Most of the human health problems and environmental pollutions are caused by the use of pesticides to improve crop production and pest control in agriculture 1. despite their fame and prevalent use, pesticides have to indicate serious warnings towards the health risks of farmers who mix and use pesticides or work in treated areas 2. Several pesticides such as paraquat, maneb, dieldrin, and rotenone are a possible hazard to humans due to their documented neurotoxicity in animals. 3. The study has been also reported that depression and neurotoxicity were caused in agriculture workers exposed to pesticides 4.

Rotenone is an organic pesticide extracted from Leguminosae plants. Now rotenone known as an environmental toxin causes neurological disorders like PD 5. Mechanism of action involves rotenone inhibits the mitochondrial complex I, as a result, reactive oxygen species (ROS) are produced which leads to oxidative stress, reducing ATP production 6, lowering the membrane potential of mitochondria 7, and finally causes cellular death 8. Oxidative stress produced by rotenone has been strongly implicated in the pathophysiology of PD 9. ROS generation is a natural physiological process in different organs. But, excessive generation of ROS can be damaging to the hepatic system 10 and nephritic system 11. The usual function the the brain is intimately associated with the normal function of the liver and kidney. Dysfunction of the liver and kidney plays a critical role in the development of neuro disorders and cognitive impairment 1213. Liver function abnormality leads to deficient detoxification thus permitting neurotoxins like manganese, ammonia, and other chemicals to pass in the cerebral circulation. Chronic kidney disease (CKD) is related to a high prevalence of cerebrovascular disorders like stroke, intracerebral microbleeds, and cognitive impairment. This condition has been found not only in patients with end-stage renal disease but also in patients with slight or reasonable CKD 14. Recently a finding suggests that liver dysfunction from mild steatohepatitis to cirrhosis and hepatic encephalopathy leads to, microglial activation and loss of Purkinje neurons in the cerebellum of patients with Steatohepatitis 15. Cognitive alterations and dementia were found in patients with chronic kidney disease 16. Increased oxidative stress in the liver of mice was found when treated with rotenone 17. Rotenone administration caused oxidative damage in renal tissue, causing overproduction of malonaldehyde and ROS, reduction in glutathione, blocking SOD, and glutathione peroxidase activity in rats.

Flavonoids as strong free radical scavengers have fascinated the remarkable interest of researchers 1819 as possible therapeutics, against diseases induced by free radical ²⁰. Quercetin (3,5,7,3’,4 pentahydroxyflavone), plant-derived flavonoids found in vegetables and fruits. They have been documented to possess several pharmacological activities involving anti-inflammatory, antiallergic, and antioxidant properties 1921. Quercetin can directly scavenge the superoxide anion and blocks numerous superoxide-producing enzymes like xanthine oxidase 222324 or the neutrophil membrane NADPH oxidase complex 25. Among the other antioxidant like Vitamin E and Vitamin C, quercetin is stronger than these 262728. Therefore, the main aim of the present study is to investigate the potential protective effects of quercetin toward histopathological and biochemical changes induced by rotenone in the liver and kidney. This study also helps in prevent subsequent neuro disorders due to compromised function of the liver and kidney. Our finding contributing to a novel approach in the treatment of neurological disorders caused by liver and kidney dysfunction through quercetin.

The present work investigated the ameliorative role of quercetin against rotenone-induced alteration in the liver and kidney of mice at various biochemical parameters and histological levels. Treatment of rotenone for 60 days leads to the increased LPO, NO also known as the inflammatory marker, and a noticeable reduction in the content of GSH, activities of catalase, SOD, and histological changes in liver and kidney were observed. The liver and kidney showed signs of increased oxidative stress. Taken together, Co-administration of quercetin decreased oxidative stress and ameliorate rotenone toxicity.

Evidence from the earlier study suggested that exposure to pesticides altered the antioxidant defense system in the brain, heart, and kidney of rats by producing oxidative stress, leading to esevere damage 363738. Rotenone a well-known environmental toxin induces oxidative stress by inhibiting complex I of the electron transport chain. Recently, in our lab, we reported that exposure to rotenone was confirmed to caused oxidative stress in both in vitro as well as in vivo mice models 17. Various studies have observed that quercetin, a natural antioxidant has strong free radical scavenging activity and sthe could protect against oxidative damage 39.

