The effectiveness of vitamin C on quinalphos ileal toxicity: a study of histological, ultrastructural, and oxidative stress markers

There is a significant hazard of human exposure to the organophosphates which is a constant threat, and they are responsible for numerous cases of poisoning and mammalian toxicity annually in non-target wildlife. The antioxidants, including the vitamin C (Vit C), have a protective effect on some organophosphorus compounds-induced organ damage. Quinalphos (QP) is one of these compounds. The investigation’s objective is to see if there was any effect of QP on the rat ileum which could be rectified by using Vit C. Three groups of 24 animals were created. As a control, the first group was given pure water. Second group subjected to oral gavages of QPs. Third group rats were given oral gavages of Vit C plus QPs for 10 days. The reaction of ileal enterocytes to food-borne QPs was marked by poorly organized microvilli, numerous vacuoles within them, disrupted nuclei with chromatin margination, disoriented mitochondria, and an expanded intercellular space. The absorptive columnar cell illustrated many vacuoles inside with herniation of microvilli, and normal goblet cells were also seen. Many Paneth cells towards the lumen of intestinal gland contained secretory granules of different sizes and shapes. The histological architecture of the ileal mucosa in the QP plus Vit C group was found to be close to those of healthy controls. The outcomes of this study suggest that administering Vit C in rats treated with QPs protects them from ill dysfunction caused by QP.


Introduction
Organophosphates are a class of insecticides that are often used to keep pests out of agricultural fields and households. They are most widely used pesticides that constitute approximately 36% of the global pesticide consumption (Samare et al. 2020). They are the first choice as pesticides as a result of their effectiveness in promptly removing pests, high biodegradability, and low bioaccumulation qualities (Rahman et al. 2004). Although they help in improving the Responsible Editor: Mohamed M. Abdel-Daim crop yield by eliminating pests, they can cause serious health problems in non-target living animals, including human beings (Chowdhary et al. 2014).
QPs (QP,O, are one of the most widely used organophosphorus insecticides worldwide (Hu et al. 2010). In developing countries, because of its simple availability, regular usage in agriculture, and a misunderstanding about the negative consequences among farmers, the hazard of QP exposure to humans seems to be very high (Chowdhary et al. 2014). Furthermore, a considerable amount of QP was already identified in food indicating its possible risk to human exposure directly or through the food chain (Kumar et al. 2016).
Because of its large surface area and physiological features, the intestine is a significant organ of the digestive system and the principal site of nutrient/toxicant exposure. Pesticide residues in water and food are becoming more prevalent at levels exceeding permitted limits raise concerns about their role in intestinal problems (Moritoki and Ishida 1977). Paneth cells are secretory epithelial cells found in clusters at the base of the Lieberkühn crypts in mammals' small intestines that generate antimicrobial agents such as lysozyme and defensins, which are bundled in secretory cytoplasmic granules (Clevers and Bevins 2013).
Elevated reactive oxygen species (ROS) generation and oxidative stress trigger an intracellular signaling cascade that boosts the expression of proinflammatory cytokines (Salzano et al. 2014). Excessive ROS production overcomes the cellular antioxidant defense mechanisms, causing lipid peroxidation, DNA damage, mitochondrial malfunction, protein oxidation, and an ATP decrease in cellular macromolecules (Salzano et al. 2014;Sies et al. 2017). Hydroxyl radicals ( • OH) attack the cell membrane causing MDA production, which binds to other cellular molecules increasing the damaging effect. Several studies have found that inhibiting the acetylcholinesterase enzyme has been associated to an increase in reactive oxygen compounds, not only in employees exposed to organophosphorus pesticides, but also in those subjected to bipyridyl herbicides like paraquat, demonstrating oxidative stress induction via a decline in antioxidant capacity and an elevation in lipid peroxidation (Ranjbar et al. 2002). ROS accelerates oxidative processes within the cell and cause lipid peroxidation in cell membranes, as previously indicated (Castro et al. 2021). It has been suggested that there is a biological consequence as a probable action mechanism of pesticide-induced lipid peroxidation on blood cells via an electrophilic attack on particular cellular components, a method of producing ROSs (Vieira and Dos Reis Martinez 2018).
Meanwhile, the formation of reactive oxygen species (ROS) is increasing, and the antioxidant system is being stressed. Then, oxidative stress disrupts cellular function, resulting in a variety of pathological disorders (Abdel-Daim et al. 2015). One of the mechanisms that protects against oxidative stress is the antioxidant system. The principal antioxidant enzyme systems are superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Glutathione is thought to be the first line of defense against oxidative stress caused by environmental factors (Bin-Jumah et al. 2021).
Vit C is a highly effective non-enzymatic antioxidant that removes free radicals from extracellular fluids and protecting biomembranes from peroxidative damage (He et al. 2019). Therefore, as a consequence, natural antioxidants in reducing oxidative damage as a factor in the histology, physiology, and pathophysiology of a number of disorders have recently gotten more attention (He et al. 2019). Antioxidants Vit C and vitamin E are accessible as nutritional supplements and are essential for practically all biological functions. Vit C is a water-soluble chain-breaking antioxidant (Teng et al. 2018). However, as a consequence of this, it has grown to be among the most widely used and least expensive non-enzymatic antioxidant compounds for preventing oxidative damage (Teng et al. 2018). Fruits, vegetables, and protein sources (particularly the liver and kidney) are the finest sources of Vit C (Znamirowska et al. 2021). It has been proven that Vit C reduces biochemical and hematological alterations produced by organophosphate pesticides in people and animals. The application of organophosphorus insecticides has been encountered to induce ileal damage by inducing major alterations in the ileal tissues, such as disorganization and enlarged interstitial spaces between them (Eddleston and Chowdhury 2016).
This study looked at the negative consequences of organophosphate insecticide QP on the ileum of rats by analyzing changes in biochemical and histological parameters that occur in this tissue because of ROSs generation and can be alleviated by using Vit C.

