N-Acetylcysteine Attenuates Monocrotophos- induced Toxicity by Mitigating Oxidative Stress and Structural Changes in Rat Liver


 The present study evaluated the effect of N-acetylcysteine (NAC) against sub chronic monocrotophos (MCP) exposure induced oxidative stress in rat liver. Albino wistar rats were divided into control, NAC treated, MCP and MCP treated groups. An oral dose of MCP (0.9 mg/kg b.wt) and NAC (200 mg/kg b.wt) was administered for 28 days. We observed high oxidative stress generation on MCP exposure in liver tissue as evident by significant increase in lipid peroxidation, protein oxidation and decreased glutathione content followed by altered activities of superoxide dismutase, catalase and acetylcholinesterase. Sub chronic MCP exposure caused an array of cellular and structural alternations in lipids and proteins of liver tissue as depicted by the FTIR, histopathological and electron microscopic analysis. N-acetylcysteine attenuated the loss of glutathione and prevented lipid peroxidation and protein oxidation. Pre-treatment of NAC also restored histological and ultra space structural alternations. So NAC protects oxidative stress and tissue damage induced by sub chronic MCP exposure in rat liver; suggesting the therapeutic and antioxidant potential of NAC.


Introduction
Monocrotophos (MCP) insecticide is used to control sucking and chewing pests of fruits and vegetables. Despite its restricted to use label, it is used extensively in various crops like rice, cotton, soybeans, sugarcane, maize, groundnut, etc. (Kumar et al. 2018;Dash and Osborne 2020). Due to its wide spread use, MCP possesses major environmental risk factors for nematodes, arthropods, shes and mammals including humans. Monocrotophos has been detected in water, soil, as well as in agriculture products (Waite et al. 1992). Exposure of MCP and its residues is known to induce hepatotoxicity (Arora 2009), nephrotoxicity (Malik et al. 2021), neurotoxicity (Sankhwar et al. 2016), genotoxicity (Mahboob et al. 2002) in mammals and in vitro toxicity (Siddiqui et al. 1992). The toxic effects of MCP exposure are attributed to the synergic effects of acetylcholinesterase (AChE) inhibition and generation of reactive oxygen species (ROS).
The liver has long been considered as major target organ of MCP exposure and its metabolites thus it is mostly affected by MCP intoxication (Hanafy et al. 1991). Studies have demonstrated that MCP exposure induces morphological and physiological changes in liver (Yaduvanshi et al. 2010;Begum and Rajini 2011). The disturbance of balance between the degree of ROS generation and antioxidant capabilities is a key factor for MCP-induced hepatotoxicity. In fact, MCP-exposure has been reported to alter the enzymatic as well as non enzymatic antioxidants inside the liver suggestive of liver oxidative stress (Yaduvanshi et al. 2010). Monocrotophos mediated oxidative stress generation is associated with increased lipid peroxidation, protein oxidation, decreased antioxidant enzymatic activity and reduced glutathione content in liver (Desai et al. 1984;Siddiqui et al. 1990;Yaduvanshi et al. 2010;Begum and Rajini 2011). Chronic MCP exposure also mediates structural changes including degeneration of hepatocytes, fatty and hydropic changes, vacuolization, brosis, congestion and in ltration of red blood cells in rat liver tissue (Arora 2009).
Antioxidants are chemical compounds capable of inducing the normal response of a biological system to reduce chemical-induced toxicity. Antioxidants primarily include natural extracts of plants and animals having chemically active groups such as thiols, polyphenols, isoquinolines, etc. These compounds are assumed to protect the biological system from oxidative stress by scavenging free radicals resulting from metabolites of synthetic chemical pesticides. N-acetylcysteine (NAC) is a thiol-containing reducing agent and possess many pharmaceutical characteristics such as anti-apoptotic, anti-oxidative, antiin ammatory and anti-neoplastic properties (Malik et al. 2007;Rakshit et al. 2009;Wang et al. 2009;Jiao et al. 2016;Mokhtari et al. 2017). N-acetylcysteine has been reported to exert an array of ameliorative pharmacological effects against pesticide exposure mediated hepatic-renal toxicity, pulmonary toxicity, neurotoxicity and reproductive toxicity (Shohrati et al. 2008;Falluel-Morel et al. 2012;Abdel-Daim et al. 2019;Owumi et al. 2021). In the literature survey, no study has reported the role of NAC against MCPinduced chronic hepatotoxicity. Further due to the persistence of MCP in fruits and vegetables, humans are susceptible to long-term exposure even if the contamination is below acceptable limits. Thus, the present study was designed to evaluate the effects of NAC against MCP-induced chronic biochemical alteration and structural changes in liver tissue of Wistar rats.

