Paraquat (PQ) has a wide range of use as an herbicide, and one of the most important agents to control weeds in a variety of crops. PQ is not only hazardous to human health but also highly toxic to plants, farm animals and man [1] [2].

Oral, inhalation and damaged skin are the main routes of exposure to the paraquat. Paraquat Mortality rate is high because of suicidal attempts by its ingestion or occupational dermal damage. Paraquat is a corrosive agent to the skin make it is easily absorbed into the body. Due to its local accumulation, the lung is the primary site of its toxic effects [3]. Additionally, chronic exposure to PQ was recorded to be accompanied with liver failure, kidney failure, and Parkinsonian lesions with lung fibrosis [4].

Paraquat is environmental pro-oxidant pollutants that induce free radical release. At high concentrations, they cause oxidative stress, a damageable process that can impair cell function, structures and has been claimed in many pathological situations [5].

Thiamine (Vitamin B1) is essential for the nervous system. It is the precursor of thiamine Diphosphate (ThDP), an important coenzyme for transketolase, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase [6]. It is thus essential for nervous system energy metabolism. ThDP is formed by pyro phosphorylation of thiamine, a reaction catalyzed by Thiamine Pyro Phosphokinase (TPK) [7].

Presently, the most widely used is Benfotiamine (BNF). Initially, BNF was used to increase blood thiamine levels in patients suffering from thiamine deficiency. It was later shown to prevent experimental diabetic retinopathy [8]. More recently, BFT was shown to have powerful beneficial effects in mouse models of neurodegenerative diseases [9] [10].

Benfotiamine (BNF), an acyl thiamine derivative, is a powerful NADPH oxidase inhibitor that protects tissue injury in many experimental models [11]. Benfotiamine has been recorded to offer protection against diabetes mellitus complications as neuropathy, nephropathy and retinopathy [12]. Benfotiamine inhibits the Protein Kinase C (PKC) pathway, thus inhibiting the NF-κB activation in diabetes mellitus patients [13]. Moreover, benfotiamine activates the transketolase enzyme that eventually blocks the production of NADPH oxidase and increases the antioxidant properties (Figure 1) [14].

Flavonoids are compounds polyphenolic in nature that are obtained from all foods of plant origin. They protect biological organs against free radical-induced oxidative damage (Figure 2) [15].

Hesperidin (HSP) is a flavanone glycoside hugely found in orange and lemon and is a cheap by product of citrus cultivation [16]. HSP is beneficially supplemented in the complementary treatment protocols. Abnormal capillary leakage, limbs pain, weakness and cramps are caused by HSP deficiency. Supplemental hesperidin also helps in reducing fluid exudation from capillaries [17]. HSP had antimutagenic activity [18], nerve health-promoting effects [19] and to protect against neurotoxicity [20].

Aim of this work is to compare the neuroprotective role of benfotiamine and hesperidin against paraquat produced biochemical, and histological changes in the spinal cord of adult albino rats.

Ethics approval

This study was approved by the local ethical committee of the Faculty of Medicine, Zagazig, Egypt.

Exposure to environmental hazards including herbicides is one of the challenges that faces the scientists to discover and measure the safety of human health especially in occupational exposure that affect the people medical social and economic sides. Oxidative stress and radicals has been recorded to be the major mechanism causing paraquat (PQ) toxicity [41].

In our study, there were highly significant increase, decreased in MAD and GSH respectively with decrease antioxidant enzyme activities in paraquate treated group indicating oxidative damage. These results are in parallel with Abd El-Rahman et al. [42] who recorded that exposure to PQ significantly increased malondialdehyde (MDA) while decreased glutathione content (GSH) with the enzyme activities in the blood, lung and dorsal aorta indicating oxidative stress and increased lipid peroxidation. Redox cycle is the key of PQ induced toxicity as it is enzymatically reduced, by NADPH-cytochrome P450 reductase [43] and NADH-ubiquinone oxidoreductase (complex I) [44], to produce a PQ free radical with the subsequent production of the superoxide radical (O₂•^–) [45]. hydrogen peroxide (H₂O₂) and hydroxyl radical (HO). It is skillfully registered that antioxidants entertain a chief role in palliating the devastating effects of oxidative stress on different tissues.

In our study, there was highly significant increase in serum PNF-H level in paraquat treated group that is indicating spinal cord damage. This result is in accordance with Adel et al. [46] who reported that serum pNF-H was highly significant increase in their Multiple sclerosis patients than controls. Neurofilaments,

**: highly significant (P: <0.001).

a major ingredient of neuronal cells, can be freely set into the cerebrospinal fluid and serum in cases of neuronal axonal injury in divergent neurological diseases such as multiple sclerosis.