Experimental design
Twenty-four adult, healthy, male albino rats of Sprague-Dawley strain weighing 200-250 gm were classified into three main groups on an equal basis, eight rats for each. The treatment schedule of each group was as follows.
Group I: Served as a control and the animals were kept in a rat cage and received distilled water orally each day at 8 a.m. for 10 days.
Group II: Animals were given 14 mg kg -1 QP [o, o-diethyl-o-(2-quinoalinyl phosphonothioate)] in 100 L pure water, which was acquired as "Suquin" 25% EC from an origin (Sudarshan Chemical Industries Ltd, Pune). QP was administered via gavage every day at 8 a.m. for 10 days. Group III: Rats were preconditioned by giving them Vit C as pure crystals (Sigma, AX 1776-1 by E. Merck Science, a division of EM industries Inc., Darmstadt, West Germany) 4 h after receiving a QP treatment of 14 mg kg -1 in 100 L pure water for 10 days at a dose of 20 mg/kg/day (i.e., 10 mg/kg bw/day (about double the human-approved therapeutic dosage)).
To rule out any diurnal variations in hormone and neurotransmitter levels, all of the animals were sacrificed between 10:00 and 12:00 h, 1 day after their last treatment. To minimize interference with neurotransmitters, the quantity of ether inhaled was kept to a minimum.

Light microscopy (LM) specimen preparation and staining
Ileal specimens were excised and stored for 24 h in 10% neutral formalin solution. After washing in a 0.1 M phosphate buffer solution, the specimen was dehydrated with alcohol in ascending grades and embedded in paraffin. Ileal Sects. (5 µm thick) were deparaffinized for 15 min with two xylene shifts before being rehydrated with progressively lower alcohol grades. To examine the general morphology, hematoxylin and eosin (Abcam, Boston, MA, USA) were used to stain the sections (Eddleston and Chowdhury, 2016).

Tissue preparation for transmission electron microscopy (TEM)
Ileal specimens were immediately preserved and fixed in a 2.5% glutaraldehyde solution (Glutaraldehyde 50% CAS 111-30-8) in 0.1 M sodium cacodylate buffer, pH 7.2, trimmed, and sliced into 1 mm 3 pieces, and stored at 4 °C for 2 h. They were post-fixed in a sodium cacodylate buffer (Shanghai Sunwise Chemical Co., Lt China) containing 1% osmium tetroxide, then dehydrated in an ascending series of ethyl alcohol and cleaned in propylene oxide. The specimens were immersed in Araldite 502 to create gelatin capsules. The capsules were heated to 60 degrees Celsius for polymerization. A JEOL ultramicrotome was used to create and pick up 100-nm sections on copper grids. After being doubly stained with lead citrate and uranyl acetate, with the help of a TEM, sections were examined and imaged (JEM-1011, JEOL Co., Tokyo, Japan) (Eid et al. 2021).