Animals and their care
Albino rats of Wistar strain weighing 150-180 g were procured from the approved animal house source and kept in polyvinyl carbon cages in well-ventilated room. All protocols related to rats experiments were according to the guidelines of the Committee for Control and Supervision of Experiments on Animals, India. Permission to use of animals was duly approved by the Institutional Animal Ethical Committee.

Experimental Design
A total of 20 rats were randomly assigned to the following four groups containing 5 rats in each group: 1. Control group: Rats received an equal volume of distilled water (vehicle) through oral gavage for 28 days, as administered to the rats of the MCP-treated group.
2. N-acetylcysteine treated group: Rats received NAC (200 mg/kg b.wt) mixed in distilled water through oral gavage for 28 days.
4. Monocrotophos + NAC treated group: Rats received MCP (0.9 mg/kg b.wt) dissolved in distilled water through oral gavage and NAC (200 mg/kg b.wt) mixed in water through oral gavage.
Dose and time of NAC treatment was based on previous studies that have shown the selected dose signi cantly effective against stress conditions (Ahmad et al. 2013;Galal et al. 2019). Dose of MCP was selected based on previous studies that have reported the selected dose to cause hepatic toxicity in rats (Nagaraju et al. 2020).
Rats were euthanized via carbon dioxide asphyxiation after last dose administration. Liver tissue was dissected out, washed in 0.9% ice-cold normal saline and was used for preparation of tissue homogenate. A part of liver tissue was used for histology and electron microscopy also.
Acetylcholine esterase (AChE) activity assay Acetylcholine esterase activity in serum and liver tissue homogenate was estimated using method of Ellman et al. (1961). The reaction mixture was read at 412 nm for 2 min. Results were expressed as nmol product formed/min/mg protein.

Estimation of lipid peroxidation
Liver tissue was homogenate was used to calculate lipid peroxidation following method of Wills (1971). Proteins were precipitated using TCA and supernatant was mixed with 0.67% TBA followed by placing it in boiling water bath to develop colour. The amount of TBARS formed was determined and used to express peroxidation of lipids as nmol malondialdehyde (MDA)/mg protein.

Estimation of protein carbonylation
The protein carbonyl in liver tissue homogenate was quanti ed after derivatization with DNPH (Levine et al. 1990). The protein carbonyl content was calculated results were expressed as nmol carbonyl/mg protein.

Antioxidant enzyme assays
Catalase (CAT) activity in liver homogenate was assayed by monitoring the enzyme-catalyzed degradation of H 2 O 2 at 240 nm for 3 min (Johansson and Borg 1988). Results were expressed as μmole H 2 O 2 decomposed/min/mg protein.
Superoxide dismutase (SOD) activity in liver homogenate was determined by measurement of the nitrobluetetrazolium reduction (Sun et al. 1988). The reaction mixture was read at 560 nm for 3 min. Results were expressed as U/mg protein.

Glutathione (GSH) estimation
Glutathione content in liver tissue homogenate of rats was estimated using DTNB as a substrate (Ellman 1959). The yellow colour developed was read immediately at 412 nm and the results were expressed as nmol/mg protein.

