).H&E 400x.

Figure 4 :
Figure 4: (a-d) Degeneration of Purkinje cells (black arrow) and mild spongiosis (star) in group VI (a), Purkinje layer cell degeneration (arrow) and mild spongiosis (star) VII (b), neuronal degeneration and phagocytosis of dying neurons by glial cells (neuronophagia) (black arrows) in group VIII (c), and degeneration and loss of Purkinje cells (arrow) and severe spongiosis (star) in cerebellar white matter in group IX (d).

Table 1 :
Treatment regimen followed for the experiment.

Table 2 :
Efect of hydroalcoholic extract of Z. ofcinale on toxicity induced by fuoride and dimethoate alone and in combination with erythrocyte antioxidant system in Wistar rats.
ZO: Zingiber ofcinale and DM: dimethoate (n � 6).Values having diferent superscripts (a, b, and c) in a column are statistically diferent from one another at a 5% level of signifcance.Values of TAS (total antioxidant status) are expressed in mM.Values of TTH (total thiols) are expressed in μM.Values of reduced glutathione (GSH) are expressed in mM.Values of SOD (superoxide dismutase) and GPx (glutathione peroxidase) are expressed in units/mg of Hb.Values of CAT (catalase) are expressed in μmol H 2 O 2 decomposed/min/mg of Hb.Acetylcholinesterase (AChE) activity is expressed in nmol of thiol produced/min.Activities of arylesterase (AE) are expressed in U/mL.

Table 3 :
Efect of hydroalcoholic extract of Z. ofcinale on toxicity induced by fuoride and dimethoate alone and in combination with erythrocyte antioxidant system in Wistar rats.−(4.5 ppm) 37.05 ad ± 3.22 7.07 b ± 2.87 4.23 cd ± 0.60 0.58 c ± 0.04 2.45 a ± 0.41 Values are given as mean ± SE of 6 animals unless otherwise stated.Values having diferent superscripts (a, b, and c) in a column are statistically diferent from one another at a 5% level of signifcance.Values of SOD (superoxide dismutase) are expressed in units/g of tissue.Values of GR (glutathione reductase) are expressed in nmol of NADPH/min.GPx (glutathione peroxidase) is expressed in units/g of tissue.Values of the advanced oxidation protein product (AOPP) are expressed in μM of chloramine-T.Values of malondialdehyde (MDA) are expressed in nmol of MDA formed/g/h.

Table 4 :
Efect of hydroalcoholic extract of Z. ofcinale on toxicity induced by fuoride and dimethoate alone and in combination with brain antioxidant system in Wistar rats.
Values are given as mean ± SE of 6 animals unless otherwise stated.Values having diferent superscripts (a, b, and c) in a column are statistically diferent from one another at a 5% level of signifcance.Values of TAS (total antioxidant status) are expressed in mM.Values of TTH (total thiols) are expressed in μM.Acetylcholinesterase (AChE) activity is expressed in nmol of thiol produced/min/mg of tissue.Activities of arylesterase (AE) are expressed in U/mL.Values of CAT (catalase) are expressed in μmol H

Table 6 :
Fluoride levels in plasma and brain on subacute exposure to fuoride alone and in combination with dimethoate (DM) and Z. ofcinale in Wistar rats.