Measurment of body weight is the key indicator of development and physical growth of the organisms. In PD gastrointestinal dysfunction is the main characteristics during rotenone administration with other symptoms such as weight loss, constipation and early satiety 40 Treatment of rotenone could be linked with late in gastric emptying which is the cause of reduced body weight of animals41. Rotenone administrated rats have been shown reduced body weight continuously during admistration of rotenone and significant decreased in body weight were found at the end of 4^th week42. Supporting these finding in our present work, we observed a significant reduction in body weight in rotenone administrated mice. These outcome significantly reversed by co-treatment with quercetin along with rotenone.Our study consistence with other published data in which quercetin has been found to Prevents body weight loss against toxicity of superparamagnetic iron oxide nanoparticles in rats 43

The liver is the primary target of ingested oxidants and also the main tissue involves in the protection against oxidative stress. Liver dysfunction is associated with increased levels of serum enzymes, which are the suggestive sign of cellular leakage and loss of functional integrity of cellular membrane in the liver 44. High levels of AST and ALT are well parameters to identify liver damage 45. Serum GGT and ALP levels are also related to the status and function of hepatic cells. In the present study elevated levels of hepatic markers like ALP, ALT, and AST in the serum proposed that an extensive liver injury was caused by rotenone. Indicating rotenone causes structural and functional damage to the cell membrane and enhanced membrane permeability leading to the leakage of hepatic enzymes into the blood. It is well established that rotenone administration significantly increased the serum hepatic marker enzyme 46. Co-administration of quercetin decreased the above liver function biomarkers in blood serum. Our result has been supported by another study, quercetin reduced the liver function biomarkers from acute liver injury in rats induced by composite factors 47. Administration of quercetin effectively decreased the activities of these enzymes in the present study. This can be attributed to the antioxidant property of quercetin and membrane-stabilizing property.

The kidney is the vital organ of the body it plays a major role in homeostasis and regulates the extracellular environment that helps in the excretion and detoxification of drugs and metabolites, which are harmful to the body48. For this reason, it is important for exogenous toxicants. By the negative impact of chemicals or drugs, excretions get altered which leads to nephrotoxicity 49. When the function of the kidney is altered and the homeostatic function disturbed it is unable to get rid of body excess waste5051. In a study, it has been reported that treatment of rotenone caused the increased serum urea and creatinine and MDA level, protein carbonyl content, and reduction in GSH level, and activities of, GST, SOD, MPO, and LDH have been observed 52. Rotenone toxicity also caused an enhanced level of serum urea and creatinine, which are central metabolites associated with renal health 53. At the time of skeletal muscle metabolism creatinine, is formed by the spontaneous and irreversible reaction. Increased creatinine level in kidneys connected with renal damage 54. One of the major incidents in nephrotoxicity is an increase in the level of serum urea due to alteration in kidney function55.

Research has been found that treatment of rotenone induces ROS production and structural damage in renal proximal tubule derived from OK cells 11. Another study also reported that rotenone administration leads to oxidative stress, causingan increased level of malonaldehyde, and ROS, and blocking SOD and glutathione peroxidase (GSH-Px) activity. Along with this due to rotenone treatment reduction in the membrane potential of mitochondrial and increased voltage-dependent anion channel (VDAC), caspase-3, and caspase-9 protein levels in the kidney were found [56]. In the present study increased levels of urea and creatinine were observed in rotenone administrated mice signifying severe renal damage. The enhanced degree of these kidneys functions markers in serum linked to oxidative stress and inflammation from rotenone-induced damaged [53, 57]. The serum biochemistry results obtained from this study also showed an increased level of urea and creatinine suggesting severe renal damage occurred in mice treated with rotenone. Our result supported by another study increased levels of urea and creatine were observed in rotenone-intoxicated rats [58]. Co-administration of quercetin decreased the level of urea and creatine. Consistent with our work quercetin reduces serum creatinine and urea nitrogen levels against the toxicity of a mixture of four organophosphate pesticides [59].

Excessive generation of ROS encourages oxidative stress following pesticide exposure, leads to lipid peroxidation 60. In view of the present results, mice administered rotenone displayed a significant enhancement of MDA content in the liver and kidney when compared with the control group. These results are in agreement with the earlier findings that reported enhance levels of MDA in the liver and kidney of mice exposed to rotenone 10. There was an enhanced level of the LPO, indicating an attack on membrane lipids by intracellular ROS metabolites 61. Promotion at the LPO level may be one of the primary mechanisms due to pesticide exposure 60.LPO has participated in numerous unfavorable effects in cells like enhanced membrane rigidity and osmotic fragility 62. Co-administration of quercetin protects against rotenone-induced LPO. The free radical scavenging effects of quercetin may be linked with the presence of two hydroxyl groups in the β-ring of its molecule 63. Quercetin posses antiperoxidative properties due to the presence of polyunsaturated substitution on the β-ring together with 2,3 double bond, a free 3-hydroxyl substitution, and a 4-keto group in its structure 64. Several studies reported that LPO was blocked by quercetin administration 65. Hence, in our study co-administration of quercetin effectively scavenges free radicals, blocks LPO, and protects the liver and kidney tissue from the rotenone-induced oxidative damage (Figure 2).

NO is a signaling molecule, which controls several physiological and pathophysiological processes in the immune system, nervous system, and liver 6667. It was documented that rotenone increases the content of NO 68. It was also reported that rotenone treatment increases the NO level in the liver and kidney 1769. Consistent with this study in the present study we also found increased NO content in liver and kidney increased level of NO also shown the sign of inflammation in liver and kidney induced by rotenone. Co-administration of quercetin decreased the NO content in the liver and kidney (Figure 3). In our previous study, we also observed that the level of NO enhanced in the cerebellum of mice when treated with rotenone and reduced significantly when co-treated with quercetin 70.