Fourier Transforms Infrared (FTIR) Analysis
Liver tissue samples were prepared for FTIR as described earlier (Akkas et al. 2007) and spectra was recorded in the region 4000 -400 cm -1 on an FTIR spectrometer (Alpha, Bruker Optics, Ettlingen, Germany). The obtained spectra was further analyzed in two separate regions (3800-2700 cm -1 and 1800-1400 cm -1 ) using ORIGIN® 19 software (Origin Lab Corporation, Massachusetts, USA) Histopathology study For histological analysis dissected liver tissues were stored in 70% ethanol after 24 h of xation in formalin followed by dehydration in different grades of alcohol. After dehydration, the xed tissues were embedded in para n wax after saturating with xylene and wax. Tissue sections (5 µm thickness) were cut using microtome and xed on slides using egg albumin and heat. Slides were dipped in xylene to depara nised the tissue sections and processed for Hematoxylene (stain nucleus) and Eosin (stain cytoplasm), and Van Geison (stain connective tissues) staining; and mounted with Dibutylphthalate Polystyrene Xylene and cover glasses. Slides were observed under the Nikon microscope and were photographed.

Electron microscopy
For electron microscopy tissues of the liver (1-2 mm 2 ) were xed in 2.5% glutaraldehyde and post-xation storage was done in phosphate buffer at room temperature. After primary xation tissues were xed in secondary xative (osmium tetraoxide) and were embedded in epoxy resin after graded dehydration in ethanol. Then epoxy resins were dried at 55°C for 48 h and cut into thin sections using ultra-microtome. Prepared sections were stained and examined under transmission electron microscope at Sophisticated Analytical Instrumentation Facility, All India Institute of Medical Science in New Delhi, India.

Protein Determination
Protein content in each sample was determined using bovine serum albumin as standard (Lowry et al. 1951).

Statistical analysis
All data was analyzed for statistically signi cance with help of one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Results with values for p ≤ .05 were considered as statistically signi cant.

Acetylcholine esterase activities
Signi cant depletion of AChE activities in serum and liver tissue was observed in animals exposed to MCP insecticides as compared to control rats. Pre-treatment of NAC with MCP for 28 days signi cantly elevated both serum and liver AChE activities as compared to MCP administrated group, while NAC alone treated group showed no signi cant change in AChE activity both in serum and liver tissues compared to control rats (Fig 1 & 2).

Lipid peroxidation
Monocrotophos exposure for 28 days signi cantly caused peroxidation of lipids in rats as compared to the control group. Administration of NAC to the MCP treated group signi cantly prevented lipid peroxidation as compared with MCP group. N-acetylcysteine alone treatment showed no signi cant change in lipid peroxidation of liver tissue as compared with control groups (Fig 3).

Protein Oxidation
Protein oxidation was signi cantly increased in MCP exposed rats. In contrast, prior administration of NAC to MCP-treated animals signi cantly declined protein oxidation as compared to those of MCP intoxicated animals while NAC alone treatment showed no signi cant change in protein oxidation as compared to those of the control group ( Figure 4).

Antioxidant enzyme activities
Monocrotophos exposure for 28 days signi cantly decreased the activities of SOD and CAT as compared to the control group. Pre treatments of NAC to MCP intoxicated animals signi cantly restored both SOD and CAT activities as compared to MCP alone exposed group. Administration of NAC alone showed no signi cant changes in antioxidant enzyme activities as compared to the control group (Table 1).

Glutathione content
Monocrotophos exposure signi cantly decreased the level of GSH content as compared to control rats. Treatment of NAC prior to MCP exposure signi cantly increased GSH content as compared to MCP exposed rats. Control and NAC alone administered group showed comparable GSH content (Table 1).