In our study, there was highly significant increase in serum NSE level in paraquate treated group that is indicating spinal cord damage. This result is in parallel with Shash et al. [47] who reported that NSE major component in the cytoplasm of neuronal cells, its release in the serum at significant high levels specifies neuronal scarring according to Bharosay et al. [48] who announced that endothelial cell death disrupts the blood brain barrier during stroke, and the released cytoplasmic elements from damaged neuronal tissues. Although, Fassbender et al. [49] in his studies, announced that serum NSE levels are less sensitive, and a devastating injury is required to release a detectable authentic differences. This can be explained by the timing of the sample’s extraction; also, the use of dissimilar kits.

In our study, there was highly significant increase in serum s100 level in paraquat treated group that is indicating spinal cord damage. This result is in parallel with wei et al. [50] who reported that blood sample from a PQ poisoning patient has estimated significantly increased of S100A8 and S100A9 levels. The expression of S100A8 and S100A9 was also advocated in the lung tissue of PQ-treated rat accompanied with lung destruction. Furthermore, the expression of S100 in patient serum could provide a preferential diagnostic tool and biomarker for predicting PQ poisoning in patients.

The present study showed decrease in differentiated neurons and increase in degenerated neurons. Also, neuropil hemorrhage, vacuolation, dilated congested capillaries and neurogliosis. These findings are in parallel with that declared by Bilian et al. [51] neuronal histopathological findings were moderate neuronal degeneration, edema, gliosis, due to anoxia, and damage to the central white matter and especially the neuronal tissue around the lateral and third ventricles.

Reports subsists that herbicides, including paraquat, may be fated in the mechanism of a neurodegenerative disorder (Parkinson’s Disease) specified by degeneration of dopaminergic neurons set into Substantia Nigra and pyramidal tract in spinal cord [52] Additionally, it is recorded that paraquat produces severe behavioural, electro cortical and neurodegenerative side effects when given into several areas of the rat Brain and spinal cord as well as when given systemically [53]. These side effects are prohibited by the administration of Cu-free superoxide dismutase (SOD) in the same sites, an enzyme which converts superoxide anions into hydrogen peroxide, submitting that oxidative stress is incorporated in the neurotoxicity produced by paraquat [54].

Ibi et al. [55] observed that abnormal formation of reactive oxygen species (ROS) may attribute a major role in paraquat-induced neuronal damage. In has been demonstrated that dopaminergic neurons are extremely sensitized to ROS output. Also, exhaustion of glutathione, another factor implied in sustaining both neuronal tissues centrally and peripherally into an antioxidant status, increases liability of dopaminergic neurons to nitric oxide and suppresses the mitochondrial complex I activity in cells leading to neurotoxic effects [56].

Nitrotyrosine immunostaining was strongly positive in the neuronal cells in PQ treated group. In control, BNF + PQ, and HSD + PQ groups, proportion of negative to moderate nitrotyrosine immunostaining and low morphologic alterations were observed. This results confirmed by Choong et al. [57] who stated that transgenic mice spinal cord, immunocytochemistry showed strongly immunostained by nitrotyrosine antibodies in neuronal cells, but no nitrotyrosine immunocytochemical reaction in neuronal cells were observed in the spinal cord of the control mice. Other studies have recorded that antioxidants and NOS inhibitor increase SOD life span in neuronal cells in transgenic mouse model. The therapy with antioxidant agents may suppress the neurodegenerative process in human amyotrophic lateral sclerosis, also it has been recorded to reduce of nitrotyrosine production.

Nitrous oxide, free radical, has many useful functions when produced in vivo by neurons and endothelial cells [58]. But, excess NO produces cytotoxicity [59] Nitric oxide significantly interacts with O₂·⁻ to give ONOO⁻ [60] Peroxynitrite is pro-oxidant interacting with SOD to form a strong nitrating agent, submitting the nitration of tyrosine of cellular proteins resulting in cellular dysfunction and death [61].

Watanabe et al. [62] reported the induction of nNOS and SOD after spinal cord ischemia, with most motor neurons showing crucial death. This result recommended that induction of both nNOS and SOD with production of NO₂-Tyr. It was also found that the nitrotyrosine immunopositivity rises with development of anterior horn cell degeneration and generation of reactive astrocytes. These results suggest that oxidative damage in SOD transgenic mice starting the degeneration of motor neurons and neuronal death.