Estimation and processing of parameters
Animals were sacrificed via cervical dislocation at the end of the investigation, and the ileum was extracted to analyze several oxidative stress indicators.

Ileum homogenate's antioxidant situation
Ileal tissues were homogenized in a solution of 100 m M Tris-HCl (pH 7.4) and stirred for 12 h at 4 °C at 12,000 g. Measuring ileum antioxidant properties such as GPx yielded the supernatant (Salameh et al. 2015) and CAT activity (Aebi technique) (Craig 2019). Colorimetric methods were used to measure CAT and GPx in the ileum. The assays for thiobarbituric acid precursor (TBARS Assay Kit, Item No. 10009055, Cayman Chemical Company, Ann Arbor, MI, USA) was being used to screen MDA spectrophotometrically (an indicator of lipid peroxidation).

Statistical analysis
For n = 8 experiments, the mean standard deviation of averages is displayed. At a p < 0.05 level, statistical significance is determined; an ANOVA was performed to analyze the data. The statistical analysis was performed using Systat for Windows, version 13 (Systat Inc., Evanston, IL, USA).

Biochemical data analysis
To see how much Vit C interacted with the control of these markers, we looked at antioxidant levels in tissues from all rat groups (Fig. 1A, B) and inflammation ( Fig. 2A, B). QP lowered the antioxidant biomarker GPx (Fig. 1A) and Cat by a significant (p < 0.05) degree (Fig. 1B). When matched to the QP group, both antioxidant enzymes were significantly increased by Vit C. As shown in Fig. 2A, B, QP significantly increased ileum tissue injury (p < 0.05) whereas both inflammatory biomarkers were decreased as compared to the QP group.

Light microscopy (LM) results
The ileum's crypts were lined with epithelial columnar cells, goblet cells, and conspicuous Paneth cells with brilliant red cytoplasmic granules in the rat group that served as a control. QP-treated rats' crypt architecture was altered, besides a raise in secretory cells in the crypts, together with Paneth cell metaplasia and granule discharge inside the cryptal lumen. The enlarged cryptal lining epithelial columnar cells had pale vacuolated cytoplasm and pale-colored nuclei. The number of goblet cells had declined significantly, and they seemed to be mucus-depleted. In the majority of the crypts, the QP + Vit C-treated rats had intact crypts that are completely covered with epithelial columnar cells, goblet cells, some damaged goblet cells, and conspicuous Paneth cells with brilliant red cytoplasmic granules (Fig. 3A, B and C).

Transmission electron microscopy (TEM) results
A TEM investigation of the epithelium covering the ileal crypts in control rats indicated that the epithelium was largely made up of columnar absorbing cells with fewer goblet cells. Microvilli were found on the luminal surface of the columnar absorbing cells, together with basal oval euchromatic nuclei with prominent nucleoli. Its cytoplasm was densely packed with mitochondria, rough endoplasmic reticulum cisternae, few lysosomes, and a healthy intercellular space. In clusters around the base of the intestinal crypts, Paneth cells with cytoplasm filled with huge spherical granules surrounded by halos were observed. The majority of the granules had electron dense basal regions with euochromatic nuclei. The QP group's intestinal villi underwent TEM investigation, which demonstrated disrupted cell architecture and a significant enlargement of the intercellular space. Microvilli were partially lost in the columnar absorbing cells. Some cells showed pyknotic nuclei with heterochromatin margination. The columnar absorbing cells also had many cytoplasmic vacuolations of various diameters including significant lateral interdigitations. Increased lysosomes, dilated and vesiculated rough endoplasmic reticulum that might be degranulated to some extent, and enlarged mitochondria, all of them have a few short disintegrating cristae in their cytoplasm. Mucous globules of variable electron density appeared to exist in goblet cells. Granules with nonuniform sizes and electron-lucent halos around the granule periphery, a dilated and vesiculated rough endoplasmic reticulum, massive lysosomes, and enlarged degenerated mitochondria were exhibited in Paneth cells. The QP + Vit C group's intestinal integrity was similarly in correlation to the control group. The luminal surface of the columnar absorbing cells was completely covered with intact microvilli with its cytoplasm contained intact organelles, and the goblet cells seemed to be normal, being distended with homogeneous electron density mucous globules. Paneth cells were observed to have spherical shape granules. Interdigitations in the lateral cells seemed to be parallel to those seen in the control. (Figs. 4A, B, C and D and 5A, B, C and D).