Fourier Transforms Infrared analysis of lipids and proteins
The FTIR absorption spectra between 4000 cm -1 to 400 cm -1 was analysed for protein and lipid structure in rat liver. Second derivative FTIR spectra was studied in two different regions i.e. 3800-2750 cm -1 and 1800-1400 cm -1 (Fig 5 & 6). The peak assignment (Table 3) shows changes in lipids and proteins among control and treated groups. As shown in Table 2, the peaks appeared at 3297 and 3080cm -1 arise from N-H and =CH stretching of proteins (Amide A & B regions). Table 3 shows that there was a signi cant decrease in band area of N-H stretch of Amide A and B following MCP exposure. Ole nic acid band observed at 3014 cm -1 mainly arise due to C-H stretching on HC=CH groups was also found to be decreased. Peak assigned to CH 3 asymmetrical (~2959 cm -1 ), CH 2 asymmetric (~2925 cm -1 ) and CH 2 symmetric (~2854 cm -1 ) stretch of lipids were also reduced signi cantly in area in MCP-exposed group (Table 3). Slight change in peak positions was also observed for these regions ( Table 2). The treatment of NAC to MCP-exposed group restored the area of the peaks representing lipid and protein structure in the region (3700-2750 cm -1 ) towards control values (Table 3).
In 1800-1400 cm -1 region (Fig 6), sub chronic MCP exposure signi cantly affected the peak area of C=O stretch (~1652 cm -1 ) of amide A; N-H and C-N stretch (~1541 cm -1 ) of Amide B (Table 3). Treatment of MCP also caused signi cant decrease in peak area of C=O stretch (~1745 cm -1 ), -CH 2 bend (~1456 cm -1 ) and COO-stretch (~1397 cm -1 ) of lipids and fatty acids (Table 3). Slight shifts in peak positions of these regions were observed in MCP exposure group ( Table 2). Administration of NAC to MCP exposure group signi cantly prevented the structural alterations corresponding to proteins and lipids caused by MCP exposure, as evident from restoration of altered peak position and area values (Table 3).

Histopathological analysis
Haematoxylin and Eosin staining of liver tissue sections of rats exposed to MCP for 28 days showed histopathological abnormalities as compared to the control group. Control tissues exhibited normal architecture of bile duct, portal triads and hepatocyte. Hexagonal hepatocytes were arranged radiating from the central vein, separated by blood sinusoids with uniform cytoplasm. Monocrotophos exposure caused edema, sinusoidal disruption and dilation of the portal triad and central vein. Focal necrosis, in ammation, apoptosis in the hepatocyte, hyperchromatic nuclei were observed in MCP exposed rat liver sections. Administration of NAC effectively normalised the onset of histopathological changes caused by MCP exposure as evident by absence of focal necrosis, hyperchromatic nuclei, and apoptotic damage in hepatocyte, in ammation, edema and architecture. N-acetylcysteine only treated group showed normal histological architecture as that of the control group (Fig 7a-d and Table 4).
Van Geison's stain is used for differential staining of collagen and other connective tissues and detects brosis in tumours, and other pathological conditions. Van Geison's staining of liver sections of rats exposed to MCP showed hepatic necrotic plaques along with brotic generations. Replication of collagen bres and degeneration of smooth muscles of the central vein was detected in MCP exposed liver tissues. In the group pre treated with NAC histopathological analysis exhibited a signi cant reduction in brotic generation and necrotic plaques as compared to MCP exposed group. Replication of collagen bres and smooth muscle degeneration were reduced after pre-treatment of NAC to MCP exposed group. Control and NAC only treated group showed normal connective tissue arrangements in hepatic tissue sections (Fig 8a-d and Table 5).
Transmission Electron Microscopy Monocrotophos exposed liver tissue sections showed marked alterations in nuclear structure as compared to controls tissue sections. Ovulated and hyperchromatic nucleus and break in the nuclear envelope were observed as major alterations in MCP exposed rats. Pre-treatment of NAC to MCP exposed rats' maintained normal spherical nucleus and intact nuclear envelope as compared to MCP exposed animal tissue sections. Control and NAC treated group showed normal shape and size of the nucleus and nuclear envelop (Fig 9a-d and Table 6).