GSH is a non-enzymatic member of the antioxidant defense system and serves as a free radical scavenger, but it also participated in liver and kidney protection against various toxicity as a substrate for antioxidant enzymes GSH-Px and GST 71. Our finding has shown that reduction in GSH level in the liver and kidney of rotenone-treated mice are linked with its primary role in oxidative stress condition. Reduction in GSH may enhance the susceptibility of the liver and kidney to pesticides toxicity 72. The administration of rotenone reduced GSH content in the liver which is in accordance with other published studies 17. Co-administration of quercetin restores the GSH content as compared to the rotenone alone group. Quercetin enhances GSH dependent protection and prevents the depletion of thiols during oxidative stress 73. In this study, co-administration of quercetin exerted an antioxidative effect, reversing the alteration in liver and kidney levels of LPO and GSH these effects might have been contributed to the observed different organ protection. The antioxidant role of quercetin may include the inhibition of free radicle, 46 Scavenging of O2-, OH^-, peroxyl radicals, and peroxynitrite 74.

SOD is known to be the first line of defense against the harmful effects of free radicals and it scavenges ROS by catalyzing the dismutation of superoxide to H₂O₂. Studies have shown that rotenone significantly decreased SOD activities in the liver 46. The inhibition of SOD activity may be the consequence of increased flux of superoxide in cells which may be the reason for the enhanced lipid peroxidative indices in our present work. Catalase serves as a preventive antioxidant and plays a major role in the prevention of the harmful effects of LPO. In the present study, there is a significant decrease in the activities of catalase and SOD in the liver and kidney of rotenone-treated mice were found. While co-treatment of quercetin enhanced the activity of catalase and SOD in the liver and kidney. Our study has an agreement with another study where oxidative stress-induced toxicity, by doxorubicin and cyclophosphamide in rat kidney and liver protected by quercetin 75.

The toxicity of rotenone was also confirmed by histological studies. Evidence from the study suggested that histological alteration in rat liver was observed in the rotenone exposed group 69. In this study, histological analysis revealed that rotenone induces striking pathological alteration in the liver, and signs of inflammatory response were observed as indicating by Kupper cell activation and leucocytes infiltration as compared to the control group. Co-treatment with quercetin leads to appear normal architecture of hepatocytes cell and reduction in Kupper cell activation were observed. Quercetin was found to reversed histological alteration induced by a high-fat diet in mice 76. Exertion of metabolite products occurs through the renal tubular epithelium, which might lead to xenobiotic-induced cell damage and hydropic degeneration of tubular epithelium and also able to induce inflammatory cytokine to excite lymphocytes and mononuclear cells infiltrated lesion. In the kidney, rotenone showed severe damage in renal corpuscles, renal tubules as compared to control while co-treatment with quercetin restore the normal appearance of renal corpuscles and renal tubules in the kidney. Protective effects of quercetin on histological changes in kidneys were observed, toxicity induced by methotrexate in rats 77. Scanning electron microscopic study further strongly supported our histological result of liver and kidney where we found severe damage and liver and kidney and protective effects of quercetin. These results suggest that rotenone is hepatotoxic and nephrotoxic and co-administration of quercetin successfully protected liver and kidney damage caused by rotenone.

In conclusion, the outcome of the present study showed that rotenone caused alteration in liver and kidney function biomarkers together with this also altered the biochemical parameters and induced inflammatory response as indicated by an increased level of No and presence of inflammatory cells. Medicinal plants with strong pharmacological properties have been found to play a key role in the health care system of huge amounts of the world’s populations. Comprehensive research on the chemistry and pharmacology of components of plant origin are essential and this may ultimately lead to the finding of medicine that can be used in dealing with numerous diseases. Biological products with antioxidant, anti-inflammatory activities allow the prevention of cells and tissues against harmful effects of free radicals by averting or governing the procedure of harm. In our study co-administration of quercetin leads to a reduction in damaged caused by rotenone. The possible mechanism of quercetin as a hepatoprotective and nephroprotective may be due to its anti-oxidant and anti-inflammatory. These results also suggest that toxicity of rotenone not only limited to the brain as reported by other literature but also affect the other organ which further contributes in neurological disorders due to liver and kidney dysfunction as suggested by various published data. Quercetin significantly protected the liver and kidney dysfunction, suggesting that quercetin can protect liver and kidney damage, maybe it further inhibit neurological disorders associated with liver and kidney dysfunction. This study might be helpful to the pharmacologist in the development of the drug in the treatment of PD through targeting the liver and kidney also. Further investigations are required to find out molecular mechanisms of quercetin and their action against rotenone or other pesticides with adverse effects on non-target organisms.

We thank Dr. Harisingh Gour University, Sagar (M.P) India for their support.

The authors didn’t receive any financial support for the research work, authorship and publication of this article.