Chou et al. [63] have recorded that neurofilamentous gathering in the motor neurons of patients with amyotrophic lateral sclerosis, were immunoreactive with an antityrosine antibody. Many of the degenerated neurons represented positive nitrotyrosine immunoreactivity, associated the distributed reactive astrocytes in the neuropil of the anterior horns and in the corticospinal tracts were obviously immunostained for nitrotyrosine.

Cha et al. [64] described the presence of nitrotyrosine-immunoreactive astrocytes and motor neurons in the spinal cords of mutant SOD transgenic mice, when were clinically evident this noticeable result is similar to that in sporadic ALS patients [65] These results indicate that the extent of nitrotyrosine staining harmonized with the degeneration of anterior horn cells and the number of reactive astrocytes and that nitrotyrosine expression may be deleterious to the motor neuron system and be incorporated in the neurotoxic mechanism [66].

In our study, there was highly significant difference in levels of MAD, GPx, and nonsignificant difference in PNF-H, NES, and S100 levels in rats of BNF + PQ and HSD + PQ groups.

Lukienko et al. [67] found that thiamine is not diminished inside the cell by PQ exposure, advocating that no considerable amounts of thiamine have reacted with ROS produced in the presence of PQ. On the other hand, it was recommended that the oxidation of thiamine by ROS mainly produces thiochrome. They determined no increase in thiochrome release by our cells after PQ exposure.

In our study, co-administration of BFM and HSD cause improvement in levels of MAD, GPx, PNF-H, NES, and S100 levels in rats, it was nearly similar to control group levels. In these groups, pretreatment with BFM and HSD decrease and increase the MAD and GSH levels respectively with increase antioxidant enzymes activities and decrease PNF-H, NES, and S100 levels beside the improvement of histopathological changes of spinal cords.

Benfotiamine (BNF) is an artificially produced thiamine precursor with elevated bioavailability. It has advantageous effects against neurodegenerative diseases in mouse. BNF protects the cells against PQ induced toxicity with high efficacy. The main metabolites of BNF were thiamine and S-benzoylthiamine (S-BT). Treatment with it produces a giant increase in intracellular import of thiamine. Also BFT, S-BT and thiamine all safeguard the cells against oxidative stress, submitting an antioxidant effect of thiamine. Thiamine has a secondary effect likely mediated by few antioxidants signaling pathways and its thiol form [68].

Although, it can be shown that BNF was approximately as defensive agent in the presence of the above results, but also, it is shown that the additive protective effects of BFT and SuBT against PQ toxicity are related to the important intracellular accumulation of thiamine. Thiamine protects the devastating effects of ROS either by direct or indirect antioxidant effects. Direct antioxidant effects has been recorded by [69] that thiamine and ThDP have free radical scavenging activitiesin vitro. Sambon et al. [70] showed that thiamine and ThDP presented suppressive effects on superoxide production. The probability of a protection against PQ by a direct interaction between thiamine and superoxide should be appraised.

Hesperidin supplementation during exposure to PQ has remarkable decreased lipid peroxidation and increased GSH in spinal cord. The decrease of oxidative stress in blood was associated with normalization of antioxidant enzymes activities. In the same way decreased oxidative stress in tissues as lung and dorsal aorta was accompanied by improvement of the histopathological changes in both tissues. The modifier role of HDN could be assigned to its capacity to suppress inflammation [71] by interacting with arachidonic acid metabolism, histamine release and by suppressing lipoxygenase, cyclooxygenase and xanthine oxidase [72]. Additionally, HDN has antioxidant [73] and metal chelating capacity by suppressing the superoxide derived Fenton reaction [74] The free radical scavenging ability [75] attributed to its sulfhydryl groups which interact with free radicals exactly peroxynitrite [76] and hydroxyl radical so, it is playing a major role in antioxidant defense system [77].

This study provides evidence that PQ adversely affects spinal cord tissues by increasing oxidative damage. The benfotaimine and hesperidin administration is highly effective in preventing neurotoxicity. Further studies are recommended on larger population, longer duration and other markers to assess the toxicity.

The authors declare no conflict of interests in relation to this article.

The authors gratefully acknowledge the support and help provided by all the staff of animal House, Zagazig University Hospitals.

Amin, D.M., Ameen, S.H. and Abaza, M.T. (2021) Neuroprotective Effects of Hesperidin and Benfotiamine against Paraquat Induced Spinal Cord Neurotoxicity in Adult Male Albino Rats. Occupational Diseases and Environmental Medicine, 9, 139-164. https://doi.org/10.4236/odem.2021.93011