Discussion
Monocrotophos is a class I highly toxic organophosphate pesticide. Despite its restricted to use a label, its unplanned application in agriculture is responsible for environmental exposure to humans and mammals. The residue of MCP have been reported in fruits, vegetables, dairy products, soil and water (Leuthold 2013;Mishra 2013;Kumar et al. 2014;Singh et al. 2021). Monocrotophos exposure is known to induce oxidative stress, reduction of antioxidant levels and causes apoptosis that could lead to multi-organ failure and genetic alterations (Velmurugan et al. 2013;Sankhwar et al. 2016;Mandal et al. 2020). Recent studies have suggested liver as the prime targeted organ for pesticide related toxicity (Yilmaz et al. 2017;Milošević et al. 2018;Refaie et al. 2020  Monocrotophos is a well-known AChE inhibitor and causes cholinergic toxicity inside the body. The present study shows that MCP exposure causes decline of AChE activity both in serum and liver homogenate. Inside body, MCP is readily bio-transformed to reactive metabolites which block esteratic site of acetylcholine esterase and inhibit AChE activity. Previous studies have also shown AChE inhibition in mammalian tissues following MCP exposure (Dwivedi et al. 2010;Joshi and Rajini 2012). Pre-treatment of NAC to MCP exposure recovered tissue and serum AChE level. Our results are in agreement with the ndings of previous study that has reported AChE recovery on NAC treatment in carbofuran toxicity in rats (Kamboj et al. 2008). The present study reports non cholinergic effect of NAC against MCP induced oxidative stress in mammals.
Oxidative stress is a central mechanism of tissue injury. Oxidative stress arises when the critical balance between oxidants and antioxidants is disrupted inside the body (Oldham and Bowen 1998;Birben et al. 2012;Pisoschi and Pop 2015). The disruption between oxidants and antioxidants could be attributed to free radical accumulation or antioxidants depletion or both. These free radicals may lead to further biotransformation and attack lipids and proteins' side chains. Quanti cation of lipid peroxidation and protein oxidation is a tool to investigate oxidative stress-mediated toxicity (Suntres 2002;Tarantino et al. 2009). In the present study, MCP exposure for 28 days was observed to cause lipid peroxidation and protein oxidation in rat liver that was signi cantly reversed by NAC administration along with MCP. The effects of NAC against MCP-induced damage to lipids and proteins were also evident in FTIR ndings that indicate possible change to lipid and proteins structural backbone. Reduction in band area and shift in band position of 3297 cm -1 , 3080 cm -1 , 1652 cm -1 and 1540 cm -1 are indicative of structural rearrangement of proteins inside cell. It is well known that CH 2 stretching band at 2854 cm -1 is indicator of "state of order" of biological membrane (Kartheek and David 2018). Decrease in band area at 2854 cm -1 con rmed MCP mediated alteration in biological membrane and mobility of fatty acids. Further, changes in CH 2 asymmetric and CH 2 symmetric stretching of lipids also depicts conformational disorder and lipid acyl chain exibility. Alteration in ole nic =CH band area at 3014 cm -1 indicates change in quantity of unsaturated lipids. The study shows that the decrease in peak of the amide I and II were consistent with the decline in amide A band area. Any change in the position of amide bands or decrease in peak area of amide bands may quantitatively re ect alterations in composition of protein secondary structure. This may be attributed to the increased protein oxidation and peroxidation of lipids indicating reduction in free radical scavengers due to MCP exposure in rat liver as evident by biochemical and histological studies also. The administration of NAC along with MCP effectively ameliorated these structural and molecular alterations in lipids and proteins. The results further support the various previous in vitro studies showing that NAC can directly scavenge free radicals (Custódio et al. 2002;Sadegh Soltan-Shari et al. 2007;Kamboj et al. 2008).