Discussion
Organophosphorus poisoning is a public health issue, with 3,000,000 instances of poisoning and 220,000 deaths reported each year around the world, with the vast majority of cases occurring in developing countries (Craig 2019). It is effective against a wide range of pests of cotton, groundnuts, rice, tea, coffee, soybeans, and so forth. Quinalphos has been classified as moderately hazardous pesticide by WHO but has become a matter of concern because of its potentiality and hazardous effect to nontarget organisms (Gupta et al. 2011).
In the QP group, the antioxidant enzymes, CAT and GPx, were shown to be decreased, which was linked to an elevation in MDA (a biomarker for peroxidation) that is being used to evaluate oxidative stress indicators (OxS). This is in accordance with previous findings that also revealed that an increase in ROS formation causes OxS to be generated, and it is attributed to a reduction in antioxidant defense system and an alteration in the cell's redox state. The OxS is generated when the synthesis and scavenging of oxygen-free radicals are out of equilibrium (Moretti et al. 2018). The antioxidant defense system involves enzymes like CAT and GPx. GPx is a lipid peroxidation detector as well as a free radical scavenger. The radical transformation of reactive oxygen is catalyzed by the catalytic enzyme CAT. As a consequence, any alteration in the antioxidant machinery's components will lead to the formation of ROS, leading to oxidative stress insult in cells (Singh et al. 2020). The cell's primary line of protection versus oxidation-induced tissue injury is the antioxidant enzymes. GPx and CAT activity have been lost in ileal tissues could be the mechanism whereby the QP enhances cellular disorders and oxidative stress, resulting in lipid peroxidation, degenerative alterations, and the buildup ROS within the cell (Elwej et al. 2017).
OxS changes the structure and function of cellular macromolecules, as well as the cell membrane, as a result of difficulties produced by elevated levels of inflammatory biomarkers (Jakubczyk et al. 2020). When QP ileum was in comparison to the ileum of the control group, IL-6 and TNF-α cytokines were observed to be elevated in the blood. According to the findings of our investigation, TNF-α and IL-6 may play a substantial role. The inflammatory process in the gut of patients with intestinal inflammation is characterized by elevated cytokine levels, resulting in mucosal damage (Joshi et al. 2015). TNF-α has previously been accustomed to enhance intestinal permeability in patients with inflammatory bowel disease by inducing the release of epithelial myosin light chain kinase (Gareb et al. 2020). IL-6 is a pro-inflammatory cytokine that offers a wide range of pro-inflammatory actions. It has been established that it has a significant impact on the intestinal epithelial barrier and that it modulates intestinal epithelial tight junctions via claudin-2 gene activation (Kuhn et al. 2018;Zhang et al. 2015).
The microscopic features of QP-induced cytotoxicity on the ileal mucosa were investigated in this study. The QP group's columnar cells had histopathological changes, according to LM analysis. The nuclei of the cryptal wall epithelial tissue were pale-stained and had pale vacuolated cytoplasm. Flattened and vacuolated enterocytes were identified in prior studies of QP-induced mucositis. In the QPtreated group of our analysis, the proportion of goblet cells was diminished, and they seemed mucus-depleted. Cryptal goblet cells decreased significantly in the rat's small intestine after QP injection (Gillois et al. 2018).
In the present research, secretory cells in the crypts continued to increase, also Paneth cell metaplasia and granule release into the crypt lumen were seen. These granules are easily identified in histological sections due to their intense eosin staining. Paneth cells produce such granules apically through the overlying crypt lumen after proper stimulation, where they help remove any pathogenic microbes (Clevers &Bevins 2013). The QP group's ileum was examined using TEM, which demonstrated significant ultrastructural alterations. The cell configuration of the crypts was disrupted, with broadened intercellular spaces observed. Previously, similar findings in the small intestine were reported (Abe et al. 2015).
Columnar cells in the QP group showed areas of substantial loss of microvilli, cytoplasmic vacuolations, and ultrastructural degenerative changes incorporating cell organoids. Paneth cells exhibited degenerative alterations, cytoplasmic vacuolations, and autophagic vacuoles. Among the changes were patchy disruption and dissolution of the microvilli, enlarged mitochondria, some pyknotic nuclei, cytoplasmic vacuolations, and endoplasmic reticulum dilatation. The accumulation of lipid peroxidation products caused by an increase in ROS level was essential for mitochondrial expansion and degradation; the same process could explain the mitochondrial damage seen in epithelial cells in our study (Chen et al. 2017).
Electron microscopy revealed cytoplasmic vacuolations in the ileal epithelial cells of the QP group, which might be caused by dilated organelles (rough endoplasmic reticulum and mitochondria), autophagic vacuoles, or hydropic degeneration. On a microscopic level, the injured cells have vacuoles in the cytoplasm with no apparent borders (Moriarty 1969).
Atypical Paneth cells with non-uniform granule diameters and electron-lucent halos at the granule periphery were identified in the QP-treated group. In the small intestine, Paneth cells are important for microbial growth regulation because they produce a variety of chemicals, such as the Wnt protein, that help to maintain the surrounding intestinal stem cell compartment, modulate stem cell proliferation, and build crypt architecture (Mei et al. 2020). Antimicrobial-containing granules discharged into the crypt lumen penetrate the mucus layer and extend outward towards the lumen, protecting the crypts from potentially hazardous microbes. Antimicrobials produced by Paneth cells have been shown to have a significant impact on the bacterial community in the small intestine, lowering total microbial numbers and altering the bacterial composition (Wehkamp andStange 2020, Yokoi et al. 2019).
The histological structure of the ileal mucosa was virtually entirely recovered in the QP and Vit C-treated group. The presences of intact microvilli with a consistent and well-defined brush border, as well as goblet cells packed with mucous granules, were confirmed by TEM analysis. The enzyme paraoxonase, which is concerned in the detoxification of organophosphorus insecticides, has been reported to enhance Vit C activity (Robea et al. 2020). Other minor antioxidant molecules, such as glutathione, urate, and beta-carotene, can be produced from their respective radical species (Medithi et al. 2021). Vit C is a water-soluble antioxidant that has been demonstrated to lower oxidative stress and neutralize ROSs by donating a hydrogen atom to make an ascorbyl-free radical that is adequately stable (Arruda et al. 2013).