Oxidative stress due to toxic insults is also attributed to alteration of cellular antioxidants such as SOD and CAT (Kaur and Sandhir 2006;Lasram et al. 2014). SOD and CAT are the main enzymes that assist the elimination of oxidants formed during the bio-activation of pesticides and function as a rst-line of defense against oxidative stress (Turkmen et al. 2019). These enzymes convert superoxide radicals and peroxides radicals into water and oxygen, thus removes ROS (Bhattacharjee and Sil 2006). In relation to this, we found decrease in SOD and CAT activities in rat liver suggesting decreased scavenging of free radicals. The depletion of cellular antioxidants following MCP exposure has also been reported by various studies in liver tissue (Rao 2006;Yaduvanshi et al. 2010;Begum and Rajini 2011). The depletion of antioxidant enzyme activity reported in this study could be tracked to their overutilization in quenching free radicals produced during bio-activation of MCP. N-acetylcysteine-induced augmentation of CAT and SOD activities in the MCP exposed group could be a result of reduced lipid peroxidation and protein oxidation. The nucleophilic and oxidant quenching properties of NAC could have played a role in the positive concomitant loop manner by reducing ROS generation. Being an antioxidant, NAC treatment might have prevented the overutilization of CAT and SOD and could have restored their activity.
Intracellular GSH is a thiol-containing non-enzymatic antioxidant and plays pivotal role in detoxi cation and metabolism of both endogenous and exogenous substances (Eraslan et al. 2009). It is an important index of cellular antioxidant status and also a biomarker to detect oxidative stress (Yurumez et al. 2007). The present study shows that MCP exposure was associated with severe depletion of cellular GSH levels.
The decline of GSH content is in concordance with various in vivo and in vitro studies that have implicated GSH depletion in MCP-induced toxicity (Siddiqui et al. 1990;Rao 2006;Dwivedi et al. 2010). Further, NAC has been reported to increase cellular GSH levels both in vivo and in vitro studies against pesticides-induced oxidative stress in rat tissues (Mostafalou et al. 2012;El-Bini Dhouib et al. 2015;Galal et al. 2019). We also observed signi cant augmentation of GSH content by prior oral administration of NAC to the MCP exposed group. In fact, NAC is a source of cysteine that is a precursor of de novo GSH synthesis (Sadowska et al. 2007). After the incorporation of NAC into cells, NAC is readily deacylated to deliver intact cysteine for GSH generation. Further NAC has direct antioxidant properties that might have reduced free radicals and have protected GSH from oxidation.
The histopathological and electron microscopy assessment of liver tissue further con rmed the biochemical and structural changes following MCP exposure for 28 days. The ndings of the present study showed noticeable degenerative and ultra structural changes in liver sections which are direct shreds of evidence to oxidative damage and apoptosis reported in the study. Ultra structural changes like nuclear membrane breakage, shrinking of nucleus and constriction of chromatids as seen in our study are testimony to MCP-induced apoptosis and lipid damages. Further, the intact nuclear membrane and proper shape and size of nucleus in NAC pre-treatment group marked its anti-apoptotic and antioxidant potential against MCP toxicity. Histopathological ndings of the present study are in agreement with previous studies that depicted oxidative stress as the main vehicle for MCP-mediated toxicity in liver tissue (Karami-Mohajeri et al. 2017). Administration of NAC prior to MCP exposure reduced histopathological alternations. Histological restoration of liver architecture by NAC administration to MCP treated group indicates its anti-in ammatory and antioxidant potential. The ameliorative potential can be mainly traced to its antioxidant properties (Abdel-Daim et al. 2019).
The ndings of the present study suggest that NAC signi cantly ameliorates oxidative stress-mediated MCP hepatotoxicity in rats. Reduction of lipid peroxidation, protein oxidation and augmentation of antioxidant enzymes is a major protective evidence of NAC against MCP exposure. In addition, the results depicted that NAC also attenuates pathogenesis, apoptosis and ultra structural anomalies of the liver. The ameliorative potential of NAC and other potent antioxidants is still obscure and more scienti c approach via in vivo and in vitro investigation is warranted to identify the molecular mechanism and their effectiveness.

Declarations
Ethics approval: The study was duly approved by Institutional Animal Ethical Committee .
Consent to participate: Not applicable Consent for publication: Not applicable.
Availability of data and materials: Not applicable.