Fig. 5
The ileum is imaged using TEM in all groups of rats. A Control rat's group displaying the Paneth cells (P) of the small intestine showing basal nuclei (N) with prominent nucleolus and large secretory granules (SG) with a protein core surrounded by a halo of polysaccharide-rich material. Paneth cells displayed orderly, stacked rough endoplasmic reticulum (RER) cisternae around nuclei. (× 3000). B QP-treated rat's group exhibiting intestinal epithelial cells (Ep) with many lipid vacuoles (V) inside, herniation of microvilli (Mv), and a goblet (Go) cells. Also, atrophic pyknotic nuclei (N) are seen. (× 3000). C QP-treated rat's group exhibiting abnormal Paneth cells (P) that contain granules of non-uniform size with electronlucent halos in the granule periphery. (× 6000). D QP + Vit C-treated rat's group demonstrating Paneth cells (P) of the ileum with nuclei (N) and prominent nucleolus and large secretory granules (SG) are more or less similar to the control and contain, more or less, intact organelles (× 3000)

Study limitation
Despite these findings, there are many limitations in this research. Our data are still observational, which is essential. Furthermore, based on these findings, it is currently unclear to pinpoint the upstream mechanism by which Vit C protects the ileum. As a result, concentrating more on these systems, utilizing animals or cells will be more beneficial. The Vit C dose employed was based on a previously tested ileal protective dose in the toxicity of organophosphorus chemicals. However, more research employing a dose-response curve is strongly advised. Identifying alternative pathways that regulate inflammation and oxidative stress could also be a promising area for future research. Lastly, this study focused solely on Vit C's ability to protect against quinalphosinduced ileal toxicity. However, examining this effect on small intestinal structure and function, as well as all assessed markers at different time intervals could be more informative. Moreover, more research into the therapeutic effects of different portions of the small intestine specimens on rats with pre-existing toxicity will add to our understanding of this impact and the mechanisms of action of this antioxidant.

Conclusion
From our study, we conclude that though the QP administration caused ill toxicity in rats, it resulted in changes in serum biochemical parameters, proinflammatory cytokines, and ultrastructural analysis. This may be primarily ascribed to the fall in the activity of antioxidative systems activity; there are significant but subtle changes in the cellular oxidative system resulting in more widespread, as yet, unquantified cellular damage. Vit C supplementation attenuated QP-induced changes, protecting the ileum against QP intoxication. More research into the precise estimation of such cellular destruction, as well as any recommended therapy aimed at such damage, could help us supplement the existing standard of care treatment, resulting in a better prognosis for organophosphorus poisoning.